abstracts from the international, congress on natural products research

88 SYMPOSIA SPEAKERS

S : 0 1 AFLATOXINS: THEIR BIOSYNTHESIS, !IOI.ECULAR RECOGNITIOK AND POSSIBLE ROLE IN SATURE T.E. Cleveland, D. Bhatnagar, J. Yu, Southern Regional Research Center, ARS, USDA, New Orleans, LA, and P-K Chang, Department of Cell

THE CHEMISTRY, BIOLOGICAL ACTIVITY AND SIGNIFICANCE OF s:02 1 FUMONISINS AND OTHER MYCOTOXINS FROM FUSARIUM I MONILIFORME Ronald D. Plan n g , USDA. Agricultural Research Service, MWA, National Center for Agricultural Utilization Research, Peoria, IL 61 604 (USA)

The fungus Fusarium moniliforme and several closely related species are found on agricultural commodities world-wide, especially maize. Although these species of fungi are associated with stalk and ear rot in maize, they can frequently be isolated from completely asymptomatic grain. Cultures of F. moniliforme grown on corn are hepatotoxic and cause liver cancer in rats. Fumonisin B,, the diester of a C20 amino penta-ol and tricarballylic acid are believed to be the cause of this hepatotoxicity and carcinogenicity. Since fumonisins were isolated and characterized with the aid of a short-term cancer initiation-promotion bioassay in 1988, there has been considerable work on this new mycotoxin family. Cultures of the fungus on maize can produce very high levels of fumonisins (2,000-6.000 parts-per-million). High levels (50-300 ppm) of fumonisins that can occur in naturally contaminated maize can cause significant animal toxicoses, especially equine leukeoencephalomalacia and porcine pulmonary edema. Detectable levels of fumonisin B, (0.05-1 ppm) can be found in most asymptomatic maize. The chemistry of fumonisins will be reviewed along with details of the proposed mechanism of toxicity in animals and plants. Laboratory cultures of F. moniliforme and other closely related species also produce sizeable amounts of several other mycotoxins. They may also have important implications in animal diseases associated with consumption of naturally contaminated animal feeds.

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SYMPOSIA SPEAKERS 89

s :04 -

MARINE TOXINS: HOW THEY ARE MADE AND WHY Jeffrey L. C. Wright, Institute for Marine Biosciences, National Research Council of Canada, Halifax, Nova Scotia, B3H 321.

Many aquatic organisms, like terrestrial organisms. produce metabolites that do not appear to play a role in primary metabolism. Such compounds are described as secondary metabolites, and marine toxins, or compounds that contaminate seafood resulting in human poisoning, can be classed as such a group of secondary metabolites. These toxins are not produced by seafood such as shellfish or finfish, but rather are the biosynthetic products of microorganisms that are ingested by, or associated with, these higher organisms. The chemistry of these toxins is varied and unique, and includes massive polycyclic ethers, linear structures containing cyclic ethers, macrocycllc lactones, as well as small heterocyclic structures. In cases where biosynthetic studies have been performed, the novelty of the structures is reflected in their biosynthesis. and unique assembly mechanisms must be proposed to account for the creation of these complex structures. The biosynthesis of several toxin groups will be reviewed, the long-range implications of these studies discussed, and in the case of the diarrhetic shellfish poisoning (DSP) toxins, a possible role for these toxins in nature will be presented.

S:03 I

THE VALUE-ADDED ROLE O F TOXIN DERIVATIVES IN MEDICINE, MARICULTURE, AND PUBI,IC HEALTH Daniel G. Baden

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S:O5 BIOLOGICALLY ACTIVE ALKALOIDS FROM POISON FROGS ;Lphn W. Daly, Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases,

THE USE OF SELECllVE PHORBOL ESTERS TO ELUCIDATE PROTEIN KINASE C FUNCTION

S:06 1 Fred J Evans, The School of Pharmacy, 29-39 Brunswck Square, London WClN IAX

The toxic plant derived diterpenes known as the phorbol esters have a range of biological effects in mammalian test systems ranging from their well known in vivo effects of tumour-promohon and induchon of inflammation to m v i m effects including amongst others induction of hyperplasia, mitogenesis, Epstein-Barr viral early antigen expression, differentiation and platelet aggregahon Their receptor(s) is believed to be the isotypes of protein-kinase C (PKC) which are broadly clarified into the group known as c-PKCs or calcium dependent forms, the n-PKCs or calcium independent forms and the a-PKCs or atypical group Accordingly, the phorbol esters are potentially powerful tools wth which to investigate the involvement of the various isotypes of PKC in several disease conditions In this communication several natural phorbol denvatives from plants of the Euphorbiaceae and Thymelaeaceae have been inveshgated for their abilihes to bind to PKC isotypes, induce transiocahon to the cellular membrane and achvate the membrane associated kinase These results have been correlated wth the various known toxicological effects of the phorbol derivatives used

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SYMPOSIA SPEAKERS 91

S:O7 Natural Products with Irmnanodulating and Antineoplastic Activity Gerhard Franz Institute of Pharmacy, University of Regensburg, 93040 Regensburg/Ger many

THE EhfERGING ROLE OF SMALL CYCLIC PEPTIDES IN CELLULAR S:08 1 BIOLOGY

Jim McAlpine, D-47P, AP9A, N e w Leads Discovery, Pharmaceuncal Products Division research and Development, Abbott Laboratones, Abbott Park. Illinois. 60064

Small cyclic peptides and peptolides have been isolated from virtually every type of organism Compounds of this type have been found in antibiotic or anticancer screens for decades but more recently they are being discovered wlth a variety of biochemical and pharmacological screens Some of these agents consist of solely "natural" and D aminoacids and others include unusual moieties wlth the unnatural aminoacid commonly an essenhal part of the pharmacophore They have been found as enzyme inhibitors, receptorfligand antagonists and transcripfional actwators Lipophilic side chams presumable act to anchor some in the membrane whereas in others the cyclic pephde appears to act as the anchor for a pharmacophoric side cham peptides can hold the ubiquitous peptide bond in an "abnormal" configurahon and use it to effect unique cellular processes

Simple cyclic

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S:09

THE CURRENT AND POTENTIAL INTEREST OF MARINE SUBSTANCES AS DRUGS

J.F. VERBIST Research group "Substances Marines a Activite Biologique (SMAB)", Faculty of Pharmacy, BP 1024,44035 Nantes Cedex 01, France.

Four points of view are dealt with ' Some general points on the interest of marine organisms as a source of new drugs. * Some successes . Protamine (heparin antagonist), Cephalosporin C (antibiotic), h a - A (antiviral drug) and Ara-C (antitumor drug). * Some disappointments : 15-epi-PGAZ (raw material for the semisynthesis of prostaglandins), I-methylisoyanosine (adenosine-like drug), Girodazole (antitumor drug) and perhaps Didemnin B (antitumor, antiviral and immunosuppressive drug) * Some current developments . Bryostatin 1 (antitumor compound), Dolastatin 10 (antitumor compound), Bistramides D and K (Antitumor and antimalarial compounds), Discocermolide (immunosuppressive compound) and Manoalide (anti-inflammatory compound). Eight other substances are also briefly reviewed.

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NOTHING IS PERFECT, NOT EVEN HERBAL REMEDIES Peter de Smet. PhD Drug Information Center, Royal Dutch Association for the Advancement of Pharmacy, Alexanderstraat 11, 2514 JL The Hague, The Netherlands

s:10 j There are many types of herbal remedies, ranging from self made teas prepared from self- collected herbs to officially registered drug products. The use of these herbal remedies entails both indirect and direct health risks. The former type occurs, when a herbal remedy with unclear therapeutic potential delays or replaces an effective form of conventional treatment. This may result when a headstrong patient puts too much trust in the healing powers of nature or when a herbal practitioner is overly optimistic. The direct nsks of herbs fall into four categories. -TvDe A reactions which are pharmacologically predictable and usually dose-dependent; - TvDe B reactions, or idiosyncratic reactions, which cannot be predicted on basis of the principal pharmacological properties and which do not show a correlation between dose level and risk of toxicity These reactions occur in only a minority of the population, but they are often serious and potenbally fatal; -Twe C reactions which develop during long-term therapy. These reactions are well-described and could be anticipated; and - TvDe D reachons which consist of delayed effects, such as carcinogenicity and teratogenicity. While herbal healers and consumers will readily recognize type A reactions, it may be less easy for them to identify type c. and it will even be more difficult to detect a type B or type D reaction. Safety claims cannot always be based on traditional empiricism as not all herbal remedies are firmly rooted in traditional medicine Quality assurance is another major problem. Consequently, we need to widely disseminate the available facts, insitutue and enforce quality assurance and conduct research to establish benefivrisk ratios of most herbs.

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SYMPOSIA SPEAKERS

s: 12

93

OVERVIEW OF SNAKE VENOMS CHEMISTRY

RECENT DEVELOPMENTS IN MARINE Takeshi Yasumoto Faculty of Agriculture, Tohoku University, Amamiya, Aoba, Sendai 98 1, Japan

TOXIN STUDIES S: l l 1 In ciguatera studies, a persistent question exists as 10 whether a single toxin or a single

group of toxins is responsible for the wide range of clinical symptoms observed in patients. Alternatively the question may be asked whether hundreds of fish species listed as toxic in the literature contain the same toxin(s). After determining the structure of ciguatoxin (CTX), we tried to answer the question by isolating as many toxins as possible from toxic moray eels and the causative dinoflagellate Gumbierdiscus foxicus. Five toxins isolated shared with CTX the same structural and pharmacological features. Gambierol and maitotoxin differed from CTX chemically and pharmacologically.

Unlike other ciguatoxic fish, trigger fish cause severe intoxication involving fatality cases. Palytoxin existed at a high concentration in the liver of the fish collected in Micronesia, explaining the high mortality rate. Palytoxin occurred even in small herbivores such as surgeon fish. The question as to the origin of palytoxin in these fish was answered by our identification of palytoxin in an epiphytic dinoflagellate Ostreopsis siamenris.

Sharks have been suspected to contain different toxins because of the severity of the intoxication and the absence of temperature reversal sensation. Recently as many as 188 people were hospitalized in Mallagascar after eating flesh and/or liver of a single shark. The mortality rate was 30%. The causative toxin(s) in the remnant was shown to differ from C T X s .

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s : 1 3 COMPOSITTON, MEDIClNAL USES AND PHYTOCHEMICAL ANALYSIS O F GINKGO BILOBA EXTRACTS Teris A van Beek

Ln the last 10 years the sales of phytopharmaceutical leaf preparations of Ginkgo biloba (maidenhair wee) have increased in Europe to several hundred million US $ per year. This has stirred the interest of many botanists, pharmacologists, biochemists, phytochemists and plant cell biotechnologists.

Ginkgo biloba is the sole survivor of a once large class of plants. 'his special taxonomic position gives rise to some peculiar botanical properties and some unusual secondary metabolites. 'Ihe most unique ones are a number of highly oxjdised terpene trilactones (ginkgolides and bilobalide). They are the only known natural products with a tertiary butyl group. Other more common products present in leaves are flavonol glycosides and esters thereof, biflavonoids, organic acids, steroids and sugars. Small quantities of highly irritating and allergenic alkylphenols may be present too.

The two main indications for prcsaibing extracts of ginkgo leaves are cerebral insufficiency (forgetfulness, dizziness, tiredness) and problems with the peripheral blood circulation (intermittent claudication), mostly in older people. Many well performed clinical trials with a partially purified extract confirm its use although some critics remain. Both the flavonoids (24%) and the terpenes (6%, selective and potent Platelet-Activating-Factor antagonists) are considered to be of importance for the activity.

Thc analysis of the fiavonoids poses no problems but for the ginkgolides it is different matter. A simple, reproducible and selective clean-up method is not yet avalaible. Some different approaches for the final quantitation will be discussed, e.g. HPLC with W-, RI- or MS detection, GC with FI- or MS detection or NMR. Application of these methods on leaves of different origin has shown that leaves differ up to a factor 300 in their terpene content and that standardisation of phytopharmaceulicals is therefore a must For qualitatively screening extracts a TLC method which makes use of silica gel plates impregnated with sodium acetate seems to hold promise. The impregnation facilitates both the separation and detection. The impregnation technology could be transferred to MPLC columns for a fast prep-scale purification of the terpene trilactones.

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AWARD RECIPIENTS

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96 AWARD RECIPIENTS

A:-) 1 A MAlTER OF SOME SENSITIVITY J David Phillioson Department o f Pharmacognosy, The School of Pharmacy, University of London,

LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY: A NEW ANALYIICAL STRATEGY FOR A RAPID SCREENING OF CRUDE PLANT EXTRACIS. Jean-Luc Wolfender, lnstitut de Pharmacognosic ct Phytochimie, Ecole de Pharmacie, Universite de Lausannc, CH-1015 Lausannc, Switzerland

A:02 1 Liquid chromatography-mass spectrometry (LC-MS) is new and powerful analytical techniquc The cornbination of liquid chromatography (LC) with mass spectrometry (MS) offers the possibility of taking advantage of both chromatography as a separation method and mass spectrometry as an identification tool LC-MS plays diffcrent rolcs in phytochernical analysis It can be used as a "universal" detector for LC, especiall) for compounds having weak chromophore that arc difficult to analyse by conventional LC- UV Furthermore the selectivity of MS detection, in addition to the rcsolution of chromatographic separation, permits a precise assignment of quantification on non isomenc co-eluting compounds in extracts In addition to its sensitirie and selcctivit) of detection, LC-MS provides important mass spectroscopic dormation on metabolites of interest This on-line information is strategic for the early rccognition of know compounds in an extract In this respect, the combination of LC-MS data with those of LC-UV can be of particular intercst due to their complementanty phytochemists to avoid thc timc-consuming isolation of common natural products and, at the samc timc, to localise lnteresting new ones As no universal interface for LC-MS is yet avulablc hvo complementary techniqucs thermospray (TSP) and continuous flow FAB (CF-FAB) habe been employed They hare permitted the analysis of a broad range of plant metabolites (100-3000 amu) both interfaces for the analysis of crude plant extracts will be prcsentcd and the complemcntanty of the LC-UV and LC-MS detection methods will be discussed The usc of thesc combined LC techniques as a rapid and cfficient screening method and their rolc for the carly recognition of metabolites in crude plant extracts will be espcciallq cmphasied

These on-line data permit

In this communication, vanous examples of utilisation of

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AWARD RECIPlENTS 91

I MARINE NATURAL PRODUCTS RESEARCH: A LOOK I N T O THE D I V E BAG

n

While the s e a r c h f o r b i o a c t i v e n a t u r a l p roduc t s i n t e r r e s t r i a l ecosys tems has been c a r r i e d ou t w i th no tab le success du r ing much of t h i s c e n t u r y and w i l l continuq t o be p roduc t ive i n the f u t u r e , e x p l o r a t i o n of mar ine sou rces of n a t u r a l p roduc t s has b a r e l y begun. The p o t e n t i a l f o r new chemica l and b iomedica l i n s i g h t s from t h e s e v i r t u a l l y untapped r e s o u r c e s is he igh tened by t h e f a c t t h a t two- th i rds of t h e e a r t h i s water and t h a t unexamined b i o t a e x i s t i n a l l h a b i t a t s and a t eve ry t r o p h i c l e v e l . The l e c t u r e w i l l i l l u s t r a t e t h i s theme wi th examples drawn from a mid-Pac i f ic vantage p o i n t .

CENTRIFUGAL PARTITION CHROMATOGRAPHY - A PREPARATIVE TOOL FOR THE RAPID SEPARATION OF PLANT-DERIVED NATURAL PRODU<JTS

A:04 I Andrew Marston Institut de Pharmacognosie et Phytochimie. &ole de Pharmacie. Universid de Lausanne, CH-1015 Lausanne-Dorigny (Switzerland)

Techniques for the partition of a solute mixture between two immiscible liquid phases have undergone radical changes since the emergence of the Craig apparatus for countercurrent distribution some fifty years ago. By introducing the concept of a spinning coil describing planetary motion around a cenaal axis, it has been possible to construct compact insuuments for performing centrifugal partition chromatography (CPQ [l]. Certain of these instruments have proved ideal for the separation of natural products. especially in cases involving biologically active material and unstable or readily decomposable compounds [2]. Lack of a solid chromatographic support is the most important advantage of CPC for these samples.

Operation with two pumps (for separate delivery of mobile and stationary phases) increases the versatility of the system, allowing gradient elution and conml of stationary and mobile phase volumes.

CPC is perfectly suited to the fractionation of crude extracts and has the ability to handle both apolar and highly polar samples.

While numerous applications of the method have already been described, there is continuing scope for further exploitation of CPC, both in exploring separation capabilities and in development of instrumentation.

[ 11 N.B. Mandava and Y. Ito, Countercurrent Chromatography - Theory and Practice.

[2] A. Marston and K. Ilostettrnmn. J. Chromatogr. 658. 315 (1994). Marcel Dekker, New York, 1988.

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YOUNG INVESTIGATORS

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YOUNG INVESTIGATORS 100

y:o1 I Schizotrin A, a Novel Antimicrobial Cyclic Peptide from a Cyanobacterium

lnna Pergament and Shmuel Carmcli*

School of Chernisuy. Raymond and Beverly Sackler Faculty of Exact Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, ISRAEL

diffcrent amino acid profile, share a common segment; a prolinc residue attached to thc 2-hydroxy-3-amino-long chain acid residue. It is possible that this segment is res biological activity of thcse compounds.

MOLECULAR MECHANISMS OF ACTION OF MICROCYSTINS AND NODULARINS, POTENT MARINE AND FRESHWATER TOXINS - MRC Protein Structure and Function Group. Department of Qiochemisuy, University of Alberta, Edmonton. Alberta, Canada T6G 2H7

Microcystidnodularin peptide hepatotoxins are equipotent inhibitors of eukaryotic type-1 and -2A protein phosphatase catalytic subunits (PP-lc and PP-2Ac) and potent Liver tumor promoters. We have identified an important functional difference between the molecular mechanisms of these cyanobacterial toxins and their intracellula phosphatase targets. Microcystins interact with PP-lc and PP-2Ac by a two-step mechanism involving rapid binding and inactivation of phosphatase catalytic subunit, followed by a slower covalent interaction between a nucleophilic phosphatase residue and N-methyldehydroalanine in the heptapeptide toxin. In contrast to the microcystins. following rapid inactivation of PP- Ic/PP-ZAc by nodularins, the equivalent N- methyldehydrobutyrine residue in these pentapeptide toxins is unreactive and does not readily form a covalent bond with PP-lc/-ZAc. These results are consistent with our determined NMR solution structures of microcystins and motuporin (a hydrophobic nodularin analogue), which indicate a striking difference in the relative positions of N-methyldehydroalanine versus N- methyldehydrobutyiine in the toxin peptide backbone. The implications of these findings will be discussed with respect to the occurrence. role and fate of microcystinshodularins in the marine environment.

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YOUNG INVESTIGATORS 101

Y :03 NOVEL TECHNIQUES FOR GROWTH CHARACTERIZATION AND PHYTOCHEMICAL ANALYSIS OF PLANT CELL SUSPENSION CULTURES.

Y:04 INVESTIGATION OF ENDOPHYTIC FUNGI OF THE NORTHWEST

ANTICANCER DRUG, TAXOL PACIFIC YEW TREE, TAXUS BREVIFOLIA, AS A SOURCE OF THE

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ORAL PRESENTATIONS

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104 ORAL PRESENTATIONS

CALYCOPTERONES: THREE NOVEL CYTOTOXIC BIFLAVONOID CONSTITUENTS OF CALYCOPTERIS FLORIBUNDA. W o e F Wall,1 Mansukh C. Wani,' Fekadu Fullas,' John B. Oswald.' Dan M. Brown.' Thawatchai Santisuk,2 Vichal Reutrakul? Andrew T. McPhail? Norman R. Farnsw~rth.~

0:l j John M. Pezzuto? A. Doughs Kinghorn? arid Jeffrey M. Besterman6 ' Research Triangle Institute. P.O. Box 12194, Research Triangle Park, North Carolina 27709

*Royal Forest Herbarium, Bangkok 10900, Thailand 3Mahidol University. Department of Chemistry, Bangkok 10400, Thailand

5Universlty of Illinois at Chicago, Department of Medicinal Chemistry and Phannacognosy, College of

6Glaxo IN., Research Triangle Park. North Cardina 27709

Three novel billavonoids (1-3) and a bmwn flavone (4',5dihydroxy-3,s,6,7-tetrarnet~~hvone) (4) were isolated by cytotoxicily-guided fractionation of a 50% MeOWCHClg-sduble extract of the flowers of

Calywpteh lloribunda (Combretaceae). The structures of the new c~npounds (1-3) were elucidated by the application of ID- and 2D- NMR spectroscopic techniques, with x-ray crystallography being used to ascertain the structure of calycopterone (1). The structural features of the calywpteronas appear to be unprecedented in billavonoids in that ring A-2 is not aromatic. is functionalized, and has an exocyclic double bond. Compounds 1 4 were evaluated for cylotoxkity in a number of solid tumor cell lines. Details of isolation. spectral, and biological properties will be presented.

(Supported by UO1 CA52956 from

Duke University, Department d Chemistry, Durham. North Carolina 27706

Pharmacy, Chicago. Illinois 60612-7231

0 The National Cancer instiiute) R PI R1

C.bCOQlmW(l) Mc H Ma L~<rlysoQlsoorOl Ma Mo It LDamalytlbrap(mnr(J) H I1 HI

0:2 ISOLATION AND STRUCTURE ELUCIDATION OF NOVEL NATURAL CYTOTOXIC AND DNA-DAMAGING AGENTS

A. A Leslie Gunatilaka George G. W g a n . Vandedan da

State University, Blacksburg. Virginia 24061-0212 (USA), and R. K. Johnson, SmithKline Beecham Pharmaceuticals, K i n g o f Prussia, Pennsylvania, 19406- 0939 (USA).

o f Chemistry, Virginia Polytechnic Institute and

The discovery of novel lead compounds for development as anticancer agents is a challenging task As one approach to this challenge, we have used a highly selective yeast-based bioassay for DNA-damaging agents; this assay will also detect inhibitors o f topoisomerase I and Il. The use o f this assay, coupled with standard cytotoxicity assays, has led to the isolation of various alkaloids. naphthoquinones, cytochalasins, pterocarpans, and other compounds from a variety of terrestrial and marine organisms. The basic principles of the assay will be described, and examples o f the compounds that have been isolated will be presented.

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ORAL PRESENTATIONS 105

I ~

BORTIFACIENT AND TOXIC COMPONENTS OF PONDEROSA PINE NEEDLES. 013 bale R. Gardner, Russell J. Molyneux, Lynn F. James, Kip E.

USDA/ARS/Poisonous Plant Research Laboratory, 1150 E. 1400 N., Logan Utah, 84321 (USA)

anter, Bryan L. Stegelmeier and James A. Pfister.

Ingestion of ponderosa pine needles by cattle during late term pregnancy is known to induce abortions. The abortion is characterized by weak uterine contractions, incomplete cervical dilation, and excessive uterine hemorrhage. Calves maybe born alive, but are small, weak and their survival depends on prompt intensive care. It is believed that pine needles are sometimes toxic causing depression, decreased appetite, rumen stasis and may lead to death in 6ome cases. In spite of research efforts over the last 20 years the toxic/abortifacient components remained unknown. Using a bovine-assay directed approach we have isolated and characterized an abortifacient active compound of ponderosa pine needles. Needles from ponderosa pine trees were collected, processed and chemically extracted and fractionated. Individual fractions were tested in feeding trials with pregnant cows. A single active component was eventually isolated and identified as isocupressic acid, a labdane diterpene acid. This compound was tested in feeding trials with five cows at dosage levels ranging from 66 to 152 mg/kg. An effective abortifacient dose was estimated to be 100 mg/kg (given 2x/day for 2 to 8 days). Similar labdane acids isolated from ponderosa pine needles may also be abortifacient, while the abietane type acids were found to be toxic causing depression, decreased appetite if administered at high enough dosages.

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q--- ISOLATION AND CHARACTERIZATION OF VASOACTIVE

Donna B. Farlee and John P. N. Rosazza1. Division of Medicinal and Natural Products Chemistry. and Center for Biocatalysis and Bioprocessing, College of Pharmacy' and Department of Obstetrics and Gynecolo&. University of Iowa, Iowa City, Iowa 52242, Department of Animal Science'.. Iowa State University, Ames. Iowa 50011 and USDA-ARS, Fort Keogh Livestock and Range Research Laboratory3. Miles City. MT 59301

Ingestion of Ponderosa pinc needles initiates a premature parturition in late pregnant beef cows. Recently, blood plasma obtained from cows fed Ponderosa pine needles was shown to elicit a vasoconslrictive response when perfused through the microvasculature of an isolated placentome (i.e.. site of maternal-placental attachment and nutrient exchange). Furthermore, cows fed pine needles exhibited a profound decrease in uterine blood flow concomitant with the induction of a premature parturition. Utilizing the in vitro perfused placentome of the late pregnant cow as a bioassay wc have conducted a series of fractionation, soxhlet extraction and chromatographic separation techniques to isolate the vasoactive component in pine needles. Spcctral (l13- & 13C-NMR. IR, high and low resolution FAE3 mass spectrometry), GC/MS, MS/MS, and chemical methods including saponification and derivatimtion were used to identify diesters of myristic and lauric acids with 1.14-tetradecanediol.

CONSTITUENTS OF PINUS PONDEROSA Mohsen S. Al-Mahmoud', Stephen P. Ford'.. Robert E. Shorts.

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0:5 1 NEW BIOACTIVE N A T U R A L PRODUCTS FROM TURKISH MEDICINAL PLANTS

BIlqe SENER Gazi Universi ty Facu l ty o f Pharmacy Department o f Pharmacognosy P.K.143 06572 Maltepe-Ankara( TURKEY)

Turkey has a very r i c h f l o r a and a long h i s t o r y o f h e r b a l medicine p rac t i ces .The i d e n t i f i c a t i o n o f t h e a c t i v e compounds w i t h i n h e r b a l mixtures becomes an important a r ea of r e sea rch d i r e c t e d toward t h e development o f l e a d s f o r novel medicinal drugs. Our ex tens lve s t u d i e s with Turkish medicinal p l a n t s , we have i s o l a t e d and c h a r a c t e r i z e d a l a r g e number o f t h e n a t u r a l p roduc t s p re sen t and have submit ted t h e s e t o pharmacological s c reen ing i n o rde r t o l o c a l i z e t h e a c t i v e compounds. Our cu r ren t r e sea rch programme on Fumaria, Corydal is , Veratrun, &rxus, Senecio, Consolida, F r i t i U a r i a , S ~ h y t u n and Amaryllidaceae p l a n t s ha5 r e c e n t l y l ed t o t h e i s o l a t i o ? and s t r u c t u r e e l u c i d a t i o n o f new a long with known n a t u r a l compounds. During t h e cour se o f t h e p a s t few yea r s , some o f t h e s e l e c t e d medicinal p l a n t s have been eva lua ted for t h e i r a c t i v e c o n s t i t u e n t s through an a c t i v i t y - d i r e c t e d f r a c t i o n a t i o n procedure. They have been screened for t h e i r a n t i - p l a t e l e t , an t ihype r t ens ive and ant i inf lammatory e f f e c t s . I n t h i s s tudy , da t a w i l l be presented on t h e above mentioned a s p e c t s and t h e s t r u c t u r e s o f new b i o a c t i v e compounds belonged t o a l k a l o i d s , t e rpeno ids and sapon ins w i l l be discussed.

1% 1 RECENT RESULTS ON NEW CONSTlrIJENTS FROM THE CHINESE HERBAL MEDICINE De-quan Yu Institute of Materia Medica,Chinese Academy of Medical Sciences and Peking Union Medical College,Reijing 1000M.P.R.China.

China is noted fo r herherbal medicine with a long history of tradit ional medical practice.Modern studies m the effective principles by Chinese scientists have been quite extensive.

Recent years in our laboratory,about 80new constituents were isolated and identified ftun Chinesemedicinal p1ants.A large portion ofthem has biological activities to various exIents.ln this paper,we showed a brief account of some recent results.From Altemisia and Chrysanthenm species,we isolated six sesquiterpenoids,two of them showed significant anti-inflammatory activity;Six new polyepoxyl diterpenes having strong immunosuppresive activity were isolated from Tripterygium species:Two novel structuresof triterpene compounds were dicovered from Ganoderma spore,Its showing a heptoprotective activity; From Hypodematium species,two novel structures were identificd:More than do7en novel protoilludane norsesquiterpenoid esters wereisolated which also have some biological activities .From plygala species,we isolated a new bisdesmoside tritcrpene saponin containing eight carbohydrate residue.The structures ofall compounds given ahove were determined mainly by spectral data especially modern NMR techniques.

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0:7 MODULATORS OF THE RYR-I Ca2+ CHANNEL FROM IAMHELLA BASTA. -, Department of Chemistry; Matthew Mack, Edmond

~ ~~

Topoisomerase II-Mediated DNA Cleavage by Adociaquinones from the Sponge Xestospongia sp.

Gisela P. Conception,' Tommaso A. Foderaro,' Glenn S. Eldredge: Louis R. Barrows' and Chris M. Ireland'

1) Marine Sciences Institute, University of the Philippines, Diliman, Quezon City, Philippines and 2) the Departments of Medicinal Chemistry and Pharmacology and Toxicology, University of Utah, Salt Lake City, UT 84112

A family of adociaquinone alkaloids was isolated from an orange sponge (Xestospongia sp.) collected at Cape Bolinao in the Philippine Islands. The structures of these metabolites were established using spectroscopic methods.

Members of the family showed varying degrees of cytotoxicity towards HCT 116 human colon tumors cells. Pharmacological investigations indicate that the cytotoxicity is likely due to formation of topoisomerase I1 cleavable complexes. Evidence will be presented that cleavable complex stabilizations do not involve drug-DNA intercalation.

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ORAL PRESENTATIONS

CIRCULINS A AhD B, NOVEL HIV-INHIBITORY MACROCYCLIC PEPTIDES FROM THE TROPICAL TREE Chassalia parvifolia Kirk R. Gustafson, Ian C. Parsons, Yoel Kashman, John H.

Cardellina 11, Michael R. Boyd Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute-Frederick Cancer Research L Development Center, Bldg. 1052, Rm 121, Frederick, Maryland 21702-1201 ( U S A )

Anti-HIV bioassay-guided fractionation of an extract from the tropical tree Chassalia parvifolia provided circulins A and B as active constituents. Circulins A and B were shown to be novel, macrocyclic peptides containing 30 and 31 amino acid residues, respectively. Their amino acid sequences and cyclic structures were definitively established by a combination of endoproteinase digestion, derivatization, N-terminal Edmar. degradation and FAR mass spectral analyses. Circulins A ar.d B are currently the iargest known naturally occurring peptides in which the primary anino acid chain is covalently cyclized via peptide bonds. They exhibit some sequence homology with the human B-Cell antigen CD22 and they share key structural features with the defensin Class of linear peptides. For circuilns A and B, the effective concentration that provided 5 0 8 cyt-oprotection (EC,,) against in vitro HIV infection ranged from about 40 to 250 nK, depending on the particular virus strain and host cell lir?e used.

D:9 1

CHEMOTAXONOMIC STUDIES OF Calophyll um EXTRACTS FROM THE NCI COLLECTIONS 0:lO I Tawnva C. McKee, Michael R. Boyd

Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, Frederick Cancer Research L Development Center, 91dg. 1052, Rm 121, Frederick, MD 21702-1201 ( U S A )

Calanolide A, a recently disclosed HTV-inhibitory ccumarin from the Malaysian rainforest tree Calophy.llm lanigerum var. austrocoriaceum, represents a novel subset of HIV-1 reverse transcriptase inhibitors. Based on this uniquc biological activity, calanolide A has been committed to preclinical development by the U . S . National Cancer Institute. In ordcr to establish adequate supplies of the compound for preclinical and clinical studies, a survey of the NCI Calophyllum collections was undertaken to distinguish those extracts which contain calanolide-type coumarins, and to identify the coumarjns present. Results from these studies will be discussed, including the isolation of costatolide fron the latex of Calophyllum feysmdnnii var. inophylloide, currently being evaluated as a possible alternative to calanolide A for drug development.

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~ ~ ~ ~~~~~~ ~~~~ ~~ ~~ ~~

ANTIMALARIAL ACTIVITY: 311 1 1 COLLECTION AND INVESTIGATION OF MARINE ORGANISMS FOR THEIR POTENTIAL AS SOURCES OF NEW LEADS

FOR ANTIMALARIAL COMPOUNDS Gabriele M. Konig, Anthonv D, Wr ioht

Department of Pharmacy, Swiss Federal Institute of Technology (ETH), Zurich, CH-8057 Zurich, Switzerland, Cindy K. Angerhofer and John M. Pezzuto, Program for Collaborative

Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago,Chicago,lllinois, USA.

4 total of 44 specimens belonging to the Phyla; Phaeophyta (2). Rhodophyta (3), Chlorophyta (l), >hordata (3). Porifera (28), Coelenterata (6) and Mollusca (1) , were collected from the Caribbean ind the eastern seaboard of Australia, during 1992-1993. From each of the collected samples I small piece ( 2 g) was taken and a dichloromethane and methanol extract prepared. These !xtracts were then assessed for their cytotoxicity and antimalarial activity. Of the tested samples w r , three Porifera and one Rhodophyta dichloromethane extracts demonstrated effects that vere considered positive in terms of selective antimalarial activity. Bulk extraction of one of the 'orifera and the Rhodophyta specimens was then undertaken. The dichloromethane extracts ibtained were then fractionated by vacuum liquid chromatography. A portion of each of the esultant fractions ( 5 mg) was subjected to further biological screening and the active fraction urther purified to yield a series of compounds, some of which are proposed to be responsible for i e observed antimalarial activity of the initial dichlorornethane extracts.

ANTILLATOXIN AND MALYNGAMIDE H, POTENT ICHTHYOTOXINS FROM THE MARINE CYANOBACTERIUM LYNGBYA MAJUSCUU Jimmy Orialq, Dale Nagle and William H. Gerwick College of Pharmacy, Oregon State University, Corvallis, Oregon 97331 (USA)

0:12 1 Biological evaluation of the crude organic extract from the marine cyanobacterium L. majuscula, collected in Curacao, showed a pronounced ichthyotoxic effect. Bioassay- guided fractionation on silica gel, Sephadex LH 20 and ODS silica gel led to the isolation of a new cyclic depsipeptide, antillatoxin, as the most potent principle. Further, a new and less potent ichthyotoxic amide of 7-methoxytetradec-4(E)-enoic acid (Malyngamide H) was found to coexist in the cyanobacterium. The structures were established by a combination of 2D NMR techniques, chemical degradation and mass spectrometry. This presentation will report on the isolation, structure elucidation and biological evaluation of these new metabolites.

Malyngamide H

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I10 ORAL PRESENTATIONS

D:13 DISCOVERY OF THE CURACINS, NOVEL ANTIMITOTIC AGENTS FROM THE TROPICAL CYANOBACTERIUM LWGBYA MAIIISCCIU

iham H. Gerwick, Hye-Dong Yoo, Dale Nagle, Jimmy Orjala, Mary Ann . .

Cancer Treatment, National Cancer Institute, National Institutes of Health, &the&, Maryland 20892 (USA)

We recently reported on the planar structure and biological activity of an extremely potent antiproliferative agent (ID50 = 1-10 nM in various cell lines), curacin A (I), obtained from a field collected cyanobacterium (blue-green alga), Lyngbyu mujuscula (J . Org. Chem. 1994, 59, 1243-1245). The relatively simple structure of curacin A is remarkable in its juxtaposition of thiazoline and cyclopropyl rings. Its antiproliferative effects are explained by its potent inhibition of tubulin polymerization through binding to the colchicine site. This presentation will report on our continuing efforts to determine the relative and absolute stereochemistry of curacin A, as well as of five naturally-occurring analogs, curacins B-F. The cytotoxicity and in vivo antitumor effects of these new antimitotic agents will be presented. Laboratory cultures of this microalga retain their capacity to produce curacin A.

PARTIAL STRUCTURES AND BIOLOGICAL PROPERTIES OF PRYMNESINS ISOLATED FROM THE PHYTOPLANKTON PHYMNESIUM PARVUM omoii -*I, Masayuki satake':! and Takeshi Yasumoto'z

0:14 1 *]Japan Food Research Laboratories. 52- 1 Motoyoyogicho. Shibuya-ku,Tokyo 15 1 ; '2Faculty of Agriculture, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi. Aoba-ku, Sendai 981, Japan

Massive fish kills by a bloom forming phytoplankton Prymnesruni purvum. has been known in many parts of the world since 1930s. Especially in recent years the organism is posing a serious threat to salmon culture in Norway. Though number of reports have been made on the biological activities of the ichthyotoxin named prymnesin, the toxin has never been purified successfully. Hence chemical structure o f the toxin has remained totally unknown.

Recently we succeeded to isolate extremely potent two hemolytic compounds, prymnesin- I and prymnesin-2, and demonstrated that they were loo0 times more potent than plant saponin. Though morc than 10mg of each prymnesin was obtained, their extremely low solubility in NMR solvents forced us to prepare N-acetates for IH-NMR measurements (COSY, HOI4AHA. NOESY). Further isolation of 1.5mg each of 13C-enriched(ca 5%) N-acetylprymnesins from 200L culture enabled us to measure I,C-NMR (BBD, DEPT) and HSQC,HMBC spectra. Consequently 96 carbon signals of N- acetylprymnesin-2(2 methyls. 24 methylenes, 10 olefinic methines, 53 other methines, 7 quaternary carbons) have been assigned. Together with the IH-NMR data the polyhydroxy-polyene-polyether- glycosidic nature of prymnesin-2 was disclosed for the first time. N-acetyl prymnesin-l probably contains an additional pentose and'hexose. In FAB-MS and ESI-MS. [M+H]+ ions were observed at m/z 2,266 for prymnesin-1 and at m/z 1,972 for prymnesin-2. When tested on Tunichfhys ulhonubes. the ichthyotoxic activity was enhanced 4 folds by addition of CaClz(2mM) to water. Thus, Ca ion was suggested to play an important role i n fish kills. lchthyotoxicity of prymnesins in the presence of Ca ion was comparable to those of the brevetoxins. Both prymnesin-l and prymnesin-2 induced Ca ion influx into C6 rat glioma cells at 70-XOnM concenuation.

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ORAL PRESENTATIONS 1 1 1

~ I FROM Portieria hornemannii WITH A NOVEL IN VITRO ANTITUMOR RESPONSE PROFILE

Richard W. Fuller, John H . Cardellina 11, Jaroslaw Jurek, Paul J. Scheuer, Belinda Alvarado-Linder, Mary McGuire, Glenn N. Gray, Jorge Rios Steiner, Jon Clardy, Ernani Menez, D. John Newman, Kenneth M. Snader, Michael R. Boyd Laboratory of Drug Discovery Research and Development, Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute; Natural Products Branch, DTP, DCT, NCI, Frederick Cancer Research L Development Center; Program Resources Inc./Dyn-Corp, Frederick, Maryland 21702-1201; Department of Chemistry, University of Hawaii, Honolulu, HI; Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY; The Smithsonian Institute, 1000 Jefferson Drive SW, Washington, D.C. (USA)

Eleven halogenated monoterpenes related to halomon have been isolated from the red alga Portieria hornemannii ( 8 from the Philippines, 3 from Hawaii). Structures were assigned on the basis of spectral analyses (primarily NMR and MS) and the compounds were evaluated in the U.S. National Cancer Institute's in vitro human tumor cell line screening panel. The structure of isohalomon was confirmed and its absolute configuration established by x-ray crystallography. The results of these studies have provided interesting preliminary insight into the

N E W POLAR TOXINS FROM MARINE DINOFLAGELLATES BELONGING TO THE GENUS PROROCENTRUM ma, Y . Oshimab. J.M.Curtisa, J.A. Waltera, A.S.W. deFreitasa and J.L.C. Wright= aNational Research Council of Canada, Institute for Marine Biosciences, 141 1 Oxford Street, Halifax, Nova Scoua, Canada B3H 321; bDepamnent of Applied Biological Chemistry, Faculty of Agnculture. Tohoku University, 1-1 Tsutsumidori Amamiyamach, Aoba-ku, Sendai 981, Japan.

Various Prorocentrum species of marine dinoflagellates have been associated with thc production of shellfish toxins, in particular a group called the diarrhetic shellfish poisoning (DSP toxins, a class of unusual polyether metabolites. These lipid-soluble compounds are phosphatasc inhibitors that can induce powerful biological effects in eukaryotic cells, and several analogue: and ester derivatives have been isolated and idenhfied over the years.

Recently, we have examined the very polar butanol soluble fractions, which also showec potent activity in the mouse bioassay. Following standard bioassay-guided fractionation procedures, several active compounds have been isolated from the butanol-soluble fractions of the methanol extracts of both Prorocentrum lima and Prorocentrum rn aculosum . These compounds appear to be the agents responsible for "fast acting toxicity" in Prorocentrum species, a biological effect that was reported some time ago by various groups.

The general isolation strategy, some highlights of the structure elucidation, and mode of action of these new polar toxins will be presented.

0:16 1

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0:18

PURIFICATION, cDNA CLONING AND ELICITOR INDUCTION OF ANTHRANILATE SYNTHASE FROM RUTA GRAVEOLENS L.

0:17 I J. Bohlmannl.3. V. DeLuca2, U. Eilertl and W.F. Martin3 ’lnslilul fiir Pharmwmtische Biologic, l l J Braun~chweig, Mendelssohnsu. I , D-38106 Braunschwcig. FRG 21nstiult de recherche en biologic vegktaie, 4101 est me Sherbrooke. Monubal H I X 2B2, Canada 31nsulut far Genet&. TU Braunschwcig, Spielmann SU. 7. D-38023 Brdunschweig. FRG

Anthranilate Synthase (AS) catalyzes the first specific reaction in the biosynthesis of tryptophan. In R. gruveolens AS also provides the direct precursor for the biosynthesis of two groups of elicitor inducible alkaloids, acridone epoxides and furoquinolines. Elicitor induction of AS is limiting for the level of elicitor inducible alkaloid accumulation in in virro systems of the Rutaceae. Therefore R. graveolens provides an excellent system to study the regulation of substrate flux between primary and secondary metabolism at the site of this key branchpoint enzyme. AS was purified from plants and cell cultures of R. graveolens. SDS-PAGE and silver staining of the purified enzyme revealed two protein bands of 61 and 62.4 kD, respectively, which correspond with the glutamine dependent AS (AS-Gln) activity. By gelfiltration a molecular weight of 65 kD was determined for native AS- Gln. Two different cDNAs were isolated for AS component I of R. graveolens. The deduced amino acid sequences show homology to each other and to the two AS1 genes from A. thuliana. I n viiro translated AS1 protein is directed into the stroma of isolated chloroplasts by amino terminal transid peptides. AS1 from Rum graveolens complements the rrpE and rrpED deletion mutant strains of E . coli. The native molecular weight for the recombinant AS-Gln was determined by gelfiltration to be 63.8 kD. The expression of the two AS1 isoenzymes in response to elicitation was investigated by northern analysis. By making recombinant fusionproteins with the two R. graveolens AS1 cDNAs we are establishing the tools for the immunological and biochemical characterization of plant AS isoenzymes.

BIOSYNTHESIS OF CYTOTOXIC LIGNANS IN SUSPENSION CULTUKES OF LINUM ALElJM

T. Smollnyl, M. Petersenl, H. Wichen2. A. Shahsavari3, and A.W. Alfermml

llnstitut f i r Entwicklungs- und Molekularh’ologie der Pflanzen, Heinrich-Heine-Unive~i~t Diisseldorf, D-402 5 Dusseldorf (Germany); lATO-DLO, Haagsteeg 6, NL-6700 Wageningen

Etoposide and teniposide are important drugs using in the treatment of dangerous types of cancers like acute lymphocytic leukemia and others. Both compounds are semisynthetic derivatives of podophyllotoxin which is still extracted from plants of Podophyllum species collected in the wild, as total synthesis is not economic. To avoid limited supply of the drug material, a production ofpodo- phyllotoxin by an alternative source like plant cell cultures would be desirable. Seeds of Linwn album known to contain podophyllotoxin were collected near Teheran. Suspension cultures were established using MS medium supplemented with 0.4 mg/L NAA and cultivated in the dark at 25 OC. A number of lignans and lignan precursors were isolated from cell cultures: a- and A-peltatin, matairesinol and coniferin/coniferylalcohol were identified by cochromatography (TLC, HPLC) podophyllotoxin, 5-methoxypodophyllotoxin, 5’-demethoxy-5-methox~ophyllotoxin, laricire- sinol and pinoresinol additionally by mass spectroscopy. Several cell lines containing different amounts of lignans were established, podophyllotoxin (up to 0.3 96 of cell dry weight) being the main product. Experiments were started to identify the enzymes involved in the first part of lignan hiosynthesis leading to coniferin. Activities of phenylalanine ammonia lyase, cinnamic acid CoA ligase, and of cinnamic alcohol dehydrogenase (CAD) were identified in our cell cultures. CAD activity was quantified measuring the back reaction from coniferyl alcohol to coniferaldehyd. The enzyme was isolated and partially purified. The characteristics of this enzyme as well as its activity during the culture cycle of the cells together with the accumulation of lignin and lignans in the cells will be presented.

(The Netherlands); 5 Research _ - Institute of Forests and Rangelands, Teheran (Iran)

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IS STRICTOSIDINE MORE THAN JUST AN INTERMEDIATE IN THE BIOSYNTHESIS OF INDOLE ALKALOIDS? T.J.C. Luijendijk. P.R.H. Moreno, L.H. Stevens. R. van der Heijden and

BIOSYNTHESIS OF HOP (HUMULUS LUf ULUS L.) BITTER ACIDS n s J.C. Scheffer, Karin W.M. Zuurbier, Suen-Ying Fung and Roben 0119

2 Division of Pharmacognosy, Leiden/Amsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden. The Netherlands

Ripe cones of the hop plant, Hwnulus lupulur L. (Cannabinaceae), contain large amounts - up to 20% - of so-called bitter acids. These compounds are prenylated derivatives of phloroglucinol and consist of a-acids and @-acids, e.g. humulone and co-humulone, and lupulone and co-lupulone, respectively. We have tested a new hypothesis concerning the formation of the aromatic intermediates in the biosynthesis of the hop bitter acids. We propose that acylphloroglucinols are the first aromatic intermediates which are subsequently prenylated to yield the a- and !.-acids. Two phloroglucinol derivatives, 2-(3-methylbutanoyl)-1.3,5-benzenemol [=phlorisovalero- phenone; PIVP] and 2-(2-methylpropanoyl)-1,3.5-benzenemol [=phlorisobutyrophenone; PIBP]. were indeed detected i n cone extracts of several hop cultivars using HPLC with photodiode m a y detection, LC-MS and GC-MS. The 4-prenyl derivatives of PIVP and PIBP (called compounds X and co-X. respectively) were also detected in the extracts. For our studies on the aromatic precursors of the a- and P-acids, we first synthesized PIVP, PIBP, X and co-X, and subsequently we developed chromatographic systems (TLC, HPLC and GC) for the analysis of these compounds in the presence of the hop bitter acids. I n vifro studies with protein extracts from hop female flowers showed that chalcone synthase activity was present, as naringenin was formed from malonyl-CoA and p-cournaroyl-CoA. When the latter substrate was replaced by isovaleryl-CoA or isobutyryl-CoA. PIVP and PIBP, respectively. were found to be formed. The formation of naringenin, PIVP and PIBP in various extracts obtained during the development of hop flower buds into ripe cones was compared.

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0122

BIOSYNTHESIS OF 4-HYDROXYBENEOATE V I A COENZYHE A ESTERS OF 4- COUMARIC ACID AND 4-HYDROXYBENZOIC ACID IN LITHOSPERHUH ERYTHRORHIZON CELL CULTURES pelf Ltieche r, Susanne somner and Lutz Heide Univereitrt Freiburg, Inst. Pharm. Biol., 79104 Freiburg, Germany

The formation of 4-hydroxybenzoate ( 4 H B A ) is a central step in shikonin biosynthesis in Lithoepannum erythrorhizon Sieb. Bt Zucc. (Boraginaceae) (1). POT the mechanism of the biosynthesis of benzoic acids in plants, several different hypotheses have been proposed. We inventigated 4HBA synthesis in cell cultures of t . erythrorhizon. In cell free extracts, the enzymatic formation of 4- coumaroyl-CoA (4CACoA) by 4-coumaraterCoA ligase could be demonstrated, an well as the conversion of 4CACoA to 4HBA. The latter reaction required only NAO as COfactOr. Using radioactively labelled 4CACoA it could be proved that 4CACoA is an intermediate in the conversion of 4-coumarate (4CA) to 4HBA. wheeeae 4- hydroxybenzaldehyde is not. 4-Hydroxybenzoyl-CoA (4HBACoA) and acetyl-CoA could be identified as products of the reaction. The CoA estere were rapidly hydroly- zed enzymatically, probably by thioesteraaes, resulting in the formation of the free acids. These data indicate that the 4HBA formation in L . erythrorhizon proceeds via a mechanism analogous to the B-oxidation of fatty acids. All enzymes involved in the conversion of 4CA to 4HBA remain in the supernatant of a 100,000 x g centrifugation. In a cell fractionation experiment,the enzymes could be loCalized in the cytoeol. Thus we can conclude that the 4HBA forming enzymes are different from those of fatty acid metabolism, localized in mitochondria and glyoxysomes. Although a 8-oxidation mechanism involves three different enzymatic reactions, we did not detect intermediates between 4CACoA and 4HBACoA so far. Purification of the enzymes involved should provide further evidence for the exact reaction mechanism and the number of enzymea involved. (1) H. Inouye et al. (1979) Phytochemistry 18x1301-1308

FROM PHYSOSTIGMINE TO PHENSERINES: NOVEL AGENTS WITH POTENTIAL FOR THE TREATMENT OF ALZHF,IMERS DISEASE

Phenserine. the phenylcarbatnate analog of physostigmine, and N1- norphenserine are potent, long-acting and selective inhibitors of acetylcholinesterase. A comparison with their antipodal isomers in assays measuring inhibition of cholinesterases showed the activity of physostigmines and phenserines to reside almost entirely with the (-)- (3aS)-enantiomers. This differed markedly in the "-nor series where optically pure (+)-(3aR)-NI-norphenserine had considerable inhibitory potency, although less than the (-)-(3aS)-enantiomer. Synthesis of these compounds and details on their biological evaluation will be presented.

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THIOAMIDE ANALOGUES O F RAS, ANTITUMOR CYCLIC HEXAPEITIDES FROM RUBIA

Yukio Hitolsuyanagi, Jin Suzuki, Yuji Matsumoto, and Koichi Takeya d Department of Pharmacngnosy, Tokyo College of Pharmacy, Horinouchi 1432-1. Hachioji, Tokyo 192

03 (Japan)

c y c k hexapeptide RA-VII (1) isolated from the roots ofRubio okone ;md R. cordifdie (Rubiaceae) attracted much attention because of its potent an t i t umor act ivi ty a n d the unique structure incorporating an unusual cydoisodilyrosine moiety. 1 is the most potent and abundant (as a result of the isolation process containing 0-methylation of concomitant RA-V 2) congener among R A s and is now evaluated clinically in Japan as an anticancer agent. However, the contents of 1 and 2 in the plant is low (total - 0.01% of dry roots). so the supply problem is one of lhe obslacles for its wide clinical use. A solution for this seems to develop a more potent alternative for 1 obtainable through minimum chemical manipulations. The lack of proper functional groups in 1 rcstricls an ordinary approach such as acylation or alkylation. Thus we have focused our attention on the thioamidation of the backbone peptide bond of 1. Substitution of a thioamide for a peptide bond is considered to be a 'isosleric replacement' since the structure of the thiopeptide unit is very similar lo the peptide bond. Treatment o f 1 with 2 mole equivalents of Lawesson's

All-2

$--$-M OR TWS \

reagent afforded a mono- (3) and a dithionated product 4 in 80 and 3 % yields, respectively. Their structures have been elucidated by spectroscopic methods to

~ 5 3 ( , D I R I I I P388 KB

b e [ 3 1 4 , CS-NHJRA-VII and [3Y4; C S - N H ; 6P1, CS-NHJRA-VII, respectively. In v i m antitumor activity of the analogues were evaluated against ':R-Ma'X-Y-o 0'w'3 o'w23

NOESYPH correlations and H-NMR coupling constants obtained for 3 and 4 o.ooo5~ O,OOtl are almost identical to those of 1. suggesting that these lhionated analogues retain the bioactive conformation. Since the activity of [3Y4, CS-NHIRA-V " R ~ M e . X m Y * s 0.w17 0.w17 (S) prepared from 2 in the same manner is also enhanced, thioamidation ofTyr-3 o.ooo88 0 . ~ 1 5

carbony1 constitutes a ready access to more promising RA analogues.

P388 and KB cells. The adivity o f 3 was doubled, and 4 retained activity. The 2: R-H. X-Y-0 0.0327 0.008

R . ~ ~ , X.S, ".o

I: R-H. x-S, Y - 0

3:24 I STKUC~UWAC~TVITY RELATIONSHIPS OF THE ANT~MALAR~AL AND ANTIFEEDANT D-SECO LIMONOID GEDUNIN.

-, John T. Amason*, Tony Durst*. Cindy K. AngerhoferA. John M. Pezzuto". Feliz Uck-. *Ottawa Carleton Institutes of Biology and Chemistry. University of Ottawa, Ottawa, Canada, K I N 6N5. "Program for Collaborative Research in the Pharmaceutical Sciences. Department of Medicinal Chemistry and Phmacognosy, College of Pharmacy. University of Illinois at Chicago, Chicago, Illinois 606 I2(USA) -San Jose Succotz, Belize.

An investigation of the structure/activity relationships of gedunin was conducted to determine Lhe moieties responsible for its antimalarial and antifeedant activities so that this information could be used in the development of a more active antimalarial and/or antifeedant insecticide. Fourteen derivatives of gedunin were prepared and screened, along with five other limonoids closely related to gedunin, for antimalarial (PlarmodiumfofciporurnJ and antifeedant (Osm'nio nubilofis) activities. The results and conclusions from the in viuo antimalarial screening and the neonate life cycle studies (Osrriniu nubilofis) will be presented.

q %CO CH,

o@

GEDUNIN

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ORAL PRESENTATIONS 1 I6

~

PHARMACOGNOSY AND MEDICINAL CHEMISTRY OF NPS R-568, A CLINICAL CANDIDATE FOR THE TREATMENT OF HYPERPARA- THYROIDISM: A TALE OF MODERN DRUG PRODUCT

I F. B W ' , Bradford C. Van Wagenen, and Eric G. DelMar, DEVELOPMENT. Department of Medicinal Chemistry, NPS Pharmaceuticals, Inc., University of Utah Research Park, 420 Chipeta Way, Salt Lake City, Utah 84108, USA.

NPS R-568, N-(3-(2-chlorophenyl)propyl]-(R)-3-methoxy-a-phenethylamine, is a synthetic alkaloid-like compound which is a positive allosteric modulator of the extracellular calcium receptor on the surface of parathyroid cells. It5 agonist activity (in the nM range) gives rise to an inhibition of parathyroid hormone (PTH) secretion in bovine and human parathyroid cells. NPS R-568 is therefore a clinical candidate of potential utility in the treatment of hyperparathyroidism and possibly other bone and mineral-related disorders (e.g., osteoporosis). Its design and synthesis, based on the principles of what may be termed a "mild drug design" approach (incorporating "weak drug" and "soft drug" design principles), will be presented, tracing its structural roots back to the natural world. Specifically, its structural evolution and connections back to proto-alkaloidal spider toxins, plant-derived phenanthrene, benzylisoquinoline, tropeine, and quinolinelquinuclidine alkaloids, their synthetic structural analogs and congeners, and relevant synthetic medicinal approaches, will be outlined. NPS R-568 is currently undergoing clinical evaluation and development in Phase 1/11 clinical trials. Its genesis and commercial development represent the culmination of a propitious blending of pharmacology, pharmacognosy, natural products chemistry, synthetic organic medicinal chemistry, and favorable serendipity, and may point the way toward future approaches to drug product development based on natural product models.

0:25 1

THE FERN POLYPODIUM DECUMANUM, USED IN THE TREATMENT OF PSORIASIS, A N D ITS FA'ITY ACID CONSTITUENTS AS INHIBITORS OF 0:24 1 LEUKOTRIENE B4 FORMATION I

Mervi Vasance-Tuominerl, Premila Perera-Ivarsson. Shen Jia, Lars Bohlin and Wenche Rolfsen Depanment of Pharmacognosy, University of Uppsala, Box 579.75 123 Uppsala, Sweden

In the traditional medicine of Honduras, the fern Polypodium decumanum (Willd.), commonly called Calaguala, has been used in the treatment of various cutaneous disorders, including psoriasis and atopic dermatitis. In clinical trials, the efficacy of the plant extarct, which is given orally, has been confirmed with improvement of the symptoms of the disease within a few weeks of treatment As one of the pathological findings in psoriasis is elevated amounts of the leukotriene B4 in the skin, the Calaguala extracf was tested for its ability to inhibit the formation of this mediator by human leukocytes. After fractionation of the extract, the inhibition was found to be caused by the polyunsaturated fatty acids Linoleic, liiolenic and arachidonic acid. Both naturally occuring and synthetic fatty acids are known inhibitors of leukomene biosynthesis and have lately been extensively studied as potential therapeutic agents in the management of various inflammatory disorders, including psoriasis. Yet the mechanism by which they act and the smctural features that are necessary for their actions are not known. In an attempt to study these relationships, the isolated fatty acids and a group of closely related analogues were compared and their IC50 values determined in the bio-assay. The IC50 values for the acids tested were of the same magnitude (20-60 pM) except for arachidonic acid which showed stimulatory activity and 8(K) hydroxylinoleic acid which gave 30% inhibition with the highest do% tested (120 pM). A quantitative determination by HPLC of the fatty acids in different Calaguala extracts was performed in an effon to relate the fatty acid contents of the plant to its clinical effect.

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ORAL PRESENTATIONS I17

POTENT NEW CELL ADHESION COMPOUNDS FROM PLANTS. L. Musza, S. McElhiney, P. Speight, C.J. Barrow, L. Killar, A.M. Gillum and R. Cooper. Sterling Winthrop Pharmaceuticals Research Division 9 Great Valley Parkway, Malvern, PA 19355

Cell adhesion processes play significant roles in pathological condit'ions, such as chronic inflammation, cancer metastases and viral infections. Interactions between a number of cell surface glycoproteins mediate various stages of cell adhesion. One family of adhesion molecules consists of the heterodimeric integrins. There are three known leukocyte specific integrins with a common B, chain. The binding of one of these, the lymphocyte function associated antigen-1 (LFA-l), to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion to endothelial layers when leukocytes leave the bloodstream at sites of inflammation. Antibodies against B, integrins attenuate inflammation in animal models. Specific inhibitors of integrin mediated cell adhesion may have therapeutic potential as antiinflammatory and anticancer agents . In the course of screening natural products for antagonists of leukocyte integrin-mediated cell adhesion in a whole cell assay, two plant extracts were identified which inhibit cell adhesion. One extract from the root of Trichilia rubra was identified as having potent inhibitory activity in a bioassay for LFA-1:ICAM-1 mediated adhesion of JY and HeLa cells, and a second bioactive extract was obtained from the stems and leaves of Conobea scoparioides. The isolation, structures and bioactivity of these cell adhesion inhibitors will be discussed.

THE DISTRIBUTION AND SIGNIFICANCE OF POLYHYDROXYLATED ALKALOIDS WHICH INHIBIT GLYCOSIDASES Robert J. Nash, Richard Layton, Paul 1. Thomas, Alison A. Watson and George WJ. Fleet' Institute of Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, Dyfed SY23 3EB (UK), 'The Dyson Perrins Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QY (UK)

Many polyhydroxylated alkaloids of the pyrrolidine, piperidine, pyrrolizidine, indolizidine and tropane classes have been isolated from microbial fermentations, plants and insects in the last 20 years and shown to be inhibitory to glycosidases. Some have aroused interest as potential anti-viral and anti-cancer agents largely by virtue of their abilities to alter glycosylation of glycoproteins. They have also been shown to have activity against insects and plant nematodes and a few are known to be toxic to mammals. Until recently, work on the naturally-occurring compounds centered on tropical legumes but it has become clear that alkaloids with specific g1)cosidase inhibitory activity are widespread in the plant kingdom, including temperate species and are found in important food plants such as potatoes.

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SEARCH FOR NEW CALCIUM CHANNEL BLOCKING DRUGS FROM PLANTS

Department of Pharmacology, The Aga Khan University Medical College Karachi-74800, Pakistan.

3129 I Anwar H. Gilani

Calcium channel blockers (CCBs) are relatively a recent introduction to the modern therapeutics. Their use in the cardiovascular disorders is well established and the other diseases where CCBs have received attention in recent years include, asthma, migraine, mood disorders, liver damage, urinary lithiasis and hyperactive bladder and gastro-intestinal tract. Considering the wide application of CCEs, several indigenous plants were tested for the search of new CCBs. Rabbit aorta strips were suspended in 20 mL tissue bath containing Kreb‘s solution, maintained at 37OC and aerated with a mixture of 95% oxygen and 5% carbon dioxide. Following an equilibrium period of 2 h, sustained contractions were induced with high concentration of postassium (50 mM) which is known to mediate contractions through opening of voltage- operated calcium channels. Plant material (extracts, fractions or pure compounds1 were then added to the tissue bath to obtain cumulative inhibitory responses. Isometric responses were recorded on Gould 3000 recorder. The results showed that over 70% plants tested blindly, inhibited high K*-induced contractions thus showing CCBs-like activity. A few examples of plants shown to contain CCBs-like constituents include, Artemisia scoparia, Brassica compestris, Cuscuta reflexa, Cyperus scariosus. Daucus carota, Moringa oleifera, Peganum harmala and Rubia cerdifolia. These results offer challenge to the plant physiologists to ponder what is the role of abundantly available CCBs-like agents in plants? Perhaps, nature has supplied through environment, a group of substances that have therapeutic application almost more than any pharmacological class of drugs and further studies in the search of new and selective agents may yield fruitful results.

0:30 Novel, Highly Cytotoxic, Annonaceous Acetogenins from Asimina lriloba (Annonaceae) Geng-xian Zhao, David L. Smith, and Jerry L. McLaughlin, Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907

Directed by the brine shrimp lethality test (BST), our continued investigation of bioactive constituents in the EtOH extracts of the stem bark of Asim’na rriloba (Annonaceae) has now yielded two series of novel isomeric acetogenins; these are asimicin isomers: asimin, asiminacin, asirninecin and asiminocin, and the bullatacin isomers: bullatin, squamocin, motrilin, and bullanin. These newly discovered compounds exhibit their third hydmxyl groups at their C-10,28,29, and 30 positions (rather than at C-4), and show significant cytotoxicities against three solid tumor cell lines: human lung carcinoma (A-549), human breast carcinoma (MCF-n, and human colon adenocaxinoma (I€”-29). with ED50 values down to ~ 1 0 - ~ 2 pglml. (Aided by ROI grani no. CA 30909from ;he National Cbncer Instihue, NIH).

.,,101’

OE OH OH

asimicin A=threo C u = R bullatacin A=erythro C a = S

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0:31 A NOVEL HIGHLY SWEET SECODAMMARANE CLYCOSlDE FROM PTEROCARYA PAWURLIS. Edward J Kennelly, L m g Ca, LIM Long, Lisa Shamon, Myung-Sook Chung, Kyaw Zaw, Bmg-Nan Zh,* John M Pezzuto, and A Douglas

ALKAMIDE LEVELS IN ECHINACEA PURPUf7EA: DIFFERENCES BETWEEN ROOT, RHIZOME AND ABOVE-GROUND PARTS 0:32 I Nice1 B. P e w . John W. Vaii Kliiik. Elaine J. Burgess arid Graenie A. Parnieiiier

Ncw Zealand Iiwtimte for Crop and Food Research Lfd.. Plant Extracis Research Uiut. Depamnriit of Cheniisfry. University of Ofago. P.O. Box 56. Dunedin (NEW ZEALAND)

We are exploring die potential of he medicinal plant Echinocco prrrpureo (L.) Moencli. (Astcraceae or Composifae) as a new high value crop in New Zealand. The 'rooi' of Illis plani is taken for its inmunostimulatory activity. Alkamides such as 1 and 2 are characteristic components of Echinoceo species and may be imponant for their immunostimulatory activity.

The 'roof' of E. purpurea normally used is in fact bodi root and rhizome (i.e. underground stem). We have shown that he levels of alkamides differ between these two different organs. For example, the average level of alkamide 1 in roots was almost ten times the average level in rhizomes.

2 H '

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ANTIMICROBIAL AND ANTIOXIDANT ACTIVITY OF PHENOLIC DITERPENOIDS FROM

Akiko Yamasaki, Carter B. Green, Raphael K. L. Kang, Randi L. Helms, Kelly L. McAlister. Gary A Reitz and Tetsuo Nakatsu

CLARY SAGE, SALYIA s c u m 0:33 1 Takasago Institute for Inlcrdisciplinary Science. 325 N o d Wigei Lane, Walnut Creck, California, 94598 (USA) Nobuo Kato and Hitoshi Takeshita Institute of Advanced Material Study, 86, Kpshu University, Kasuga-koen, Kasuga. Fukuoka, 816 (Japan)

Sage, Salvia spp. known in folklore have been used in herbal medicines, cosmetics and foods. Many diterpenoids haw been isolated from Salvia spp. and their biological activities have been reported. We are currently working on natural products from commonly used herbs, fruits and teas. Three known ditcrpenoids including fermginol. salvipisone, acthopisone as well as one novel compound 'clariol' were isolated from the EIOAc McOH root extract of Salvio sclarea. These smaures were decided by NMR and X-ray analysis. These compounds showed fairly strong antimicrobial activity against PropionibacMum acne, Stuphylococcus oureus including methicillin resistant S. a. W S A ) and Candido albicans. We will d i m s their antioxidant activity as well.

&&&yyJ$ \ 4 , /

Ferruginol Salvipisone Aethopisone Clariol

THE ROLE OF CAPILLARY ELECTROPHORESIS IN NATURAL PRODUCTS RESEARCH Kina C. Chan, Haleem I. Issaq, Gary M. Muschik and George M. Janini Program Resources, Inc.R)ynCorp, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702 (USA)

3:34 1 Capillary electrophoresis (CE) is a powerful microanalytical technique which gives high

resolution of large molecules as well as small ions. In its micellar mode the analyst can resolve a mixture of charged as well as neutral molecules. In our laboratory, CE and micellar electrokinetic chromatography (MEKC) have been used for the separation of different groups of compounds of pharmaceutical and clinical interest. In this presentation we will show the utility of CE and MEKC as an important and usehl analytical tool in natural products research and as an alternative separation technique for analytical HPLC. CE and MEKC with W-laser induced fluorescence (LIT), and photodiode array detection have been used for the separation of anti-cancer and anti-AIDS drugs. Examples will be given on the separation of taxol and related compounds from the bark and needle ofWestern Yew; MicheUamine A and B and their monomers from the leaves of Ancistrocladacaea. Also, other examples will be presented. Different strategies for the separation of natural compounds were examined and the feasibility of using the technique for the analysis of trace amounts in real samples is explored. Also, the advantages of UV-LF detection will be highlighted.

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ORAL PRESENTATlONS 121

SEPARATION OF CAPSAICINOIDS BY COMF'LEXATION CHROMATOGRAPHY Howard Constant and Geoffrey A. Cordell

0:35f I Program or Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago IL 60612

Capsicum has been used for thousands of years as a spice and also has also been used for medicinal purposes. In the study to analyre capsaicin [I] (the parent compound of capsicum) and its &isomer (civamide) pIl in a variety of matrices. a HPLC method was devised which would not only separate capsaicin and civamide. but also separate all of the established ~ t u r a l capsaicinoids, as well as nonivamidc m. Synthetic nonivamidc has been found as a chcap adulterant in several OTC preparations and in the fwd indusuy. A brief introduction to the methods pertaining to the analysis of capsicum and the new HPLC rnechod will be presented.

0

"""r87 HO C H 3 Q T L / H

HO

Civamide (n) Nonivamide (m)

Capsaicin (I)

Determining Absolute Configurations of Stereocenters in Annonaceous Acetogenins through Formaldehyde Acetal Derivatives and Mosher Ester Methodology Zbe-mine Gu, Lu Zeng. Xin-ping Fang, Trim Colman-Saizarbitoria. Mei Huo, and Jerry L. McLaughlin* Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmad Sciences, Purdue University, West Lafayette, Indiana 47907 (USA)

Formaldehyde acetal derivatives can be conveniently prepared, on a small scale, using parent Annonaccous acetogenins which have 1,2-, 1,4-, and/or 1.5-diols along their aliphatic chains. The resulting cyclic acetal protons give NMR signals which allow characteriiation of the relative stereochemistries of the two chiral centers that originated from the diols. Less complicated (vs. the parent acetogenins) per-Mosher ester [methoxy(trifluoromethyl)- phcnylacetate or MTPAJ derivatives of the acetal derivatives can then be prepared and used to determine absolute configurations of the chiral positions which bear thc remaining free hydroxyls. Prior knowledge of relative stereochcmical relationships then permits assignments of absolute configurations to additional chiral centers along the chain of the molecules. This method has been particularly useful in solving the absolute configurations of several non- adjacent his-THF (tetrahydrofuran) and mono-THF acetogenins, viz., bullatanocin, (2.4-cis and rrons)-bullatanocinoncs, bullatalicin, (2.4-cis and irons)-bullatalicinones, squamostatin A, squamocin, gigantetrocin A, and goniothalamicin. Most of the resulting acetals (vs. the parent acetogenins) show enhanced bioactivities, and their mode of action is, likewise, via mitochondria1 inhibition. (Aided by research gram no. HOI CA30909 from the National Cancer Institute, N I I f ) .

0:36 1

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0137 ANALYSIS OF SULFOLIPIDS FROM HETEROSIGM AKASHIWO BY MASS

M. Keusaen. S.W. Ayer, J.M. Curtis, P. Thibaulf and J.A. Walter SPECTROMETRY AND NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

0:38

The flora of North America includes a large number of medicinal plant spedes, the vast majority still remaining unexplored, phannacologically and chemically. Bash Columbia possessses many specles of hlgher plants which have not been systematically screened for their medicinal properties although native peoples have used many species in traditional herbal remedies. In our ongoing studies, we have identified a number of antibacterial and antifungal phenols, quinones, flavonoids, terpendds and halogenated compounds from spedes of Bhru, Blnua, Moneses. -, :eenothus,Mand -. Their chemlsby and antibiotc activity will be discussed.

ANTIMICROBIAL METABOLITES FROM BRITISH COLUMBIAN

Geeta S- R. E. W. Hancock. and G. H. N. Towers MEDICINAL PLANTS.

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ORAL PRESENTATlONS 123

I ISOLATION OF A NOVEL BIOACTIVE NATURAL PRODUCT FROM THE FUNGUS PYRENOPHORA TERES

0:39 I D.C. Manker*, J. Breinholt, T.L. Bachmann, C. N. Rosendahl, R. Nielsen and M. Heide *Novo Nordisk Entotech, 1497 Drew Ave., Davis, CA 95616; Novo Nordisk A/S, Plant Protection Division, Novo Alle, DK-2880 Bagsvaerd, Denmark

In the course of screening for fungicidal and insecticidal metabolites, a novel bioactive compound was isolated from the culture broth of t h e ascomycete fungus Pyrenophora teres. The novel metabolite, BK226, possesses both insecticidal a n d antifungal activity. The bioactive purification was followed using second instar larvae of t h e lepidopteran Trichoplusia ni . The compound was determined to contain a n acyl tetramic acid moiety with molecular formula C26H3gN04. BK226 was isolated a s the sodium sal t of the tetramic acid moeity which could be converted to the parent compound upon acidification. The compound was found to inhibit the growth of a number of insects including Spodoptera exigua, Helicoverpa virescens and Drosophila melanogaster. In addition, BK226 also inhibited the growth of a number of fungi belonging to the classes Oomycetes, Ascomycetes an Deuteromycetes.

NEW BIOACTIVE METABOLmES FROM THE COPROPHILOUS FUNGUS CERCOPHORA SP. Authrine C. Whvte and James B. Gloer*, Department of Chemistry, University of Iowa, Iowa City, Iowa 52242. James A. Scott and David Malloch, Department of Botany, University of Toronto. Toronto. Ontario, Canada, M5S 1Al.

Antagonism among members of fungal communities has frequently been observed and reported. Chemical investigation of secondary metabolites responsible for such antagonistic effects could lead to the discovery of new natural products with antifungal activity and possibly other effects. During our continuing search for antifungal agents from antagonistic coprophilous fungi, organic extracts from liquid cultures of Cercophora sp. (JS 166) were found to have antifungal and antibacterial activities. Studies of these extracts afforded a known member of the trichothecene class as well as several new, unrelated aromatic metabolites with varying dcgrees of antifungal activity. The stlllctures of these compounds were determined primarily by mass spectrometry and ID- and 2D- NMR spectroscopy. Most of the antifungal activity of the culturc extracts could be accounted for by these metabolites. To our knowledge, these are the first novel natural products to be reported from a member of the genus Cercophora. Details of the isolation, structure determination, and bioactivity of some of these compounds will be presented.

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0141 ABIDJAMIDE A: A NEW CYCLIC PEPTLDE FROM THE ASCOSTROMATA OF EUPENICILLIUM SHEARII. Gilbert N. Belofske and James 9. Gloer*. Department of Chemistry, University of Iowa, Iowa City, IA 52242. Donald T. Wicklow and

BIOMIMETIC SYNTHESIS OF CYTOI'OXIC PYRID0[2,3,4krJACRIDINE ALKALOIDS. School of Chemistry, Tel Aviv University, Ramat Aviv 69978, Israel.

Gary Gellerman. Amira Rudi and Yoel Kashrnan, 0:42 1 Over 40 pyrido[2,3,4-kl)acridine alkaloids have been published over the last decade.

This group of alkaloids is currently of great interest due to their significant biological activities. Almost all have been reported as having significant cytotoxity. Other activities. namely, regulation of cellular growth and differentiation, affection of CAMP-mediated processes, inhibition of lopoisomerase II. anti-HIV activity and others have also been reported. Our discovery of six compounds of this group of alkaloids (e.g. eilatin and norsegoline) possessing interesting biological activities. brought us to the synthesis of these compounds.

A new biomimetic synthesis of pyridoacndlnes is the subject of this presentation. The synthesis of several pyridoacridines and a possible biogenesis of a few others will be reported.

NOIC5IGOI Ih'k

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0~43 -

S E M I s m A E S I s OF TAxOL FROM m T URAL PRECURSORS

0 ~ 4 4 1 STUDIES OF MODIFIED TAXOLS: THE SOUTHERN HEMISPHERE M. 0. Chordia, A. G. Chaudhary, J. M. Rimoldi, K. A.

Neidigh, M. Gharpure, and A. A. L. Gunatilaka Department of Chemistry. Virginia Polytechnic Institute and State University. Blacksburg, Virginia 24061-0212 (USA).

The diterpenoid tax01 (1) is in clinical use for the treatment of ovarian cancer, and an active search is being conducted in several laboratories for analogs which have improved

bioactivity. As a part of this search. we have investigated the effect of changes on the southern hemisphere of taxol on i u biological activity. including deacetylation at C-4 and changes to the oxetane ring and the C-2 benzoate. A summary of these studies will be presented.

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0145 1 SHOm. E m a m m w syNlHEsIs OF PpERCmE. rn m E m C m u UNSATURATED AWDE FROM PIPER NICRUM. Aw RELATED COMPOUNDS

PO Box (ooo. Fmiaicton. Neor B-wick. canad.. E3B 6W and Gcorgc M. Strunt. canedlan Foreat Scrvlce.

Concise. emcient syntheses of the unsaturated lnecctlcfdal amMe plpercide (1) and the related compound piperolein A. (2) from Prper n4nun as well as their nor-homdoguee have been developed. The key step wad a modlflcatlon of the aldol-Orob-type fragmentatlon sequence recently reported by Sakai et aL (1993)

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POSTER PRESENTATIONS 253

I

COUMARIN PRODUCTION IN VIVO AND IN VITRO BY CORONILLA SPECIES. P251 I I

G. Innocenti". R. Caniato'. A. Piovan". R. Filippini' and E.M. Cappelletti' "Dipartimento di Scienze Farmaceutiche- Universitd di Padova- Via Marzolo,b 35131 Padova 'Dipartimento di Biologia - UniversitA di Padova- Via Trieste, 75 - 35121 Padova

Following previous researches on Cornnilla species as possible natural sources of biologically active furocoumarins, C. scorpioides Koch and C. varia L. have been investigated both in vivo and in vitro.

In vivo all the plant organs of C.scorpioides contain psoralen and the hydroxycoumarins umbelliferone, scopoletin and daphnoretin, previously reported only in seeds. Moreover, the dihydrofurocoumarin marmesin. i.e. the immediate biosynthetic precursor of psoralen. was found in leaves and roots. No furocoumarin was found in the different plant parts of C. varia; only the three hydroxycoumarins were detected.

In vitrn, no coumarin compounds (not even the very widespread hydroxycoumarin Jmbelliferone) were detected in both callus and medium, at least in the first subculture of C. varia.

C. scorpioides, on the contrary, sinthesizes in vifro (callus and medium) umbelliferone. marmesin and psoralen, but fails to produce scopoletin and daphnoretin. C. varia seems to lack !he genetic infomation responsible for furocoumarin biosynthesis, differently from another species lot containing psoralen in vivo, C. viminalis Salisb. for which expression occurs under the same xllus culture conditions.

However further research is needed in order to establish the biosynthetic capabilities of C. {aria under different culture conditions.

ISOLATION AND STRUCTURE ELUCIDATION OF SESQUITERPENE POLYESTERS FROh A Judit H o h m a ~ , Gdbor Nagy. Zuber Dini, h e Math&. Department of Phamcognosy. Albert S m t GyBrgyi Medical University. H-6720 Szeged, POB 121, Hungary; Gdbor Gimther, Department of Pharmaceutical Chemist0 Albert Szent-GyBrgyi Medical University. Szeged. Hungary; Gyula Argay. Alajos K i l r d n . Central Research Institute fo Chemistry, Hunganan Academy of Sciences, B u d a p t , Hungary.

Sesquiterpene esters based on the dihyd~o-&h~garohran skeleton have long been known as active compounds against insecb In recent years these compounds have also aroused interest in consequence of their cytotoxic and anlitumaur promote activities. In continuationof our research into biologically active metabolites from the Eu0nymu.s genus (Celarrroceae), fiv new (1.3, 5. 7.8) and three known (2, 4. 6 ) dihydro-!3-agarofuran polyesters have been isolated. Various chromatograph methods. including CC. preparative TLC and HPLC. were applied to the lipophilic extract o f the fruit from Euonymu sachalinensi~ to yield compuodc 1-6 and 8 . and to that from Euonynius nanw to yield 2, 4. 5 and 7. The stmclures wer elucidated by means of spectral analysis ('H NMR. 'H,'H COSY, "C NMR. DEPT. NOESY. COLOC and mas spectroscopy). The absolute configuration of 8 was deiennined by X-ray crystallography. This IS the first report on th occurrence ofcompounds 1-8 in these plant species. ( l l u s investigationwas suhsidixd by National Scientific Research Fun OTKA grant F.5128.)

P252 E u O N w U S SPECIES

R' R' R' R' .OAF

1 H Ac H Bz 2 Ac Ac H Bz 3 Ac Mebu H Bz

5 Ac Ac H Nic 4 AC BZ n BZ

6 Ae Bz OH Bz 7 iBu iBu OH Bz OBz

8

Ac=acayl. iBu= isohutyroyl. Mebu = 2-methylbutyroyl, Bz= benzoyl. Nic=nicotinoyl. Fu= 3-furoyl

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254 POSTER PRESENTATIONS

COMPARATIVE STUDY or TRADITIONAL EXTRACTS WITH SUPER CRITICAL FLUID ONES OF SOME LAM/AcEA€ SPECIES GROWING IN TEMPERATE BELT

, . ~ ._c...,l . , ~ ~

Hunganan Academy 2 SUM& (1{-2163 VAcratM). Hungary

The Mediterranean Lamiaccae Species Melissa ohianalis L (melissa), Salvia olfidnalis L (sage) and Lavandula angustrlolia Mill. (lavender), culllvaled in Hungary, under fhe temperate bell. produce less essential oils than in lheir coun(ry of origin. Such drfterences have been proved by the comparative studies of the volatile oil confenf and non-volataile. melhand and Water exlraclable fractions of strains oblained via bolanic garden seed exchange

Addifional differences m n be observed. if super -1 fluid e m (wW carban dioxide) was used. If turned out fhel more remarkable dmerences are in h e wafer steam d i s W M o n s than in h e methanol - water extrads. As far as the quanlily concerned by carbon dioxide ext-adion. lavender (0.17-2.06 mU100 ml). sage (1.13-1.4 mu100 ml) and melissa (0.030.06 mV100ml) was me decreasing order of drug oil confent. The most remadable difference belween the carbon dioxide fraC(i0ns and the wafer disWed samples is in raw of linalyi acetatdinalool. If was always much higher man in the first case while in the second case if remained always below the values of boa sage and lavender. The oils of m e l i differenbated m dtronellal m f e n t ; it was three times more man obtained wim carbon dioxide extraction where geranial was me mam mmponent.

Of the polar. non-vdataile fradhns. obtained afler carbon dioxide, followed by diluted ethanol extraction. of drugs. me fofal Ravonoid content. expressed in hyporoside showed me following order sage (0.91 X). lavender(0.49 %). rnelissa (0.30 %). in h case of tannins. expressed in pyrogalld. melissa (7.50 %). sage (4 37 %). lavender (1.87 %) WEIS the order. The o a u m of msmarinic add was general, varying between 7.00-1.2% m me crude extrads.

I

The Determination of B a l c a l i n and S t a b i l i t y of Promethazlne i n

p254 1 Xiao tr Qing F r l 21 Ke Syrup

L i u Een and Chnng Y l l i n q

Hubei C o l l e g e of T r a d : t i o n a l Ch ,nes~ M e d i c i n e .

Wuhan. P.R Ch lna

x i a o E r Qlng Fe 21 KP s y r u p c o n s i s t s of s e v e r a l k i n d s of

Chinese h e r b s end p romethaz inc . It has been used t o t r e a t

c h i l d r e n cough f o r many y e a r s . One of major components.

B e r c a l i n . in t h e sy rup was s e p a r a t ~ d b y po lyamlde column and

de te rm ined b y t r i p l e wave leng th spec t rapho tomet ry . The

r e c o v e r y is 9 7 . 3 % . C V 1 s 1 . 1 . * ( n = 6 ) . A l s o t h e s t a b i l i t y of

promethaz inc in t h e ch ines? h e r b s y r u p was s t u d i e d b y t h e

method of s t a i r c a s e p so thermal. It w a s showed t h a t t h e Chinese

h e r b s y r u p promoted t h e d e g r a d a t i o n of promethaz ine . t 0.9, t h e

t ime k e e p i n g 909 t h e c o n t e n t o f promethaz ine . l a 2 5 days i n

t h e s y r u p . The s tudy can o f f e r 6 o m E s t a n d a r d t o c o n t r o l

q u a l i t y of the Chinese m a t e r i a medlca s y r u p .

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FOSTER PRESENTATIONS 255

P255 TRITERPENIC ALCOHOLS AND ESTERS AS ANTI-INFLAMMATORY

S. Sosa', R. Della Loggia', A Tubaro', H. B&erz, St. S a d , 0. Isaac). PmcIpm oF C ~ L E M ) ~ ~ O F F I C N ~ S ~ O W E R S

IS DINOPHYSISFORl7I (DINOPHYCEAE) INVOLVED IN DSP TOXICITY IN THE NORTHERN ADRIATIC SEA? Aurelia Tubaro ", Silvio Sosa", Laura Sidari* Paola Nichetto*, Sara Cok*, Giorgio Honsell+, Roberto Della Loggia'

Institute of Pharmacology ad PharmacOgnosy, University ofTncste, 1-34100 Trim (Italy) Laboratory of Marine Biology - Strada Costiera 336,34100 T r i m O d y )

+ Faculty of Agricolture, University of Udinc ,33 100 Udim (Idy). Diarrboeic Shellfish Poisoning @SP) IS a gastrojcnteritis d by h@On of shellfish Wntmhtd by toxic algae belonging to the genus Dmnophysis. Since 1989, DSP problems have bem raorded also along the Adriatic coasts and monitoring p r o g m were started to follow tbe pheuoma~a We foaucd 0x1 tbc Northern Adriatic Sea and, in parhcular, on thc Gulf of Trieste. During a d y m m i t o a program, 6m.1 August to November 1993 an exceptional DSP outbrcak was mrdcd. Tbc PhytoplanldOD pnscnt in scawatcr was rculrdcd in ~a~nppl~ collected, by vertical net-hauls, in a mussel farm. AU over thc sampling period, D. /owl and D. cmrdora wm tk most

abundant than D. cuuabta and prescntcd a quite diffnent tcmporal distribution.

counting their techac. D.fortii was the most abundant species andthcalgal pnsarc~ in musJcLs comlaccs ratbcrwcll with the algal presence in seawater. Mussel toxicity evaluated at Yapumoto's mawc biogssay started about three wedu f i r D. f i r m appearance in seawater, confimung p h w 0bsaMtiarr f i r such long Mod of contamination. 'Three wcdcs arc required also for mussel ddo~catioo. No evidat taxicity cornlation was obscrval tor D. cauduta, whose maximal prcscnce in seawater followed thc p k ofmusrd

in mussels. Tbe concentration of thc toxins corrclatcs rather well with the mwc bioauay toxicity and d e d tbc higbest mussel contamiaation since 1989. It is noteworthy tbat the Northem Adriatic Sea is the only site in Europe whcrc thc DSP phaommon is related to tbe presence of D. firtii. whereas along the other European coasts DSP is caused by other DfnophFis spades such as D. acumtmla. D. acuta, D. norvegtw .

abundant sp ies , representing always more than 97 % of the td dhd%dhU . D. fimi w a ~ always much more

The presealce of dinoflagcuates was d t o d also in the stocnach of mwcls (A4ytilIu ga l lop~nc ia l f s ) by

Using 811 ELISA m y , the p n ~ e n c ~ of thc moJt important DSP t o h , OLadaiC add .nd DTX-I, w a ~ aLF0 &&d

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STRUCTURES OF INSECT TOXINS FROM CONIFER ENDOPHYTES John A. F i n d b , Sentsetsa Buthelezi, Martin Frenette, Guoqiang Li and Luis M. Pcfia-Rodriguez

OF MARINE INVERTEBRATES FROM THE

N. Govindan. and David G. I. Kingston. U. S. VIRGIN ISLANDS.

Division of Science and Mathematics, University of the Virgin Islands, St. Thomas, U.S.V.100802, and Department of Chemistry, Virginia Polytechnic Institute and State University. Blacksburg, Virginia 24061-0212, USA.

As part of a research program in marine natural products chemistry at the University of the Virgin Islands, we carried out the extraction and bioassay of 86 sponges, gorgonians, tunicates, and mollusks in a mechanism-based bioassay involving genetically altered yeast strains. Of these 16 have shown differential activity between the mutant and wild-type yeast strains indicating the presence of potential DNA interacting agents. Another 24 have shown general antifungal activity. Our continuing efforts towards the isolation and characterization of active compounds from some of the bioactive extracts will be discussed.

P258

The spruce budworm, Chorisroneuru fumifermn, is Canada’s most serious forest pest and, despite a variety of strategies aimed at controlling its depredations during the last quarter century, it remains a major economic insect problem.

Elucidation of the role of toxins produced by micro-organisms associated with host tree foliage would have an important bearing on our understanding of spruce budworm population dynamics which is essential for planning effective control measures. Furthermore, insect control by manipulation of the fungi and/or fungal metabolites which may be among the tree’s natural defence systems is an attractive and exciting possibility.

Bioassay directed fractionation of extracts of endophytic fungal cultures originating from conifer needles has led to the isolation and identification of several new metabolites which are toxic to cells and/or larvae of the spruce budworm. The structure elucidation of these compounds using spectroscopic methods will be presented.

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P259 NOVEL METABOLITES FROM THE NUDlBRANCH APLYSIA PUNCTATA. John A. Findlay, Guoa iane Li, and Peter E. Penner, Department of Chemistry, University of New Brunswick, Fredericton, NB, Canada E3B 6E2

Two novel compounds and five known sesquiterpenes have been isolated from the nudibranch&!y&punctata collected in Sardinia, Italy. The structures of 1, a novel bicyclic sesquiterpenoid ketone related to the perforenones from Laurentia oerformat& and 2. a-new chlorinated bicyclic diether. have been assigned based on DEPT, COSY, HETCOR, NOE and HMBC, NMR experiments, and MS data.

1 2

Also isolated were laurinterol, debromoisolaurinterol, laurenisol, plus a known bromochamigrene and a known aromatic sesquiterpene alcohol.

P260 TRACE ANALYSIS OF HARMAN ALKALOIDS IN PASSZFLORA ZNCARNA TA BY REVERSED-PHASE-HPLC

Anne Rehwald. Beat Mcier. and Otto Sticher Dcparrrnenr of Pharmacy, Swiss Federal Institute of Technology (HTH) Zurich. CH-8057 Zurich. Switzerland

Passiflora incarnata L. (Passifloraceae) has a widespread use as sedative and tranquilizing herbal drug. It contains flavonoids and traces of harman alkaloids, which are both considered to be the active principles. In the last fifteen years a series of heterocyclic amines showing potent mutagenic and carcinogenic activity have been discovered. Since pcarboline alkaloids like harman and norharman are among them, it is evident that the alkaloid content of P. incamafa should be as low as possible and is limited by various monographs. A reversed-phase HPLC method for the trace analysis of harman alkaloids in P. incarnata is presented. It comprises extraction of the plant material, purification and concentration of the alkaloid fraction by combined solid-phase extractions on Extrelut and cation- exchange cartridges, and subsequent HPLC analysis using diode-array and fluorescence detection. The method was found to be sensitive and selective and revealed good recovery. Seventeen different samples of P. incarnata were screened for harman alkaloids applying the presented method. Only one sample yielded, on the level of detection limit. a possible content of approximately 0.1 ppm of harman, all the other samples contained no alkaloids. Taking these results into consideration, the analysis of harman alkaloids in P. incarnata is regarded as not necessary for routine analytical work such as identity and quality control of plant material, extracts and preparations. The efficiency of the method was demonstrated by the analysis of Peganurn harmala seeds.

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EX261 1 NEW BIOLOGICALLY ACTIVE CONSTITUENTS FROM DILLENIA PAPUANA

Andre'. Anthony D. Wright'. Topul Rali2, and Otto Sticher' 'Department of Pharmacy. Swiss Federal Institute of Technology (LTH) Zurich, CH-8057 Wrich, Switzerland

2Chemisuy Depanment. University of Papua New Guinea, Port Moresby. Papua New Guinea

In a biological screening of several traditional medicinal plants from Papua New Guinea, the lipophilic extract of Dillenia papuana (Dilleniaceae) demonstrated significant antibacterial activity towards Bacillus subtilis, Escherichia coli and Micrococcus luteus. Biological activity guided fractionation of this extract yielded four new compounds (1,2,4,6) and two new natural products (3,5) as active principles. Together with these oleanene-type triterpenoids, the known lupene-derivative betulinaldehyde was also isolated. Structure elucidation was carried out by means of spectroscopic methods, including El-mass, UV, IR and a variety of 1D and 2D nmr experiments (1H-'H-COSY, HMQC, HMBC and NOESY experiments). All of the acids showed antibacterial activities.

1 R, =a-OH. R,=O 4 2 R1=O, R,=P-OH 3 R , = H p , R 2 = 0

5 6

GLYCOLIPIDS FROM THE BLUE-GREEN ALGA FISCHERELLA AMBIGUA

-h, Gabriele M. Konig, Anthony D. Wright and Otto Sticher P262

Depanment of Pharmacy, Swiss Federal Institute of Technology (ETH) Zurich. 8057 Zurich. Swilzerland.

In our search for metabolites from Fischerella ambigua (Nag.) Gom. (Stigonemataceae), a nitrogen-fixing cyanobacterium, we encountered several monogalactosyl diacylglycerols. Glycolipids are involved in several membrane functions. Some of them also possess antialgal, anti-inflammatory, and haemolytic activities. Additionally, they are used for chemotaxonomic classification of cyanobacteria. Extracts of the freeze-dried algae were fractionated by a combination of chromatographic methods and upon acetylation a series of monogalactosyl diacylglycerols were isolated. Regioselective enzymatic hydrolysis followed by GClMS studies revealed three pure galactolipids (1-3) together with 5 mixtures of galactolipids. These mixtures contained "eukaryotic" galactolipids which possess C16 fatty acids at the sn-1 position. This finding of "eukaryotic" glycolipids in cyanobacteria emphasizes, once more, the unique position of cyanobacteria in the plant kingdom.

R2 1 : R' = lineoyl R2= palmitoyl 2: R' = oleyl R2 = palmitoyl 3: R1 = lineoyl R2= palmitoleoyl

HO Ho&o&o

OH 'R1

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p263 ~~

DISCOVERY OF PLANT-DERIVED NATURAL PRODUCTS TEAT INHIBIT PEORBOL ESTER-INDUCED ORNITEINE DECARBOXYLASE (ODC) ACTIVITY AND REDUCE ODC mRNA EXPRESSION IN CULTURED MOUSE 308 CELLS

~~

CL!hh&a W. Mar. S.K. La. L. Luyengi. Z. hfbwambo. L. Cai, H.H.S. Fong. A.D. Kinghorn. and J.M. Permto. Department of Medicinal Chemis~y and Pbannacognosy, College of Pharmacy, Univenity of Illinois at Chicago. Chicago, JL 60612, USA

Ornithim decahoxylase (ODC) is a key enzyme in the biosynthesis of polyamines and is highly inducible by growth-promoting stimuli and tumor pmmotors such as 12~-tetradccaooylphorbol - l3-~~~e (TF'A). Both ODC activity and thc resulting polyamims are essential for cellular proliferation. Thus, agents that inhibit polyamine synthesis may be viewed as candidates for cancer chemotherapy and chemoprevcntion. The screening of approximately 300 plant exuacts for inhibition of TPA-induced ODC activity using cultured mouse 308 cells led lo the identification of about IS active leads. Subsequently. bioassay-guided fractionation permitted the isolation of dcguelin (IC,,, = 0.3 ng/ml) and tepbrosin OC,, - 3.9 ng/ml). mtcnoids from Munduleo srricro (Leguminosac). and la-hydroxymapmunic acid 3-p-hydroxybcnzoate (IC5,, = 22 ng/ml) and Zu-hydmxymapmunic acid 2.3-b~~-p-hydroxybenate (IC5,, - 10 nglml), tritelpcncs fmrnMoprounco africano (Euphorbiaceae). All four of these compounds have also been shown to inhibit 7 . 1 2 d i m e t h y l b c ~ ( o ) a n ~ c e ~ - induced premoplastic lesions in mammary organ culture. In additiok using inhibition of TPA-induced ODC acuvity as a monitor. several active compounds were isolated from Euphorbia quinquiocos~oto (Euphorbiaceae) and Dirco occidentolis (Thymelaeaccac) with IC,, values in the range of ~ 0 . 8 - 200 ng/ml, respectively. whose sUuctures are reported elsewhere at this meeting. Interestingly. all of thcse isolates act as TPA-antagonists. Moreover. using Nonhern blot hybridization and slot blot cxperiments. dosedependent inhibition of TPA- induced ODC mRNA expression in mouse 308 cells could be demonsuatcd for each of these compounds, indicating an influence on the transcriptional regulation of TPA-induced ODC activity. Further studies include invesogauon of the effect of these agents on the expression of the mitogcn-inducible proto-oncogenes c-myc. c+s and c-iun. which are involved in intranuclear transcripuonal control. (Supponed by POI CA48112 aNarded b) the Nauonal Cancer IIW~NIC)

~~~ ~

NATURAL PRODUCTS D E M D FROM PLANlt THAT DEMONSTRATE POTENT IN€UBCI?ON D- 3 A I OF HIV-1 REVERSE l"SCRIPTASB 1 LU* , L. C d . H. H. S. Fong'. K. Ingolfsdoni?. H. Wagner'. L-2 Lid. A. D. Kinghorn',

I. M. P m l o l and G. A. Cordell' A =Majirind Chemistry and Fbmmxgnosy. University ollllinois at Chicago. Chicago, IL 60612, USA; '-1 of pharmacy. University of Iceland. R&javik. Iceland; 'Inqtitute of Pharmaceuiical Biology, University of Munich, 8OOO Mum& 2. Gmnany.

In our continuing cEfons to identiry natural plodud madiacsd inhibition of human immumdcfidw virus-1 mcrse aansCripcasC ( I w - 1 RT), active inhiitax wae idcnIi6cd. Swc-dc (1). a new flavwoxanthone glucosidc isol;ltcd from k r l i o j l d r e h r m a . l a p ' add 3- (2) and 2a-hydmqInaprOunif acid 2,3-bis-p- hydm*nzoalc (3). tritnpavS isdatcd fmm tbe mots cfMcrpame0 uficm~u, and pmtolichsterinic add (4) isdatcd fmm Ihe Lichen Ceamio islrmdco. wm found u, be m t inhibitors of the DNA polyucras aaivily of HIV-1 RT. These wmpmrlsmwerc~xicagainaavarictydauwuaULina. S w d f m d m 'dc had a 507'0 inhibirory dose 0%) in the HIV-I RT sysccm of 30.9 @d (42.9 m. Ip-Hydroxympxnk acid 3p.hydrox3tam?ate and Za-hydmxymprcunic a c i d 2 , 3 ~ i ~ ~ ~ ~ ~ ~ I ~ v a l ~ c f Z . Z ~ d ~ . 7 ~ a n d 2 . 6 ~ d ( 3 . 7 ~ , ~ , a n d

ah lo id fapunhe chlondc previodyjudgcd as one dthe m0s1 potent plant DccMdary mctabdils tsrrd in (his syaem which had anIC,valueofS@ml(13 pW. ll~~kinaicmChanismrofHIV-1 RTinhiiitionmediatcdbyUlacmmparndswae elucidated. (SupportcdinpartbypO1 CA481IZandRO1 CAZ0164awankdbythcNationalCanoerIndi~)

pmtolicheaainic aEid Wan IC, of7.9 @II (24.4 phQ. Tbgc values can be ampad with thc pmtoberbauv '

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p:266

PI265 1 FIAVONOID A N D TERPENOID CONSTITUENTS FROM DRAGON'S B L O O D OF DRACAEh'A C I N N A B A R 1

Mohamed Masaoud, Helmut Kippager, Uwc Himmelreich, Andrea Ponel. Jijrgcn Schmidt and Guntcr Adam Iiistitutc of Plant Biocliemistiy. P.O.Box 250, DO6018 l-lalldSaalc, Germany

NEW ANTIMALARIAL DRUGS OF KENYAN PLANT ORIGIN: A

Gakuniu DMN-, Dossaji SF, Laur D. Waterman PG, Gray AI, TRADITIONAL APPROACH

From Dragon's Blood of Drocoeriu cirtnnbori Hall. fil. (Agavaceae). a medicinal plant of Yemen. four homoisotlavans. three flavans. two dihydrochalcones. 4,4'-dihydroxy-2'- methoxychalconc, 7.4'-diliydroxyllavonc, 7-hydroxy-llavan-4-one. five sterols and three tii1erpenoids have k e n identified. Three of these compounds. 7-hydroxy-3-(~-hydroxy- 4-mcthoxybenzyl)chroman, (2S)-7,3'-dihydroxy-4'-methoxy~av~n and 4-hydroxy-2- methoxydihydrochalcone. arc new. Furthciinore. two compounds of new structure type.

a billavanoid named cinnabarone composed of a dihydrochalcone and a dcoxolctrahydl-oclialcoiic moiety, and a triflavonoid, were isolated and their structures established hy iims spectroscopy and two-dimensional I l l - , 13C-NMK studies.

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POSTER PRESENTATIONS 26 1

p:268

VARIATION AMONG KNOWN KALMNOL AND NEW KALIHINENE P:267 1 DFTERPENES FROM THE SPONGE ACANTHELLA CA VERNOSA

J Rodrieuez'. R M Neito , L M Hunter', M C Diaz', P Crews', E Lobkovsky', J Clardy' 'Department of Chemistry and Blochemishy and hstl tute for Marine Sciences, University of California, Santa Cruz, CA 95064, 'Department of Chemistry, Cornell University, Ithaca, h?', 14853-1301

Seven new diterpene constituents were isolated from the sponge Acmihellu cuvemosu investigated from Fijian locations Their structures were established based on a combination of spectroscopic data, x-ray crystallographic data and biogenenc arguments These new diterpenes, of the kalihinane family, can be divided into the kalihinene (compounds 4-6) or the 6-hydroxy- kalihinene (compounds 7- 10) frameworks Also present were three known compounds, kalihinol A ( I ) , isokalihinol F (2) and kalihinene (3) It i s proposed that the kalihinane type diterpenes are a chemotaxonomic marker for A cuvemosu Its diterpenes can be divided into three biogenetic families based on the stereochemistry of the decalin ring forming process The presence of different members of these biogenetic types might be used to chemically distmguish various populations of A cwemosu The compounds, I , 3 and 7 were major constituents in both the bulk collection extracted after collection and in 15 specimens held in a controlled aquaculture system for seven months before extraction discerned among these samples based on the content of compounds 1, 3-5 and 7

A subtle chemical difference could be

SOME PHARMACOLOGICAL ACTIONS OF DRIED L U P EXTRACT OF ACANTHUS MoNTANUS

' Several uses have been indicated for &anthus montanui @fees) in tropical M n c a In Southern Nigena and part of Congo, the leaves are applied to a sort of boil on the finger @hitlow') and to maturate absccsser' No information on studies of h e pharmacological rtchvilies of he plani was found in avrulable literature The pharmacological actions of the dned l ed extract of the plant are currently being s t u d i d in our laboratory Here we report 11s anti-inflammaiory action on carragenaan-induced paw ocdema in rates and its effects on the isolated guinea-pig ileum and rabbit duodenum W h K s montanui extract in doses of 100, 200 and 400mgkg body weight p o showed significant lnhibitory cKeci on carragenaan-induced rat paw ocdema (using Ugo Basile Plethysmomeicr) The cNect WIU

comparable to the effect was comparable to the effect of 4mgkg indomcthacin p o The dned extract (0 02 10 2Omg/ml) exhibited a dosedependent relaxant eNect on both the rabbit duodenum and guinea-pig ileum IO'M to 6 5 x I O W ) was produced by 0 6, 2 0 and 6 Omg/ml of the dned extract Phytochemical screening' of the dned e x h a d of Acanthus montanus revealed h e presence of alkaloids, saponins, tannins, glycosidcs and simple rugan The test was negative for anhaquinoner The results showed that h e acanhus monlanus extracl possessed ann-inflammatory, histamine anlagonist and smooth muscle relaxant properties progress

A nonzomparative dosedependent mhibition of the agonist effect of histamine (6 5 x

Further investigations of the pharmacological activities of the plani IS in

1 Odebiyi, 00 & Sofowora, €A (1978) Lloydia, 41, 234

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POSTER PRESENTATIONS

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INTERLEUKIN-5 RECEPTOR ANTAGONIST NOVEL LABDANE-TYPE BIS-DITERPENE FROM A TABLETOP PINE PINUS TABUUFORMIS (PINACEAE) JL4Lb.u. D. Bennett, A. L. Breen. L M. Chaiken, D. S. Eggleston, K, Johanson, A. J. Freyer, C. R. Haltiwanger, M. E. Hemling, R. P. Hemberg, R. Matico. M. A. Mentzer, and J. W. Westley Research and Development, SmithKline Betcham Pharmaceuticals, King of Pmssia, PA 19406-0939.

Interleukin-5 W 5 ) plays an important role in the development of asthma via its influence on eosinophil production and function. We configured an ELISA-based plate assay to measure the binding of IL-5 to its receptor using a recombinant chimeric receptor (Fc-IL-5) and biotinylated ligand A variety of natural product extracts w e n screened using this assay and a plant extract from Pinus rabulifonnis (Pinaceae) collected at a local arboretum was identified as active. Fractionation led to an active component which has been idcntifitd by X-ray crystallography to be a novel labdane-type bis-ditepne with an IC50 value of 30 pM. Thc isolation. s ~ ~ c t l v c determination. and biologcal activity of this compound, which we have named tabalenic acid, will be presented.

THE STANDARDIZATION OF HERBAL EXTRACTS USING HIGH PERFORMANCE LIOUJD CHROMATWRAPHY WITH RP C18 C O L U ” -

Jerome Elliot, Lizhi Lian East Earth Herb Inc., P.O. Box 2802, Eugene, OR 97402 (USA)

Growing use and understanding of natural medicine among consumers has inspired the herbal products industry to conduct more intensive research in the am of standardization of herbal extracts. This paper describes the standardization of six commonly used herbal extracts by HPLC method, using only one RP c18 column. The standard compounds for these extracts are epigaUoca!echin gallate (green tea), ginsenasides Rgl, Rbl, Rb2 and Rd (ginseng). daidzcin and daidztn (kudzu), ephedrine and pseudocphedrine (ephedra). hydrasthe and berberine (goldenseal). and schisantherins A and B (schisandra). Each of these chromatograms prtsents a fingerprint not only for quick identification of the quality of raw herbs or extracts. but also for quantitative analysis of these compounds The term “standardization” is problematic; there is st i l l argument as to which constituents are worthy of standardization. This paper presents some examples for discussion.

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Ah'TITUMOR ACTIVITY OF SOME DITERPENOIDS FROM SALVIA UILTIURRHIZA. Shi Yong R f i , Seung Ha Lee. Jae Sung Noh, Sang Un Choi, Chong Ock Lee and Jong Woong Ahn. Korea Research Institute of Chemical Technology, P.O. Box

P3 J 9 . . Daedeog-Danji, Taejeon. 305-606, Korea.

As a part of screening studies of Korean medicinal plants for the cytotoxic activity against some kinds of cultured human tumor cell in vitro. the MeOH extract of the root of Salvia miltiorrhiza (Labiatae) was found to exhibit a marked activity. The serial activity-oriented fractionation of the MeOH extract of Salvia miltiorrhiza was led to the isolation of twelve kinds of constituents as active origins of the antitumor property. A l l isolates belonging to the abietane diterpenoid were exhibited marked cytotoxic activity against five kinds of cultured human tumor cell lines(A-549. SK-OV-3, SK-MEL-2. XF-498 and HCTl5. ) , in vitro.

LNXV' AND 4 VITB WjRATIVE STUDY OF TREIOKEWIC WYCWJXINS G.M. LaekeMn , A . Musuku , H.1. Selala2, 8. Neefl 6 P. Schepens2 ' Fac. Wan. Sciences, Catholic Univ. of Leuven, E. Van Evenstraat, 4 , B-3000 Leuven - Belqiu.

kpt. Warp. Sciences, University of Antuerp, Universiteitsplein, 1, B-2610 Vilrijk - Belqiu.

Treiorqenic mycotoxins are a group of neuronally active compounds produced by lold contaminants in fwd- and animal feedstuffs. The aim of this study was to validate an lodel by cumparinq h&.g and h&u activity of mycotoxins. At the Same time we uanted to investiqate the mechanism of action of these metabolites.

Penitrei A (PA) and Tryptoquivaline A (TA) uere intraperitoneally injected in Swiss mice and their effects compared to those of GH1, a new mycotoxin. PA and TA uere more tremorgenic than GHl. They also enhanced the tuitch contractions of the electrically stimulated quinea-pif ilew In vitro yielding a destabilized baseli e. Glll had no influence on this model. Atropine (10- II) inhibited partially and tetrodotoxin (3.10- I!) completely the enhanced twitch contractions. Bowever, these antagonists had no influence on the destabilized baseline. Baclofen, a WAB agonist potentiated the inhibition by atropine, but qave no conplete depression of the enhanced twitch contractions. The G A M A antaqonist bicuculline did not affect the electrically induced twitch contractions of the quinea-piq ilew enhanced by PA or TA. Cumulative dose response curves (CDR) with acetylcholine shifted to the left in presence of PA and TA. PA and TA enhanced also the contraction maxima of the acetylcholine contractions.

These results s w t an interference of the metabolites investiqated with cbolinerqic neurotrmlission. The coaxially stimulated ilew of the guinea-pig seens to be a valuable lodel uhich can help to distinguish between trenorqenic and non tremorgenic mycotoxins. It can replace the expensive hk@ experiments in order to study interference of these metabolites with neurotransaission.

B

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130

'I5

FOSTER PRESENTATIONS

HYPOGLYCEMIC ACTIVITY OF JUGLANS REGIA AND GALEGA OFFICINAUS. HJW, P. Declercq and G. Laekeman Faculty of Pharmnceulical Sciences. Catholic University of Leuven (KUL), Van EveartRst 4.

P6 INHIBITORY EFFECTS OF TAR/Lx4CUM O F F I C I N m AND CICHORIUM ENDIMA ON ADP-INDUCED PLATELET AGGREGATION HJk&', H. Deckmyn'. S. De Reys'. P. Declerckl and G. Lnekeman'.

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p8

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E F F E n S OF G U A Z W U M F O L L 4 BARK EXTRACT ON CHLORlDE SECRETlON IN RABBIT DISTAL COLON M r . H hmpler and M Heinnch ImtiNt f i r Phamazeuhsche Biolwe, Uruveraly of Freiburg Schanzlem I . 79104 Frelburg

P:7 1

PROCYANIDMS FROM BYRSONIM CMSSIFOLL.1 BARK F GelRI, M Hetnrtchl, D Hunkler2. and H kmplerl G z t f i r pharmazeubsche Biolwe, Schanzlestr 1. D-79104 Fre ihrg Germany

-

h z u m ulmfilio Lam. (slmuliacee) is - lmda the m e of Wak aky - used by the Mixe Indm of O a u u (Mexico) to treat diarrhoea Similar n m are lmovm h m &a m a s of Mexico. Smce dianhoa is unully associated with a dnstic loss of Wlta md CledrOlYtcf, it LS of mtae&!I to cvalurte such a plmt for its p0mti.l mti-ry dT&. A mll hovm physiologiul mdbod for the in vlho dudy of intest i~l ion ennrport ir the m u x u of rabbit distal colon mouuted in UI Using chsmbsr. The opm-circuit trPnsepithelial voltage CV,) md mistance &) wcn measured ~d the equinknt short-circuit c w t &=v&) m s calculated. The short-circuit cllrrcnt is equivalent to the clcctragenic ernscpithelial ion mtt.

Chlonde secretion ms stimulated by PGh (via CAMP, as e.g. in cholm) or by cholm toxin The M e d &anohc cnde extnd of the bark was dissolved in dimeihylsulpboxide md w . 6 added to the muMlul side of b e bathing solution. 'Ihe data are p m t e d as meam f S M . Pabed md UplLEd 1-l& Wth a rienifiunce k V C l Of

P < 0.05 was used. Ibe e m d (400 pdml. m u c o d ) mcreased sigdcmtly the

PGE+duced negative short-circuit c w t p6.0 (f 5.78)

ui increase in secdon. In Iowa uroccatntioaa (20, 40, 100,200 pdd) the innarc in negntive nbort-circuit c m t did not mcb r(.tir(iul si@unce. In m e of the uroceutntioar tntsd M inhibitm of ssretion ms necn Cholcn toxjn-mdd secretion (OJ6 pM. muuwl) m6

inhibited wmpletely by a wwntmii.m of200 pdml aude utnd. if the atmt ms added to the mucorrl b.thing solution prior to adding the toxin. Since Guorvm ulmi/dlo Lurk extnd did not d&mre the F'GE+ducod asretion a direct inhibition of in~cellulu secretory mechmismr of the cAMp-mcdi.tcd pathmy m s rather unlikely. Thus the extncl may a d mdirrctly on the sscrdory pmceps by binding to cholm toxin or to the toxin r&ep(or of the mtaocyks.' The Stmuli.cee md also the p u s Gvanrmo are well h o w for being rich in tmuuns. 'Ihir p u p of wrnpounds m y thus be responsible for the ohsaved d€&. Further smlics to idmtfy the active principle(s) and to &and& the mode of adim are in pmgess

pA/cm'. n-l I]. This uuupectcd result m y be intapreted as

1 21nstitut f i r Organische Chemie, Universilit Freiburg D79104 Freiburg Germany

Ryrsonirno crmyolia bark [B. crassfolio (L) H.B.K.. Malpigh~acae] is widely used in Mexico and Guatemala against gastrointestinal disorders and skin infections. The ethanol-water extraa of the bark showed good anivity in an nematodicidal model with Caenorhobdilis elegans as test organism Furthermore good antiinflammatory activity \MS

observed in the HET-CAM-assay and the cyclwxigenasei~bition-as~y. The ethanol-water extract after removal of the ethanol was suaphsively extraxed with CHzC12 and ethyl aoetate The ethyl acetate-soluble part wds separated by CC on Sephadex LH 20 with ethanol and mixtures of ethanol. water and a t o n e to yield 6 6anions which were funher purified by CC on MCl gel with methanol 35%. We obtained 2 known monomeric flavan-3-01s and 3 known dimers from the ethanol eluate and I known dimer and a new h e r &om the fraction eluted with ethano1:water:acetone 42,5:42.5:15 off the sephadex column. The monomeric and duneric compounds were identified as epicatechin, 3-O-galloylepicatcchii4epica(echin-[4fl~S~-epi~~ 3-&gUOylepica- techin-[4R+8]-epicatechin, epicatechin-[4~-t8]-3'-O-gall~lepicatechi~ 3U-galloylepicatechin-[4R~8]-3'-0-galloylepicatechin. Their structures were established by FAB-MS and IH-NMR specIfoscqry of the free phenolic compounds and IH-NMR of the products of thiolytic degradauon in comparison with literature values. The new trimer exhibited the (M + If)' ion peak in the eleclrcspray ionization mass spearurn at d z 1171. Fragmentation due to cleavage of the intcfflavanoid bonds produced sequena ions at d z 881. 291 (cleavage of the lower bond) and at d z 441 and 731 (cleavage of the u p p r bond). These findings suggested the presence of- 3-0- galloyl-flavanoid units (catechin or epiouechtn) in the upper positions and a catechin or epicatechin unit in the lower position. The H-3 NMR signals oorresp3nding to the top and the middle flavan unit were shifted downfield to 5.50 ppm and 5.56 ppm, respectively. confirming the position of the galloyl groups. Separation of the thiolytic cleavage products by flash chromatography on MCl gel and subquent CC on Sephadex LH 20 yielded epicatechin. 3-0- galloyleptcatechin, 3-O-ga l loy lep icarechn~~~ l th ioe ther and a dimeric thioether. Desulfurization ofthe latter uith h e y nickel yielded 3 - O - g a l l o y l e p i c a t e e ~ ~ 4 ~ ~ 8 ~ 3 ' - 0 - g a l l o y l e p i c a ~ ~ n . IH-NMR specIra of the cleavage products were consistent with those of authentic samples. The vimer was thus characterized as 3U-galloylepicateehtn-[48-18]- 3'-O-galloylepicatechin-[4~~8]~picatechin.

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IDENTIFICATION OF NON-SIEFZOIDAL HUMAN PROGJSlERONE RECEPMR LIGANDS FROM SECONDARY h f E T A B O m OFlHE MARJNE ALGA CYMOPOUA BARBATA

-t. Robert B. Stein?, TI S. Bergertt. William FenicalS, Teodoro Ianirot, Dale E. Maist. Antonio T o m and Mark E. Goldmant tLigand Pharmaceuticals Inc., 9393 T o m e Centre Drive, San Diego. CA 92121 *Scripps Institution of Oceanography, La Jolla, CA 92093 #Present Address: Nanogen Inc., 4510 Executive Drive, San Diego, CA 92121

The cotransfection assay is a novel functional assay employing cells hansiently-transfected with plasmids coding for both intracellular receptors and reporter genes. Using this assay, natural product extracts were tested to identify compounds that modulate intracellular receptor activity as measured by changes in reporter (luciferase) gene activity. A crude extract of the marine alga Cympolia b a r b was discovered to weakly mimic the actions of progesterone (enhanced luciferasc expression) in cells aansfected with the human progesterone receptor (hPR). Purification of the extract guided by the cotransfection assay yielded two diastereomers of cyclocymopol monomethyl ether posessing opposing pharmacological activities. The antagonist, 3R-cyclocymopol monomethyl ether (LG 100127). blocked progesterone-stimulated transactivation with an ICso value of 549255 nM in the cotransfection assay. The agoniss 3Scyclocymopl monomethyl ether (LG100128). had an efficacy similar to that of progesterone and an ECM value of 35f2 nM. Both diastereomers displaced PHIprogesterone from baculovirus expressed hPR. LG100127 and Uj100128 each interacted with the human androgen receptor but did not interact with human glucocorticoid receptor. estrogen receptor, vitamin D receptor or retinoid receptors. These cyclocymopol monomethyl ethers represent the first naturally occuring non-steroidal compounds to interact with the hPR.

P9 1

A VEW CLASS OF TUMOR PROMOTER FUMONISIN ISOLATED FROM FUSARILN MONILIFORME Ronald F Vesonder 1, Harukuni Tokuda 2. Akio lwashima 2, and Hoyoku Nishino

I Mycotoxin Research, National Center for Agricultural Utilization Research 181 5 N Street. Peoria. IL (USA), 2 Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 602, Japan and 3 Cancer Prevention Division. National Cancer Center Research Institute. Tokyo 104, Japan

Fumonisin i s a water soluble polyhydroxv-amino compound produced by cultures of Fusarium noniliforme isolated from naturallv contaminated corn The tumor promoting activity of this :ompound was tested by a shon term i n t r _ o assay for tumor promoters (Epstein-Barr virus early mtigen induced by Raji cells) and w by a two-stage carcinogenesis experiment on mouse skin n these studies. we found that fumonisin induced the early antigen in Raji cells xoduced the papillomas on mouse skin w All of these results indicated that fumonisin is a iew class of tumor promoter, that is, structurally different and novel from common tumor promotei

P:10 1 University

and

CH, CII, I I

Cl1,-(CIIL)~-CH-Cll-CI I-CH,.Cl I -CH,-CII-(CH,) , -CH-CH2-CII-C~~-~II~ I t I I I I

K A TCA OH 0 1 1 0 1 1 NH,

TCA = COOH,CH(CO,H)CI~,CO,II

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CHARACTERIZATION OF VANICOSIDES IN POL YGONUM PENSYLVANICUM BY HPLCNEGATIVE ION-FABMS and MS/MS. Michael L. 7immerrnann.'~* Albert T. Sneden.' and Teny L. Sumpterz 'Department of Chemistry. Virginia Commonwealth University, P.O. Box 842006. Richmond, Virginia 23284-2006 (USA) and 2Philip Morris USA, P.O. Box 26583. Richmond, Virginia 23261 (USA)

Investigations of ethanolic extracts of Polygonurn pensylvanicum guided by bioassays for protein kinase C inhibitory activity led to the isolation. through exhaustive solvent partitions and chromatographic separations, and structure elucidation of the glycosides. vanicosides A and B. Hydropiperaside. a related glycostde. had been prevlously isolated from Polygonurn hydropiper by other investigators. and was also tentatively identified as a component of P. pensylvanicum. In addition, several other minor constituents of the extract of P. pensylvanicum were also found to be related glycosides. but were not isolated in amounts sufficient to fully elucidate their structures. In order to obtain additional quantities of vanicosides A and 6. as well as the related glycosides. a more efficient extraction procedure was developed An HPLC system was developed which allowed baseline separation of the individual glycosides in the extract on an analytical scale, and has been applied to preparative Scale separations of the individual glycosides. Simultaneously. this HPLC system was coupled to a reverse geometry, four-sector tandem mass spectrometer. allowing examination of indivldual components of the extract by negative ion-FABMS and MSlMS techniques. Vanicosides A and B could be positively identified using this technique which gave fragmentation patterns characteristic of the structures Two different fragmentation patterns were observed. one resulting from the traditional fragmentation of the sucrose moiety into two monosaccharide units and the other resulting from sequential fragmentation of individual esters Examination of other components of the extract confirmed the previous identification of hydropiperoside. and provided significant structural information about the other related glycosides

EFFICIENT ANALYSIS AND INVESTIGATION OF EUGLOBALS IN 1 EUCALYPTUS SPECIES USING LC/API-MS ldidori Takasaki. Takao Konoshima. llutsuo Kozuka and Kei j i Hashimoto

Kyoto Pharmaceutical University, Yamashina-ku, K y o t o 607. Japan.

Re have already reported the isolation and structual elucidations of 12 euglobals (acylphloroglucinol-mono or -sesquiterpene skeletons) from Euca- /yptus g/nbU/us. Eucalyptus genus (Myrtaceae faoi l y ) , having 7 subgenera, is one of biggest genera. About 650 species are belonging to this genus. For searching novel beneficial natural products and studies on the che-

motaxonomy of Eucalyptus genus, the investigation of the distribution of euglobals which have unique structures will be interested. To confirm the existence of euglobals and to search for new euglobals,

the analyses by LC/API-MS are efficient and convenient. From E: grandis. E: tereticornis, E: incrassata and L bfakefyi, six new euglobals were isolated together with several known euglobals, and their structures and biological activities were elucidated. Furtheremore. 45 species (belonging to three subgenera) were analyzed

by LC/API-MS. In our experiments. it was confirned that euglobals were existent in the species belonging to Symphyomyrtus subgenus. On the other hand, in 15 species belonging to Corymbia and Yonocalyptus subgenera. the existence of euglobals were not confirmed. In this presentation, the details of the analyses of euglobals by LC/API-YS will be discussed.

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ISOLATION, STRUCTURES AND ANTI-TUMOR-PROMOTING ACTIVITIES (CANCER CHEMOPREVENTION) OF ROTENOIDS FROM AlORPlA F;pJTfC6?sU Hiroki Teradal, Takao Konoshioal, Midori Takasakil, Mutsuo Kozuka', llarukuki Tokuda2 and James R. Estes'

PA3 1 'Kyoto Pharmaceutical University, Yamashina-ku. Kyoto 607, Japan, 'Kyoto Prcfectural University of Medicine, Kamigyo-ku, Kyoto 602. Japan. 'The University of Oklahoma at Norman, Norman, Oklahoma 73019, U. S. A.

As a part of screening studies for anti-tumor-promoters (cancer chemopreventive agents), many plant extracts were tested using an in vitro assay system indicated by inhibitory effects on Epstein-Barr virus (EBV) activation induced by tumor promoters. ninosae) collected in Oklahoma, U.S.A. exhibitcd remarkable inhibitory sffect on EBV activation. From this extract, active rotenoids (amorphispi ronone and tephrosin) were isolated and their structures were determined sy physicochcmical methods (2D-NUB, difference NOE spectra and X-ray analy %is ctc.)as shown in formulae 1 and 2. These rotenoids ( 1 and 2) exhibited

strong inhibitory effects on EBV activation induced by TPA. Furthermore, compounds 1 and 2

O \oMeo~e on two-stage carcinogcnesis test.

The extract of A00rphe fruticose (Legu~

hN J ,.o 0 \ OH / exhibited effects (more significant than 80% inhibitory inhibition: \ /

Me0 OMC 1 2

Pl ISOLATION, STRUCRURES AND ANTI-TUMOR-PROMOTING ACTIVITIES (CANCER CIIEMOPREVENTION) OF LIGNANS FROM COLEOGY'E RANOSLWIYA Takeslii Tatsumoto'. TaJaoKonoshima', Nidori Takasaki I ,

Wutsuo Kozuka' and Harukuni Tokuda' 'Kyotci Pharmaceutical University. Yamashina-ku. Kyoto 607, Japan zKyoto Prefcctural University of Medicine. Kamigyo-ku, Kyoto 602, Japan

Some North American Rosaceous plants cxhibited the inhibitory effects on Epstein-Barr virus (EBV) activation induced by TPA well known as a strong tumor promoter. From the aerial parts of Co/eo.q.ym re~osissi0a col lected at southcrn desert of Utah,U.S.A., three active new lignans were isolated together with thirteen known compounds (flavonoids, triterpenoids and phe- nylpropanoids). And their structures were determined by physicochemical methods (ZD-NYR and difference NOE spectra etc.) as shown in formulae 1, 2 and 3. In these compounds. Iignans 1 and 3 exhibited strong inhibitory effects

on EBV activation induced by TPA. Furthermore, compound 1 exhibited re- wo? nlOH n ~ ~ ~ ~ - "o

markable inhibitory effect ~ (more than 40% inhibition)

" o i l / *cn,oH om " " O n oCn, on two-stage carcinogencsis test in vivo.

OH

Oc*, oc*, X H , on on

I I 2

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P15 ISOLATION, STRUCTURES AND ANTI-HIV ACTIVITIES OF NElP CUCURBITACINS FROM cum14 YEXICAM TAk-ao Konoshima'. Yoshiki Kashiwada2, Midori Takasaki'.

POTENT HEMOLYnC ACTION OF SEA ANEMONES PREVALENT ALONG THE WEST COAST OF CANADA.

P:l6 1 u. Cline and *Micheal W. Wolowyk Faculty of Pharmacy and Pharmaceutical Sciences University of Alberta Edmonton AB T6G 2C5 CANADA

The cytolytic action of sea anemones have been extensively studied more than any other cnidarian. This action is brought about by polypeptides and proteins (cytolysins) of molecular weight ranging from 10 - 30 KD. These cytolysins arc also known for their cyto- and cardiotoxicity.

In our search for cardiac stirnulatory peptides from extracts of sea anemones, we obsewed that aqueous extracts of nine of the ten species tested elicited strong hemolytic activity. Hemolysis occured in erythrocytes of rat, guinea pig, pig, dog and humans at concentrations as low as 0.1 mg/ml of the crude extracts.

W e have also shown that phospholipids, one of which is sphingornyelin inhibits hemolytic activity of these extracts in a dose dependent manner and at concentrations of as low as 1 pg. Some of our results including elecwon micrographs showing structural changes to the red cell membranes after hemolysis will be reported.

* Deceased January 31 1992.

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HAMACANTHIN A AND B. NEW ANTIFUNGAL BIS-INDOLE ALKALOIDS FROM THE MARINE SPONGE HAMACANTHA SP.

Sarath P. Gunasekera, Peter J. McCarthy and Michelle Kelly-Borges Harbor Branch Oceanographic Institution. Inc., 5600 US. 1, North,

Fort Pierce, Florida 34946

P17 1 In our search for biologically active substances from marine organisms, an ethanolic extract

from a deep water marine sponge Hamcantha sp. inhibited the growth of Candida albicans. This new species of Horncantha was collected by manned submersible off the southeast coast of Madeira at a depth of 548 m. An ethanol extract of the fromn sponge yielded an extract that was partitioned between EtOAc and water. The EtOAc-soluble fraction on bioassay-guided cc over Si gel followed by reversed-phase hplc furnished two new antifungal constituents hamacanthin A (1) and hamacanthin B (2).

Both hamacanthin A and B showed significant antimicrobial activity against Can. albicanr, Cryprococcus neoforrnns and Bacillus subrilis. The delails of smcture determination and biological activity will be presented.

I I

STRUCTURE / ACTIVITY RELATIONSHIPS OF MSAL NORDITERPENOID ALKALOIDS

Kip E. Panter' and S. W. Pelletier'. 'U. S. k p t . of Agric., Agricultural Raearch Service. Western Regional Research Center. 800 Buchanan St., Albany. CA 94710 (USA). U. S. k p t . of Agric.,

OCCURRING IN TOXIC RANGELAND LARKSPURS (DELPHINIUMS?) Gaw D. Mann crs'. P18 1 Agricultural Research Service, Poisonous Plant Research Lab. I150 East 1400 N o a Logan, UT 84321 (USA), 'Institute Tor Natural Products Research, University of Gaxgia. Atbens. GA 30602 (USA)

Ihe mahylsuccidimidoanUuanoyllymctonine (MSAL) norditerpertoid alkaloids methyllycawnitine(fiigure, R=OMe). nudicauline (figure, R-ow and 14d~tylnudicadine (figure. R-oir) have been shown to be the primary toxic anslitucnts present in rangeland larkspurs responsible for the poisoning of range land cattle. Utilizing a m o m bioassay, nine naturally occurring and synthetically derived SlnMUral analogs of the toxic MSAL norditerpcnoind alkaloids were loxiwlogically evaluated. The ~~xiwlogical data indicate spbcific StruCTural characteristics to be important factors in the k ~ e d toxicity of MSAL norditerpenoid alkaloids.

17

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p-19 CONCENTRATIONS OF TAXOLS AND RELATED TAXANES IN THE NEEDLES OF DIFFERENT TAXUS CULTlVARS

A . ' I H a l a m , Edward M. Croom, Jr., Wlodzimierz J. Kopycki, Alpana S.

Rescarch Institute of Pharmaceutical' Sciences, Department of Phmacognosy, Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, University, MS 38677 (USA)

The taxane content of the needles of 57 cultivars obtained from five major nurseries in the United States is presented. The analysis of taxol (I), and 5 other related manes, lO-desacetyl-7- epi-mol(2). cephalomannine (3). 10-desacetyl taxol (4). baccatin m(5) and 10-desacetyl baccatin nI(6) was carried out using an hplc method developed in our laboratory. The results of these analyses showed that the total 'useful' manes varied between 0.0232 - 0.1 13%. with tax01 and 10- desacetyl baccatin I11 being the most abundant manes. The survey identified eleven cultivars with taxol content ranging from 0.028 to 0.062% and 10 cultivars with lo-desacetyl baccatin III ranging from 0.028 to 0.066%; cephalomannine and 10-desacetyl tax01 each represented approximately 30- 50% the concentration of taxol in most cultivars. Therefore, the needles of ornamental Tuxus represent a significant, rich and renewable source for taxol and other related taxanes.

Joshi, Mahmoud A. ElSohly and James D. McChesney

INVESTIGATION OF FUNGI ASSOCIATED WITH TEE PACIFIC YEW TREE TAXUS BREVIFOLIA Andrea A. Stierle and Gary A. Strobel, Department of Plant Pathology, Montana State University, Bozeman, MT 59717; Donald B. Stierle, Department of Chemistry and Geochemistry, Montana Tech, Butte, Montana 59701 (USA)

Tax01 is one of the most promising anticancer agents currently under investigation by the National Cancer Institute. It is isolated primarily from the inner bark of the slow growing Pacific yew tree Taxus brevifolia in extremely small yields (<0.02% dry weight). The pharmaceutical potential of this drug has elevated the status of the yew tree from nuisance weed suitable for burning to precious commodity. Unfortunately this change in status does not alter the underlying dilemma: there are not enough yew trees to satisfy the growing demand for taxol. A number of different strategies have been explored to solve this problem, including tissue culture, semisynthesis, and total synthesis.

We have explored the possibility that an endophytic microorganism might also synthesize taxol. During this investigation we have isolated a fungus which produces a compound that corresponds to authentic taxol by spectroscopic comparison and monoclonal antibody reaction. The isolation and characterization of taxol from this fungal extract will be discussed.

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P21 1

A RENEWABLE SOURCE OF TAXOL FROM THE NEEDLES OF CULTIVATED CHINESE YEW TAXUS MAIREI.

Hangzhou, Zhejiang 310013, P.R. China. p22 I L i - H u W , Zhejiang Academy of Medical Science, 60 Tian Mu Shan Road,

Taxol. a diterpenoid, has shown exciting promise in clinic trails of treating breast and ovarian cancers. Because of the high cost and low yield of its total and partial synthesis the main s o m of taxol continues to be the bark of the yew tree. Removal of the bark kills the tree. This has caused considerable concern to environmentalists and ecologists over the damage done to the terrestrial ecosystems and the threat to biodiversities. Therefore, the search for alternate sources of taxol has become a high priority.

Here we report the production of taxol by extraction from the needles of cultivated Chinese yew, Taxus mairei , which is easily cultivated and grows rapidly. Experiments determined that the approximate yield of taxol was O.W6% dry weight of the needles, which is better than the yield of taxol from both the needles of Chinese T. cuspidata and T. chinensis. Furthermore, to our knowledge it is the first time tax01 has been isolated from T. mairei, a practical renewable source of taxol. Details of the isolation and anticancer activity of taxol and other compounds wiU be presented.

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p.23 SUPERCRITICAL FLUID EXTRACTION OF DIFFERENT

Marta L Colombo 1 and Andrea Mossa 2 CLASSES OF METABOLITES FROM NIGW DUSCENA L. SEEDS

PANICEINOL, A NOVEL INHIBITOR OF PROTEIN KINASE C FROM A MARINE SPONGE OF THE ORDER OF HAPLOSCERIDA

F24 1 I;iaproe W C m , Alan J. Freyer, Glenn A. Hofmann, Mike Mattern, Mark E.

Hemling, Mary A. Mentzer. Brad K. Carte', and John W. Westley. Research and Development, SmithKline Beecham Pharmaceuticals, P. 0. Box 1539, King of Prussia, PA 19406-0939

Protein Kinase C (PKC), a serinefihreonine kinase, is an important enzyme in a number of vital physiological functions. This enzyme, with its different isoforms exhibiting various specific regulatory mechanisms, is an ideal target for pharmacological intervention. Bioassay guided natural product screening led to the discovery of an ethyl acetate extract of a marine sponge of the order of Haploscerida from Pohnpei, FSM. Fractionation of the extract by repeated silica column chromatography gave three pure active compounds. Their structures were determined by spectroscopic analyses to be two known compounds, panicein 83 and panicein A hydroquinone, and a novel hydroxyl analog. The-most potent compound, paniceinol, inhibited PKC with an IC50 of 2.8 pM. The biological activity and spectroscopic studies of the three marine natural products will be discussed.

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A NOVEL DITERPENOLIGNAN FROM TAXUS BREVIFOLIA

Robert L. Arslanian, David T. Bailey, Michael C. Kent, Steven L. Richeimer, Qun Y. Zheng

Hauser Chemical Research, 5555 Airport Blvd., Boulder, CO 80301

Hauser, the only GMP producer of bulk paclitaxel (taxol), uses bark from the pacific yew (Taxus brevifolia Nutt.) as starting material in its paclitaxel isolation and purification process. brevifolia has been isolated a novel diterpenolignan, which we have named brevitaxin. coumarinolignans have been isolated and identified, to our knowledge, this is the first report of a diterpenolignan. discussed.

Also from the bark of T .

Although flavanolignans, xantholignans, and

Structural elucidation will be

q Plant Research

IDENTIFICA~ION OF CALYSTEGME I32 IN SOLA NUM DIMlDIA 7 U M . A POTENTIAL CAUSATIVE AGENl OF CRAZY COW I)ISEASE Russell J. Molyneux,' Lynn F. James' and Robert J. Nash' ' Wesrem Regional Research Center. ARS. USDA. Albany, California 94710 (USA); * Poisonous

L.aboratory, A R S . USDA. Logan, Utah X4321 (USA); ' AFRC Institute of Grassland and Environmental Research. Aberyslwyth, Dyfed SY23 3EB (LK)

The noflropane alkaloids calystegin A3, UI, and BZ have recently been discovered in the bindweeds Calysregia repium and Convolvulur orvensis and shown to be potent inhibitors of p-glucosidase and a-galactosidase. Inhibition of glycosidases causes lysosomal storage diseases in livestock and ingestion of calyslegines might therefore be expected to have similar effects. While the bindweeds have not been implicated in poisoning episodes, Solmum dimrdrorum causes "Crazy Cow Syndrome", charactenzed by neurological signs suggestive of a cerebellar disorder, including slnggenng. incoordination, and epileptiform seizures precipitated by a variety of stimuli. Lesions include severe cellular vacuolationand degeneration o f h k i n j e cells. Extraction of leaves and fruits of fresh.% dimidiatum, collected from San Angelo, Texas in an area where the syndrome occurs. and of herbarium samples. gave an alknloid fraction which, when analyzed by GC-MS, established the presence of calystegin B, and related alkaloids. It therefore seems probable that this syndrome is a manifestation of an induced lysosomal storage disease, caused by inhibition of P-glucosidase, possibly exacerbated by inhibition of a-galactosidasc.

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LARQE-SCALE P-8 FOR TAX0 L FROM NEEDLES OF TAXU 8 BP. P27 I --&aka V. Rao, Medicinal Chemistry Dept. College of Pharmacy, BOX J-100485, University of Florida, Gainesville, FL. 32610.

The renewable, needle biomass of T. m edia var. Hicksii has been proposed as the future source of taxol nearly four years ago. However, other than examination by analytical HPLC, no procedure for the isolation of the taxanes has been published. The increased presence of waxes, chlorophyll etc. and the co-elution of unrelated taxanes with taxol are said to be additional problems to overcome for a procedure.

We have developed a process based on reverse-phase column chromatography of the chloroform- (or dichloromethane-) solubles of the methanol extract of the needles, without any prior treatment, and using aqueous acetonitrile (or methanol) as the eluent. The taxanes of interest crystallize directly from the fractions. Extracts from 200 lb quantities of the needles of T. media var. Hicksii and of T. floridana were processed by using columns of 6"x6 .

A comparison of the two species shows that, as far as taxol procurement is concerned, the needles of T. floridana are clearly superior to those of 3. medi a var. Hicksii. Besides taxol (0.01% from fresh needles), the former yields 10-deacetylbaccatin I11 (0.05%), and two other oxetane-type taxanes. On the other hand, TC media var. Hicksii has taxol (0.01%), accompanied by 6 other 11,4/20-diene type taxanes, which are at present unusable.

~ ~ ~~

REGULATlON OF SOME CHORISMATE UTILIZING ENZYMES AND FORMATION OF 2.3-I>YHYDROXYBENZOlC ACID A F E R ELICITATION OF CATHARANTHUS ROSEUS CELL SUSPENSION CULTURES Paulo R.H. Moreno and Roben Verpoorte Division of Pharmacognosy, LeidedAmsterdam Center for Drug Research, Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands

p2$ A The biosynthethic pathway for phenolics and alkaloids has as a branch point chorismic acid that yields phenylalanine, the precursor of most phenolic compounds, and L-tryptophan, early precursor of indole alkaloids. The regulation of phenolics and alkaloid biosynthesis in the stress response was studied by assaying the activity of the following enzymes: chorismate mutase (CM), anthranilate synthase (AS). isochorismate synthase (ICS), tryptophan decarboxylase (TDC), and phenylalanine ammonia lyase (PAL). Cell suspension cultures of Cuihurunrhus roseus (L.)G.Don (Apocynaceae) were elicited with a cell- free filtrate of Pyrhiwn uphunidermurum. After elicitation, AS and TDC were induced, resulting in an increase of tryptamine i n the cells. An increased amount of phenolic compounds was found in the culture medium, however, neither CM nor PAL were induced after the elicitor treatment. The major phenolic compound was identified as 2,3-dihydroxybenzoic acid (DHBA). This compound could not be detected in non-elicited cultures. On the other hand, the presence of the glucoside of DHBA has been reported in the leaves of C. roseu. In cell suspension cultures this glucoside could not be detected. The accumulation of DHBA could be related with the induction of the enzyme ICS. In bacteria DHBA is synthesized from chorismate having isochorismate as intermediate. Thus, after elicitation of C. roseiu suspension cultures the activity of some chorismate utilizing enzymes is induced, leading to the formation of hyptamine and DHBA. However, the phenylpropanoid pathway seemed to be inhibited by elicitation.

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Bl6SYNTHETIC AND CATABOLIC ENZYMES OF 3-HYDROXY-3 METHYLGLUTARYL-COENZYME A IN CATHARANTHUS ROSEUS . van der H e w . R. Verpoone’ and J.A. Duine” Division of Pharmacognosy. LeidedAmsterdam Center for Drug Research

Department of Microbiology and Enzymology, Delft University o

-q Leiden University, P.O. Box 9502, 2300 RA Leiden, The Netherlands.

Technology, Julianalaan 67, 2628 BC Delft, The Netherlands.

Mevalonate i s a central building block o f a vast array o f plant primary and secondary metabolites. as phytosterols and indole alkaloids. At present much research i s focused on the regulation of the mevalonate biosynthesis in order to manipulate the accumulation of the mevalonate derived metabolites. An important intermediate in the biosynthesis of mevalonate i s 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA). Mevalonate i s formed from HMG-CoA in a reaction catalysed by the well-characterized enzyme HMG-CoA reductase. However, HMG-CoA is also a substrate for two other specific enzymes. i.e. HMG-CoA lyase and 3-methylglutaconyl- CoA hydratase. Furthermore i t i s converted by non-specific enzymes such as 3’-.nucleotidase. I t was found that in plants the formation of HMG-CoA from three molecules of acetyl-CoA could be catalysed by one single protein (TJ Bach er al., Lipids 26 (1991) 637-648). In yeast and animal cells, the formation of HMG-CoA requires the activity of two enzymes: acetoacetyl- CoA thiolase and HMG-CoA synthase. The enzymes involved in HMG-CoA metabolism are now being studied in Caiharanrhus roseus. a model system for studies on the regulation of secondary metabolic pathways, i.c. the formation of indole alkaloids. Spectrophotomeuic and some newly developed HPLC methods were used to assay the activities of these enzymes in various C. roseus tissues, and cell and tissue cultures. Acetoacctyl-CoA thiolase and HMG-CoA synthase have been panially (co-)purified from C. roseus stems, 3-methylglutaconyl-CoA hydratase and HMG-CoA lyase have been partially purified from C . roseus Suspension cultures.

IHOW TO USE DISSIMILATION CURVES TO OBTAIN DRY WEIGHT OATA FROM A SINGLE FLASK CONTAINING A SUSPENSION CULTURE DURING P3Q I THE CULTURE PERIOD? d Jan SchriDsemq and Robert Verpoorte

Leiden/Amsterdam Center for Drug Research, Division of Pharmacognosy. Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden. The Netherlands.

Dissimilation curves are very useful for the characterization of the growth of a suspension culture in a single culture flask (Schripsema et al. 1990). In the present study the method was further refined in such a way that dissimilation curves can Serve to obtain dry weight data from a single flask during the culture period.

The utilization of double-necked Erlenmeyer flasks is essential. On one neck of the flask a normal silicon stopper is used, while the other is hermetically closed with a rubber stopper. A number of normal Erlenmeyer flasks, which just contain medium, are used as blanks to determine evaporation. Before inoculation the evaporation rate of the double- necked flask is determined relative to the blanks. For inoculation the opening with the rubber stopper is used, thus avoiding any change in the relative evaporation of the flask, compared to the blanks.

The dissimilation curves then obtained give an accurate ‘estimation of the dissimilation at any moment of the culture growth. Using these accurate data a dry weight curve can be calculated. Some other factors influencing the dissimilation curves will also be discussed. e.g. changes in the elementary composition of the biomass, carbon dioxide levels In the flask and oxygen limitation in suspension cultures.

J. Schripsema. A.H. Meijer. F. van ken, H.J.G. ten Hoopen and R. Verpoorte. Plant Cell Tissue Oraan Cult. 22. 55-64 (1990\.

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ISOLATION, IDENTIFICATION AND BIOSYNTHETIC STUDIES OF ANTHRAQUINONES. PHYTOALEXINS FROM AN ELICITED CINCHONA ROBUSTA CELL SUSPENSION CULTURE Jan SchriDsem3, Ana Ramos-Valdivia and Robert Verpoorte Leiden/Amsterdam Center for Drug Research, Division of Pharmacognosy. Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA Leiden. The Netherlands.

Upon elicitation with a Phytophtora cinnarnorni preparation cell suspension cultures of Cinchona robusfa How. (Rubiaceae) produce a number of anthraquinones, which under normal conditions are not produced. As was shown previously (Wijnsma et al. 1985) anthraquinones act as phytoalexins in cell and tissue cultures of Cinchona species.

A gradient HPLC system was developed using a RP-18 column and as eluents 3% acetic acid (A) and acetonitril (6). A linear gradient increasing from 20 to 100% B in 40 min was used. The final condition was maintained for 10 min. This system not only yielded an excellent separation, but was also very suitable for LC-MS. By diode array UV detection and by thermospray MS (both positive and negative ion mode) seventeen different anthraquinones were detected, all characterized by a high number of substituents (4-6). The substituents found were hydroxy and methoxy groups and in some cornpounds a 2-methyl substituent was present. The main components were isolated by preparative HPLC and the exact position of the substituents was determined by NMR.

Anthraquinones were also isolated alter elicitation with concomittant feeding of [5-"C]- mevalonate, a proposed precursor. The labelling percentages of the anthraquinones were compared with those from sterols. biosynthetically derived from the same precursor, which were also isolated.

R. Wijnsma. J.T.K.A. Go, I.N. van Weerden. P.A.A. Harkes, R. Verpoorte and A. Baerheim Svendsen. Plant Cell ReD. 4: 241-244 (1985).

P31 1

P:32 1 EXTENSIVE 2D-NMR MEASUREMENTS OF SEVERAL NATURAL PRODUCTS

Gabriele M. Konig and Anthonv D. W riaht Department of Pharmacy, Swiss Federal Institute of Technology (ETH), Zurich, CH-8057

Zurich, Switzerland.

3ve natural products, four marine derived (1-4) and one from a lichen (5) were analysed by both I D and 2D (lH, %) NMR. Experiments employed included; COSY, NOESY, XHCO. HMQC, i M B C and INADEQUATE. The results of these measurements enabled the 'H and l3C NMR lata for compounds 1 (allolaurinterol) and 2 (diisocyanoadociane, adociane) to be reported and ully assigned for the first time. For compounds 3 (tetrachloromertensene). 4 and 5 (atranorin) t was evident that in the original reports of their 13C NMR data, in each case, two carbon 'esonances had been incorrectly assigned, these are now corrected on the basis of our latest neasurements.

1

2

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p- 3 4

THE COMBINED APPLICATION OF A SHIFT REAGENT

SOLUTION OF NEW NATURAL PRODUCTS FROM THE TROPICAL BROWN ALGA DICTYOPTERIS DELICATULA

AND 2D-NMR METHODOLOGIES TO THE STRUCTURE P:33 1 Gabriele M. Kbnig and Anthonv D. Wriaht

Department of Pharmacy, Swiss Federal Institute of Technology (ETH), Zurich, CH-8057 Zurich, Switzerland.

From the marine brown alga Oictyopteris delicatula Lamouroux three new and one previously reported sesquiterpenes (1-4) were isolated and characterised. For compounds 1 and 2 it was necessary to use extensive PD-NMR methodologies in combination with the lanthanide shift reagent C~H3D27EuF2106, (Eu(fod)s)) to enable all relative stereochemistries to be assigned. Proton shifts within compound 3 upon the addition of Eu(fod)s were shown to be unpredictable by classical lanthanide-induced shift (LIS) methods. Application of 2D NOESY measurements were applied to NMR samples of 1 and 2 containing Eu(fod)s to further demonstrate the enhanced role of this lanthanide shift reagent in the structure elucidation of natural products containing hydroxyl functions.

GLYCYRRHET IN I C ACI D MONOGLUCURONl DE @CGR) :ANEW

KenJl Mizutanl'. Harukunl Tokuda' , Takao Konoshima' , CHEMOPREVEW I VE AGENT AGA I NST ORGAN-SPEC 1 F I C CANCER.

Glycyrrhetinic acld monoglucuronlde (MGGR). a oleanane type trlterpene. manufactured by the enzyme hydolysls of glycyrrhlzln showed cancer preventive effect In three different cxperlments. MGGR and its related compounds tested were examlncd for thelr -__ In vitro anti-tumor promotlng effect on Epsteln-Barr virus early antigen activation induced by the tumor promoter 12Utetradecanoyl-phorbol-13-acetate and In vivo same effect on two-stage mouse skin and lung carclnogenesis. I t has been demonstrated that thls compound possesses potent anti-tumor promotlng actlvlty In several carcinogenesls models. The role of this compound as food addltive may be of new classes of chemopreventlve compound and promising tools in human population.

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CANCER PREVENIVE ACTIVITY OF COlCIS SEMEN EXTRACT AM) P35 1 ITS CONSTITUENT. I Harukunl Tokuda' . Takao Konoshlma' , Mldori Takasakl'

Mutsuo Kozuka' ,Aklo Iwashlma' and Hoyoku Nlshino' 'Department of Blochemlstry, Kyoto I'rcfectural University of Medicine. Kyoto 602. 'Kyoto i'harmaceutlcal IJnIverslly. Kyoto 607, 'Cancer Preventlon Dlvlslon. Natlonal Cancer Center Research Instltute, Tokyo 104, Japan

The seed of Colcls semen Is one of the Important Chinese herbal medicines usrd as a dlurellc and an anti-Inflammatory drug, and a lso used In lhe treatment. of paplllomas In Japanese f o l k mcdlclne. The exlracl. of seeds are uniuue to lea and may be active as chemoprevenl Ive agcri1.s for prol.cc:l Ion against cancer. We tested methanollc extract and a-monollnolcln. one of constituenls of seed using a model of mouse chemlcal and ultravlolel radlatlon carclnoyenesls. Oral admlnlstratlon of extracts of seed inhibited the formation of pap1 I lomas Induced by 7. 12-dlmethylbenz(a)- anthracene and 12-0-letradccanoyI~horbol-13-acelate or UV light. From these results. I t was suggested that lhc seeds of Coisis semen are effecl Ive as an antl-tiimor promotcr. Thc combine observations seem Lhal I.hls malerlal 1s morc extensively as one of the agents for purpose of cancer prevent ion.

CHEMOPREVE~ION OF SKIN AND LUNG CANCER RY GNUXNIA IRIDOID Shlnichl Ueda'. Harukuni Tokuda'. Aklo Iwashlma' and Hoyoku Nlshino'. 'Department of Natural Product Chemistry.

Faculty of Pharmaceutical Sciences, Kyoto University. Kyoto 606-01. *Department of Biochemistry, Kyoto Prefectural Universlty of Mediclne, Kyoto 602, 'Cancer Prevention DIvIslon, National Cancer Center Research Institute. Tokyo 104. Japan

P36 I

Edible Gardenla Jasmlnoides fruits. whlch have bccn usrd as a food additlve and a crude drug contain various lridold lypc monoterpene glucosides including genlposide. Genlpln. lhc aglucono of geniposide. remarkably inhlbited the tumor promotion by 12-0- tetradecanoylphorbol-13-acetate (TPM and n-buturlc acld on RaJl cells. Gardenia iridold glucosides. tarennoslde and geniposldlc acld also showed the anti-tumor promoting actlvlty. On mouse skin carclnogenesis Initiated by 7. 12-dlmethylbenz(a)anthraccne and mouse pulmonary carclnogenesls Inltlated by 4-nitroqulnollnc-l-

tumor promotlon. Of the irldolds so far examined. genlpin has the highest actlvlty l o Drevent tumor promotion. These resul ts suggest that Irldolds in edible plants may be useful in preventing cancer.

oxide, gcnlpin was significantly effectlvc to prevent CoZCHJ

50 HO genlpin

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p-37 RECEYIDR BI"G SNDIES ON AYLIRVHlIC CRUDE DRUGS - II. TLUUiINALlA BELLERXA ROXB -,' Jerry Cott,' James Silverton,' Beena Bhat! and Sukh D ~ v . ~ "IA,

CHZOH THE CONIFER CAMBIUM AS A SOURCE OF E-CONIFERIN - METHODS FOR ASSESSING YIELD. Rod Savidae, Faculty of Forestry and Environmental Management,

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Pharmaceutical Biology. University Cenm for Pharmacy. A. Deusinglaan 2. 9713 AW' Groningen, The Netherands [HJW]; Institut fiir Pharmazeurixhe Biologie. Heinrich-Heine-Universitit, UniversiUitsstraLk 1. 40225 Dibseldod. Germany [IM. CMP, US, GW]; Department of Radiobiology, University of Gmingen. Bloemingel 1. 9713 BZ Groningen. The Netherlands [HHK. AWTK]

Recently. we studied the cytotoxicity of flavonoids and sesquiterpene lactones from Arnica species against ltwo human tumour cell lines (1). As flavonoib are considered to act as potenrial modulators of the biological activity of other ~naaual) poducts. we investigated the effea of four flavones (apigenin. luteolin. hispiddi , eupafolin) and four flavonols (kaempfeml, quercetin. 6-methoxykaempfml. paculetin) on the cytotoxicity of helenalin in the human lung carcinoma cell Line GLC4. Cytotoxicity was W e d using the m - a s s a y . The cells were a p e d to the drugs and drug combinations for 2 h (serum-free). washed and incubated for 4 days. Cell growth was quantified by the cmversion of MTT due to cellular mirochondrial activity. At non-toxic concmtratims. M e e n 0.1 and 10 w, a l l flavonoids reduced the helenalin-induced cymloxicity. The IC50-value of the sesquiterpene lacme helenalin could be increased up to 150% when the flavonoids were added. No dose dependency was found in the concentration range tested.

1 W d b q W. Merfo~l 1. Palk i t s CM. kbmidt TJ. Willuho G, Van Uden W, P m N. Kampioga HH. Konings AWT. Cytotoxicity of flavoooids and sesquiterpe lanones from hiu species %aim b e GLGl and COLO 320 tumour cell lines. Planta Medica 1594. io press.

I I

Anemisinin. a sesquiferpene lactone isolated from Anemiria mnua L. (Asteraceae). and its derivativa conlain an endopemxide bridge. This structure is thought to prodoce free-radical species in d blood cells i n f d with malaria parasites. We determined their cytotoxicity to a murine Ehdich ascites 0319) and a human HeLa S3 cancer cell line, using the MTT w a y and the clonogenic assay. The MTT m y canna distinguish growth inhibition from cell killing. while the h e r detects actual cell deah Using both assays tc test a certain drug may give information about the mode of its cytotoxicity. ie. p w l h inhibition vermu ceU-

,killing activity. The endopx ides anemisinin and the dimer of dihyhanemisinin showed higher cytotoxicity in the MTT assay BI compared with the clonogenic assay. Thus the cytooxic effect of these compounds is predominantly ascribed to p w t h inhibition and not 10 cell killing. For anemisitene and eupatoriopicrin, h i c h possess an exccydic methylene with alkylating pmperties, both tests yielded comparable d u . indicating cell killing. For the refemce drugs cisplatin and d o x d c i n the MlT assay showed lower cymoxicify than the clonogenic assay. This may be explained by the metabolic activity of cells that are clonogenic dend. Our experiments show that the rapid M?T assay m o t always be used cu a substitute for the time-consuming clonogenic assay. when evaluating new drugs wih lespect to their cell- killing activity.

... I

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p42

P41

ANALYSIS OF PARTHENOLIDE IN TANACETUM PARTHENIUM: COMPARISON OF

Yara Anderson, Rein Bos, Herman J. Woerdenbag. Henk Hendriks, Hans-Peter R. Bootsma and C- J. de Weed

AN HPLC AND A GC METHOD

ASPOCHALASIN D AND ITS LACTONE FROM ASPERGIIJHJS FIA VJPES Robert L. C~~DIMII, Chicago College of Pharmacy, Midwestern University, 555 31st Street, Downers Grove, Illinois 60515 (USA), College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, Ohio 43210 (USA) and Larry W. Robertson, College of Pharmacy, The Ohio State University, 500 W. 12th Avenue, Columbus, Ohio 43210 (USA)

Aspergllusmips is known to produce a number of secondary metabolites and carries out some useful microbial transformations. Screening of A. fluvips broth culturs revealed significant antimicrobial and antimetabolite activity against a variety of fungi and bacteria, including Pseudomonus Numerous attempts were made in the development of separation methods applied to culture filtrate extracts and extracts of A. J7~1~ipes mycelia. Three metabolites were isolated, purified and identified from a culture filtrate extract. The antibiotic activity of these compounds accounts for some of the total activity present in the culture filtrate. The production of dihydrogeodin by other aspergilli is known but not reported for A. flmips. This work reports the isolation and identification of two potent mycotoxlns of the cytochala~an class, aspochalasin D and its novel lactone, from culture filtrates ofA.j7uvipes. Characterizations were made using high field proton and carbon-I3 NMR spectroscopic methods and high resolution mass spectrometry.

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p43 CHEMICAL STUDIES ON CUIYWGIANOSIDES. POTENT ANTI-LEUKEMIC TRITERPENEGLUCOSIDESWlTa 14.18-CYCLOAPOEUPHANE SKELETON v . 1 ToshihiroFujioka,z Kunib idehfk~hi .~ Ih-Sbeog che43 and Kuo-Hsiung Let' . .

ISOLATION AND STRUCTURE ELUCIDATION OF XYLOBUXIN, A NEW LIGNAN FROM XYLOPIA BUXIFOLJA, ANNONACEAE.

Anne Wahl, and Andrt Cavt, Laboratoire de Pharmacognosie. URA 1843 CNRS (BIOCIS). Facultt de Pharmacie, 92296 Chltenay-Malabry Cedex (FRANCE)

P44 1 The stem barks of Xylopia buxifolia (Annonaceae) collected in Madagascar have

been reinvestigated for their chemical constituents. Besides some known isoquinoline alkaloids and 3.4-dimethoxybenzoic acid methyl ester, a new lignan, xylobuxin, bearing a benzofuran skeleton, has been isolated.

Structure determination has been achieved by high field NMR techniques, mainly homonuclear (COSY) and heteronuclear (HMQC. HMBC) correlations and NOE. A careful analysis of NMR spectra allowed the determination of the dihydrofuran ring configuration.

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~ ~~~ ~~~~~ ~~

PIPEROGALIN, NEW PRENYLATED DIPHENOL FROM PEPEROMIA GALIOIDES, PEPERACEAE.

Valtrie Mah~ou, , Reynald Hcquemiller. h d r t Cavt, Alcira Angelo', Alain Fournet., and Paul-Henri Ducrot**, Labornroire de Phnrmncogmsie, URA 1843 CNRS (BIOCIS), Facuhe de P h a m i e . UniversifC de Paris XI, 922% Cmenay-Malabry Cede& France; 'lnsrilulo Boliviano de Biologia dc Ahura (IBBA). CP 171, La P a Bolivia: 8*Labornroirc DCSO. Ecok plylcchnque, 91128 Palaiseau Ceder. France.

Neutral extracts from Peperom'a galioi'des H.B.K. (Piperaceae) have been investigated for their antiparasitic properties against Leihmaniasis and Chagas disease. The interesting in vitro activity of the petroleum ether extract on k i s h m ' a ssp. and Trypanosoma cruzi prompted us to investigate its chemical constituents and resulted in the isolation of three major phenolic compounds. Structural assignments resulted from high field NMR measurements and homo or heteronuclear (HMBC, HMQC) experiments. Pipemgalin 1 is a new prenylated diphenol with a geranyl moiety. Grifolic acid 2 and grifolin 3 are isolated for the first time from higher

p45-1

plants. OH

/ H3cr H3C p"G,,c A OH

2 : R = O H . grifolic acid 3 : R = H, grifolin 1 : piperogalin

SYNTHESIS OF C1-C12 FRAGMENT OF ANNONACIN THE MOST CYTOTOXIC P46 1

ACETOGENIN OF ANNONACEAE OF TYPE A Xavier F d . Bruno F i g a d t d and Andd Cavd Labratoire de F'harmacognosie. associ6 au CNRS (BIOCIS), Facult6 de Warmacie 92290 CMlcnay-Malabry @ance)

Annonacin. a monotetrahydrofuran acetogenin of Annonaceae. isolated from A . muricata, A . densicoma, A. squamosa and Goniothalamus giganteus possess four hydroxyl groups at C4, ClO. CIS C20, whose absolute configurations have been recently elucidated as all R for C4, c l5 . C20, by the NMP analysis of its Mosher's esters. For ClO, the stereochemical relationship remains unknown.

To date, annonacin is also the most cytotoxic acetogenin of type A : ED50 &g/mL) 10-5 for 9PS a n c 10-3 for KB.

In order to completely elucidate the stereochemical relationships of the asymmetric centres of thf molecule, and to have an access to larger amount of product for further biological studies, we havf envisionned the total stereocontrolled synthesis of annonacin. The retrosynchetic scheme is based on i disconnection between C12 and c13, and requires to prepare in a stereocontrolled manner both synthons We disclosed herein the preparation of the C1-C12 fragment bearing carbons C4, ClO and C34 with wel: defined absolute configurations.

0

-5 5 annonacin

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p47 SPECIFIC BLOCKADE OF SEROTONIN RECEPTORS BY NEUTRAL ANTHRAQUINONE AGLYCONEW FROM RUBIA CARDIFOLIA RADIX. Anwar H. Gilani, K.H. Janbaz, A. Lateef, M. Zaman and A. Suria.

INHIBITOR(S) OF ARACHIDONIC ACID METABOLISM IN EXTRACTS OF OAT IAVENA SATIVAJ SEEDS Sheikh A. Saeed and Anwar H. Gilani Department of Pharmacology, The Aga Khan University, Medical College, Karachi- 74800, Pakistan

P48 1 The Colloidal oat (Avena sativa) fraction is known to possess beneficial properties in relieving itch, sun burn and other inflammatory conditions of the skin. Since metabolites of arachidonic acid IAA) e.g. prostaglandins are known to be involved in skin inflammation due to burns, contact eczema and exposure to U.V. radiation, we have evaluated to what extent extracts of oats inhibit AA metabolism. Oat seeds (Aventa sativa) were ground (wi th a moulinex grinder) into a fine powder. Batches of powdered oats (30 g each) were macerated 1 :10 with 5 0 m M phosphate buffer pH 7.4 or 96% ethanol for t w o hours. The extracts were centrifuged and tested against AA metabolism using bovine PG synthase and human platelets assayed as described previously (1,2). The results obtained showed the inhibitory activity of buffer and alcoholic extracts of Avena sativa on PG biosynthesis and AA metabolism by platelet homogenate. The buffer extract showed strong inhibitory activity, like wise the ethanolic extract also inhibited PG biosynthesis and AA metabolism by platelet homogenate. However, the ethanol extract of oat seeds showed strong inhibitory activity against thromboxane A-2 (TXA-2) production whereas phosphate buffer extract had no effect on TXA-2 or 12-HETE (the lipoxygenase metabolite). The detection of inhibitor(s1 of AA metabolism in Avena sativa may explain the anti-inflammatory effects of oats on certain inflammatory condition of the skin.

1.

2.

Saeed, S.A., Simjee, R.U., Mahmood, F. and Rahman, N.N. ( 1 993)J. Pharrn. PhafrnaCOl.

Collier, H.O.J., McDonald-Gibson, W.J. and Saeed, S.A. ( 1 9761Brit. J. Pharrnacol. 58: 45: 715-719.

193-1 99.

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p:50

NAKIENONES A-C AND NAKITIUOL, NEW CYTOTOXIC MITABOI~I IXS FROM AN OKINAWAN SYNECHOCYSTIS-ASSOCIATED C O M L

P:49 1 (ACROPORA SI'.) OVEKGROWTH Dale G. Naele, Mary Ann Roberts, and William H. Genvick, College of Pharmacy, Oregon State University, Corvallis, Oregon 9733 1 (USA).

This paper reports the structural elucidation and biological evaluation of several newly discovered CI I cyclopentene- and cyclohexene-containing metabolites isolatcd from an Okinawan microalgal-associated coral reef kill. Dead and necrotic branches of stony coral (Acmpora sp.), which were completely covered with a gray-black mat of microalgae (Synechocystis sp.), were extracted and shown to contain a series of chemically reactive secondary metabolites. These compounds and an unusual acidcatalyzed rearrangement of one metabolite, produce a series of compounds which are sbucurally similar to the didemenones A-D, isolated from Didemnwn sp. and Trididemnum sp., ascidians known to harbor unicellular algal symbionts. Additional results, including an investigation of the microalgae associated with this phmomena and a discussion of the similarities between this chemisby and that produced by other marine organisms, will also be presentcd.

INTRACEREBROVENTRICULAR BUT NOT INTRATHECAL

ANTINOCICEPTIVE EFFECTS IN THE MOUSE. 1 ~ 2 ~ 1 ~ A D M I N I s m A m o N OF m R P E m G L Y c o s m s HAS STRONG

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P51 INTRACEREBROVENTRICULAR ADMINISTRATION OF SMILAXINS

44. W. Suh. 1M. H. Woo. Q. C. DolY. S. Choi. [Dept. Pharmacol., Coll. Med., Hallym Univ., Chunchon, *Dept. Food and Nutr.,

PRODUCES ANTINOCICEPTION IN THE MOUSE. 1R.K So ne. ~~

k. d J - Y . H. &om. h d o n g Natl. Univ. Andong, 3Coll. Pharm. Hyosung Women's Univ.. Hayang, and 4ColI. Phann. Yeungnam Univ. Kyungsan, Korea.

Smilar sieboldii is a climbing shrub which grows in Korea, Japan, and China. We have recently solated that new spirostanol glycosides named smilaxins A, B and C from this shrub (Woo et al.. 1992). These compounds are mainly composed of laxogenin and tigogenin with different number of iugar moiety attached. In the present study, we examined the antinociceptive effects of these smiluxins ldministered intracerebroventricularly in ICR mice. Tail-flick test was used as an analgesic assay. h o n g smiluxins tested, smilaxin B, in which 3 sugar moieties are auached to C-3 of laxogenin. , h o w 4 the strongest antinociceptive effecf whose effect was dose-dependent. Smilarin A, in which 2 ,agar moieties are attached, was weak in the production of antinociception. In terms of structure elationship for the antinociceptive activities, we also obselved that the weak antinociceptive effects was hown when oxygen function was detached from C-6 position, although 2 sugar moieties were intact at :-3 position. Our results suggest that smilm'n derivatives have antinociceptive effect when they are idministered supraspinally. Further studies are needed to delineate their antinociceptive mechanism. Ref. Woo et al.. J. Nat. Prod. 55:1129-1135, 1992)

P52 ANT-HIV PHOTOAClIVE TERTHIOPIIENES: PROUI.EMS AND PKOSPI:cI'S.

Terry Connclly (4), Tony Durst (4). and Cesar M . Coinpadre (5) 1) Botany J>cparrmcnr. Brandon University, Brandon. MU R7A 6A9 2 ) Div. of Med. Microbiol., U. of British Coluinhia, Vancouver, BC V5Z 1M9 3) Ottawa-Carleton Institute of Biology. IJ. of Ottawa. Ottawa. ON KIN 6N5 4) Ottawa-Carleton Institute of Chemistry, U . of Ottawa. Ottawa, ON KIN 6N5 5 ) Dept. Biopharni. Sci., IJ. of Arkansas for Med. Sci., Little K o c k , AK 72205

;irlrs ( I ) . James 11. IIudson ( 2 ) , I.. Harris ( 2 ) , J . l h o r ArndSon (3), 1 Robin 1 . M

Terthiophcnes are three-ringed sulphurantaining heterocycles distrihuted widely among species of the Asteraceae. They have a wide range of insecticidal, antibiotic. and antiviral activities mediated by their long-UV wavelcngth photoactivatcd production of singlet oxygen. Their impressive activity (at concentrations as low as 12 n g h l or 30 nM) against human immunodeficiency virus .- 1 is influenced profoundly by tlie nature of side-chain suhstitucnts and the prescnce of serum in thc bioassay medium. and some thiophenes also show cytotoxic activity. Wc have deterniined some of the structure-activity relationships for these clfects and contrasted the antiviral activity with cytotoxicity and whole-animal toxicity in order to identify synthetic derivatives of the natural lead coinpound. alpha-tcnhienyl, which are selective anti-IIIV agents with low nontargct toxicity.

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p53 CRYPTOLEPICARBOLINE, A NOVEL INDOLOQUINOLINE- CARBOLINE DIMERIC ALKALOID FROM CRYPTOLEPIS SANCUINOLENTA. M.HM Shard and P.L. Schiff, Jr., Department of

p54 FYVONOIDS OF LITSEA CLAUCESCENS (LAURACEAE). Jos; k Lopez and Wagner Barillas, Insti tuto de Investigaciones Farmackutica? (INIFAR), Facultad de Farmacia, Univcrsidad de (;osta

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PI55 BIOTECHNOLOGY OF Solonum chrysorrichum. Schldh (Solanaceae). yillaneal*, Mariana Me&= **, Victor Navarm., Gabriela Rojas *, C? Mpez-Sancha*** Marta RodrlguQ***,Carlos Arias*** ; *Centre de

.

PLANTB USED IN MEXICAN TRADITIONAL NEDICINE WITH BEDATIVE, HYPNOTIC AND ANTICOEnmLBANT EFFECTB AND THEIR CLINICAL TRIAL PACILITIEB Jaime-. Centro de 1nvestigaci6n Biomgdica del Bur. INBE. Argentina 1 Xochitepec, Nor. N&xico CP 62790.

P56 1 An ethnobotanical study of plants used in Mexican traditional medicine was made. The source was the national inquiry done by the IMSS-COPLAMAR health program (1983-1985), which contains the information obtained from physicians of rural communities, about the medicinal plants most frequently used by local healers. Plants used for the treatment of problems related to the nervous system were selected. The information was classified in different groups according to the reported use: A) anticonvulsants, B) sedatives and C) hypnotics. From the 2 2 4 2 inquiries, a total of 81 different vegetal species used to treat mental disorders were obtained. In relation with these, the use of medicinal plants as sedatives was the most frequently mentioned by the healers, representing 63.7 %. The same vegetable species could be assigned to more than one group use. For this reason anticonvulsant use, was also referred to in 51.8 % and plants considered in the hypnotic group represent 27.1%.

A group of 2 6 interesting plants were obtained, from which Ternstroemia pringlei from Theaceae family, Ruta chalepensis and Citrus aurantium from Rutaceae family were at the head of sedative, anticonvulsant and hypnotic group respectively.

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I

FURTHER ALKALOIDS OF TELITOXICUh4 GLAZIOVII.

Altoona, Pennsylvania 16601-3760 and Alan Freyer, SmithKline Beecham Pharmaceuticals, UW- 2940,709 Swedeland Road, P. 0. Box 1539, King of Prussia, Pennsylvania 19406.

Tefitoxicwn gluziovii (Menispennaceae) is a woody vine indigenous to the Amazon basin. Chemical investigation of the nonphenolic fraction of this plant revealed the presence of four aporphine alkaloids: teladiazoline, telazoline, lysicamine, and 0-rnethylmoschatoline. The oxoaporphine splendidine was also isolated from this fraction recently. The phenolic fraction of T. gluziovii yielded the azafluoranthene alkaloid telitoxine. Details of the isolation and identifkation of other rare alkaloids from the neu!d and phenolic fractions will be presented.

D. Menachery, Hope Mandell, and Shawn Desaw, Penn State Altoona, 3000 Ivyside Park,

CONSTITUENTS OF THE ESSENTIAL OIL OF GRAVEOLENS.

Yan-Yi Han, You-Zuo Zhou and Chao-Mei Yu, Zhejiang Academy of Medical Science, 60 Tian Mu Shan Road, Hangzhou, Zhejiang 310013. P.R. China; Ru-Jun Yan and Ding-Zhi Jia. Hangzhou First Traditional Chinese Pharmaceutical Works, Hangzhou, P.R. China.

1 Essential oils of plants are widely used in perfumes, fragrances, spices, and medicinals. During the course of our search for bioactive natural products, we examined the essential oil of Pelargonium graveolens L'Herit (Geraceae), obtained by steam distillation of its fresh leaves, and found it to have anticancer activity. In further clinic studies, suppositories formulated with this essential oil exhibited total therapeutic effect in 70.4% of 135 cases of cervical cancer.

This essential oil, which possesses a characteristic rose-like smell, was separated by fractional distillation using a 2.8 (I.D.) x 100 cm column. The bioassay revealed that the fraction (in 44% yield) with b.p. 80-85 "C / 3 mm Hg showed improved anticancer activity. This fraction was further separated by repeated normal phase chromatography, and three major fractions A. B and C were isolated. Purity examination indicated that C was a mixture, while A and B were pure compounds. These two compounds were further identified as citronellyl formate and citronellol on the basis of spectroscopic analysis.

In vivo anticancer testing showed that citronellyl formate, citronellol and fraction C were all quite active. Of the three samples, citronellyl formate was the most active, and was 10% more effective than the original oil. Details of the isolation and anticancer activity of these compounds will be. presented.

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p-0

I

?RENYLATED FLAVANONES FROM DERRIS LAXIFLORA

ISOLATION OF XYLOSYL TAXANES FROM TAXUS MAIREI

Chemistry and Pharmacognosy, Purdue University. West Lafayette, IN 47907, U.S.A. C.-t. Chang. C.-y. Lee and Y.4. Yang. Union Chemical Laboratories, Industrial Technology Research Institute. Hinchu, Taiwan, Republic of China.

Y.-c Liu and C.-j Chang*. Department of Medicinal P59 1

Tax01 possesses unique mechanism of action and remarkable anticancer activity. The major obstacles in further development of these taxane drugs are their limited availability from plants. As part of an investigation of new sources for taxol and related taxane, the taxane compositions of various part5 of Tarus muirei have been analyzed using high performance liquid chromatography in conjunction with a highly compound-specific and sensitive tandem mass spectrometric method. We have quantitatively determined and reported the isolation of taxol, cephalomannine. 10-deacetyltaxol, 10- deacetylcephalomannine. baccatin 111. and 10-deacetylbaccatin 111 from needles of Tarus mairei. Here we report the quantitative determination of three xylosyl taxanes by H P X and isolation of taxanes from the needles. The resu1t.s indicate that Tarus mairei may provide renewable source for tax01 and related manes . ( Supported by National Cancer Instituk, R01 CA55118 )

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p:62

Pyridine Alkaloid Biosynthesis in nine Nicotiniana species. J Brent Friesen, Departement de Chimie, Faculte des Sciences Exactes et Appliquees, Universite du Tchad, N'Djamena. E. Leete. (deceased February 8, 1992) Natural Products Laboratory, Department of Chemistry, University of Minnesota, Minneapolis, MN 55455.

P:61 I A systematic study of (2'-"C) nicotine demethylation in excised tissues from roots, young leaves and mature leaves on fine species of tobacco plants showed that demethylation activity varies widely according to tissue source and species. Mature leaves from N grandiflora display a high demethylation activity of exogenous nicotine (10% Absolute Incorporation and 100% Specific Incorporation) despite the fact that nornicotine is practically absent in the leaves. N alata roots exhibited significant demethylation (5% At) while mature leaves from N glutinosa showed significant demethylation (10% AI) compared to the roots (O.S'?'o AI).

A systematic study of pyridine alkaloid biosynthesis from (2-'H) nicotinic acid supported the general observation that pyridine alkaloids are produced in the roots and translocated to the aerial parts of the tobacco plant. However, mature leaves from N alata (Lime Green) exhibit significant alkaloid biosynthesis activity (0.1 A1 and 56% SI) even though nicotine is normally present in low amounts in the plant leaves.

ANTI-TUMOR-PROMOTING ACTIVITY AND ANTIOXIDATIVE E F F E C T S OF P E N D U L O N E , A N I S O F L A V A N Q U I N O N E F R O M W I S T A R I A B R A CH Y B 0 T R Y S T h o Konoshima (1). Midori Takasaki (1). Mutsuo Kozuka (1). Toshihiko Osawa (2).

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p63 ANTIOXIDATIVE ACTIVITIES OF EUGLOBALS AND THEIR RELATED PHLOROPHENONES Midori Takasaki ( I ) , Taka0 Konoshima (1). Mutsuo Kozuka ( I ) . Toshihiko Osawa (2).

"W

A PHYTOCHEMICAL SURVEY ON

Pedro M. Abree Secqio de Quimiea Orginica Aplicada, Faculdade de Citncias eTecnologia da Universidade

Nova de Lisboa, Quinta da Torre, 2825 Monte da Caparica, Portugal and Eurico S. Martins

Centro de Botinica do lnstituto de Investigaqio Cientifica Tropical, Tv. Conde da Ribeira 7, 1300 Lisboa, Portugal

MEDICINAL PLANTS OF GUINEA-BISSAU P44 1

In the course of a field work carried out by the authors, near Contuboel, north of Guinea-Bissau, several medicinal species of Mimosaceae, Leguminosae, Rubiaceae, Meliaceae, Anacardiaceae, Combretaceae, Euphorbiaceae, Caesalpiniaceae, Zingiberaceae, Apocynaceae, Verbenaceae and Lorantaceae, were collected. The local therapeutic uses are described in accordance with the depositions of the native quacks obtained on the occasion of the gathering of plant material. As many medicinal plants found in Guinea-Bissau also grow in other areas of tropical West Africa and Asia, a phytochemical survey of previously studied species is presented, based on published data.

Preliminaries results of our phytochemical research on Parkiu biglobmu, Khaya senegalensis, Pterocarpus erinaceus and Anthostenla senegalensis are reported.

This work was supported by JNICT under research contract PBIC/C/CEN/I 023/92.

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BIOSYNTHETIC INVESTIGATIONS OF TIIE EUDISTOMINS IN THE TUNICATE EOIlISTOMA OLIVACEUM. Guo Q Shen and Bill J Baker, Department of Chemistry, Florida Institute of Technology, Melbourne, FI, 32901

P65 1 l h e tunicate Eudrsiomu olrvuceum produces a series of P-carboline derivatives, the eudistomins,

ihich display a variety of pharmacological activities, including potent antiviral and antimicrobial ctivity and cytotoxicity The eudistomins are produced by the tunicates in exceedingly small uantities We have undertaken a biosynthetic investigation of the eudistomins in an effort to better nderstand metabolic process leading to their production Tryptophan and proline were shown to tbel both eudistomin H ( I ) and I (2). while tryptophan was demonstrated to be a specific precursor of udistomin I Ornithine and arginine were not utilized significantly by E. olrwceum Eudistomin I is tbeled by tryptamine, to the exclusion of eudistornin H. Bromotryptophan and bromotryptamine are electively incorporated into eudistomin H. Based on these incorporation results, a pathway to the udistomins in I;. olrvuceum wll be presented

u I. hudistomin I[ R = Hr 2. Eudistomin I R -- H

ISOLATION AND CHARACTERIZATION OF PTEROENONE, A FISH DETERRENr FROM THE PELAGIC ANTARCTIC PTEROPOD CLIONE ANTARCTICA Wesley Yoshida.l.2 Patrick Bryan,) Rill J. Raker,] and James B.

AcClintock.’ ‘Department of Chemistry, Florida lnstitutc of Technology, Melbourne. Florida 32901, Department of Chemistry, University of Hawaii, Honolulu, Hawaii 96822, and ’Department of liology. University of Alabama at Birmingham, Birmingham. Alabama 35294

An intriguing relationship between an antarctic hyperiid amphipod and its unwitting guest, a lteropod, was reported recently by our group The amphipod HyperieNu dilufutu, a common prey of everal antarctic fish, is capable of grasping Cliorre unfurc/rcu (= C/iotie Irmucinu) from the water olumn and positioning it on its back where the chemically defended mollusc serves to prevent lredation of the amphipod. This defensive property was demonstrated by the rejection of mphipodhollusc pairs. as well as the mollusc itself, by the usual amphipod fish predators. Utilizing hese fish predators as the basis of a bioassay guided isolation has lead us to the chemical principle esponsible for chemical defense in C. unfmc/icu. We report here the isolation and chemical structure his substance, which we have named pteroenone. Pteroenone is a f3-hydroxyketone polyketide, tiogenetically derived from four propionate and one acetate units.

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P67 EVALUATION OF CHEMOPREVENTWE AGENTS THAT STIMULATE PROTEIN KINASE C ACXIVITY BUT INHIBIT TUMORIGENESIS

P:6$ BIOASSAY-GUIDED ISOLATION OF INDOLE ALKALOIDS FROM PESCHIERA

M., X. J. Ma, R. Mukhcqcc', N. R. Famsworth, G A Cordell, A. D. Kinghorn, and ~ E T A THAT REVERSE WLTIDRUC-RESISTANCE

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EVALUATION OF THE W O T O M C MECHANISM MEDIATED BY THE DITERPENE LACTONE 1 3 - M E T H O X Y - 1 S O X O Z O A . Lisa Shamon, &-So0 Lee, A. Douglas Kinghorn and John M. Pevuto Program for Collaborative Research in the Pharmaceutical Sciences, Department of

Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illmois, 6C612, USA

As reported elsewhere at t h s meetinf, a new diterpene lactone, 13-methoxy-15-oxozoa atlin, was isolated from an ethyl acetate extract o the Zimbabwean medicinal plant Parinari curatrl&ia on the basis of bioactivity-directed fractionation. The isolate demonstrated significant cytotoxicity against several human tumor cell lines. Therefore, more detailed studies designed to examine its mechanism of achon were performed using cultured human hormone-dependent breast cancer cells (ZR-75-1) as a model system. Treatment with 13-methoxy-15-oxozoapatlin blocked cells in the G2+M compartment of the cell cycle, but the morphology of dibutylyl CAMP-treated astrocytoma cells in culture was unaffected. Thus, this accumulation does not appear to be due to antimitotic activity. As judged by spectrophotometric procedures, the compound formed adducts with nudeophiles such as glutathione and pmercaptoethanol. Subsequent to reaction with the latter, an adduct was isolated and structurally characterized, which indicated that the compound undergoes a Michael-type addition. As antici ated, relative to the parent compound, the cytotoxic respowe mediated by the adduct was reducecf 13- Methoxy-15-oxozoapatlin was not mutagenic with Salmonella typhimurium strain TM677. These and additional data presented here suggest that the cytotoxic activity of 13-methoxy-15-oxozoapatlin is mediated b covalent reaction with a cellular component (such as a sulfhydryl-containing protein) by means of a Kichael-type addition. (Supported by grant UO1 CA52956 awarded by the National Cancer Institute)

‘Lee, 1.-S., H.-8. Chai, T.E. Chagwederq N.R. Farnsworth, D.D. Soelarto, G.A. Cordell, J.M. Pezzuto and A.D. Kinghorn. Rearranged rnt-kaurenoids with cytotoxic activity from Pannari curatcllifolia, Thirty-fifth Annual Meeting of the American Society of Pharmacognosy, Halifax, Nova Scotia Canada. July 31-August 4,1994.

P69 I

CHEMISTRY OF LIGUSTILIDE, A BIOACTIVE PHTHALIDE DERIVATIVE FROM LIGUSTICUM PORTERI, A MEDICINAL PLANT OF THE SOUTHWEST U.S.

P70 I John J. Beck and Frank R. Stem&, Department of Chemistry, Colorado State University, Fort Collins, Colorado 80524.

Ligustilide has been obtained from Ligusticum porteri Coult. & Rose (Apiaceae) and from total synthesis. Its in vitro chemical reactivity has been examined and will be discussed.

1,6 addition 7

cycloaddition or radical reaction

Z-Ligustilide

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T71 CHEMlSTRY OF THE ANTARCTIC SPONGE ISODICTYA ERINACEA. Byoungho Moon and Bill J. Baker, Department of Chemistly. Florida Institute of

The chemistry of Antarctic marine invertebrates is being investigated from both an ecological and biomedical standpoint. Several interesting bioactive natural products have been isolated from sponge specimens from Antarctica. As part of our ongoing search for new compounds fiom Antarctic marine organisms, we have investigated extracts of the polychaete sponge, I.wdictya erinacea, collected at McMurdo Sound, Antarctica. We have isolated several highly polar aromatic heterocycles from the MeOH extract. Isolation and structural identification of these compounds will be presented.

TWO NEW NATURALLY OCCURRING PYRROLE DERIVATIVES FROM THE TROPICAL MARINE

SPONGE AGELAS OROIDES

M. Konig, Anthony D. Wright

P72 1 Department of Pharmacy, Swiss Federal Institute of Technology (ETH), Zurich, CH-8057 Zurich, Switzerland, and Anthony Linden, Laboratory for Crystallography. Department of

Organic Chemistry, University of Zurich, Zurich, CH-8057 Zurich, Switzerland.

Continued investigation into the natural products chemistry of the tropical marine sponge Agelas oroides, collected from the Great Barrier Reef, Queensland, Australia, has yielded two new naturally occurring pyrrole derivatives, 1 and 2. The structures of 1 and 2 were determined from the interpretation of their 1 D and 2D NMR, UV, IR and mass spectral data. For 1 a synthesis is reported. Attempts to synthesise 2 applying the same methodologies used to obtain 1 are also reported. The main product of both syntheses was 3. The derivation of 3 and its X-ray structure are reported.

H

C/ 'CH3 Qc,Nb I \ Qc,kc,H I \ 0 HH3

I II I I I 11 H O o H o

I 11 H o

1 2 3

/

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p:73 1 NEW DOLABELLANES FROM THE MARINE BROWN ALGA DICTYOTA PARDALIS cf. PSEUDOHAMATA

Gabriele M. Kbnig, Anthony D. Wright Department of Pharmacy, Swiss Federal Institute of Technology (ETH), Zurich, CH-8057 Zurich, Switzerland, and Frank R. Fronczek Department of Chemistry,

Louisiana State University, Baton Rouge, LA 70803-1 804. U.S.A.

An investigation of the natural products chemistry of the brown alga Dicryota pardalis cf. pseudohamata collected from Magnetic Island, Australia, yielded five new dolabellane type diterpenes (1-5) and the previously reported compounds 6-10. The structures of 1-5 were determined from the interpretation of their 1 D and 2D NMR, UV, IR, X-ray and mass spectral data. The previously reported structures for 9 and 10 were clarified.

R H 9

R R R R1 OAc 1 H 3 H H 7 H 6 A c 4 H OAc 8 Ac 10

OAc H 2

QUlNOLlZlDlNE ALKALOID DISTRIBUTION IN THE SEEDS OF FURTHER SPECIES OF ORMOSlA (LEGUMINOSAE, SUBFAMILY PAPILIONOIDEAE). Manuel F. Baland r h l , A. Douglas Kinghornz, and J.

Michael Edwards3, 1 Department of Medicinal Chemistry, NPS Pharmaceuticals, Inc., 420 Chipeta Way, Salt Lake City, Utah 84108, USA, 2Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612. USA, and 3Section of Medicinal Chemistry and Pharmacognosy, School of Pharmacy, University of Connecticut, Storrs, Connecticut 06268, USA.

The seeds of certain species of the tropical arboreal genus Ormosia [Leguminosae (Fabaceae), subfamily Papilionoideae, tribe Sophoreae] are believed to have medicinal (i.e., analgesic and cardiovascular) properties. In a continuation of our work on legume alkaloids, the quinolizidine alkaloids from the seeds of eight species and two unidentified taxa from 30th the Old and New Worlds were examined by means of thin-layer chromatography (TLC) and capillary gas chromatography/electron-impact mass spectrometry (GC/EI-MS). The Old World species examined were the Asiatic 0. emarginata, 0. pachycarpa, and 0. semicastrata (all from Hong Kong), and the East Indian 0. calavensis (from the Philippines). The New World (i.e., Latin American) species examined were 0. macrocalyx (from Mexico), 0. costulata, 0. nobilis, and 0. paraensis (the latter three from Brazil); the two unidentified taxa also were from Brazil. Crushed seeds were first defatted with hexane, followed by sxtraction with methanol. Crude alkaloid fractions were further purified according to previously established protocols. The profiles found for the refined alkaloid extracts were then compared with that of the known 0. krugii, which was used as an internal reference.

P:74 1

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POSTER PRESENTATIONS I65

VALERIAN-DERIVED SEDATIVE AGENTS. II. DEGRADATION OF VALMANE@-DERIVED VALEPOTRIATES IN AMMONIATED HYDRO- ALCOHOLIC TINCTURES. Manuel F. Ba- ’ 1, Bradford C. Van

Wagenenl, and Geoffrey A. Cordellz, 1Department of Medicinal Chemistry, NPS Pharmaceuticals, Inc., University of Utah Research Park, 420 Chipeta Way, Salt Lake City, Utah 84108, USA and 2Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 S. Wood Street, Chicago, Illinois 60612, USA.

Aqueous extracts of the roots and rhizomes of Valeriana officinalis L. (common name: valerian) and closely related species (family Valerianaceae) have been used for over a millenium in Europe and Asia for medicinal purposes. These plants biosynthesize a series of biologically active and chemically unstable iridoid monoterpenoids termed valepotriates which are currently used in numerous pharmaceutical products in Europe (most notably in Germany) and which are sold as sedatives, tranquilizers, and sleep aids. Ammoniated hydroalcoholic tinctures of valerian root have been used historically in Great Britain and the United States since at least the nineteenth century as medicinal preparations. In a continuation of our work on the valepotriates (vide L.-J. Lin, G.A. Cordell, and M. F. Balandrin, fharm. Res. a(9): 1094-1 102 (1 991 )], amrnoniated hydroalcoholic tinctures containing the major triester valepotriates (i.e., valtrate, didrovahrate, and acevaltrate, derived from the German pharmaceutical product, Valmane@) were found to degrade rapidly under various conditions to afford primarily isovaleramide and several minor degradation products. A mechanism for the ammonia-mediated decomposition of the major valepotriates, and the generation of isovaleramide from isovalerate moieties, will be presented.

P:75 I

NTI-MYCOTIC ACTIVITY FROM CULTURES OF DUCKWEED (I.emnu minor L.) IF76 k K D INITIAI. CHARAClERIZATION OF THE ACTIVE COMPOUNDS

epartment of Biological Scienccs. Philadelphia College of Pharmacy & Science, 600 S. 43rd Street, Philadelphia, Pennsylvania 19104 (USA)

Observation of still cultures of duckweed accidentally contaminated during transfer revealed that the duckweed is releasing compounds to thc medium with fungistatic activity. Fungi will quickly overgrow the cultures in the first few days after transfer to fresh medium but will not grow in established cultures. Fungi transferred from the duckweed cultures re-established rapid growth on transfer to fresh medium. The fungistatic compounds of this plant would be of cnormous importance for competition in the aquatic environment, where fungi and other microorganisms are abundant and grow rapidly on decaying organic matter. The identification ofthe active compound(s) could also lcad to new classes of anti-mycotic agents for medicinal or food-processing uses. Cultures of duckweed in one-half-strength Hutner’s medium were collected and pooled. Approximately 6 1 of used medium was frozen, lyophilized and the residue taken up in a minimal volume of water. This solution was exhaustively extracted with ethyl acetate. The aqueous medium was re-fro7xn, lyophdiwd, the residue extracted with methanol, and the insoluble material dissolved in water. The ethyl acetate fraction was evaporated in vucuo, the residue extracted with DMSO:H,O ( l : l ) , and the insoluble material was dissolved in ethyl acetate. All four fractions were filter sterilized and used in bioassays with Succhuromyces cernjisiur and Cudidu dbicuns. There was significant inhibition o f the growth of both yeasts when exposed to the DMSO:H,O extract compared to the solvent control. Slight inhibition also occurred with the water and ethyl acetate exIracts relative to their amtrols. The isolation of active compunds was performed using bioassay on TLC plates of the extracts. Identification of activc compounds IS currently undrrway.

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166 POSTER PRESENTATlONS

p77 .

ISOLATION AND CHARACTERISATION OF PHARMACOuXiICALLY ACTIVE COMPOUNDS FROM THE BARK OF CHOEROSPONDIAS

AXILLARIS. A VIETNAMESE MEDICINAL PLANT USED TO TREAT BURNS.

An aqueous crude extract (CAE) of the plant Choerospondiar axilloris. Roxb.Anacardiaceae, which has long been used in Vietnamese wditional medicine in the treatment of burns, was studied on its anti-inflammatory and antibacterial properties. An inhibitory effect on prostaglandin biosynthesis and PAF-induced exocytosis has been shown by CAE. The potency of CAE on prostaglandin synthetase was equal to that of aspirin (ICso=75.0pg/ml), but lower than of indomethacin. CAE also inhibited PAF-induced exocytosis in human neutrophils with an ICso value of 362.5pdml. It was also found that some fractions of CAE possess an antibacterial effect in virro against the common wound pathogens Sraphylococcus aureus and Pseudomonas aeruginosa using the filter paper disc assay method.

Bioassay- directed fractionation of CAE, based on the methods mentioned above, resulted in isolation of naringenin (l), choerospondin (2). (+) catechin (3). (-) epicatechin (4), quercetin ( 5). pinocembrin-0-glycoside (6). gallic acid (7) which were identified mainly by NMR spectroscopy. mass spectromeny and TLC comparing with authentic samples. Among them, (2) and (6) dose- dependently inhibited prostaglandin synthetase in virro, whereas their PAF-antagonistic property was not considerably shown. Compounds (3), (4), (5). (6). (7) have not been previously isolated from the bark of the medicinal plant.

@ More than 80 references have been consulted in preparing this paper.

IT2rXIUiTiCXN OFPUT<EWAWI< SDSPENXONTOF~D~TKFN~- - --__

MEDIUM I'OLAIUTY CRUDE EXTRACTS AND THEIR BIOAVAILABILTY F"..- I Gil. A. MagosO, Raul G. Enriquez', Benjamin Ortiz', Ismael Le6n'; Maria L. -~ 'Villarreals, Mariana Meckess; 'Instituto d e Quimica, UNAM, Cd.

Universitaria, Mexico, D.F. 04510; OFacultad de Medicina, UNAM ibid.; gunidad d e nvestigacidn Riomkdica, IMSS, Xochitepec, Mor. Mexico.

en carrying out the prelimmar screenings of medicinal plants in search of biological evaluation

ater suspensions. For this purpose, a number of dispersing agents are used which include polymers f polivynilpirrolidone type, polyethyleneglycols, oil dispersions, alcohol, dimethylsulphoxide, etc.

1 of these normally require that a blank solution is tested to rule out additional biological effect in e model used due to these suspending agents. Therefore, we have tested a useful method for

valuating crude low-medium polarity extracts as well as pure subtances which are not soluble in ater. Aliquots of extracts are first dissolved in suitable organic solvents or mixtures of them; further

ddittion of the desired ammount of water is carried out and organic mixture is removed. This ethod was based in similar procedures reported by Famsworth (1). Three case examples were used

o evaluate such procedure where the suspensions were shown to possess the activity and proved dequate for carrying out pharmacological testings. Thus, hexanic, methylene chloride and ethanolic

a specific experimental model, a common problem encounter is the preparation of suitable

de extracts of Ipomoea stans , Arirtolochia taliscana and Montanoa fomeittosa were used for conductin1 timicrobial and isolated tissue screenings following the mentioned procedure with satisfactory

esults. The bacterial and fungi species used were: Staphillococcus aureus ATCC 29213, Bacillus subtilis TCC 06633, Pseudomona aureoginosaATCC 27853, Salmonella typhi ATCC 06539; Tricophyton ntagrocytes NRIiL1942 and Microsporum gypseum A-2605. A variable shelf life ranging from hours

o days was observed making the preparations useful for many preliminary biological testings and de. A discussion of the scope and advantages of the procedure using crude extracts and pure

~ ubtances will be presented. I.-- - - - - - __ - - __ -

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MINOR ALKALOIDS FROM THE LEAVES OF SARCOMELICOPE DOGNIENSIS HARTLEY (RUTACEAE)

. . -, Sofia Mitaku, Alexios-Uandros Skaltsounis, Michel Koch, Jacques Pusset, and Thierry SCvenet Laboratoire de Pharmacognosie de I'UniversitC RenC Descanes, U.R.A. au C.N.R.S. no 1310, Facultt des Sciences Pharmaceutiques et Biologiques, 4, Avenue de

I'Observatoire, 75006 Paris (France) I.C.S.N. du C.N.R.S., 91 190 Gif-sur-Yvette (France)

Sarcomelicope dogniensis Hartley is a medium Rutaceous tree up to 12 m high, recently described as endemic to the Plateau on Dogny in New Caledonia. We previously described the major aklaloids isolated from its leaves, which all belong to the N-desmethylacronycine series. We have now isolated two novel minor alkaloids. The structure of the first one has been established as 1-0x0- 1,2-dihydro-N-hydroxydesmethyl- acronycine (1) on the basis of its spectral data, mainly MS and NMR. The second one, 2.3-dicarbomethoxy- l-methyl-4(1H)quinolinone (2) arises most probably by oxidation of the C ring of an acridone percursor.

0

1

ALKALOIDS FROM THE LEAVES OF SARCOMELICOPE MEGISTOPHYLLA

Francois Tilleauin, Lyria Sedrati, Alexios-Uandros Skaltsounis, Michel Koch, Jacques Pusset, and Thierry SBvenet Laboratoire de Pharmacognosie de I'Universitt RenC Descartes, U.K.A. au C.N.R.S. no 1310, FacultC des Sciences Pharmaceutiques et Biologiques, 4, Avenue de I'Obsewatoire, 75006 Paris (France) I.C.S.N. du C.N.R.S., 91 190 Gif-sur-Yvette (France)

HARTLEY (RVTACEAE) P80 I

The genus Sarcomelicope includes nine species of Ruraceous shrubs or small trees, eight of which are endemic to New Caledonia. In a continuation of our chemotaxonomic study of that genus, we repon here the alkaloid contents of Sarcomelicope megistophylla. a tree easily recognized by its large pubescent leaves. The leaves afforded 0.2% total alkaloids. Fractionation on column chromatography resulted in the isolation of melicopidine, 1,2,3-uimethoxy- 10-methylacridone, acronycidine, acronydine and of a novel furo[2,3b]quinoline derived alkaloid, sarcomegistine (1) The sbucture of this new compound was deduced from its spectral data. Of particular interest were NOESY 2D IH NMR experiments which permitted to localize unambiguously the position of the substituents on the A ring. From a chemotaxonomic point of view, the isolation of a furo[2,3b]quinoline alkaloid with such a C- bisalkylated A ring is very interesting since this type of compound was so far considered as typical for the genus Haplophyllwn.

1

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p-1 SEARCHING BOLIVIAN-KALLAWAYAN MEDICINAL PLANTS FOR LEADS TO ANTI-HIV, ANTI-TUMOR AND OTHER POTENTIAL DRUGS. Samia Abdel-Malek,’ Joseph W. Bastien,‘ Thomas H. Corbett,’

P:82 NEW OKARAMINE CONGENERS, OKARAMINES D, E AND F, FROM PENlClLLlUM SlMPLlClSSlMUM ATCC 90288 m e o H W , Yoshihide Asabu, Sawao Murao, and Motoo Arai

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p8 1

THE BlOuxilCAL XXIVITIES AXI CONSTITUENE OF Pedicularis resupineta var. oppsitifolia

ISOLATION AND STRUCTURAL ELUCIDATION OF NOVEL SECOIRIDOID GLYCOSIDES FROM FRAXINUS UHllEl

L&q&&Ei. Sook-Yom Lee. Seung-Jo YOO Sahm-ymk University. College of pharmacy Chungryang P.0.b~ 118 Searl. Korea

The Plant of Pedicufaris resupinate L.var. oppmitifolia (Scrufiulariaceae) have been appeared to be used for the treatment of humtoid arthritis, mlirplant abscess(tuoor). urolith. and diuretics in oriental sedicinal books.

The methanol extracts of native plants show some effects on bile juice secretion and acute toxicity in rat and mice respectively. The hthanol fraction of the ethanol extracts and acteoside from the buthanol fraction had inhibition effect on carrageenin induced er!ema in rat p a w . enhancemmt of bile juice secretion. and hepntoprotective activity. The hepatoprotective functions sere improved on serum transaminase activities (s-CpT and s-CTJ"). liver weight, and even sleeping time in mice treated with these coopwnds. This i s the first report shown their biological activities and other effects of acteoside from this particular plants. Three different coopwnds were purified and characterized by spectroscopy (W. I R . W. M5) analysis. These compounds were Acteoside (cmpound I ) . Suaviss~lside A 1 (compound 11). and D-mannitol (ccspound I l l ) .

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170 WSTER PRESENTATIONS

P86

EVALUATION OF ANTIVIRAL ACTIVITIES OF TANNIN DERIVATIVES AGAINST HUMAN CYTOMEGALOVIRUS -we i Wane and Chang-Yih Duh Department of Microbiology, Kaohsiung Medical College, Taiwan, ROC

P85 1

CYTOTOXIC NORCEMBRENE DlTERPENES FROM THE SOFr CORAL SINULARIA INEXPLICITA

Sixty-threetannin derivatives, including ellagitannins. gallotannins andcomplexed tannins, have been evaluated antiviralactivities against humancytomegalovirus (HCMV) strain AD169. The antiviral activities(ED SO) were measuredinhibition of HCMVDNA replication by nucleicacid hybridization Cytotoxicities (CD so) of tested compounds against cultured human embryonic lung cells were assessed by an MTT colorimetric assay. The relationship between tannin structures and their antiviral efficacy (SI=CD5o/ED%) will be presentcd and discussed.

0

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~ ~~

A STUDY FOR PURIFYING MULTIGRAM QUANTITIES OF FUMONlSlNS P87 1 W i l l i a m J e H m r , Chandra Chaurasia, Roger K. Harris and Jim Greaves Midwest Research Institute, 425 Volker Blvd. Kansas City, MO 641 10

Fumonisins in corn have raised economic, human health, and science policy concerns. Examples include causing cancer and/or other afflictions in farm and laboratory animals; being implicated as causative factor in human esophageal cancer; and possibly invalidating animal studies since their rations contain significant levels of corn. Under Midwest Research Institute's contract with the National Toxicology Program, we were assigned to perform a feasibility study for purifying large amounts ( - 100 GI of pure Fumonisin 61. Research teams in South Africa and the United States have previously published methods for purifying smaller amounts of 61. They used both traditional LC and HPLC techniques in their purification methods. In addition, several other publications have developed analytical methods for detecting and quantitating Fumonisins 81, 82, and 83. MRI chemists evaluated several of these techniques to determine their utility to purify Fumonisin B1. Selection criteria included reducing time, solvent, and column media costs while increasing impurity detection. To this end, MRI evaluated supercritical fluid extraction (SFE) to partially purify Fumonisin B1. Further purification was by preparative HPLC. To improve detection of impurities in the latter HPLC step, MRI compared an evaporative light scattering (ELS) detector with the refractive index (RI) detector. Details will be discussed.

ISOLATION AND MECHANISM OF ACTION OF A HEPATOPROTECTIVE AGENT FROM THE NIGERIAN MEDICINAL

Roseline Aliyul*Z. Z.S.C. Okoye2 and W.Thomas Shier1 University of Minnesotal, Minneapolis, Minnesota 55455 (U.S.A.), and University of Jos2, Jos (Nigeria)

PLANT COCHLOSPERMUM PLANCHONII P88 1 Cochlospennwn plunchonii (Hook. f.) is a pantropical plant widely used by rural dwellers of northern Nigeria as a traditional ueatment for jaundice. Hepatoprotective activity in aqueous extracts of the rhizomes was confirnacd by demonstrating significantly reduced serum bilirubin and liver diagnostic enzymes in a CCk-ueaml rat model sysmm. Various plausible hepatoprotective mechanisms have been considered, including antioxidant and radical scavenging activities. An alternate possible mechanism, which frequently forms the basis for treating organic solvent toxicity in humans, is inhibition of metabolism of the solvent. Consistent with this, C. plunchonii extract significantly inhibited the rat liver cytochrome P-450 monooxygenase enzymes aniline hydroxylase and aminopyrine-Ndemethylasc. Inhibition of these enzymes was used as a bioassay to guide purification of the inhibitor from the hexane-insoluble fraction of the aqueous extract by polyamide column chromatography. preparative thin layer chromatography on silica gel, and fractional crystallization from aqueous ethanol (yield: 1.42% of dry starting plant material). Pretreatment with 1.5 mg of the crystalline P-450 monooxygenase inhibitor daily for 3 days protected rats from CCl4-induced hepatotoxicity. as indicated by reducing CCl~-srimulated increases in serum bilirubin. alkaline phosphatase and alanine aminouansferase to 18.3%. 13.38, and 52.9% of control, respectively.

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INITIAL STUDIES FOR DETERMLNING THE ABSOLUTE AND RELATIVF, CONFIGURATION OF THE BIOACTIVE MYCOTOXIN, FUMONlSIN B1. Thomas R Hoye,# ' ,+ and Jorge I. Jimenez.#

Department of Chemistry* and Department of Medicinal Chemistry+ University of Minnesota, Minneapolis, MN 55455

Fumonisii B1 (FBl)(l) is a mycotoxin produced by Fuarium molinifonne, an important pathogen of stored corn. FBI contaminates some food products and may function as an environmental tumor promoter. A combination of comparative chiral capillary GC analysis and Mosher ester analysis has permitted the assignment of the absolure configuration of both the C(10) carbinol center and the C(16) methyl-bearing center as R on the carbon skeleton of FBI. Ln addition, IH and 1%2 NMR spectroscopy have led to assignment of the relative configuration for the fragments C(2)€(3) as erythro (or 1.2-anri). C(3)-C(5) as threo (or 1,3-anti), and C(14)-C(15) as erythro (or 1.2-anh'). The methodology used to determine both relative and absolute configuration of FBI will be presented.

H O ~ O H COOH

Me 2 I 10 12 11 10 18 =+'OH

erythra

RIOACTIVE METABOLITES FROM COPROPHILOUS FUNGI. William R. Gamble and James B. Gloer*, Department of Chemistry, University of Iowa. Iowa City, 1A 52242. James A. Scott and David Malloch, Department of Botany, University of Toronto, Toronto, Ontario, Canada, M5S 1Al.

Many coprophilous (dung-colonizing) fungi exhibit antagonism toward other species that inhabit the same competitive ecosystem. This antagonism is typically caused by metabolites produced by one fungal species which inhibit the growth of competitors. Chemical studies of the metabolites responsible for these antagonistic interactions could lead to the discovery of new natural products with antifungal properties and other activities of pharmacological interest. Our prior investigations in this area have already yielded a variety of new metabolites that exhibit antifungal activity. Recent studies of four species (Polytolypcr hysrricis. Bombardioidia homhardioides, Geomyces sp. , C J ~ Anopodiurn rrmpullaceunt) have afforded several additional aromatic compounds that exhibit antibacterial and/or antifungal effects. Details of the isolation and structure determinatior of some of these metabolites will be presented.

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PETREL1,LNS: NEW DEPSIPEPTIDES FROM THE COPROPHILOUS FUNGUS PETRIELL4 SORDIDA. Kit K. Lee and James B. Gloer*. Department of Chemistry, University of Iowa, Iowa City, Iowa, 52242. James A. Scott and David Malloch, Department of Botany. University of Toronto, Toronto, Ontario, Canada, M5S 1Al.

Our ongoing studies of fungi from competitive ecosystems continue to afford novel biologically active secondary metabolites. An isolate of the coprophilous fungus Petriella sordida obtained from porcupine dung displayed antagonistic effects against the competitor fungi Sorahria fimicola and Ascobulus fugbaceus. Bioassay-guided fractionation techniques were used to locate ana isolate the active compounds from crude P . sordida fermentation extracts. These studies led to the discovery of four new antifungal depsipeptides. which we have called petriellins A-D. The structures of these compounds were determined primarily through application of 2D-NMR techniques, especially HMQC, HMBC, and selective INEPT experiments. FAB mass spectral data and MSMS data for the petriellins and their reaction products were also useful in detemuning the amino acid sequences of these compounds. The stereochemistry of constituent amino acid units was determined by chiral GC analysis of amino acid derivatives. Details of the isolation, structure determination and biological activity of these compounds will be prcsented.

AFLAVININE DERIVATIVES FROM THE ASCOSTROMATA OF EUPENICILLIUM CHUSTACEUM. Huiiuan Wang and James B . Gloer*, Department of Chemistry, University of Iowa, Iowa City, 1A 52242. Donald T. Wicklow and Patrick F. Dowd, Agricultural Research Service, National Centcr for Agricultural Utilization Research. USDA, Peoria, 1L 61604

P92 1 As an extension of our ongoing investigations of fungal sclerotia as sources of antiinsectan

natural products, ascostromata from members of the gcnus Eupenicillium were examined chemically. Sclerotia are long-lived physiological structures important in the life-cycle of ccrtain fungi because of their ability to survive harsh conditions. Our prior studies have shown that the sclerotia of many Aspergillus spp. contain secondary metabolites with antiinsectan effects that discourage consumption of .sclerotia by fungivorous insects. Eupenicillium spp. produce specialized resting bodies called ascostromata which appear to play ecological roles analogous to those of sclerotia. Studies of the ascostromata of E. crusfaceum (NRRL 3332) afforded two metabolites of the aflavinine class. These compounds displayed limited antiinsectan activity against the fungivorous beetle Carpophilus hemiperus, and the minor component had not been previously reported. The structures were elucidated by analysis of ID and 2D NMR data, and by comparison to data for known aflavinines. The major component was found to be present in high concentrations. and examination of both liquid fermentation and petri plate cultures indicated that the aflavinines were produced only in the ascostromata. Aflavinine derivatives were also detected in the ascostromata of other isolates of E. crusfaceum, and in those of an isolate of E. reficulisporurn. Interestingly. aflavinines had previously been reported only from the sclerotia of Aspergillits spp. The existence of aflavinines in both Aspergillits sclcrotia and Eupenicillium ascostromata is of potential significance from ecological and taxonomic perspectives. The structures of thcsc compounds, details of quantitative analyses, and a &scussion of thc results will be presented.

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p-4 ImFxtwYcuc ANIFuNGa mmmms moM A K ~ ~ T A L E I N s m y Dmel C. Dunbar fU, Mark T. Hamatm(lJ), Atice M. clark(lJ), Scott Johnson(3) it^ Paul J. Scheucr(4) Univmiiy. of Mi&&qn, Dcpt of pharmaCognoay(1) and RaKanh htitutc of Pharmaceutical S u c n ~ 2 ) , School of Phmacy,U*, h4S 38677 (3) Box 325, APO AP %555 (4)univasity of Hawaii at Man04 Dept of Ckmktry, I-IOXIO~IIIU, HI 96822

In rcccnt times thc incidence of lift thrratening systcmic my~ses h.s risen dramrticany becaust of the use ofirmnunosuppreesivc drugs m transplant patien& and autoimmune distaee, kum- cancer chcmotficrapy and most i m m the spread of the AIDS virus. There are only fivt druga currently used to treat eystcmic mycoses (amphotericin B, 8ucytOeinc. ketocwazolc, itracollrur, k and fluumazole) each of which has many shortcomings. The four drugs work on only three di&nnt molecular tarpcts. New prototype drugs are dcsperatcly needed an lcad compounds for SAR studia an to identify ncwmoleculartargcts specific to fungi An extract from a black- oflI~~gcnur Halr~hondria collected at Kwajalein Atoll, Micnmcsia, is vcry selective for fungi M it has littlt or no cyiotoxitcity against Bmral mammahan * CCn ha (iiluding healthy monkey kidney &) or bacteria Bioassay guided fixtionation wag canicd out and thc first compounds isolated am halich- o which four have bccn pmiously qorted aa hmkg mtihgal activi@(Kcman MR, Mokki TF and Faulkncr JD. J. Org. Chem. (1988) 535014-5020; 0.2cI&lml for Cundida ulbicuns, 12.5 &ml for TnchophyIon mentagropbtes). Other fi-actions fiom this sponge are as0 activc and contain fairIy diiferent types of canpounds based on cltromatographic behavior and NMR Thcsc h c t i n ~ abo exhibit littie or no cytotoxicity to mammahan . cell lines. MIC values againrrt A I D S related oppomnm . pathogem and cytotoxicity data will be reporte d for several of thae compo unds.

'c

NUCLEOPHILIC SUBSTITUTION IN THE SAMPANGINE SYSTEM:

n K. Z i a m 1 . 2 . Ashraf A. Khalill. Charles D. Huffordl2 and Alice M. Clarkl.2 Department of Pharmacognosy 1 and the Research Institute of Pharmaceutical Sciencesz. School of Pharmacy, The University of Missisippi. University, MS 38677.

THE SYNTHESIS OF 3-METHOXYSAMPANGINE. . .

3-Methoxysampangine, an alkaloid with significant activity against AIDS-related fungal pathogens, was isolated from West African me, Cleistophofis patens in O.OOO11 yield. In order to obtain a larger supply of this compound for biological studies, several synthetic efforts were undertaken. The total synthesis of this compound that we fxst reported1 suffers from a low yield (less than 1%) and is very tedious and troublesome.

We now report an efficient method of synthesis of 3-methoxysampangine by nucleophilic substitution of 3-bromosampangine. The reaction was found to be temperatun- dependent. At lower temperatures (WC), substitution of the hydrogen atom at C-4, instead of bromine at C-3. takes place. The less stable, 3-bromo-4-mthoxysampangine is f d as a product of kinetic control. At higher temperatures, substitution of the bromine atom occurs with the formation of the desired, thermodynamically more stable 3-mcthoxysampanginc.

The effects of solvent and phase transfer catalysis on the course of the nucltophilic substitution of 3-bromosampangine were also studied.

1. J.R. Peterson, J.K. Zjawiony. S. Liu. C.D. Hufford, A.M. Clark, and R.D. Rogers, /.hied. Chem., 1992.35, 4069-4077

F i s work was supported by the National Institutes of Health Grant No. A1 - 324851.

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POSTEK PRESENTATIONS 175

p-5 ANTARCTICA A FRONTIER FOR THE ISOLATION OF BlOAcIlVE MARINE NATURAL PRODUCTS

DETERMINATION OF LOBELINE IN W B E L I A INPLATA L CONTAINING DIETARY SUPPLEMENTS BY CAPILLARY ELECTROPHORESIS. Joseuh M. Betz, William R. Obermeyer, Food and Drug Administration, Center for Food Safety and Applied Nuaition, Division of Natural Roducts. Washington, DC 20204, USA

The dried leaves and tops of Indian tobacco or pukeweed (Lobeliu i & r u L., Lobeliaoeae) were used to treat asthma and bronchitis throughout the 19th century. The crude drug has teen re-4 to contain 0.24-0.49b total alkaloids, the most important of which is (-)-lobeline. Lobeline is a piperidine alLaloid which has been used as the principle active i n m e n t in a number of smoking deterrents. While this use has been largely d i s d t e d by careful clinical aials, Lobefia containing herbal dietary supplements remain available at retail health food outlets. The pharmacological activity of lobeline is similar to nicotine (although less potent), and overdoses may cause vomiting, respiratory difficulty, convulsions, coma and death. The potential toxicity of lobeline raises grave concerns over the safety of Lobelia containing supplements, especially in children and pregnant and nursing women.

Since a great many of the supplement products available contain more than a single botanical ingredient and/or are marketed as liquid extracts of one or more plants, many of the official methods for their analysis (when such exist) are inadequate. A method for the separation of the alkaloids of L. inflnto by capillary electrophoresis has been developed and applied to a number of lobelia containing products from commercial sources. For solid dosage forms, the method involves aqueous exmaion in an acidic buffer followed by electrophoretic separation on an uncoated 72 cm fused silica capillary tube (UV detection). 4 u i d dosage forms were directly injected. The running electrolyte was 25 mM sodium citrate buffer (pH 5.0). The applied voltage was 27 kV and the capi l la~~ thennostating temperature was 35 "C. This method resolved lobeline from its piperidine congeners and no interference from other alkaloids expected to be present in mixed botanical products was observed. The products examined contained lobeline levels ranging from none detected to 60 ppm.

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p97 1 STRUCTURE DETERMINATION OF THE ANTIBIOTIC UMBRINOMYCIN

Stella Huan Steven F- Klgs'hr. and Jeffrey 1- Whitney

Daniel R Schroedcr, Robert W. Myllymaki.

Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, CT 06492

Umbrinomycin was discovered and patented by Squibb 25 years ago, but the structure was never determined. Recently it was picked u p again from a Strepfornyces sp. in our screening program for antibacterials. We have now solved the s t ructure with the aid of MS analysis a n d two dimensional NMR spectroscopic techniques. Umbrinomycin has a condensed naphtoquinone ring system with a n unusual sugar- like acetal appendage. The NMR da ta obtained o n the parent compound a n d a triacetate derivative included 'H, 13C DEFT, COSY, HETCOR, HMHC, and nOe difference in DMs0-d~ and C6D6, respectively. Complete spectral assignments were made.

un H I

f Unilirinomycin CzzH2,0, MW 416

0

A REINVESTIGATION OF MAPHOUNL4 TFUTERPENES. John A Beutler, Yoel Kashman, Mark Tischler, John H. Cardellina 11, Glenn N. Gray, Peter M Blumberg', and Michael R. Boyd Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, DCT, NCI, Bldg. 1052, NCI-FCRDC, Frederick, MD 21702-1201.

'Laboratory of Cellular Carcinogenesis and Tumor Promotion, NCI, Bethesda, MD 20892. AIDS-antiviral activity and inhibition of phorhol ester receptor binding activity in two species of

Muproirneu (Euphorbiaceae) were traced to small amounts of highly potent phorbol esters of the daphnane type. The triterpenes previously isolated from this genus [e.g.,l] were found to be devoid of biological activity when scrupulously purified. Four new triterpene esters were elucidated: two [3,4] were found in M. ujricunu, while three [4,6,7]were found inM. memhrunuceu. Nmr assignments have also been made for two previously known compounds [2,S] in this group.

P:98 1

6 R1=RZ=H. R3q3-8

7 RI=H, R p - A . R p - O H

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p99 NOVEL NAPHTHOQUINONES FROM Conospermum sp. Jin-Rui Dai, Kirk R. Gustafson, Laurent A. Decosterd,

~~~ ~~

p-100 ROSENONE, A CYTOTOXIC SESQUITERPENE FROM THE GORGONIAN Subergorgia suberosa 1 Heidi Bokesch, Tawnya C. McKee, Gerald R. Leather, Doug Luster, John H. Cardellina 11, Michael R. Boyd Laboratory of Drug Discovery Research L Development, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute-Frederick Cancer Research h Development Center, Program Resources, Inc./Dyn-Corp, FCRDC, and Agricultural Research Service, USDA, Bldg. 1301, Ft. Detrick, Frederick, Karyland (USA)

Bioassay-guided fractionation (duckweed phytotoxicity) of extracts of the gorgonian Subergorgia suberosa led to the isolation of a novel sesquiterpene, rosenone. In addition to phytotoxicity, rosenone exhibited broad, moderate cytotoxicity in the NCI human disease oriented antitumor screen. Details of the isolation, structure elucidation and biological testing will be presented.

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p-101 THE SEARCH FOR MICHELLAMINE: A TALE OF THE INTERRELATEDNESS OF DRUG DISCOVERY, TAXONOMY AND NATURAL PRODUCTS CHEMISTRY

p lo2 1 1CVAIAJA'I'ION OF ANI'IIUMOR AC'I'IVII'Y OF PLANT EXIHACTS I'I(Oh1 ZAIRE.

!L N. Muaiiza. M. Alaiii, K. L. Euler. aiid L. Williains. I k ~ i n i ~ i i i e n t o f Pliariiiacological and Pliarinaceutical Sciences. College of I'liarttiacy. University of Houstoii. Iiooston, 'I'X 77204-55 15.

As :I 11:irt of our coii l i i iuit ig senrcli for poienlial bioactive substances fioiti Aliic;ii i iiidigerious tiicdicnl systctns. exiritcts l roni nii ie inedicii ial plaiits gtowi i ig iii %xiire were niinlyzed for their i i i i t i tuinor activities.

Selecled } h i l l s included Alchorrieo cordifolio (Schuni & Thoiiii) Muel l . Atg. ( I~~ i~ i l i i i r l i i acene) . A I I I I ~ I I ~ srrrrgaleirsis Pers. (Aiinonaceite). B r i d e l i o Jprriigiriro I%ei i l l i . ( IJupliorbiaceae). Eurnlypius cirriodora ( M y r t a c e ae), l lyi i ieiif if . l i if /;fi lrcidn T u l . ( I l y i i i e i iocard iaceae) . J n i r o y h a c u r c a s L. (13ulilioibi:icc;ic). Mongijero iridico L. (Ail;ic:irdinceae). M n p r ~ i i e a uJricunu Miicll. hie. (Iiulil;orbiaceae) anid Sido r/rorr~DiJoliu L. (Malvaceae).

'l'lie i i i i t icancer ac i i v i l y was evaluated againsl the Nat ional Cancer liistitute (NCI) panel of 60 liuiiiaii ce l l Iiiies derived froii i eight cancer types iricludii ig liteasf. leukemia. lurig. colori, braiii, inelarioliia. ovarian, and reiial I iuniaii iiitiiors.

l k s i i l t s regarding l l ie aiiticalicer acl iv i t ies of analyzed extracts and sonie cui i i~~i i i i i ids isolated from the sleiii bark of M. iridicn wi l l be presented.

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WSTER PRESENTATIONS 179

A CONVENIENT SYNTHESIS OF (-)-CMULTISTRIATIN FROM

tc& Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269-2092 U.S.A.

Multistriatin - a component of the aggregation pheromone of the Elm bark beetle Scolyrur mulfisfriutis. It has been synthesized in 1982. The new synthetic approach developed in our laboratory starts from levoglucosenone 1 Via the 1,4 and 1,2 Michael addition adduct of nitromethane 2. Conventional reduction of nitromethyl groups at C-2 and C-4 with tributyltin hydride and reductive removal of hydroxyl at C-2 with triethylsilane produced a branched-chain sugar 3 in 48% yield. ISubsequent elaboration of 3 via the dithiane ring opening elongation method, followed by alkylation of C-1 with ethyl iodide, and ring closure by removal of the dithiane group produced (+)-a- Imultistriatin 6 in 22% overall yield.

THE ALKALOIDS OF PICEA BREWERIAh'A

1De.partment of Chemistry. Lafayette College, Easton PA 18042 bpar tment of Chemisby, Wellesley College, Wellesley MA 02181

P104 1 - L i n J . L . e 1 and Suzanne Brendze2

Pice~ brewerianu is unusual among Piceu as i t does not produce the pipendine alkaloids pinidind oc epidihydropinidine. P. brewerinnn does. however, produce interesting alkaloids. including I-methyl-9-nor-3-grananatanone 1 and a hygroline 2. The presence of both 1 and 2 in P. brew- is of biosynthetic interest. Isolation and identification of the alkaloids of P. breweriunu will be described.

-0 0

1 2

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Pl05 1 ~~~ ~~~

PHYrOCHEMlCAL AND BIOLOGICAL ASPECTS OF FSCHSCHSCHOLTU C A L I F O ~ I C A CHAM SEEDS Carlo Bugam, Mana L Colombo I and Franca Tome 2 1 InsUtute Pharmacological Scie~CeS, UNvCnlty of Mdan, Italy 2 Depamnent of Biology, Umversrty of Milan, Italy

fiwhrcholara culifornica Cham. (Papave- family) has been extensively studied for its morpbologd characteristics (S.A.Cw4 Evolution, 1962, 16 : 278-299). Several compounds have been lnvcsugatad from the aerial or hypogeous parts of this p h t , mostly isoquinoline alkaloids (D.GuMon, N.Cappelaere and V.Simanek, Pbytochem Anal., 1990, I : 77-82); little is known about seed morphology, germination, oil compomnts and othcr metabolites. In this work data about numerical analysis performed using SEZ and weight of seeds are descnbed ; oil percentage, fany acid composition and other metabolites are also reported. The seeds are almost isodlametric and rough. The diametcr size is I .3 - I .5 nun. The seed weight Mnes between 0.135 and 0.165 10-3 mg. Seed germination is favoured by darhas; in these condmoos I t occurs 70-72 h (85%) after seed imbibition. The estimated values for crude oil on a dry basls vary between 35 and 42 %. The GC coopled with MS spccvomdry made evident the prescnceofc I6 : 0. C I8 : 0, C 20 : OandC IS : 1, C 18 : 2, C 18 : 3 fattyacids methyl esters. The C 18 , 0, C 18 : 1, C 18 : 2 were found to be major conStitucots and all of the unsaturation was due to C 18 aads. Other metabolites of E. calljornica seeds are nd lipopbilic compounds, extractable at r.t. with n-hocam. These compounds are stable in acidic medium @H = 2) and darkness, but they change in colour in the alkaline mai~um @H = 9) and under white light. Their separation (five substaaces) was achieved using HPLC quipped with photo d i d m y detector.The quantity ofthe red component drastically decreases m the plantlet and then disappean in mature plant

Two New Bioactive Triterpenoids f rom Meflu vofkenskii (Meliaceae) J.u&g. Zheming Gu. Xinping Fang. Phillip E. Fanwick.7 Ching-jer Chang, David L. Smilh. Jerry L. McLaughlin'. Marjorie I. Plummertt Department d Medicinal Chemisay and Pharmacognosy. Schml of Pharmacy and Pharmacal Sciences. 'Deparunent of Chernisuy. Purdue University, West Lafayette. IN47907. t*IWC Corporation, Princeton. NJ 08543

Meliavolin (1). a new triterpcne with an apotirucallane skeleton, meliavolkin (2). a new tetranortriterpene. together with melianin A, a known compound, have been isolated from the root bark of Melia volkenskii (Meliaceae) by using activity-directed fractionation with brine shrimp. The structures have been elucidated by spectral data. The relative and absolute stereochemistry of meliavolin (1) was determined by analysis of Mosher ester derivatives and by X-ray crystallographic analysis.of meliavolin diacetate. *I, 2 and melianin A showed pesticidal effects as well as marginal cytotoxicities to certain human tumor cell lines. (The antitumor tests were supported bv NIHNCI grant no. ROI CA 30909).

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Percealide: a Novel, Biologically Active Component from the Bark of Tercea amrkana (Lauraceae)

One Ye, Zhe-ming Gu. Lu Zeng, Geng-xian Zhao. Jerry L. McLaughlin. Department of Medicinal Chemistry and Pharmacognosy, School of Pharmacy and Pharmacal Sciences, Purdue University. West Lafayette, Indiana 47907WSA) and Soelaksono Sasrrodihardjo, Department of Education and Culture, Center of Intemniversities, Biology Division, Bandung Institute of Technology, 10 Ganesha St, Bandung 40132, Indonesia

Brine shrimp lethality-directed fractionation of the 95% EtOH extract of the powdered dried bark of Percea americnna led to the isolation of one new C20 alkane-alkene acetonyl methyl ester designated persealide. Percealide showed moderate cytotoxicity against three solid tumor cell lines: human lung carcinoma (A-549), human breast carcinoma (MCF-7) and human colon adenmarcinoma (A-549) (Aided@ researchgrant m. wl CU0909from the NatiunafCancer Institute, NIw

O H

Persealide

BIOACITVE ANNONACEOUS ACETOGENINS FROM THE BARK OF XYLOPIA

-, Z.-M. Gu. L. Zeng, and J.L. McLaughlin. Department of Medicinal Chemistry and Pharmacognosy. School of Pharmacy and Phannacal Science, Purdue University,West Lafayette, Indiana 47907

p- 08 AROMATICA. P In a continuing study of Annonaceous acetogenins from Xylopia uromrica (Annonacene), collected in Edo. Amazonas (Venezuela), three new compounds were isolated from the EtOH extract of the bark, by bioactivity-directed fractionation using lethality to brine shrimp. These acetogenins include xylopienin (1). xylomatenin ( 2 ) . and venezenin ( 3 ) . 1 and 2 are two new mono-tetrahydrofuran (THF) ring acetogenins. and 3 is an unusual acetogenin having no THF ring; however, it bears a vicinal diol and double bond which are appropriately located for it to be a biogenetic precursor of annomontacin. The smcrures were established by the application of UV, IR, MS IH- and l 3 C - W and 2D 1H-IH spectral techniques with the parent compounds and/or simple chemical derivatives. The cytotoxic activities of 1-3, against human solid tumors, will be reported. 1 and 2 are inhibitors of oxygen uptake by rat liver mitochondria. (T. Colman-Saizarbitoria acknowledges CDCH from UCV and FUNDAYACUCHO for a research fellowship; this work was aided by NM/NCI research grant R01 CA 30909).

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POSTER PRESENTATlONS 182

FROM THE! LEAVES 0 F A " O N A MURICATA

Depanment of Medicinal Chemisy and Pharmacognosy. School of Pharmacy and Pharmacal Sciences, Purdue University. West Wayette. Indiana 47907 (USA)

Zhe-Ming Gu. Lu Zeng. Geng-Xian Zhao, Yan Zhang and Jerry L. McLaughlin*

COMPARISON OF MITOCHONDRIAL RESPONSE TO SEWR.4L THEIR ACETAL DERIVATIVES. , Z.-M. Gu, J.L. Landolt. and J.L. McLaughlin.

Depanmenr of Medicinal Chemistry and Phmacognasy. School of Pharmacy and pharmacal Sciences. hrduc Univeniry. West Lafaycue. Indiana 47907 (USA)

The Annonaceous acetogenins have earlier been determined to inhibit ATP production at NADH-ubiquinone reductase in Complex I of the mitochondria1 electron-eransporr chain; the result of the present work suppons the conclusion that the bioactivities of all the aceto enins derive. at least in pan. from their action on complex I [Lewis et al., Pesric. Biocliem. P&siol. 45, 15 (1993) and Ahammadsahib et sl., Life Sciences, 53, 11 13 (1993)l. Parent acetogenins (bullatanwin; 2 .44s and rruns- bullatantxinones; bullatalicin: 2.4-cis and rrans- bullatalicinones; squamostatin A; squamocin; gigantetrocin A and goniothalamicin) and their acetal derivarives, were tested. Respiratory functions of rat liver mitochondria were measured polarographically by determination of their rates of oxygen consumption using a small oxygen electrode inserted into a semiclosed reaction cell; activiues of the compounds at the subcellular level were then compared. The data obtained revealed pronounced inhibitory effects of Complex I for all the acetogenins rested, with IC50 values in the range of 11-554 n moles/lt/mg protein; as a positive control rotenone gave Ic50 34 n moles /Lt/ mg protein. Most of the resulting cyclized acctal derivatives (vs. the parent acetogenins) shou .4 enhanced bioactivities. Correlations with other bioassays (brine shrimp lethalities and cyt6)toxicities to human tumor cell lines) were readily apparent. and relative potencies between the parents and acetal derivatives were similar in the different assays. (T. Colman-S. acknowledges CDCH from the Universldad Central de Venzzuela for a Research Fellowship; aided by NWNCI grant ROI CA 30909).

Previous studies on the seeds of Anmna muricaia (Annonaceae) involved the bioactivity-directed isolation and characterization of a number of cyloroxic and pesticidal acetogenins BS well BS one of their precursors. The leaves of Annona muricata have yielded three new Annonaceou acetogenins (1-3), named annomuricin A (1) and B (2 ) and annomuricacin (3). Their chemical smctures were deduced by MS. NMR. IR. and UV spectral and chemical methods. "le suuciures of 1 and 2 arc shown in the figure below. Annomuricins A and B are unusual. and each possesses five hydroxyl groups. Two hydroxyl groups are vicinal. one is hreo (1). and he olher is eryho (2). (Aided by research grant no. R01 CA30909 from t h e N a t i o ~ I Cancer Instilute. NIH).

Biological activites (pglml):

Compound BST(EDS0) A-%g(EDSO) MCF-7(EDm) m-29(ED50) Annomuricin A 6.25~10-1 3.30~10-1 > 1.0 >1.0 Annomuricin B 6.87xW1 1 . 5 9 ~ 10-I > 1.0 4.35~10-~ Nows: B S T Brine ShrunpTcn; A-459: Lung Canar; MCF-7: Brcrri Cancer: HT-29: Colon Cmcn.

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NOVEL ORTHOQUINONE TERPENOIDS FROM THE ROOT OF SALclA L(NIGUL.. k-soo La. Norito Kaneda, Rutt Suttisri, Abdalla M. El-Lakany,' Nawal N Sabri,' and A. Douglas Kinghorn, Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Phamacognosy, College of Pharmacy,

University of Illinois at Chicago, Chicago. Illinois 60612, U.S.A., 'Department of Pharmacoposy, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt.

Members of the plant genus Salvra (Labiatae) occur widely distributed in the world, mainly in the mo~tainous areas of the tropical and subtropical regions, and have been used as medicmal plants. Ethnobotanical surveys show that they have been used traditionally against a broad range of ailments because of their antimicrobial, anticancer, antihepatotoxic. antifertility. diuretic, and CNSdepressant effects. Previous phytochemical studies on this genus have led to the identification of several groups of compounds mclusive of diterpenoids (especially of abietane-type). tnterpenoids, and flavonoids. In this investigation of Salvta higeru. collected in Egypt as part of a collaborative study on traditional medicinal plants, two novel orthoquinone terpenoids, namely, 8-hydroxy-3-1sopropyl-7-methyl-1,2-naphthoqumone ( I ) and methyl 1.10- seco-5( 10).6,8,13-abietatetraene-l1.12dion-l+ate (2). have been isolated, which were accompanied by two other previously hown abietanc-type diterpenoids, arucadiol and pisiferal. The structures of the new compounds were enablished by spectroscopic methods.

I

A NOVEL BENZENOID FROM EUPHORBM QUINQUIOCOSTATA WITH ORNlTHlNE DECARBOXYLASE INHIBITORY ACTIVITY. Zakana H Mbwambo. Woongchon Mar, Sang Kook Lee, Elunweka N Mshu,' John M Pezzuto and A Douglas Kmghom, Program for Collaborative Research m the Pharmaceutical Sciences, Departmcnt of

Med~cmal Chenustry and Pharmacognosy, College of Pharmacy, Umversity of lllmois at Chicago, Chicago, Illmois 60612, U S A , 'Traditional Medicme Institute, Muh~mbrli Med~cal Center, Umversity of Dar-es- Salaam, Dares-Salaam, Tanzarua

Euphorbia qurnquiocosruta Volk. (Euphorbiaceae), is a tree used in Tanzania to accelerate wound healing and to treat stomach pains. An ethyl acetate soluble fraction of the methanol extract of the stem wood of this plant exhibited significant inhibition agarnst phorbol-ester induced omithine dccarboxylase (ODC) activity in cell culture. Following a bioassay-guided fractionation procedure, three novel compounds were isolated, namely, 2'-hydroxyJ'-methyl-4'.6'~thoxy-2-hydroxyacetophenone ( 1 }, 3,3',4'-trimethylellagic acid 4-O-{a-L-rhamnopysyl( I+6))-P-D-glucopyranoside (2). and enf-14~-hydroxykaur-16en-3,12dione (3). Compound 1 significantly inhibited the induction of ODC activity (1Cw 2.7 Mml), although the other two compounds were inactive (I& >200 pg/ml). The known compounds inclusive of sitosterol, sltosterol-p-D- glucopyranoside, enr-kaurane-l6,17dioI, were also inactive in ODC bioassay. Structure elucidation of 1-3 was carried out by a combmation of spectroscopic methods (MS, W, IR, ID- and 2D-NMR) (Supported by PO I CA 481 12).

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184 FOSTER PRESENTATIONS

PI13 POTENTIAL CANCER CHEMOPREVENTIVE CONSTITUENTS FROM DIRCA OCCIDENTALIS. Lumonadio Luyengi, Nanjoo Suh, Yingde Luo, Sang Kook Lee, Harry H.S. Fong, John M. Pezzuto and A. Douglas Kinhorn, Program for Collaborative Research

p:l14 APORPHINE-BENZYLISOQUINOLINE ALKALOIDS FROM THALICTRUM FABERl WITH ANTIMALARIAL A C T V Y V. Wonma&. L-2. Lin, M. You, C. K. Angerhofer, J. M. Pcuuro. and G. A. Cordell, Program

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C K. Anncrhofcra. I4 Guinaudeaub. I..-Z. Lina, K. Iikhitwitayawuda, 1’. Wongpanicha, J.M. PezzutoP. N Ruangrungsic. and 0 A Cordella

ahogram for Gdlabrative Research in the Pharmaceutical Sciences. Department of Medicinal Chemstry and Pharmacognosy. L!niversity of Illinois at Chicago. College of Pharmacy, Chicago, IL; bpar tmen t of Pharmacognosy. Faculty of Pharmacy, University of Angers. France, CDeparlment of Pharmacognosy, Faculty of Pharmaceubcal Sciences. Chulalongkom Uruversity, Uangkok,Thaland

Bisberv.ylisoquinolines comprise a class of alkaloids whch are known to occur in several plant families. Members of this class have recently been reportcd by us. and by other groups. lo i thbi t the growth of cultured malanal parasites with ICso values in the nanomolar range. As of an ongoing collaborative effort to discover new antimalarial agents from natural sources. we have isolated a series of bisbenzylisoquinolines from C)clea barbara. Srephunia erecla. Pachygone dasycarpa. Cyclea ajehenris. Hernandia pelrara. l’hallcrrwn sp., Curare candicanr. and Cocculus pendulur and tested them in vilro for cytotoxicity in mammalian cells as well as for antimalarial activity against chloroquine-sensitive and chloroquine-resistant clones of P. Jalciparum. ’Ihc isolates had a wide range of biological potency in these assays. although the majo~ity exhibited some dcgroe of cytotoxicity against human KB cells. Nearly half of the compounds tested. however. showed selecbve an t idana l activity, with z IW-fold more toxicity toward one or both P. Jalciparum clones. and the most active examples were cycleanine. cycleatjehine, cycleatjehrum. malekulatine, repandine. and ternuconine. Iletailed resulls of the biological testing will be presented. and emerging relationships between the structures. the stereochemistry and h substitution pnems of the compounds wc have studicd and [heir in viao anumalanal and cytoloxic activibes will bedixwsed.

p:116 ANALYSIS OF CAPSAICMOIDS IN OTC PREPARATIONS BY COMPLEXATION CHROMATOGRAPHY 1 Howard Constant and Geoffrey A. Cordell

Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60612

Capsicum oleoresin is fairly common in drug products and is approved for use as an external analgesic. A cheap adulterant (nonivamide) has been known to be found in capsicum preparations since the early 1960s. Using a new and stable HPLC system to separate capsaicinoids, topical analgesic creams, lotions, and gels have been analyzed for their capsaicin content. From 19 samples analyzed, 5 had nonivamide as the active ingredient, 5 had a capsaicin content lower than stated (the percentage labeled was for total capsaicinoids), and 9 had the stated concentration. The group that had the stated concentration of capsaicin could be divided into two groups: those containing capsicum oleoresin and those containing a purified capsaicin as determined by the ratio of capsaicin, nordihydrocapsaicin and dihydrocapsaicin.

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Raiendra U e h t q , Uin You, Michael Hawthorne, Cathy Thomas, Andreas Cons tan t inou , J i n f a n g L i u , C la r i s sa Gerhause r ,

FROM CHINESE CABBAGE AND ITS MECHANISM OF ACTION

Robert M o r i a r t y , John Pezzu to and R icha rd Moon. S p e c i a l i z e d Cancer C e n t e r , Department o f Med ic ina l Chemis t ry and Pharmacognosy and

B r a s s i n i n , c y c l o b r a s s i n i n and s p i r o b r a s s i n i n a re n a t u r a l c o n s t i t u e n t s i n p h y t o a l e x i n s d e r i v e d from t h e fungus i n f e c t e d Chinese cabbage. B r a s s i n i n and i ts a n a l o g s were s y n t h e s i z e d and t h e i r a c t i v i t y was de te rmined i n t h e mouse mammary g l a n d o r g a n c u l t u r e (MMOC). Under a p p r o p r i a t e hormonal c o n d i t i o n s , 7 ,12 -d ime thy lbenz (a ) - a n t h r a c e n e , (DMBA) , i n d u c e s p r e n e o p l a s t i c mammary l e s i o n s (ML) d u r i n g a 2 4 day c u l t u r e . B r a s s i n i n , c y c l o b r a s s i n i n and s p i r o b r a s s i n i n , t h e n a t u r a l l y o c c u r r i n g b r a s s i n i n a n a l o g s , i n h i b i t e d ML f o r m a t i o n i n MMOC, whereas, t h e methyl- or chloro- d e r i v a t i v e s of b r a s s i n i n were i n e f f e c t i v e i n i n h i b i t i n g t h e l e s i o n f o r m a t i o n . The effect of 1% b r a s s i n i n ( i . g . ) , p r i o r t o DMBA t r e a t m e n t , was e v a l u a t e d on t h e i n i t i a t i o n of mammary c a r c i n o g e n e s i s i n ra t s . B r a s s i n i n s i g n i f i c a n t l y i n h i b i t e d tumor m u l t i p l i c i t y i n t h e r a t s . Although c y c l o b r a s s i n i n was more e f f e c t i v e t h a n b r a s s i n i n i n i n h i b i t i n g ML f o r m a t i o n i n MMOC, metabolism s t u d i e s i n d i c a t e d t h a t n e i t h e r i n v i t r o n o r i n v i v o b r a s s i n i n was c o n v e r t e d t o c y c l o b r a s s i n i n i n d i c a t i n g t h a t t he c y c l i z a t i o n of b r a s s i n i n is n o t e s s e n t i a l f o r its a c t i o n . Furthermore t h e b r a s s i n i n i n h i b i t i o n of i n i t i a t i o n p h a s e of c a r c i n o g e n e s i s was correlated w i t h t h e i n d u c t i o n of qu inone r e d u c t a s e a c t i v i t y i n t h e mammary t i s s u e s . (Supported by N I H g r a n t PO1 4 8 1 1 2 ) .

1 CANCER CHEMOPREVENTIVE A C T I V I T Y O F PHYTOALEXIN DERIVED pi17 c Department of Chemistry, U n i v e r s i t y of I l l i n o i s , Chicago, I L 60612.

TERPENE ESTERS OF EUPHORBIALES AS POWER- ERIMENTAL ONCOLOGY AND IN CELL BIOLOGY

d Gminski, Gotz Krauter, Hanno Kretschmer, Sigrid Sosath, Peter &ha, and Erich Hecker, Center. Research Prograni Cancer Risk Factors and Prevention, Division:

echanism of Tumorigenesis, Im Neuenheimer Feld 280, D-60009 Heidelberg (Germany) In real human life multifactorial causation of cancer is the rule and classical unifactorial causation the exce tion. Essential elements of this comprehensive and current concept have evolved from in-deptR investigations of the e erimental model of skin cancer0 enesis usin as condi- tional cancerogens tumor promoters z t h e skin irritant dite ene ester (bTE) type. h e occur in certain families of the Euphorbiales (Euphorbiaceae, Xymelaeaceae, taxonomy debster, 1987). By quantitative dose res onse relations in the experimental initiation/promotion protocol of cancerogenesis recentjy Dk were identified conclusively as a new, non-classical category of risk factors of cancer (Hecker, Rippmann, 1990). As of “per se essentially non-cancero enic amplifiers” of tumorigenesis DTEs contrast mechanistically and etiologicall the classical sof tary cancerogens. Moreover, DTEs are most powerful tools in molecular cell iiology, as illustrated by current results of toxicokinetic and toxlcodynamic studies including cellular signaling circuits and receptors involved (protein kinases C ) . In the context of environmental cancerogenesis, for life-style oesophageal cancer, prevalent on the Caribbean island of Curapao, DTE were shown to be the principal risk factors (“Welensali factors” from Croron fluvem L), in conjunction with initiators from petrol contaminated drinking water. One of the typical risk factors involved, Welensali factor F1, was isolated also from the tung oil tree (Aleunfes fordii Hemsl.). DTEs of the tigliane, ingenane and daphnane types including the Welensali factors were shown to activate the expression of certain human tumor viruses, e.g. the Epstein-Barr virus. The latter is known to be associated with nasopharyngeal carcinoma (NPC) prevalent in South China. It may be concluded that uncontrolled utilization of Euphorbiales-derived environmental materials, e.g. as renewable technological raw materials (Hecker, Sosath, 1991) or as (ethnomedicinal) drugs (Hecker, Glaer, Gminski, 1991), may comprise so far undefined risks of human cancer. They are under assessment by a general strategy as developed in our laboratory. DTE originating from Euphorbiales together with the multifactorial concept of cancerogenesis have added a new dimension to the etiolom of human cancer.

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p:119 DEVELOPMENT OF AN ANTI-MYCOTIC OINTMENT FROM THE ROO'rS OF PENTAS LONGIFLORA L. Van Puyvel&, D. Hakizayezu, P. Brioen, N. Lk Kimpe, C. De Vroey, J . Bogaerts

PI120 PHYTOTOXINS FROM THE LEAVES OF LAGGERA D E C U m N S Luc Van Puyvelde', L R o s e la&, N. De Kimpe', C. Stevens', J. Van Gestel', V. Van Den Eynde' and P. Van Damme'

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1 2 1 TAGETES NATURAL PRODUCTS, ESSENTIAL OILS, COLORING SUBSTANCES AND PLANT EXTRACTIVES AS AFFECTED BY FERTILIZERS.

22 NOVEL C Y T O T O X I C LABDANE DITERPENOIDS F R O M NEOWARU ACLIMlNATISSIMA. Xianjian Ma, Ik-So0 Lec. Hee-Byung Chai, Domingo A. Madulid.. R. Brian Lamont,' Melanie J . O'Neill.' Jeffrey M. Besterman,: Norman R. Famswonh. D.

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REARRANGED ENT-KAURENOIDS WITH CYTOTOXIC ACTIVITY FROM

No- R. Famsworth, GeofFrq A. Cordell, John M. Pezzuto, and A. Douglas Kinghorn Program for Collaborative Research in the Pharmaceutical Sciences, Department of

Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, U.S.A., 'Department of Pharmacy, University of Zimbabwe, Harare, Zimbabwe.

PARINM CWTELLIFOLL4. Ik-Soo Lee, Hee-Byung Chai, Tangai E. Chagwedera,'

Members of the tropical plant genus funnuri (Chrysobalanaccae) have been used traditionally in several African countries as folklore remedies to treat ailments such as dysentery, leprosy, malaria, and venereal diseases. Biological screening studies performed on extracts of plants in this genus have shown biological activities inclusive of antibacterial, antifungal, antimycobacterial, and antimalarial e f f a , and previous phytochemical studies have led to the isolation of several flavonoids. The medicinal plant, furinan curateNrjoliu of Zimbabwean origin, was chosen as part of our continuing search for naturally occumg anticancer agents when an ethyl acetate extract of its root bark demonstrated significant cytotoxic activity in a preliminary 2 R = H screning panel of human cancer cell lines. 13-Methoxy-IS- oxozoapatlin (1) was isolated as a new diterpene lactone wth a rearranged en(-kaurenoid skeleton, which was accompanied by 15-oxozoapatlin (2), a known compound first isolated from Montunou tomentom. Compounds 1 and 2 showed greatest cytotoxicity against a human epidennoid carcinoma (A43 1) and a human glioma (U373) cell line when tested among a panel of cancer cell lines. (Supported by grant UO1 CA-52956).

1 R = O M e 0

DITERPENES FROM POLYALTHIA W E S I I . Lee, Hee-Byung C h , Kyaw Zaw, Norman R. Famsworth, D. Doel

Soejarto, Geoffrey A. Cordell, John M. Pezzuto and A. Douglas Kinghorn, Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry

and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, U.S.A.

The tropical genus Polyolfhru has been found to be the source of various types of s e a n d a q metabolites, including alkaloids. sesquiterpenes, diterpena, uiterpenes, flavonoids. sterols, and zinccontaining compounds. Several of these compounds have been shown to exhibit interesting biological activities such as leishmanicidal, cytotoxic, and anti-w effects. In this presentation, we report the isolation of three cytotoxic clerodane diterpenes, namely, 16a-hydroxycleroda-3,13(14)Z-dien-l5,16-oli& (1). a kpown compound, and two novel clercdanes, 3P,lsadihydroxycleroda~(l8).13( 14)Zdien-IS. 16-olide (2) and 4p, 16a-dihydroxyclerod-13(14) Z-en-15,16-olide (3). from the stm\ bark of P. bornesri (ANxxlaccae). This plant was collected in the Philippines as part of a collaborative search for naturally occurring antineoplastic agents. Among a panel of human tumor cell lines, compounds 1-3 were most potently eytotoxic for the prostate (LNCaP) and glioma (U373) cell lines. (Supported by grant UOI CA-52956).

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PI1 25 ISOLATION AND IDENTIFICATION OF A NEW CEPHEM COMPOUND FROM PENICILLIUM CHRYSOCENUM BROTH EXPRESSING DEACETOXYCEPHALOSPORIN C SYNTHASE ACTJVITY

INHIBITORY EFFECTS OF DIFFERENT EXTRACTS AND ISOLATED COMPONENTS FROM PETALONlA FASCIA (MULLER)

G.lf.Mahran, M.M.Saleh, M.M.El-Sherei and M.H.Grace KUNTZE ON A I V REVERSE TRANSCRIPTASE

Pharmacognosy Dept., Faculty of Pharmacy, Kasr el Aini, University of Cairo, (EGYPT)

Bio-assay guided fractionation of the methanol extract of the brown alga Petalonia fascia (Muller) Kiintze on human immunodeficiency virus type-1 reverse transcripLase led to isolation and characterization of three active components, 1,8 dihydr- o x y 3 methoxy 6 methyl anthraquinone [ I ] , 1 , 8 dihydrnxy anthraquinone [ 2 1 and 2 ' , 4 ' dihydroxy 6' methoxy chalcone [ 3 ] . Component 2 and 3 showed 50% inhibition of the enzyme activity at a concentration of 70 and 25 ug/ml respectively, while component 1 showed slight activity.

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MOUSE SKIN CARCINOGENESIS Govind J. Kavadia’ , Barukuni Tokudq‘, Takao Konoshima3,

Akio IwashimaL and Hoyoku Nishino4 ‘Department of Medicinal Chemistry, College of Pharmacy and Pharmacal Sciences, Howard University, Wshington, D.C. 20059, ’Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 6 0 2 , 3Kyoto Pharmaceutical University, Kyoto 6 0 7 , 4Cancer Prevention Division, National Cancer Center Research Institute, Tokyo 104, Japan

As a part of screening studies for cancer chemopreventive agents, we have recently reported anti-tumor promoting activity of a number of natural and synthetic phlorophenone, anthraquinone, anthronelanthranol and bianthrone derivatives. In continuation of these studies, we have examined a number of naphthoquinone derivatives. The products evaluated consisted of monomeric, dimeric and tetrameric naphthoquinone derivatives occurring in the plants of Dioswvros genus. Several synthetic naphthoquinones were also evaluated. The compounds tested were examined for their in vitro antitumor promoting efkect on Epstein-Barr virus early antigen activation induced by the tumor promoter 12-0-tetradecanoylphorbol- 13-acetate and in vivo the same effect on two stage mouse skin carcinogenesis. We present the initial report that part of these compounds showed potent anti-tumor activity.

OLIGOMERIC PROANTHOCYANIDINS CONTAINING ONE OR TWO DOUBLE

Al iou BaldB’, Luc Pieters’. Tess De Bruyne’, Herbert Kolodziej’, Dirk Vanden Berghe’, Magda Claeys’ and Arnold Vlietinck’

I Universitb de Conakry. Guinee-Conakry (present address) ’ Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1,

INTERFLAVANOID (A-TYPE1 LINKAGES FROM PAVETTA OWAR/€NSIS

6-2610 Antwerp . Belgium; ’FB Pharm, Freie Universitat Berlin, Germany

Pavetannins 87 and 88, two new trimeric proanthocyanidins, have been isolated from the stem bark of Pavetta owariensis. along with the known tetramers cinnamtannin 82 and its positional isomer, pavetannin C1, and the pentamer, pavetannin D1. NMR and mass spectral data established the structure of the pavetannins as epicatechin44R-8, ZR~-.7)-ent-epicatechin-(40-.8. 20-.0--7)- ent-catechin, epicatechin-(4&-8, 2C-O-.71-epicatechin-(4l3-.8, 2l3-0-.7)-ent-catechin, epicatechin- (4C-61-epicatechin-(4l3+8, 2l34-71-epicatechin-(4l3-8)-epicatechin. and epicatechin-(4&+8)- epicatechin-(4B-8, 2~-0-.7)-epicatechin-~4~-8l-epicatechin-~4l3-8~-e~icatechin, respectively. Pavetannin 87 and 88 represent rather uncommon trimeric proanthocyanidins in that they have two double interflavanoid [A-type) linkages in each molecule, representatives of this class of natural products being to date aesculitannins C and D only. The naming of cinnamtannin 82 was revised to epicatechin-(4l3-.8)-epi-catechin-(4R-8, 2R+O-71-epicatechin-(4R-.8)~epicatechin, according to its structural presentation. Indeed, oligoflavanoids with an A-type unit require special anention regarding configurational assignment at C-4 by the a/& nomenclature to prevent ambiguities. According to IUPAC, the a or R denomination of the stereochemistry at C-4 must always be related to the normal, convenfional orientation of the parent molecule. It must be retained, even i f for graphical reasons this parent unit would be rotated and represented in a non-conventional orientation. Overlooking this rule has caused a lot of confusion throughout the existing literature [I-21.

H. Kolodziej, D. Ferreira, G. Lemibre, T. De Bruyne, L. Pieters and A. Vlietinck, On the nomenclature of oCgoflavono/ds with an A-type unit, J. Nat. Prod. 56, 1 199 (1 993) A. Bald6 et dl. , J. Nat. Prod. 56, 1078 119931

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ANTI-COMPLEMENTARY ACTIVITY OF CRA TA€GUS SINAICA Boiss. A.A. Shahat', T. De Bruyne', A. Lasure'. B. Van Poel'. k . Pieterh'. F.M. Hammouda'. S.I. Ismail', A.J Vlietmck'. 'Dept of Pharm. Sciences. University of Antwerp. UniverSiteitSplein 1 , 8.2610 Antwerp. Belgium 'Pharm. S C ~ . Div.. NRC. DOKKI. Cairo, Egypt.

r

1 Crataegus spp are famous In folk medicine for their use in minor forms of coronary heart disease, minor forms of heart lallure and cardiac arrythmia I 1 I We studied the anti complementary activity of n-butanal, ethyl

I acetate and water fractions of the f r u m and leaves of C sinaica Boiss using a hemolytical assay according to ' Klerckx et a1 121 Progressive fractionation led to the isolation of flavonoids and procyanidins The air drted leaves and fresh fruits were extfacted exhaustively wi th 70% and 80% aceton respectively

The concentrated resldues were delatted wi th ether and exlraLted successively wi th ethyl acetate and n butanol The butanol and ethyl acetate Iractions were chromatographed on Sephadex LH2O w i t h ethanol From the ethyl acetate fraction of the fruits the procyanidins 8 2 and B5 were isolated as well as an A dimer and a mixture of flavonoids 10 glycosidesl The butanol fraction of the leaves yielded a B frimer and a mixture of C-glycosides Structures were elucidated using 'H and ''C NMR and mass spectroscopy

The actwtty of the different fractions towards the classical ICPI and alternative (API pathways of the complement sytem are shown In table 1 For the most active compounds studies are carried out concerning their mode of action

Fraction CP IC, bglmli AP IC, balmll

Fru8i. EtOAc A dims, 0 gt"eorde. > 1000 > I000

8 2 2 ,1000 Lsauee EtOAc 2 4 5 , 656 0

0"OH 21 2 300 3 2 1 20 1

4 7 0 > 1000 C glycosid- I 2 6 5 0 >I000

Tab 1 Anti complementary activlty of subfracltons of C sind,ca Boiss

I l l Arnmon ti P T , Handel M. 1138811 PI. Med C3141, 313-322. 121 Klerckx J.. Beukelman C , vanDyk H.. Willers J 119831 J Imm Mefh.63.215-222.

VA1,IDATION OF Eh'ZIMATIC ASSAYS FOR TIIE BIOSCREENIh'G OF NATURAL PRODUCTS Maria Giiiiiea' and Josefina Parellada'.

t Department of I'liarinacy and Phariiiaceutical Technology. Scieiices. University o f A lca l i de Henares. E-28871, Madrid. Spain.

D e p a r t i i l e n t o f Nutrition and Food

The bioscreening of a large number of samples require fast, easy, reliable and low cost assays. Bioscreening by enzimatic inethods rely on the selective recognition of substrates. Coinercially available chromogenic substrates allow easy and photometric transduction of enzynie products. Monitorization of the enzimatic reaction rate correlates to the biological activity of the sainple. The main challenge for the realiability o f the enziinatic method chosen i s imposed by the complex coinpositioii of real saiiiples Interferences in the bioassay can be envisaged if the coinpounds present in the satnple react cheinically with the substrates or/and modify the phisico- chemical conditioils of the medium during the assay. Special attention has to be paid to these interferences i f absorbance nieasiireinents are correlated 10 biological activity due to the low selectivity of IIV-Visible deterininations.

We have fomd severe interferences in the enzinia~ic hioasssay o f phenolics, specially with the Ilavono~d coiiipouiids. in the hioscreening made with the proteases trypsin (E.C. 3.4.21.4), chyniotrypsin ( t . C . 3.4.21. I ) . pancreatic elastase (E.C. 3.4.21.36) and leucine aininoprptidase (E.C.3.4. I I . I ) . using I)-nitroanilidetlerivative suhstrates. This work presents the strategies involved in the optimization of the enzimatic bioassays for the validation of our results. Acurancy. repeatihility and reproduclhility of the the inethods wi l l be shown in the preseiice of chemically defined coinpounds and crude vegetable extracts froin Citru., ( . h i / , A/7wlarrchirr o v d i s and ffJfhJphy//f4In pc//ci/fm,7. Protocols and results for the bioscreening of nientioned cainples wil l he presented.

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A RAPID QUANTITATIVE M E T H O D FOR T H E ANALYSIS OF AMARYLLIDACEAE ALKALOIDS BY CAPILLARY COLUMN G A S CHROMATOGRAPHY

-aBastos". Xu Lia, N.P.Dhammika Nanayakkaraa, Charles L. Burandt, Jr.a, James D. McChesneya*b aResearch Institute of Pharmaceutical Sciences, bDepartment of Pharmacognosy, School of Pharmacy, University of Mississippi, University, MS 38677. USA

Plants belonging to the family Amaryllidaceae have heen widely used in traditional medicine throughout the world. Many members of this family have bcen chemically analyixd and Amaryllidaceae alkaloids have been identified as the major class of compounds responsible for the biological activity. Amruyllidaceae alkaloids are a diverse class of naturally occuning bases belonging to 5 major structural types and over LOO have bcen isolated and identificd. Some of these compounds have been shown to possess a wide spectrum of useful biological activities including antiviral, mtineoplastic. immunostimulant, analgesic and insect antifeedenL Galanthamine. a common alkaloid in this family, has shown cholinesterase inhibitory activity and is currently undergoing clinical trials For veatment of Alzheimer's disease.

Although different analytical techniques have been described to quantitate some of these :ompounds, a rapid, reliable and quantitative method for analysis of complex mixtures of the llkaloids in plant extr~cLs is still not available.

We describe here a rapid, reprodhciblc capillary CG-MS tcchniquc to identify and quantitate the :ommon mgjor alkaloids of the family Amaryllidacex.

QUANTITATIVE UETERMINA rlON OF P O W P H Y L L O T O X I N RELATED COMPOUNDS IN PODOPHYL/.L'.if SPECIES B Y ItIGfI PERFORMANCE l.lQUlD CHROMATCGRAPHY lair0 K Dastos'. Wlodzimierr 1 Kopycki'. Charles I. Burandt. Jr'. NP Dhammika Nanayakkara'. and

p: 132 I James 11 McChesnevLb Research Institute of Pharmaceutical Sciences. "Uepartrnent of Pharmacognosy, School of Pharmac~.lJniversity of dississippi. I!niversitv. MS 38677 l!SA

'he drugs etoporide and teniposide have shown broad antitumor activiry and are cuncnllv used for the t reamcnt of :SIICU!BI cancer and small cell lune cancer 8odophyllotoxin ( I ) . which i s isolated irom !he plant Podophyllum rmcdii Wall ex Royle collected from the wild. Dt 3 the dccline oi Podophyllum eniodii in the wild. new sources or a svsternatic cultivation of the plants are required 1c v x i ihc future demand for lhesc drugs Several other naturnllv o c c m n g arylterralin Iignans. such as podophyllotoxr. iycoside I VII ) . 4-deme1hylepipodoplivilotoxin I 111). ~-demeth~lpodopn).Ilotoxin (VI) and epipodophyllotoxin (11) can c chcrntcally modified 10 produce these drugs In an effon to find new sources and lo identify environmen~al factors hat alfect !he production of these compounds. uc are screening scwra l wild and cultivated podophyllum populations hat are growing under different ecolugical condilions In this papcr we describe a rapid and sensitive reverse phase 1PI.C method using a TaxilQ column and gradicnl elution for Ihe routine determination of podophyllotoxin and SIX

elated compounds (1. 11. V. V, VI, VII & VIII) in Pcdophyllum plant extracls

Both drugs are commerciallv produced from the an i te t ra l in Iignan.

R, = OH R , - H R,= H R, - H R, ~ H R , - OH R, - OGl

R, = H & - O H R, - O H

R, - H R, - H R, - H

& - H

R . - I { R , - M e R. - H R , - H R . - H R , - H & - O H R , = M e & - O H R , - H R . - H R , - M e R . - H

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~~

NEW CYTOTOXIC CEMBRANE DITERPENOIDS ISOLATED FROM T H E CARIBBEAN GORGONIAN OCTOCORAL EUNICEA MAMMOSA.

University of Pueno Rico, P.O. Box 23346. UPR Station, Rio Piedras, Puerto Rico m, Ivette Pifia G6rnez. Abimael D. Rodriguez

0093 1-3346 P b v i e r J . During the past three decades the isolation, structural elucidation and synthesis of marine natural products have become the focus of attention of many organic chemists because of the significant cytotoxic and antitumor activity shown by most of these compounds. As part of our long term interest in exploring the marine fauna from Puerto Rico, we have recently studied the secondary metabolites of the gorgonian octocoral EUNICEA MAMMOSA. Twelve novel cembrane diterpenes were isolated from the CHCI3 extract of this gorgonian, all of which belong to a new series of cembranolides hereon known as the UPROI IDFS . These compounds were isolated and purified using chromatographic methods and their structural elucidation was achieved using NMR, MS, IR and UV spectroscopy. Some of the UPROLIDES have shown strong cytotoxic activity against several

5133 1 TWO NOVEL 19-RESJDUE PEI'TAlBOLS ISOLATED FROM CULTURES OF A MUSSEL ASSOCIATED FUNGUS & & e n W, AVQ (l), Donald Brewer (l), Pierre Thibault (l), Ziba R. Fathi-Afshar (2). and Anita Gandhi (2) (1) Institute for Marine Biosciences, National Research Council of Canada, 141 1 Oxford Street, Halifax, Nova Scotia B3H 321 (Canada) (2) SynPhar Laboratories Inc., #24 Taiho Alberta Center, 4290-91A Street, Edmonton, Alberta T6E 5V2 (Canada)

In the search for novel biologically active natural products from marine fungi, the antibacterial activity exhibited by extracts of the culture broth of a fungus isolated from blended tissues of mussels, Mytilus edulis (collected in Ship Harbour, Nova Scotia). was investigated. The fungus has been identified as a Tricho&nna species.

high molecular weight peptaibols (linear peptides in which the C-terminal carboxyl group is reduced to an alcohol, the N-terminus is acetylated, and which contain a high proportion of a- aminoisobutyric acid). Analysis of the peptaibol mixture by HPLC coupled with ionspray-MS revealed the presence of two major, and four minor, components. Final purification of the peptaibol mixture was achieved by reversed phase HPLC. The two major components, c h a r a c t e d on the basis of MS and NMR data, were determined to be novel 19-residue peptaibols (C88Hi&N22023 and C8gH148N22@$, each of which contain phenylalaninol as the C-terminal alcohol. The isolation and structure elucidation of the two novel peptaibols, and details concerning their antibacterial activity, will be presented.

Through bioassay guided fractionation, the antibacterial activity was traced to a group of

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ISOLATION AND CEARACI'ERIZATION OF FIVE NEW DOLABELLANE DITERPENES FROM THE CARIBBEAN GORGONIAN EVIVZCEA LAcrrmATA. Eduvlels G- Cynthla C o d e z . and Abhnael D. Rodriguez' Unlverslty of Puerto Rlco . P.O. Box 23346. UPR Statlon. Rlo Pledras. Puerto Rlco 00931-3346

P135

The Caribbean gorgonian Eunicea lacinniata is known to produce compounds possessing the dolabellane carbon skeleton. Palomino1 and Isopalominol , two such compounds, were isolated from previous investigations conducted in this research group. As part of our ongoing effort to isolate biologically active new compounds from the marine fauna of Puerto Rico , the study of Eunicea lacinma was recently re-opened and five new compounds containing the dolabellane carbon skeleton were isolated. One of them possessed a conjugated enone functionality which was located on the 11-membered ring and two of these compounds were found to &st as palr of diasteromeric alcohols. All the structures were established using 1-D and 2-D NMR techniques such as 'H-NMR. 13C-Nh4R. COSY, CSCM. NOESY, and INAPT. The details of these characterizations will be discussed.

P136 1 THE PARTIAL SYNTHESIS OF BIOLOGICALLY ACTIVE NOVEL CEMBRANOLIDES Jvette C. Rfia Gbmez and Abimael D. Rodriguez University of Puerto Rco, P.O. Box 23346 UPR Station, FUo Pledras, Puerto Rico 0093 1-3346

Cembranoid diterpenes are a widespread class of natural products found both in plants and animals. The cembranolides. which are characterized by a 14-membered ring fused usually onto an a - methylene- y- lactone ring, are particulary interesting due to their wide spectrum of biological activities. For that reason we have undertaken the partial synthesis of a series of novel cembranolides using well-known readily available marine natural products as starting materials. Our intent has been to prepare new molecules with enhanced biological activity using tandem transannular cyclization reactiohs. Many of the new compounds thus synthesized have already been screened for biological activity in an effort to understand their mechanism of action and/or structure- activity relationships. These studies could eventually lead to the development of new more powerful target-specific drugs.

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1 CRITICAL MICELL CONCENTRATIONS OF TRITERPENOID SAPONINS

Jorg Bartlakowski, Thomas Schopke, Karl Hiller IIurnboldt University of Berlin, Departmelit of Phannacy Goethestr. 54, D-13086 Berlin, Germany

Saponins are natural products, which usually occur in plants atid have some characteristic properties such as hemolysis of erythrocytes and formation of foam. These are used in several methods, which were established in order to quantify saponin action. A further characteristic feature of saponins is tlie decrease of surface tension of their solutio~is, which can directly be measured by means of teiisiometer by LECOMTE DU NOG'Y. Triterpenoid saponins from Bellis peremis, Hedera hefix, Prinlula vrris, SOrricula rwuyac.4 arid Glycurr/Ia &aha were isolated and tlie surface activity of their solutions was measured. The graphical interpretation of the concentration-tension-curves showed similarities with the behavior of Tween 80 solutions: 1. decrease in tension due to rising concentrations of the agent; 2. in higher concentrations of the agent furtlier saponin addition causes only very little tension decrease (saturation of the surface). After surface saturation the occurance of micells is supposed (critical micell coilcentration CMC). Measured CMC-values range from

111 case of brllissnpoiiiri 2 a loser' liglit scattering ~~ieasure~nent Coilfinned the OcciimJce of paxticles i r i sapoilin solutions with concentrations over the CAIC

g/L to 10-2 g/L.

pi38 I A PERFUSION MKI'HOD FOR IN\TSTIGATING ABSORPTIOX PROPERTIES OF DRUGS IN ISOLATED RAT SMALL INTESTINE

I\lartiri Xorta, Thomas Schopke, Karl Hiller Humboldt-Universitiit zu Berlin, Ma t l i e~ r i a t i s c i r -No t~ i~~~se~ i~c l i~ t l i c l i e Fakultat I, Institut fiir I'hanriazie. Goetliestr 54, D-13OSb Berlin (Gennany)

A perfusion method for tlie determination of absorption rates in isolated rat small intestine was created.

Aiological research in intestinal absorption liiritetics of drugs knows some in bitso methods using artificial or natural membranes. Tliere are unekerted and everted sacs, which are easy to haidle but not so accurate in results. Moreover there exist more complex perfusioti models where segnienb of isolated intestine are kept alive serving as diffusion barriers. The apparatus described in this paper is a two conipartment chamber derived from a prototype by DARLISGTON and QUASTEL [I] which was reduced in scale and combined with ari IiPLC analytical ~riethod. It allows exact analytics together with little loss of material arid produces absorption curves that cat11 directly be compared. After validation B standardized method for the charactelizatiori of the GI vitro absorytioii of drugs i11 the fonii of (~ i ig x uin-l x The irietliod described sliows accuracy a i d is applicable as tests with tlieopliyllitie iiidicate.

was developed.

1 Darlington, W. A,, Quastel, G . H. (1953) Arch Biophy?. 43, 194

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EFFECTS OF TRITERPENOID SAf’ONNS ON THE INTESTINAL ABSORFTION OF DRUGS.

Thompr S c b k e , J6rg BartlnkowsLi, MPrcio Norta. Karl Hiller Humboldt-Universtitit m Berlin, Mathemstisch-Natunvissellschsftliche Fakultit I, Institut firr PhnrmPEie. Goeheatr. 54, D-13086 Berlin (Germany)

The influences of saponins on the absorption of drugs bave bee0 under discussion for a long time. On the one bmd evideue exists &at SrpWiDs M able to increase the intestinal, rectal md d absorption of differeat drugs. On Ihe other band a cornlation with the critical micell conceatrntion (CMC) is supposed. Despite this correlation no investigations of the CMC of 6aponins have been underIakea before starting absorption studies. For this reason we initially determined the CMC values of a number of triterpenoid spponins and mixtures of them belonging to different structure type& T b a studies indicated CMC values of about lo2 g/L in the case of mixtuns of saponins and of about 10’ to lo4 g L in the case of pure paponins. I n vim0 absorption studies wen performed on rat s d l intestines With indow(hnciw and theophylline as modell compounds. Using uneverced sac4 no significant changes in the absorption of indomethacine were caused at snpanin concentrations above cmc. Apart from the u n e v d pacs a modell besed on the apparatus of DARLINGTON and QUASTEL [ 11 was applied for further study. Using u-hede.rh (acidic monodesmoside) and saponin mixtures obtained from Soniculo europour (neutral monodesmosides) at concealrations of 50 mgL aud 100 mgL (both above cmc) neither an inrease nor a decrease of the absorption of tbmphylline was observed.

1 Darlington, W. A,, Quastel, G. H. (1953) Arch. Biophys. 43, 194

NEW POLYACETYLENE GLUCOSIDE WITH IN MlRO OVER GROWTH ACTIVITY FROM BIdenspUosa (ASTERACEAE)

Laura Alvarez’, Guillermo DelgadG. Maria Luisa Villar~eal~ and Daniel Alonso3

El40 1 ’Deparlamento de Quirmoc Orpanica, Divisi6n de Estudios Supenores de la F a d a d de Ciencias Qulmicas

c Mustriales de la Universidad Autbnoma del -do de Morelos. Av. Universidad 1001, Cuernavaca, Monlos. Medco. lostituto de Qutmica de la UniWrsidad Nacional Aut6noma de Mk~ica. Apdo. Postal 70-

213. Mexico 04510, D. F.. 3Centro de Invcnigaci6n Biomedica del Sur. IMSS. Xochitepec, Morelos. Mexico.

Bidens pilosa L. (Helianthcae, Coreopsidinae) is a widely distributed pantropical weed. The leaves and roots of this plant are used in Mexican traditional medicine for treatiq stomach disorders and diabctcs. It is also known by its antimicrobial properties, since it normally accumulates the &biotic compound I-phenylhepta-1,3.5-triyueeiyne. Previous examinations on this spccies resulted in the identification of lipophilic polyacetylenes, tlavonoids, and aurm and chalcone glumsides.

The present commuoication deals with the. isolation and strudure determination of a novel polyacetylene glumside: ~ - D g l u c o p y r a n o s y l - J ~ y ~ 0 ~ ~ ( ~ - ~ - 8 , 1 0 , 1 2 Q i y n e (see formula), which was identified by W, R EMS, ‘H- and ’3C NhfR, and chemical methods.

This compound WBS found to be responsible for the in v i m over-growb activity in the cells OVCAR 5 and UlSO SQC-l when tested in its ability to modify the growth rate of various human cell lines in culture. This proliferative activity m l d be of significance for the previously reported cocarcinogenicity found in this plant.

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p1 41 TRITEHPENE SAMNINS FROM CYCLAMEN COUM

Ihm -*. Ayeen Yuriiker*. Nevin Tnnker**. Anthony D. Wright***, Otto Sticher*** * Hacettepe University. Faculty of Pharmacy. Dzprtmmt of Phannacognosy. TRMIOO Ankara.

p: A NEW FURANOII) I)IlERPENOII) OF NEO-C1,EROI)ANE TYPE

FROM TEUCRIUM CHAMAEDRYS h &*. Erdal Bdir*. Anthony D Wright**. Otto Sttcher"

* Hacrttrpe University. Faculty of Pharnmcy. Department ot F'harmawgnnsy. TR-06100 Ankara, TURKEY ** Swiss F r & d Institute of Technology Zunch (ElHZ),

Depsrtment of Pharmacy. CH-8057 7unch. SWITZERI AND

142 1 Teucrium L. species (Lamiaceae) are widely used in traditional folkloric medicine. Teucriun

chamdrys L. is one of the most investigated species. It is used as stimulant, tonic, diaphoretic, diu retic and against stomach pain. From the water soluble part of the methanolic extract of the aeria parts of T. chornnedrys a new clerodane type diterpenoid [I 1 was isolated together with the phenyl propanoid triglycoside teucrioside which has been reported from the same plant (1).

The structure of 1 was elucidated primarily through the application of ID and 2D-NMR tech- iiques and established as 15,16-epoxy-2~,6a,18-hihydroxy-19-nor-neo-cleroda-4,13( 16), 14-hien- 20.12-0lide.

( I ) G:A. Grnss. M.F. Lahloub. C. Anklin, H.-R. Schulten. 0. Stichcr. Ph)~oc / ien i i~ r~27 , 1459 (1988).

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INFLUENCE OF VARIOUS TECHNIQUES IN TLC-SEPARATION OF PLANT POLYPHENOLS FROM CRUDE MEDICINAL PLANT MATERIAL Jari Summanen, Heikki Vuorela and Raimo Hiltunen

Pharmacognosy Division, Department of Pharmacy, P.O. Box 15, FIN-00014 University of Helsinki, Finland

reported to contain p k t polyphenols it has been found dif icult to separate compounds properly because of tailing and strong bounding to the stationary phase. For a good chromatographic separation of phenolic compounds it is necessary to find out as selective a system as possible for TLC by choosing the right analytical techniques. This prompted us to test various TLC-techniques and optimize the parameters for phenolic reference substances.

Traditional TLC-technique in which the separation is based on capillary forces was tested using vertical and horizontal development. The other tested technique was overpressured liquid chromatography (OPLC) in which the mobile phase is forced trough the station phase using a constant pressure. Differences between various stationary phases cellulose, reverzphase (RP 2,8 and 18) and silica (normal-phase) as phase materials were studied to examine their influence on tailing and bounding of compounds. The volume of gas phase of different development chambers, saturation time of chamber andor TLC-plates and flow rate in OPLC-technique were parameters that had an important role in the separation procedure. The "PRISMA" model was used to optimize the mobile phase composition.

Cellulose as a stationary phase gave a good resolution and Rfvalues were higher when the plates were saturated prior to use. For tested polar molecules the RP-18 seemed to be superior to other TLC stationary phases in traditional TLC-technique. When using reversed phase the optimum pH was an important parameter for good separation in addition to the selected solvent composition. OPLC-technique was found to be the most rapid method for the separation. However the suitable pH for the separation caused problems in the form of breaking the impregnation of cellulose-plates. Due to this silica was found more suitable for OPE-separation.

When identif 'ng compounds by thin-layer chromatorphy (TLC) From medicinal plants

Pl44 I TRISUBSTITUTED SPERMlDlNES FROM POLLEN OF QUERCUS DENTATA Maria Bokcrn. Ludger Witte+. Victor Wray*. and Meurcr-Grimes

Lehman College. De aTmKnt of Biologiul Sciences. The C. Universiiy of New York. 250 Bedford Park Blvd West. Bronx NY 10468 L! S. A: f h d l u t filr Runoueulwchc~io,~~dcrTechnwchrn Universitil Bnunschwei Mendkohnstr. 1: D-3300 Braunxhweig, F. R. G.; *Geaellschn lbr BroCcchnologische Forxhung (GBF). h&sscherc&r Weg I. D-3300 Braunxhweig. F. R. G.

The reproductive organs of plants are known to contain bydroxycinnamic acid amjdes [1,2.3]. The distribution and structures of these compounds constitute useful raxowmic charauen 14.51. We report here the sfmCtll~+s of three novel and one known hydroxycinnamic acid conjuagtes of spermidine. The substances werc isolatcd from a methanolic pollen extract by preparative HPLC. The structures were elucidated by ZD-H1-NMR (COSY), FAB- MS, and El-MS. and identified as tri-caffeoyl-spermidine [M-HI- = 630, di-affeoyl-mono-paumaroyl- spcrmidine m-H] - = 614, mon~feoyldi -pcouoyl -spermidine [M-HI- = 598, and tri-p-cnumaroyl- spermidme W-HI- = 582. Tricoumaroylspermidme was previously described from floral p a l s of Rosaceac [5]. All four compounds are the major phenolic WnStituenIS in pollen of uisuhstituted spermidine derivatives in the plant kingdom.

111 Martin-Tanguy. J., F. Cabanne. E. Perdrizet. andC. Martin. 1978. Phytochemistry 17: 1927-1928. [2] Meurer. B.. V. Wray. L. Grotjahn. R. Wiermann. and D. Strack. 1986. Phytochemistry 25: 433435 131 Meurer, B.. V. Wray, R. Wiemrann. and D. Strack. 1988 b. Phytochemistry 27: 839-843. [4] Meurer. B.. R. Wiennann. and D. Sfrack. 1988 a. Phytochemistry 27: 823-828. 151 Strack. D.. U. Eilen, V. Wray. 1. Wolff, and H. Jaggy. 1990. Pbytochemistry 29: 2893-2896.

species. This ia the second repon of

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LICORICE CHALCONES, A S E W CLASS OF POTENT 45 ANTIMALARIAL COMPOUh'DS MING C M E N ' Y ~ , , THOR G. THEANDER3>4, S 0 R E N BR0GGER CHRISTENSENS, LARS HVIID3.4, A N D ARSALAN

Centre for Medical Parasitology, Department of Clinical Microbiologyl, National University Hospital, and Statens Seruminstitut2, Copenhagen, Department of Infectious Diseased, National University Hospital, and Institute for Medical Microbiology and Immunology4, Copenhagen University, Copenhagen, Department of Organic Chemistry5, The Royal Danish School of Pharmacy, Copenhagen, Denmark

K H A R A Z M I ~ *

A

Malaria is a major health problem in many parts of the world affecting millions of people. The spread of chloroquine resistant parasites to many malaria endemic areas and the development of multiple drug resistant parasite emphasize the urgent need for the devclopnienr of new antimalarial drugs. Here we report that licochalcone A, purified from Chinese licorice roots and a number of synthetic related oxygenated chalcones inhibit the growth of chloroquine-resistant of the human parasite Plasrnodiiirrz falcipurum in vitro and control lethal infection of Plasmodium yoelii in mice. These findings might provide the basis for a new class of antimalarial drug.

The r a m of a Z m h sp m ( Sam Chand !98 ) , coUecred on Erromango Island (Vanmu ) , led to the isohon oivanous flavonoids . Woids and acetophenones Three of these l&er are novd nslural products O-merhvl oclandrenolone (1) and the wo

corresponding Rcms-diols (2 and 3 Thar smumru were deauced h m rhar s p d data,

oclandrenolone as starung matend mRlalV 2D T - H NMR ~ r p m e m ~ , a d cornkmed by ch-4 ~ O r r d a a O n S UMq

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POSTER PRESENTATIONS 20 I

El47 ALKALOIDS FROM BRWSVIGU JOSEPHINAE William E. Campbell, Shaheed Mathee

NOVEL LIMONOIDS FROM THE SEED OF EKEBERGIA CAPENSIS Oulcie A.Mulholland ana Serge E.Iourine El48 1 Natural Products Research Grow. Department o f Chemistry. dnivers i tv of Nata l , King Geor.ge V Avenue,.Durban, 4001, South Afrjca

Ekebmgia cupem& Sparrm i s a member of the Meliaceae family whose range s t r e t c h from t h e Southern Cape to the Sudan and Ethiopia. In Natal the t r e e i s used f o r medicinal and magical uses by the Z u l u people. An e x t r a c t from the bark i s used t o t r e a t coughs and r o o t e x t r a c t s a re used i n the treatment of dysentery.

We have ext rac ted severa l complex novel limonoids of thetypsshown b e l o w from t h c hexane e x t r a c t of the seeds o f t h i s species . e luc ida ted using 2D NMR and HR MS techniques.

S t r u c t u r e s of these compounds were

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SNOIlVlNBSWd W l S O d 202

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BIOACTIVE AND OTHER LIMONOIDS OF TRXCHILIA EMETIC4 (MELIACEAE)

-.A. A. Leslie GunaUlaLa. Ermias Dagne and David G. I. Kingston Instituto de Quimica. Umversidade Esiadual Paulista, C.P. 355.14.800, Araraquara S b Paulo. Brazil; Depamneot of Chemistry, University of Addis Ababa Ethiopia and Depamnent of chemistry; Virginia Polytechnic Institute and State University, Blacksburg. VA 24061-0212.

Bioactivity-guided firactionation of the bioactive methyl ethyl ketone extract of the bark of Wchilia emerica utilizing a medmism-based yeast bioassay for DNA-modifying agents (1) afforded two bioactive Umonoids. Nymanla 1 [I]& TI-3 [2], along with the inactive Ilmonoids, Rohitika 3. Drageana 4. and 'McNUn C. Nymania 1 dm showed aalvity In the Vero monkey cell cytotoxicity assay. ?his constitutes the first report of the DNA-modifying activity of limonoids.

'El 51

1. Gunarilaka. A. A. L.. Samaranayake. G.. Kingston, D. G. I.. Hofmano. G.. and Johnson, R. K.. 1. Nar Prod. 55. 1648 (1992).

ISOLATION, STRUCTURE ELUCIDATION AND SYNTHETIC STUDIES OF El 52 I NEW TAXOIDS FROM TAXUS BRE'WFOUA. Ru Chen. and 2 David G. I. Kingston. Department of Chemistry, Virginia Pofitechnic Institute and

State University, Blacksburg, Virginia, 24061-0212.

The western yew, T a w brevifolia, was for many years the source of the important anticancer drug taxol, but it also contains many other natural taxoids. These compounds are of interest both from a structural viewpoint and also as a starting point for the synthesis of taxol analogs. As a complement to our ongoing studies on the chemistry of taxol, we have investigated T. brevifoliu extracts and have isolated some new taxoids. Among these are the A-norbaccatin analog (1) and the baccatin V analog (2). The isolation and structure elucidation of the new taxoids will be described. We have investigated synthetic modification of 1 by acetylating the hydroxy positions and comparing the NMR data to those of known taxoids. The structure of 2 has been verified chemically by semisynthesis from the known taxoid 10-deacetylbaccatin IIL

1 2

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plant material. Tne SET extracts have been canpared with the cmpsition of products gained by traditional steam distillation and solvent extraction by means of GC, WLC, TLC-densitanctry, IR, CC-IR, GC-MS techniques.

p1 54 ACID CATALYZED INTER CONVERSION AND ARTIFACT FORMATION OF PECTENOTOXINS, POLYETHER MACROLIDES INVOLVED IN DIARRHETIC SHELLFISH POISONING.

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PI55 I MASS SPECTROMETRIC INSIGHTS INTO THE BIOSYNTHETIC ORIGIN OF SULFUR IN ESPERAMICIN

L &%m€&&~ Kevin J. Volk, pike S. Lee, Edward H. Kerns,

Kin S. Lam, Judy A. Veitch, Salvatore Forenza Bristd-Myers Squibb Pharmaceutical Research Institute

5 Research Parkway, Wallingford, CT 06492

The coordinated use of tandem mass spectrometry and high resolution mass spectrometry in the initial phases of natural product isolation is a vital component of the rapid structure elucidation process of complex natural products at Bdstol-Myers Squibb. Simllar to the structure elucidation of unknown isolated natural products, sample constraints such as limited quantities and complex mixtures are typlcally encountered with biosynthetlc studles. Furthermore, the difficulty of demonstrating incorporation of labeled species and the expense of the labeled precursors add to the challenge.

Here we describe the use of tandem mass spectrometry with fast atom bombardment and electrospray ionization to evaluate the incorporation of 34S in esperamicln. Previous substructure analysis of esperamicin using tandem mass spectrometry MS/MS) and high resolution mass spectrometry provided detailed structural information. &omparison of the substructures generated from the natural compound (32S) with those of the labeled spedes (34S) allowed for the rapid quantitation and location of the incorporated atoms from small scale (100 mL) fermentations.

The utility of this systematic approach for the evaluation of sulfur incorporation in a complex natural product is demonstrated. The utilization of substructure analysis schemes will be reviewed. The capability of rapidly distinguishing the number and location of S4S atoms incorporated into esperamicin from microgram quantities of material will be illustrated.

ABILITY OF DIFFERENT FLAVONOIDS TO INHIBIT

Jean-Michel AUGEREAU. Marie BILLON. Mannette LECOMTE SANOFl RECHERCHE - BP 137 - 31676 Labkge cedex. France

Facultt des Sciences Phannaceuttques 35 Chemin des Mm-chers - 3 I062 Toulouse &ex Alan LALE, Jean-Marc HERBERT SANOFl RECHERCHE - 195 Route d'Espagne - 31000 Toulouse

- Tissue factor (TF) is an ubiquitous membrane-anchored glycoprotein &it initiates blood coagulation by forming a complex with circulaung factors VII and Vlla. Under normal circumstances. monocytes do not express TF activity but in some pathological situations. when tbey are exposed to inflammatory mediators. they can acquire procoagulant propeniees. Interleukm-lp (ILI) increased in a dosedependent manner the expression of TF on che surface of human monocytes. 65 natural flavonoids of different classes (Ikvone. Ikvonol, flavanone, dihydroflavonol and bfflavmoids) were screened for their hability to inhibit (he procoagulant activity of adherent human monocytes slimulated by ILI

I R of the different wmpounds had an acuvity between I04M and 10-xM but the most active product was a biflavonoid (hinokillavone) with an ICSO value of 5.10-8M. The results lherefore show that hinokiflavone is a potent inhibitor of IL-1-induced TF expression on human monocytes and suggest that this class of compounds might constitute an interesting approach in the prevention and veament of thrombosis.

in vitro.

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F L A V O N O I D S C O N S T I T U E N T S F R O M P O L L E N G R A I N S OF P H O E N I X DACTYLIFERA I..

':157 1 -1; 1. Schmidt ?; F. M. Hammouda I ; G . Adam I ; S. 1. Ismail I; M. M. El-Missry I I - Chemistry of Mediciiial Plants. National Research Ceiive. 1231 I Cairo. Egypl

- Iiislilule of Plaiii Binclieinislry. I'.O.Box 250. 1)-06018 IlalWSaale, Grriniuiy

Pollen pa ins of Phonii.r (hcfylifera L.. which belongs to family Palmae are used in the Egytian folk medicine f'or treatment of both male and fcmale infei-tilicy [ I 1.

Recently, we have reported the presence of the brassinosteroid 24-epi-castasterone in the aqueous methanol extract and diffeicnt phytosterols in the hexane extract[2].

Here. we report on the presence of flavonoids. From ethyl acetate extract (1.5 g) of pollen grains. isorhamnetin, 5.7.3'.4'-tetrahydroxy-6-metlioxy llavone could be identified. From butanol extract (I0 6). isorl~amnetin-3-1utinoside and quercetin 3,7-diglucoside could be identified by means of TLC. UV, El and FAB MS as well as IH and

This is the first rcport of presence of isorhamnetin 5,7,3',4'-tetrahydroxy flavone, isorhamnetin-3-ru1inosidc and qucrcetin-3.7-diglucosidc i n pollen gtains of Phornix chctylifera L.

References [ I 1

I21

NMR.

El-Ridi. M. S. and Aboul Wafa, M. J. Roy. Egypt. Med. Assoc.. 30. 124 (1947) Zaki. K . A,; Schmidt. J.; Haminouda. F. M. and Adam, G . Planu Mcdica Ahstr. of 41" Annual Congress on Medicinal Plant Research, Diisseldorf, 31. August 1993, p. 37

PA58 MECHANISTIC STUDIES ON A POLYKETIDE SYNTHASE - CMETHYLSALICYLIC ACID SYNTHASE PamBhoeal. Chris Child. Johnathan B. Spencer. Peter M. Jordan.

Current work centres around feeding the polyketide producing enzyme. 6- methylsalicylic acid synthase with intermediates and novel substrates in order to elucidate its enzymic mechanism. Kesults from his work will thus further the knowledge of antibiotic producing enzymic pathways.

Polyketides are produced by bacteria, fungi and plants. They are used in human and veterinary medicine whert: they act ils antibiotics. chemotherapeutic and growth promoting agents. Other polyketides have been found to be imponant as fungal aflatoxins or as plant pigments and flavour compounds.

6-methylsalicylic acid is the first intermediate product in the route to the synthesis of the antibiotic patulin. It is synthesised by 6-methylsalicylic acid synthase. which utilizes the substrates acetyl coenzyme A. malonyl coenzyme A and NADPH. The enzyme has been successfully punfied from Periicilliurn parirlunt and possesses a homotetramer structure with a subunit size of 180.000 Da.

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p:l59 SCREENING INDIAN MARINE SPONGES FOR ANTIBIOTIC ACTIVITY K. Padmakumar, R. Robert Vethanayagam, Department of Aquatic Biology and

Preparations of Sdviae fofiurn and Thujae herba with a reduced Amount of ThuJone PI60 1 M T d n . G. Hunirhlepcr I %; of phrm. T~choolG and B~ophsmucy. Sc- & B ~ h m c r . S&@ICC. Gmnmy

Thujone is a charaaeristic plant-constituent of the drugs Salviae filium and Thujue herbu. Because of its to*c properties after internal applicahn prepamions of &/vine / o h m and Thujue herbu sbould contain only smdl caucnts of ulujone (I). lk aim of this investigation was to find cocditions for exvanion which minimize the anmint of thujone in such extracts. Percolation, digestion and distillation were chosen as procedures to obtain e x m which am comparable to industrial production and water, ethanol 30 % VN and ethanol 90 % VN as solvents for exvaCtim Salvlae filium was in accordance with the specification of the Geman Pharmawpoeia DAB 10. Ex quality of %jae

chromatographic methods (2). The cut drugs were permlaced with water, ethanol 30 % V N or *ham1 90 % VN, digested with ethanol 30 % VN at a temperature of 80' C or extracted accoTding to the method given for essential4l- dislillation AAer filfrdfion che percolam and the exuact obtained From digestion could be used directly for cbc meaSurement of thujone. Thc essential oil obtained by steamdistillation was diluted with toluol in a ratio of 1 to 10. The content of thujone was determined by capillary gas-chmmatography with FID according to (3). The total content of the essential oil was in Sulviue filium 2.2 % (m/V) and in Thujue herbu 3.8 % (m/V). Distillation gave the highesl amounts of thujone in bxh drugs: 7.57 mg thujone per g drug for Salviue folium and 7.59 mg ulujooe per g drug for Thujue herbu. Percolations with water decreased the wntent of thujone 38fold (Salviue follum) and l2fold (Thy'ae herbu) respectively. Also, the percolates obtained with ethanol 30 % VN mtained a r a d u d CaDtent of thujone (1.97 mg thujone per g drug for Salviae filium and 2.79 mg thujone per g drug for Thujue M u ) . In wnclusion, the usual procedures for extraction diminish the content of thujone in preparations of WI& f o h n and Thujue herbu. In combination with the conditions for the optimum extraction of the medical active co& a procedure can be derived, which givm optimal medical preparations of solviae fi l ium and ntujae herbu. ( I ) Hall RL, 2. Natl. Acad. Sci. (Wash) (1973) 458459; (2) Baumm I ct al., DAZ 127 (1987) 2518-2522; (3) Teguneier M., Ilamischfcger G.. Pharmade 49 (1994) 5 6 5 8

herbu, which was hmed fmm a controlled cu l t iw io~ was determined by known pharmacogncsn 'c and

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PI62

EVA1,UATION OF PURE COUMARINS USING TIX DENSITOMETER, SPECTROPIIOTOMETER AND IIPLC WITH YIIOT0DIOI)E ARRAY DETECTOR

Pharmacognosy Division, Department of Pharmacy, P.O. Box 15, FIN-00014 University of Helsinki (Finland)

p 161 Moura H. Vuorelu, P, Vuorelu und R Hiltunen 1 o.

Coumarins are natural products widely distributed in plants and displaying a variaty of biological activities (1-3). In the course of current strategy used for isolating coumarins from plant material, quality control tests for the extraction fractions and the isolated pure compounds are always needed. The coumarin character is strongly indicated by its UV absorption. To date, the UV spectra of coumarins could be obtained by using different combined chromatographic and spectroscopic UV off/on- lines. In the present communication, a comparison of the U V off/on-lines of twenty seven pure coumarins was carried out using TLC-densitometer, spectrophotometer and high performance liquid chromatography (HPLC) with photodiode array detector (DAD). Regardless of the structure of the coumarins, a common Amax and/or A,,,,,, can be detected. In most cases, a shift of 5k7.46 and 2f5.76 n m was observed by T1.C-UV and HPLC, respectively with referense to the spoctrophotometer. The intensity of the absorption bands were not the same among the different UV off/on-lines. This may be atmbuted to the photomode, the interaction between the analyte and the mobile phase or the stationsy phase. In addition, the individual sensitivity of the detector is subjected to solvent effect which could be a reason for the presence or absence of certain bands. However, the data obtained from the different UV off/on-line techniques is very useful for the preliminary identification. References I . Thastrup, 0.. Fjalland, B., Lemmich, J. (1983) Aciu phurmucol. ei roxicc~l. 52,246-253. 2. Call, T.C., Green, J . (1956) Proc: Moni. A c d . Sci. 16, 49-51. 3. Woo, W.S., Lee, C.K., Shin, K.H. (1982) Plunra Med. 45. 234-236.

OPTIMIZATION OF THE rIP1.c ANALYSIS OF BIOGENIC AMINES

CULTURE I,INES 'IYPICAI, I!V PEUCEDANUM PALUSTRE PIANTS AND CELL

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AUXINS STIMULATE HYOSCYAMINE PRODUCTION IN TRANSFORMED El63 1 ROOT CULTURES OF HYOSCYAMUS MUTICUS

L. Vanhala, M. Eeva, R. Hiltunen and K.M. Oksman-Caldentey

Pharmacognosy Division, Department of Pharmacy, P.O.Box I5 (Fabianinkatu 35). FIN-00 140 University of Helsinki, Finland

The transformed root cultures of Hyoscyamus muticus (Egyptian henbane) are usually cultivated in hormone-free Gamborg B5 medium in which the production of hyoscyamine is high, on average from 100 to I20 mgl. Present investigation shows that transformed roots tolerated well exogenous auxins in concentrations 10 nM - 5 pM. The auxins added to the medium were indoleacetic acid (IAA), naphtaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). The growth of the mots was only slightly inhibited by 1AA and NAA, but strongly by 2,4-D at concentrations above 0.5 pM. Hyoscyamine production was stimulated by IAA and NAA in the concentration range 10 nM - 5 pM, optimum being at 0. I - 1 jiM, where the hyoscyaminc content in roots was more than double compared to the roots growing without auxin addition. The concentrations of 2,4-D above 0.1 pM were toxic tc the roots, and inhibited completely the hyoscyamine production. Addition of auxins did not increase the release of hyoscyamine into the medium. It has been postulated that transformed roots are more sensitive to auxins than normal roots. Nevertheless, our findings show that they tolerated well exogenously applied growth regulators, and we suggest that the endogenous auxin production in transformed roots is not sufficent for maximal hyoscyamine production.

~ ~ ~~~~~~

BIOLOGICAL ACTIVITY OF RETINOL, RETINOIC ACID AND RETINOAES IN V u R o Marja Salo (1). Jari Kiviranta (I) , Vesa Knuutila (2). Lauri Kangas (2) a n d w Vuorela (1)

(1) Department of Pharmacy, P.O.Box 15, FIN-00014 University of Helsinki, Finland (2) Orion-Fannos Pharmaceuticals, Turku, Finland

Dietary retinol (ROH), usually as palmilate esters, is metabolized by hydrolysis followed by oxidation to retinoic acid (RA). They are essential in growth promotion and epithelial differentiation and maintenance. RA can inhibit chemically-induced carcinogenesis of a number of cancer types, but its use is limited by its narrow therapeutic ratio. The biological activity of ROH. RA and a series of esters of RA was studied. The toxicity was measured by brine shrimp (Arremia salina) bioassay and the anticancer effect by cell culture assay with human cancer ovarii cells and potato disc assay. The toxicity as EC, ranged from 10 pM (RA) to 420 pM (ROH). EC, values of rednoates ranged between 25 pM and 297 pM. The anticancer effect as IC,, was 9 pM for RA and 47 pM for ROH. IC, concentrations of retinoates were about the same magnitude as those of ROH ranging between 31 and 52 pM depending on the size of the ester. The effect of the retinoates is related to the hydrolysis rate of the ester. The results of the potato disc assay indicated similar activity of the compounds. The lower biological activity and toxicity of ROH and retinoates compared to those of RA would indicate that retinoic acid is the active compound, and that ROH and retinoates require oxidation or hydrolysis, respectively, for biological activity.

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P165 1 I

IS A PROTEIN KKNASE C AN ACCURATE INDICATOR OF MITOTIC ACTIVITY IN PLANT CELLS? Jennifer L. Aytonl, Peter Premdas' and

Department of Biology, Department o z b t University P&rborough, Ontario K9J 7B8

PKC is an enzyme tha! catalyses phosphorylation reactions within cells and it is believed that directly before cell division PKC binds to the membrane. Therefore, a decrease in cytoplasmic PKC activity should indicate an incrrase in mitotic activity. However, this study i n d i m that it is not always the case, at least in actively dividing plant cells. AlZiwn sufivwn bulb mot tips, including promeristem, were incubated in various treatmmts including inhibitors and stimulators of mitosis for a total of 21 hours. One treatment was 2OmL of l 0 p m xanthotoxh (a mutagen and mitotic inhibitor) which decrrased the rate of phosphate transfer (PKC activity) in comparison to the control. ?his would indicate that mitosis was stimulated when in fact it was retarded. Therefore, assaying for PKC activity docs not ng.essarily yield information rtgarding the effects upon mitotic activity of a given treatment in plants cells, or it is masked by diffaent activities.

' ' 2

INFLUENCE OF CONCENTRATIONS OF DIFFERENT MICROELEHENTS IN THE SOIL ON THOSE IN THE PLANT, AND ON PRODUCTION OF p1 66 I FURANOCOUMARINS xa.n., 'S. Dudkq, SK. Renke and .K. Glowniak

%hemistry and 'Environmental Studies, Trent University, Peter- borough, ON K9J 7B8, 'Pharmacy, Medical Academy, Gdansk, Poland, and 4Pharmacognosy, Medical Academy, Lublin, Poland.

High dosages of metal lons included in Sudbury soils were investigated, in comparison to different controls, on the influence of their uptake by leaves of Ruta graveolens and on the biosynthesis of defense chemicals such as furanocoumarins. The highest concentration8 investigated wars 2700 ppm of Nl, 2300 ppm of Cu, 116 ppm Co, I1 ppn Cr, 24 ppm Cd and 400 ppm Pb. The direct reactions of these lons on secondary metabolite8 and enzymes involved in their production vere observed after collection of leaf samples follovlng 3, 18, and 36 h, 8nd 3 , l and 14 days of treatment. Concentrations of xanthotoxin, bergapten and inperatorin vere measured by HPLC following the extraction of these counarins from two different plant compartments: leaf interior and leaf surface. The leaf surface was examined for the first tine, after we had previously proved that plants extrude protective compounds to the surface under stress conditions, such as UV irradation and infection. If these defense mechanisms were nonspecific for stress signals, the toxic level of metals should switch on the production of secondary metabolites.

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Production of Anthocyanins and Other Flavonoids in Acer saccharum and Acer mbrum in Response to Stress

Jeff Lynch and Alicja Zobel. Department of Chcmistty, Trent University; Peteborough, Ontario. K9J 7B8

Ravonoids arc found ext&ly in the western diet and have long been pckvcd as compounds that contribute merely to plant pigmentation. A resurgence of interest in fivonoids is currene ex@ by medical researchers who are interested in the radical scavenging abilitia of these compounds. In this experiment, the protectory role of these tri- phenolics to plant celb was investigated via a comparison of the contrasting genetic expreion during ontogend Visible in leaf colouration each autumn when Acer saccharurn bccomcs red and Acer rubrum becomes green. Qualitative and quantitative 6ndings indicated that all of the autumn leaves of both species had an increased concentration of flavonoids. The results suggested that the biosynthesis of flavonoids is stress induced and is an immediate and non-specific defensive response. We postdate that the hydroxyl groups of the flavonoids enable them to serve as scavengers in the vacuole and on the surface of plant cells.

Polymethoxylated flavonoides from C- cuv Balady

Mahassen A. Abdel -Al im. WS. R-Hamouly. E.A. Aboutable' and Mona Herra

Depamneni of Chemlrtry of xarurai Products. VsOonal Research Cenrer. DOLLI. Cairo. Egypr

'Pharmacagmry tkpamnenr. Faculty of pharmacy. Cairo Univcniry, Egypt

ABsTRAm

sirosterol, 5-0-desmerhyl nobllerlne. and two new polymerhoxylated flavonoides namely 3.5.6.8.4-pentamethoxy and 3.5.6.8.3'.4'- heramethoxy (3 fIavones wcre isolated. Structure ehcldaflon of the new flavonoides was achieved through mass spectroscopy. IHNMR and WNMR analyses.

they are not oxygenated at position 7.

From the petroleum ether exmctof the leaf of the tltled plant. 0-

The new compounds are of special inrerest because uncommonly

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NEW CUCURBITACIN GLYCOSIDES FROM PlCRlA FEL-1 ARRAE P I b 9 ] Youjun lm and zhongliang cheq S h a n w lnstituie of Matena Mcdica. Chmesc Academv of Sciences, 3 19 Yue-yang Road, Shanghai 20003 I . Cliffla

The plant Picna fel-lanae Lour belongs to che farmly Scrophulanaceae 11 has since long k n used for aeameni of h i Inflammaho& bcctma lnfcchon and snakebite m folk d c m e A chermcal mveshgallon of Ctus plant &mdcd seven cemcyclic mtcrpcne glycosides 1-7 1- 3 and picfcltarraegenms hare been isolated from same plant previously The biologml tests showcd that I and 2 poszn potent ~ O t o x l c action and 2 has analgesic cffoM in MVO

0 0

I R'=€I, R2=Rha-\yl-

2 RI-H. R ~ = R I ~ - ~ I ~ - 3 R=Rha-glu- .I R ~ ~ H . R ~ - ~ J . I - 6 R=glu- 5 R'=H, R2=glu-

7 RI-OH, R ~ = giU-

Studies on Chemical Conmtuents of- Rap ?A70 I

Yong Wang, A m la0 , Hong cheng Wang and*Yusuo FUJimOlO (Shangha Inslilute

of Matena Medca, Academa Suuca, Shanghill20003 I, ChIna,College of Phar- macy, Nihon University, 7-7, Narashoda , Funabashi, Ctuba 274, Japan)

A new C I 9-dterpenoid alkaloid, 2-Hydrow-3deoyacoruttne( 1 ) along wth SLY know

compounds Songoramme, have been isolated from the roots of Acornturn karakohcum Rap f ie structure of ( 1 ) was determmed by 1 D and 2D-NMR

Acomtme, 3-Deowacornlme, Indaconmne, Chasmacornme, Songonne and

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pl71 Entornochemical Analyses of the lmae of AnaDhe venata Butler (Lepidoptera, Notodontidae). Department of Pharmacognosy, College of Health

p-172

Composite Qian liebao capsule is made from pure traditional Chinese medicines. Pharmacodynamic studies showed that Qian liebao capsule remarkably restrain the digiti pedis swelling in mouse by carrageenin, that the high and middle dose of them can treat and prevent prostatauxe in mouse by testoterone propionate and that they can certainly treat rat's prostatitis by carrageenin and mycoplasmosis. Acute toxicity test revealed that Qian liebao capsule is not toxic, the high dose is 5000g/kg in mouse. Several results of subacute toxicity test are normal.

Pharmacodynamic studies of composite Qian liebw, capsule for treating pstntauxe and pmtatitis. Liu Shantzboa, Chao Daode, Chang Yilin, Yu

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214

P:l73


CHANGES IN THE ORGANOSULFUR COMPOUNDS RELEASED FROM GARLIC DURING AGING IN WATER, DILUTE ETHANOL OR DILUTE ACETIC ACID L a r r v e n d a n d n - Y u J . W a c g Murdock Madeus Schwabe. I%., P.O. Box 4000, Springville. Utah 84663 (USA)

p. 74 OKADAIC ACID CLASS OF PROTEIN PHOSPHATASE INHIBITORS - PHARMACOPHORE MODEL AND SYNTHETIC ANALOGUES Ronald I. O m , Cherie A. Taylor, Adam McCluskey and Paul Alewood*

Our approach to confirm the pharmacophore model by synthesising target compounds will be discussed. Synthetic pcptides (cyclic and non-cyclic) have been synthesised and side chains predicted by the pharmacophore model have been added.

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ALPHITONIA ZIZYPHOIDES ENHANCES THE PLATING EFFICIENCY

LYMPHOID CELLS CELL LINES AND THE SURVIVAL OF NORMAL

a, Premila Perera-lvarssona, Kjell-Olov Grdnvikband Lars Bohlina a Division of Pharmacognosy, Department of Pharmacy. EMC, Box 579, S-75123 Uppsala. Sweden bSection of lrnmnology and Cell Culture, National Veterinary Instiiute. EMC, Box 580, S-75123 Uppsala. Sweden

An aqueous fraction of the crude extract of Alphitonia zizyphoides A. Gray (Rhamnaceae) was found to enhance the growth of T- and Bcell hybridomas, and the survival of normal lymphocytes in vitro. The fraction, termed AZH/c, was obtained from the red inner bark of this rainforest tree found throughout the Pacific. This bark is used to prepare a water decoction against various diseases (mostly inflammatory) and as a tonic by the healers of Western Samoa. T-hybridoma cells cultivated at low cell densities in 1 % Foetal Calf Serum (FCS) containing medium supplemented with 10 pg/ml of AZWc show a plating efficiency equal to media supplemented with 10% FCS. This is 30 times better plating efficiency than cell media containing only 1% FCS. In addition, AZHk appears to support the survival of bone marrow cells and normal lymphocytes in the absence of antigenic stimulation. The enhanced growth of a 8-cell hybridoma is also paralleled by an increased production of monoclonal antibodies in cultures containing low cell densities. Investigation of the chemical nature of AZH/c indicates condensed tannins to be responsible for the observed activity.

PHARMACOLOGICAL INVESTIGATION OF ALPHlJONlA ZlZVPHOlDES p:176 1 RESULTING IN THE ISOLATION OF FIVE NEW TRITERPENE SAPONINS Premila Perera-lvarsson and Lars Bohlin

Division of Pharmacognosy. Department of Pharmacy, BMC.Eox 759. S-75123 Uppsala. Sweden

The healers of Western Samoa use a decoction of the red inner bark of Alphitonia zizyphoides, A. Gray (Rhamnaceae) to treat a variety of disorders. including inflammation, postpartum haemonhage, wounds and cough. It is also used as a tonic. In this study the muscle relaxant and anti inllammatory properties of an ethanol extract prepared from the bark have been investigated. In a hippocratic screening the ethanol extract showed strong muscle relaxant activity. A bioassay guided fractionation using the isolated guinea pig ileum as a model to monitor muscle relaxant activity resulted in the isolation of five triterpene saponins. These were identified by spectroscopical methods as Zizyphoisides A-E. The extract showed activity in the following anti-inflammatory test systems: in viwo inhibition of the EPP induced rat ear oedema. and in vilro inhibition of prostaglandin biosynthesis. PAF induced exocyiosis and LTE-4 biosyihesis

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ACTIVITIES OF THE TWO NEW NATURAL TOXINS ON THE AGRICULTURAL PEST INSECTS AM) CANCER CELL GROWTH.

M. A. Malek’ and R.H. Wilkins Department of Agricultural and Environmental Science University of Newcastle upon Tyne NE1 7RU United Kingdom.

Two new compounds were isolated and purified from the seed oil fractions of Annona squamosa Linn. The newly isolated compounds were found to be toxic measuring against the mortality of the larvae of Tribolium castaneum Herbst, Plutella xylostella Linn., Spodoptera littoralis Bio., Artemia salina Linn. and adults of Tribolium castaneum Herbst., Drosophila melanogaster Meig. and Nilaparvata l u g e n s (Stal). A 50% inhibition of the growth of human lung carcinoma (A549); human colon carcinoma (RT29), normal mouse fibroblast (3T6) and Chinese hamster ovary normal epithelial cell lines occurred on the treatment with newly isolated natural toxins.

Abstract

h x species (PolygoMceae) are traditional d i f s used against gastmintestinal and other kinds of diseases (1). b y are originally all over the mrld ( 2 ) and m of than are dcscrited as an officinal dmg i n hoRlepthic ~11113copoeias.

A rapid characterization of the roo= of different h x species GUI k carried m t by the derenrdnaticn of d u y m w c acid a d physcion as literature indiwtfs ti%? presence n s p . the abserre of physcim in the investigated ~ L E S (1).

5 ) and a brizcntal TLcchamber. A mixture of ethylacewte:ethanol:mter (103t17+13) or etky?%?tate: isopropm1:water (lW17+13) s3rved as mbile m, thc migration distance had an ammt of 3.5 an. Ihe resulted spots *ere detected m irc~unt of their color and under 1- welmth W-light (335 rm) (3, 4 ) .

By the a i d of the &scribed conditions the sqmation of duyso&nic =id and physim is optirrally perfonred wi th a very h r t nnning tim ( 5 ndn). ’Iherefom a fasL d e t e m t i m of *ys icna ta in ing or $ysciorr-frce ~JEX spxies is passible. b i d e s qllantities of 0.5 4 can te detected.

(1) hers WM der Ram. Praxis (1978), P.H. List, L. Hbrhmmr (eds.), 4th ed., vol. 6, Springer. Berlin. kidelkerg, New York, 192-203.

(2) G. m, IllusXrierte Flora vcn Mitteleuropa (1981). 1I.J. k r t , E.J. Janer, J .W. Kahreit, W. khul tzdbte l , G. k@itz, ILL. k k r (eds.), 3rd cd., vol. 3, Paul h a y , Berlin, I b h r g , 352421

Mthnolic mt-extrixts m e prepared and =prated by ?LI: using HFILI: p la t e s (NJ F

Refer-:

(3) A. Koch, Lj. Kmus (1992). h n n . 7x8. Wiss. 5/137, 6 , 250-253. ( 4 ) A. Kmh (193). m. A p t h . Ztg., 133, 26, 239-2387.

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SYNIIWIS OF NEW HEPARINOIDS BASED ON NAWRAL SEAWEED- AND BACTFRIA- F'OLYSACCHARIDES

Institute of Pharmacy. University of Regensburg, UniversmssuaSe 31. 93040 Regensburg (Gemany) p 1 79 SusanneAlban. Gerhard F r m

Fa more than 50 years heparin is the drug of choice in the clinical pre- and postsurgical prophylaxis of thrombotic events. However it could not completely eliminiate the incidence of thrombosis and pulmonary embolism and shows severe side effects such as bleeding and beparin-associated cbrombocytopenia. Further. this glycosaminoglycan is inhomogenous in its molecular weight (MW). its degree of s W n @ S ) and its substicution pattern with the consequence of a considerable variability of its physiological activities. Because the synthesis of the macromolecule is not possible under cconomic considerations heparin has to he isolated fmm biological material with the risk of severe contaminations such as bovine spgifome encephalopathie (BSB). For these reasons an imponant field of rescarch are alternatives Lo heparin. which are commonly known as heparinoids. In (he presented work, currently available genuine neutral polysaccharides from seaweed and bacteria, i.e. laminar& p U u l a n and curdlan were chemically modified and examined for their anticoagulant ppenies. In order to evaluate the swctural requirements for a heparinoid, the following aspects were investigated: the dependence of the anticoagulant activity on the DS. the MW, the type of glycosidic bonds, the substitution pattern of the glucose units and the degree and kind of glycosidic branchig. The examination of the anticoagulant activity of the various synthesiied glumsulfates in the classical coagulation assays results in the following suucture-activity-relationship: An anticoagulant effect requires the presence of a minimum of sulfate groups and the activity increases with increasing DS until an optimum. Depending on the assay representing the influence on the dislinct stages of the coagulation cascade. the MW has a dlfferent influence on the activity. Regardless of (he MW and the DS, a more unlform charge distribution on the glucose molecule instead of the preferred substitution of the primary C6-OH-group improves the anticoagulanl effect. The results of the branched derivatives demonstrate that, apart liwn b e charge density. b e polymer size a d Lhe patern of the anionic substitution. there is also a correlation between the flexibility and the three-dimensional swcturc of the molecule and the physiological properties. Fianally. by the combined variation of the diverse paramelcrs it should be possible 10 find the optimum structure for a heparimid synthesized using natural sources.

217

P. 80 Fe q y e t i d variability and psibilities of chdcal selection to dmiiush the !mc COntent in cbc h e q plant.

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PI82

CONTRIBUTION IN PHYTOCHEMCAL STUDIES OF BALANlTES PA81 1 AEGYPTL4CA

KINOBEON A, A NOVEL RED I'IGMENT, PRODUCED IN SAFFLOWER TISSUE CULTURE SYSTEMS Tsutomu Kanehira, Sachio Wakayama, Kazuhito Kusaka,

2 -*, Britt H., Prof. Keita A., AG Mahamoud, Sanogo F., Gunnvor B., and Maiga A. *Departement de Medecine Traditionnelle, Programme SSE Volet Plante Sauvages. BP. 1746 Bamako, Mali.

Balanites aegyptiaca is a medicinal plant of Mali. It can be found in different parts of Sahel. Different parts of this plant are used in medicine. For example: leaves, in a decoction, are used against stomach ache, the bark of the root is used against headache, the thorns are used as a pomade when there is inflammation, the fruits are used in alimentation to treat dryness and the roots are used to make soap and as an insecticide. These different uses motivated us to conduct chemical studies on the different plant parts. We found it contains saponosides, mineral elements, proteins, glucides, tannins, vitamin C, alkaloids 0.20%, sterols, terpenes, mucilages and carotenoids. This is the first part of our results. Chromatographic studies continue.

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Pl84

STRUCTLRE AND MODE OF ACTION OF SOME MITOTIC POISONS WMCH ARREST THE MITOTIC SPINDLE FORMATION IN SEA URCHIN EGGS Akio Kobayashi, Shin-ichiro Kajiyama, and Kazuyoshi Kawazu p.83 1 Okayama Uruversity, Tushimanaka 1-1-1 Okayama, 700 Japan

The mitosis is one of the most highly regulated events of ccll division cycle , and the studies on the mitotic spindle formation could give the important clues to elucidate the proliferation mechanisms of eukaryotic cells. But so far, few is known about the mechanisms because of its complexity and the lack of proper chemical probes. In this view point. we have k e n searching for the specific arresters of mitotic spindle formation using the sea urchin egg assay system for more than 10 years and found some unique compounds which induced the abnormal spindles in the M phase cells and arrested the mitosis. For example, T-I(l), a metabolite of Pseudomonus sp., induced barrel-shaped spindles with swollen centrosomes. Macbccin 1 (2). an antineoplastic agent. caused large and overstabililed spindles. A novel mitotic arrester Nostodione A (3) , a highly unsaturated compound isolated from a blue-green alga, afforded small spindles with low birefnngence density. In this congress the authors will report the unique bioassay methods with sea urchin eggs as well as the possible mode of operation of these mitotic arresters.

EFFECT OF SESQUITERPENE LACTONES ON MYCELIAL GROWTH OF PLANT PATHOGENS

ti T-1 (1) Macbecin I(2) Nostodione A (3)

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El86

THE FATE OF HYDROGEN DURING THE BIOSYNTHESIS OF DOMOIC ACID FROM 2-13CD3-ACETATE BY THE MARINE DIATOM PSEUDONITZYCHIA PUNGENS FORMA MULTISERIES. Una P. Ramsev, John A. Walter, Donald J. Douglas, and Jeffrey L.C. Wright Institute for Marine Biosciences, National Research Council of Canada, 141 1 Oxford St., Halifax N.S., Canada B3H 321

PA85 1

CHEMICAL CONSTITUENTS FROM THE CYANOBACIERIUM OSCILLATORIA AGARDHII Judv Needham, Anna E. Brison, Donald J. Douglas and Jeffrey L. C. Wright

Domoic acid is a neuroexcitatory amino acid responsible for an outbreak of shellfish poisoning in eastern Canada in 1987, and more recently implicated in the deaths of aquatic birds on the West Coast of the U.S.A. The Canadian source of the toxin was identified as the diatom Psuedonitzchiu pungens forma rnultiseries. From our [1,2-13C]-acetate labelling studies it appears that domoic acid is biosynthesized in the diatom from an activated citriceacid cycle intermediate which condenses with an isoprenoid chain. We have investigated the timing of this event and the validity of the pathway using [2-13CD3]-acetate and [1,2- 13CI-acetate added at different stages of the growth cycle.

Cyanobacteria are tentatively suspected as a possible cause of death of several horses in Eastern Canada. A number of cyanobacteria were isolated from the horses' trough water and analyzed for the production of known toxic compounds. During the course of this investigation a family of four unusual hydrocarbons of medium polarity were isolated from cultures of the freshwater cyanobacterium Oscillaroria agardhii. The compounds were purified by reversed-phase HPLC and their structures were elucidated on the basis of ID and 2D NMR data. These metabolites appeared after 1 week of culture growth and peaked at 3 weeks. After 4 weeks of culture growth the concentration of these compounds in the cells was substantially reduced. The structures of the compounds will be presented and their biogenesis and function will be discussed

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p. $8

BIOSYNTHESIS OF STILBERICOSIDE IN ZWLVBERIGA ALATA.

Dpt. of Organic Chemistry, The Technical University of Denmark, DK-2800 Lyngby, Denmark.

1 Lotte Doe Frederiksen.

SANANGOSIDE, A NEW PHENYLETHANOID ALLOSIDE FROM SANANtiO RACFMOSW. Ssren Rosendal Jensen Fax: +45 4593 3968

The biosynthesis of the irodoid glucoside stilbericoside (l), isolated from Thunbergiu alara (Acanthaceae), has been investigated. Deuterium labelled deoxyloganic aicd (2) and 8-epi- deoxyloganic acid were fed to T ulufu and the former was incorporated into stilbericoside with retention of the label at H-6, H-7 and H-8. Further studies with deuterium labelled precursors have elucidated the most probable biosynthetic pathway to be as depicted in the Scheme.

p4:-j$-po 4 - qo4-oQo o w 110 o o c I 1 0 oaf m c o o l c

(2) (1)

HO

0 Sanangoside

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STUDIES ON THE SPERHICIDAL AND CERVICAL MUCUS MODIPYINQ A

PROPERTIES OF NEEW ( D C INDICA) SEED EXTRACTS PI89 Saniav G a u ( * ) , G. D%%e:y*y S. N . Upadhyay ( * ) and

Nat iona l I n s t i t u t e of Immunology, N e w Delh i , Ind ia . ** CONRAD, Eas te rn V i r g i n i a Medical School , Norfo lk ,Virg in ia , USA.

AG. P.Talwar ( * )

- - ~ T l T C T T C N ~ F L 3 ~ ~ C T ~ E T C A i ~ m D x r O r A m N - A Q m S - - EXTRACTS OF MONTANOA TOMENTOSA:: IS IT OR IT IS NOT PRESENT k790 I IN WATER INFUSSIONS?.

'Instituto de Quimica, UNAM, Cd. Universitaria, Mexico, D.F. 04510; 0Facultad de Medicina NAM; §Department of Chemistry, University of Toronto, Toronto, Ont. M5S 1A1, Canada. wo conflicting reports appear in the literature concerning the analytical detection of auradienoic acid I in aqueous extracts of Montanoa tomentosa (MT) obtained by preparation f infusions according to popular procedures i.e. herbal teas. The first report (1) describes the ucceshl identification of compound I in the aqueous extract of MT while the second (Z), entions the failure to detect the titled compound in the same preparation. Since the

lucidation of the uterotonic effects of this plant have not been completely understood, a orroboration of the presence of kauradienoic acid in water infussions of this plant seemed ecessary. Both investigations rely on the use of analytical HPLC but we found minor though rucial differences in the methodology of sample preparation which explain the contradictory esults. Thus, it was found that the method used in report (2) may be inadequate for the urpose of representatively detecting the kaurenic compouds. Several precautions are tressed out concerning ultrafiltration and sample prepurification through solid phase xtraction. In addition, the 500 MHz spectrum of the whole organic extract of the water fussion was used as a rapid and accurate analytical tool for such microscale detection while

Is0 confirmed the original findings reported in (1). ~ 1) Ra61 G. Enriquez, Laura I. Escobar, Maria L. Romero, M. Antonio Chhvez, 1. Chromatog., 58 (1983) 297-301; (2) Xiaoping Dong, Matthias 0. Hamburger, Geoffrey A. CordeU and L arry H. S. Fong, Planfa Medica, 55 (1989) 185-187.

pi 'Ralil G. Enriquez', Gil. A. MagosO, Ismael M n * , William F. Reynolds§;

k

I_ - ___ -. -- - ____ _ ~ _ _ _

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ALBIFLOMANTHINE - A NEW CRINANE ALKALOID FROM P-I91 1 HAEMANTHUS ALBIFLOS JACO. (AMARYLLIDACEAE) Francois Till-, Genevibve Baudouin, and Michel Koch Laboratoire de Pharmacognosie de I'UniversitC RenC Descanes, U.R.A. au C.N.R.S. no 1310, FacultC des Sciences Pharmaceutiques et Biologiques, 4, Avenue de I'Observatoire, 75006 Paris (France).

The recent description of the strong antiviral activity of bulb extracts of Haemanthus albiflos Jacq. prompted us to investigate the alkaloid contents of this Amaryllidaceae species. The dried bulbs afforded 1.85% crude alkaloids. Fractionation on column chromatography led to the isolation of the known albomaculine, haemanthamine, haemanthamidine (as a mixture of 6 a - 6p epimers), galanthamine, lycoramine and of the novel alkaloid albiflomanthine (1). The structure of this latter was deduced from its spectral data, mainly from 2D NMR experiments : COSY, HETCORR and COLOC. The absolute configurations of both the ethylene bridge and the hydroxy group at C-11 were established by CD.

The isolation of albiflomanthine (1) seems interesting from a biogenetic point of view. Its oxidation level at C-ring may lead to consider two orrhodioxygenated C-6-ci and c 6 - c 2 units as its biogenetic precursors. This oxidation level is commonly encountered in several classes of Amaryllidaceae alkaloids. In contrast, it is described here for the first time in a crinane derivative bearing a 1.2-double bond.

~ ~ ~

OCCURRENCE OF PHYTOECDYSTEROIDS IN SILENE SPECIES MBria BBthori (l), R. Lafont (2), J. P. Girault (3), -6 Jr .(1,4) El 92 I (1) Inst. of Pharmaccgnosy of the A. Szent-Gyorgyi Medical University, P.O.

BOX 121, H-6721 Szeged, Hungary, (2) Ecole Normale SupBrieure, Dept. du Biologie, Lab. de Biochimie et Physiologie d e Wveloprnent, CNRS UA 686. 46 rue dUlrn, F-75230 Paris Cedex 05, France, (3) Universit6 RenB Descartes, Lab. de Chimie et Biochirnie Pharmacologiques, CNRS UA 400, 45 me Caints-PBres. F-75006, France, (4) Inst. of Ecology and Botany of the Hung. Acad. Sci. H-2163 VB&t6t, Hungary The discovery of the occurrence of the insect moulting hormones. ecdysteroids in plants, very often in much larger quantities than in animals, has stimulated the regular studies of plant sources resulting in also chemotaxonomic results. To date approx. 130 related compounds have been described from 120 plant families. Approximately 25 % of the ecdysteroids was isolated from the species of Caryophyllecwae family. The genus Silene has proved to be one of the richest in these compounds. Round 40 % of the spedes tested contained ecdysteroids in varying quantities. In all cases where the presence of ecdysteroids could be verified, R ecdysone is the major compound. In addition to it 20-hydroxy-scdysone, 2desoxy-, 22- desoxyadystemids and their benzoates have been isolated in preparative scale. The compounds were identified by UV., IR. NMR-spectroscopy. These compounds can have significance. mainly, from the taxpnomic point of view. Of the approximately 50 species, Silene altaica. S. anneria, S. bmhuice. S. catholica, S. cilieta, S. coeii-msa. S. flamscens, S. ga//ica, S. iatifo/ia, S. longicaiydna, S. micmpetaia. S. multicaulis, S. nutans, S. otifes, S. pygmee, S. schumecheri, S. sendtneri, S. tatanca, S. wa///cf?iane, S. zawadzMi contained B-ecdysone mainly by TLC and semiquantitative densitometry detectable quantities. The percentage distribution of Ikcdysone content among the organs of some Sihsna species, e.g.: S. otites, proves that the generative organs (inflorescence. fruit) are the richest in ecdysteroids. followed by the leaves and stems.

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PI93 -

VARIATIONS IN THE ELEMENT ACCUMULATION PATTERN O F PLANTS AND POSSIBLE CORRELATION WITH SECONDARY METABOLITES Imre Mdthe Jr.(l,2), Akos Mdthk(3)

ANTI-IN'PLAIMATORY ACTIVITY OF " B O U L D I A W I S SEEM EXTRACTIVE8

AJAYI-OBE 00; MOODY JO; AZIBA P Department of Pharmacognosy. College of Medicine, University of Ibadan, Nigeria Newbouldia laevis Seem (Bignonaceael is a tree of medium size used in several West African communities for the treatment of dysmenorrheoa, snake bite, epilepsy, septic wounds and rheumatic swellings(1). Previous pharmacological studies of N. laevis revealed that it inhibited the tonus of the rabbit ilewn(2). Four pyrazole alkaloids were also recently isolated from the root bark(3l.The ethnomedical claims,of the ef,fectiveness of N. laevis in the treatment and management of rheumatoid arthrltm and other inflennnetion-mediated diseases however lack any scientific evidence. A bioactivity-monitored phytochemical examination of a l l the plant parts using carrageenan-induced rat paw oedema assay protocol resulted in the location of the highest anti-inflammatory actlvlty in the chloroform-soluble portion of the methanolic extract of the stem bark(63.5% lnhibltlon) from which two chromatographic fractions BFII(66.30 inhibition) and BFI11180.2% inhibition) were obtained. The significant activity (p < 0.05) ,of BFII and BFIII when corn ared to aspirin (88.1% inhibltlon! not only lustify the folkloric claims gut also shows that N. laevis merits further phytochemical studies to isolate and characterize the active constituents.

References 1.Burkill HM (1385). The Useful Plants of West Tropical Africa Ed 2 Vol 1, Royal Botanical Gardens, Kew. 2. Correra da Silva AC, Costa AS., and Paiva MQ (1965) An. Fac. Farm. Porto, 25 35-50. T'Adesanya SA, Rene N, Fontaine C and Pais M (1334), Phytochem.35, 1053-1055.

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I DITERPENOIDS FROM ISODON ERIOCALYX VAR. LAXIFLORA

PI195 Han-Dong Sun', Zhong-Wen Lin', Fang-D1 Niu', Pei-Cling Shen', Lu-Tai Pan', -' 1 and Geoffrey A Cordell'

'Laboratory of Phytochemistry. Kunmino Institute of Botany, Academia Sinica, Kunming, 650204, Yunnan. P.R. China;%uizhou Institute of Tradional Chinese Medicine, Guiyan, Guizhou, P.R. China;'Program for Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharrnacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 6061 2, USA.

Three new diterpenoids, laxiflorins A (11. B (2) and C 131, together with four known diterpenoids eriocalyxin B (41, maoecrystal A (61, maoecwstal B (61 and oridonin (71, were isolated from the leaves of lsodon eriocalyx var. laxifloru, and their avuctures M d NMR spectral data were assigned by a combination of one- and two-dimensional NMR techniques and computer modeling calculations. Laxiflorin C (3) displayed weak cytotoxic activity.

Pl96 CYTOTOXIC DITERPENOIDS FROM ISODON MEGA THYRSUS

Han-Dong Sun', Zhong-Wen Lin', Fang-Di Niu'. Cono-Ze Lid, Heebyung Chai', John M. I Pezruto' and Geoffrey A. Cordell'

'Laboratory of Phytochemistry, Kunming Institute of Botany, Academia Sinica, Kunming, 650204, Yunnan. P.R. China;'Program for the Collaborative Research in the Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosv, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 6061 2, USA.

A new diterpenoid, megathyrin A I1 ), together with three known compounds, rabdocoetsins B (21, C 131 and D (41, were isolated from the leaves of lsodon megathyrsus, and their structures and NMR spectral data were assigned by a combination of one- and two-dimensional NMR techniques. These compounds displayed potent cytotoxic activity.

MrgrthyM ( I ) Rnbdocoetria B (2) Rnbdoeoesin C (3) Rnbdococbln D (4)

Acknowledgement: This work was suppported, in part, by a grant from the Division of Cancer Treatment, National Cancer Institute, Bethesda. MD. USA.

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226

P.197


ISOWTION OF POTENTUL ANTI-ESTROGENIC COMWUNDS FROM PLANTS C. T. C d . T. C. M. Tam2, H H. S. Fongl and I. M. Permtol

Deparrmcnt of Medicinal Chemistry and Parmamgnasy. College of Pharmacy. University of Illinois at Chicago, Chicago. Illinois 60612. USA zDcpsrlmmt of Chemistry. The Hang Kong University of

M. S. J a n e L. Cai, H. H. S. Fong, A. D. Kinghorn, H. Wagner, A. Probstle, R. Bauer, and J. M. Pezzuto, Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, IL 60612, and Institute of Pharmaceutical Biology, University of _. Munich, Karlstr. 2g, 80333 Munich, Germany

A variety of agents known to inhibit cyclooxygenase activity also demonstrate anti-inflammatory effects. Moreover, certain anti-inflammatory agents such as indomethacin and piroxicam have been shown to inhibit the promotion of tumorgenesis. Thus, in our search for novel cancer chemopreventive agents, an in wifro system has been utilized to evaluate the potential of plant extracts or pure compounds to inhibit cyclooxygenase activity. Reaction mixtures are comprised of 0.1 mM sodium phosphate buffer, pH 7.4, containing phenol (1.0 mM), hemin (0.01 mM), arachidonic acid (0.6 mM), and microsomes derived from sheep seminal vesicles (0.7 mg/ml) as a crude source of cyclooxygenase. Initial reaction rate is measured oxygraphically at 37oC and inhibitory activity is obtained by comparing the rate of oxygen consumption in the presence of test samples with that observed with solvent (DMSO) only. We have thus far evaluated 346 plant extracts and identified 16 as active (>75% inhibition at 7 pg/ml). The inhibitory activity of two active plant extracts was confirmed to be identical when the conversion of arachidonic acid to prostaglandin E2 was assessed by HPLC analysis. Bioassay-directed fractionation was then undertaken with Cassia quinquangulara (Leguminosae). As a result, resveratrol and frons-3,5,3',S'-tetrahydroxy-4-methoxystilbene were isolated as active principles. These compounds exhibited ICso values of 16.7 and 20.1 pM, respectively. Activities of this magnitude are considerably lower than that of indomethacin (ICso 1.1 pM) but higher than those of piroxicam (ICsO 130 pM) and aspirin (1'250 893 pM). Additional testing of the chemopreventive activity of these active isolates is currently undeway. (Supported in part by PO1 CA48112 awarded by the National Cancer Institute).

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P2Oo

A COMPARATIVE STUDY OF THE SEPARATION OF NATURAL COMPOUNDS BY MICELLARELECTROKINETIC CHROhL4TOGRAFWY AND HIGH PERFOR- MANCE LIQUID CHROMATOGRAPHY Haleem J. Issaq Program Resources, Inc./DynCorp, NCI-Frederick Cancer Research and Development Center, P.O. Box B, Frederick, Maryland 21702 (USA)

High Performance Liquid Chromatography (HF'LC) is a well established analytical and preparative separation technique which found a wide application in natural products research. Capillary Electrophoresis (CE) and Micellar Electrokinetic Chromatography (MEKC) are e5cient and rapid microanalytical separation techniques which offer high resolution, up to a few million theoretical plates, of large as well as small neutral molecules and charged compounds. In this pmeatation, the utility ofCE, and especially MEKC, in natural products research will be given with emphasis on the advantages, disadvantages and capabilities of both HF'LC and MEKC. The effect of micelle and organic modifiers concentration and type on MEKC separations will be discussed and examples will be presented, Finally, comparison of detection modes, solvent consumption and sample size will also be compared and commented on.

CORALLINA VANCOWERIENSIS ACETONE POWDER (CVAP): A VERSATILE BROMINATING CHEMOENZYMATIC REAGENT.

A crude acetone powder preparation of Corullinu vuncouveriensis (Rhodophyta: C~rallina~eae) has been found to serve as a convenient source of bromoperoxidase enzyme. The Corullinu vuncouveriensis acetone powder, or "CVAP", catalyzes the bromination of a variety of aromatic and alkene substrates in the presence of H2@ and bromide in phosphate buffer. Conversion of substrate to product(s) is usually rapid and in high yield. The reaction conditions are also significantly milder and the reagents easier to handle than chemical halogenation reactions. Compared to pwified bromoperoxidase, the CVAP preparation shows a significant;y greater stability over time. CVAP may thus be useful as a general source of bromoperoxidase. and may extend the utility of this enzyme as a chemoenzymatic reagent. Applications of CVAP in the preparation of brominated synthetic precursors or analogues of natural products will be prcsented.

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INTESTINAL REIAXANT EFFECT OF THE ME'MANOLIC EXTRACT AND PURE COMPOUNDS FROM DODONAEA VILscosA

Aleiandra Raja ', 3, S i k a Cruzz and Rachel Mata3.

P:201 1 'Facultad d e Quimica, Universidad Autonoma de Queretaro, Queretaro, Mexico. 2Seccion de Terapeutica txperimental, CINVESTAV, F" 3Facultad d e Quimica, IJuiversidad Nacional Autonoma de Mexico, Coyoacan 045 10, Mexico D.F. Mexico.

The intestinal smooth muscle relaxant effect of Dodonaea viscosa (I-.) Jacq. (Sapindaceae) was investigated employing isolated rat and guinea pig ileum preparations. The mcthanolic extract of the aerial parts of this species displayed a concentration- dependent inhibition of spontaneous and electrically-induced intestinal contractions. Bioactkity directed fractionation resulted in the isolation or four active compounds: ent- 15,l6-epoxy-9-a 11-labda- 13( 16), 14-diene-jfl, 8a-dial, ltautriwaic acid and two flavonoids, sakurattetin and a kaempferol derivative. All of these compounds inhibited the ileum coutractiotis in a concentration-dependent manner. The smootb muscle relaxant effect elicited by the methanolic extract and the isolated bioactive substances from D. i~isco.sa, tends to support tlie use of the plant as an antiespasmodic agent in Mexican tradicional medicine.

ISOLATION BY HPLC AND PRELIMINARY STRUCTURAL ELUCIDATION OF THE MINOR CONSTITUENTS OF THE PIiYTOGROWH INHIBITOR RESIN GLYCOSIDES FROM IF'OMOE4 TRICOLOR

I Mmaph-. Adolfo Perer-Diaz and Rogelio Pereda-Miranda' Laboratono de Fitcquimrca Depananiento de Fannacia Facultad de Quimica UniversiQd Nacional Autoiioma de Mexico Apanado Postal 70-265 Coyoacan 0451 I D F Mexico

We prenously demonstrated that the Ipomoea resin glycosides are potent rnhibitors of plant growth and primarily responsible for the allelopathic interference exhibited by these species The major phytogrowth inhbitor present in

the resins of I tricolor (I violacca) was Uven the trinal nanie of tncolorin A ( I ) and displayed a broad range of biological actiwties. includmg antimicrobial. cytotoxic and inhibition of the protein kinase C activlty Three addtional compounds were isolated from the bioactive resin of this spcies and their isolation in a pure slate as successfully achieved by a high resolution preparative HPLC niethcdology The preliminary structural elucidation stu&es of these minor bioactive compounds indcated a siniilar ~ ~ Y C M I C structure to those isolated previously from other Ipomoea species. havlng the carbonyl group of the aglycone linked to an oligosacchande core to form a niacrocyclic ester as i n tricolorin A (I) Details of the isolation and the sfructural elucidation of these glycolipids wlll be presented

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p203 MITOTIC STIMULATION OF LYMPHOCYTES A h 9 AGGLUTINATION OF NEOPLASTIC CELLS BY A LFLTIN FROM LENTINUS EDODES

P204 CANCER CELL AGGLUTINATION AND BIOPHYSICOCHEMICAL PROPERTIES OF THE LECTINS FROM ASTERINA PECTINIFERA

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P205 THE EFFECTS OF EPHEDRA SINENSIS EXTRACTS ON MURINE PERITONEAL MACROPHAGES

Aqueous and organic extracts of Ephedra sinensis were prepared from the whole plant and administered at a dose of 5-20 mg to male inbred mouse, BALB/c for 4 days. A dose-dependent increase of the phagocytic activity was induced in the mouse peritoneal macrophages. At the same time there was an increase in the percentage of 3H-adenosine bound to the macrophages. However, when the macrophages were treated with 2.5 and 10 mglml Ephedra extract in vitro, there was a dose-dependent reduction in the amount of 3H-adenosine bound, and a diminution in the phagocytic activity of macrophages which occurred between 0.75 and 3 mdml. The reduction in 3H-adenosine bound by macrophages and their phagocytic activities in vitro might be due to the toxicity of E. sinensis extract at high dosage. This study revealed that there was a correlation !between the phagocytic activity and the 3H-adenosine binding activity of mouse peritoneal macrophages treated with E. sinensis extracts.

THE CYTOTOXICITY OF POISONOUS CHINESE MEDICINAL P:206 I HERBS 2 Kevin Wing-hon Lai, Jin-hua Jia, Yao Xie and Ken Wing-keune Liu.

Departments of Anatdmy, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong

Four commonly used poisonous herbal medicines, Periploca sepium, Croton tiglium, Phytolaccca acinosa, and Trypterygium wiljordii were purchased from China and the aqueous extracts of the whole plants were prepared. Their cytotoxicity was evaluated using in vitro cultured cell lines, including mouse fibroblast L929 cells, human melanoma B16 cells. Chinese hamster ovary EM9 cells and human leukemia HL-60 cell line. Of all the extracts tested, P. sepium was the most cytotoxic, followed by C. tiglium, P . acinosa and T. wiljordii. The mode of cell death caused by these extracts was studied using morphological observation of chromatin condensation and DNA electrophoresis and the results were correlated with the induction of apoptosis in these cells.

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p207

23 1

NEUROPHARMACOLOGICAL ASPECTS OF THE POLYCYCLIC

SPONGE CRAMBE CRANBE. GUANIDINE ALKALOID CRAMBESCIDIN 816 FROM THE MARINE

P.Govoni(3) ( 1 ) Dipartimento di Farmacologia 40126 Bologna, Italy ( 2 ) Dip.di Chimica delle Sostanze Naturali,80131 Napoli.Italy ( 3 ) Istituto di Istologia e Embriologia , Parma , Italy.

Several papers reported the isolation of a number of cytotoxic guanidine alkaloids from tissues of a marine sponge Crambe Crambe. These include the simple and less active bisguanidine alkaloids crambescins A , B, C1 and C2 (the original names of crambines has been recently changed to crambescins) and the more complex crambescidins 800, 816, 844, isocrambescidin 800 and crambidine all structurally related to ptilornycalin A.These last are highly cytotoxic and antiviral compounds whose characteristic structural features are a pentacyclic guanidine moiety linked through a long- chain w-hydroxy acid t o a hydroxyspermidine or spermidin unit, As many marine toxines were found to affect the ionic channels, we started to study the biological property of crambescidin 816 on cell cultures NG 108 ( neuroblastoma x glioma ) to evaluated the effect on the cytosolic calcium.In these conditions crambescidin 816 produced a dose depent inhibition of calcium elevation. Moreover it has been evaluated its activity "in vivo" after i.c.v. administration in the rat of 100 fmol, 1 and 10 pmol .The alkaloid induced dose-related neurotixic effects 3-6 min after the injection, characterized by bizzare posture, hyperthermia and hyperalgesia. Brain areas from rats were examined histologically.

P208 THE EFFECTS OF BFTULlNlC ACID AS A I,WJhaUDCUITDR Oh THE W E EPIDERMAL CELL.

Kyung-Jee V m ( K i m ) . 0.5. Yim. S.Y. Lee. and John R. Porter' Sahm-Yook Vniversity, College of Pharmacy. P.O. Box 118 Seoul 139-742. Korea ' Philadelphia College of P h a m c y and Science (3rd S. st. Philadelphia. Pa 19104

Betulinic acid .as isolated and purified I r a the plant of Lyzoprs maadrimus (Labiatee). The effects of betulinic acid as a iuunmdulator an the -68 epidemal Langerhms cells(LC) had been exposed with U I traviolet B(W6) rsdistIon(2OOJIm') fms sun lam^ fluorescent FSZO tubes were studied in order to support mti-CD3!4dh-induced T-cell mitogenesis.

Eplde-I cells(EC) were exposed t o L'B radiation and cultured for 6h when LC were recorverd by treating with betulinic acid. en-ratad. and assayed for simultaneous expression of I - A antiens ud I C M I by incorporatim of k thymidins.

LW-irradisted LC that had been treated with betulinic acid exhibited levels of I-A antigens c-reble t o those on untrsated WB-irradiated LC. The cytotoxicity assay mas carried a t for their ability t o stimlste Ip@xblast-fotration hi& is natural killer(FiK) cell-mediated cytotoxicity against hwan t m r targsts(erythmleukwic cell line K-562) .hen added t o cultures of resting priphsral blmd I~m~kcytes.

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Pz1

and Raymond J. Andersen

Ingenamine (I)', ingamine A (2) and B (3), and madangamrne (4). novel cytotoxic pentacyclic alkaloids. have been isolated from thc marine sponge Xesrospongia lngens collected in Papua New Guinea. Their swctures have been elucidated by extensive spectroscopic analysis,

Ingenamine (I) , ingamine A (2) and B (3) represent the frst examples of a new class of pentacyclic alkaloids. The biosynthesis can be formally envisaged to arisc from an intramolecular '4+2' cycloaddition reaction as shown below. The discovery of ingemnine (1). ingamine A (2) and B (3) funhcr support Baldwin and Whitehead's p ropod for the biosynthesis of the mamamines*.

Madangamine A (4). another cytocoxic alkaloid isolated from the same sponge wich novel carbon skeleton. will also be presented.

Continuously well growing root cultures of Phytolacca acinosa were established From leafexplants on hormone-free Linsmaier and Skoog medium, cultured in large scale in shake flasks in dim tight at 26'. The selected clone consists mainly of a white roothair-

' 4 + 2 ' fil cycloaddi l ion

, 2 x=oH Ingarnine A 3 X=H Ingamine B

1. Kong. F.; Anderscn, R.J.: Allen. T.M. Tefrohedron [err. 1994,35, 1643. 2. Baldwin. J.E.; Whitehad. R.C. Tetrahedron Lerf. 1992,33. 2059.

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p211 IRlDOlO GLUCOSIDES FROM JASMlNUM HEMSLEW Takao Tanahashi, Atsuko Shirnada. Naotaka Nagakura, Kenichiro Inoue.1 Tetsuro Fujitaz and Cheng-Chang Chena

NEW MACROLIDES FROM CULTURED MARINE DINOFLAGELLATES OF GENUS AMPHIDINIUM Masami Ishibashi, Masashi Tsuda and Jun'ichi Kobayashi Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060 (Japan)

Amphidinolides are a series of cytotoxic macrolides, isolated from the laboratory-cultured dinoflagellates Amphidinium sp. which are living inside of Okinawan marine flatworms Amphiscolops sp. These macrolides are full of chemically unique structural features; (i) amphidinolides possess a variety of new carbon-skeletons different from each other, which are produced by one genus of microalga, and (ii) many of them contain odd-numbered macrocyclic lactone rings, which are seldom found from natural products of terrestrial origins. We therefore have continued mass cultivation of the dinoflagellates and further investigation on the extracts. In this presentation we report isolation, structures, and cytotoxicity of new macrolides which we have recently isolated from the dinoflagellates.

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INHIBITION OF BACTERIAL MUTAGENESIS BY CITRUS FLAVONOIDS Mario Calomme, LUC Pieters, Arnold Vlietinck and Dirk Vanden Berghe Department of Pharmaceutical Sciences, University of Antwerp,

p-13 I Universiteitsplein 1, 8-261 0 Antwerp, Belgium

The antimutagenicity in the Salmonella I microsome assay of the Citrus flavonoids naringin, hesperidin, nobiletin and tangeretin against a range of chemical mutagens, including benzotalpyrene, 2-aminofluorene. acridine orange and quercetin, which require metabolic activation, and the directly acting mutagen nitroquinoline N-oxide, was investigated in order to evaluate their potential beneficial effect as chemopreventive agents against carcinogenic risk factors in the human environment. It could be concluded for the chemical mutagens tested ( 1 ) that tangeretin is a quite general antimutagen for indirectly acting mutagens. but that a large molar excess is necessary; (2) that hesperidin also has antimutagenic properties, but not to the same extent as tangeretin; (3) that naringin and especially nobiletin sometimes act as an antimutagen. but that in other circumstances they may enhance the mutagenicity of certain mutagens; 14) if hesperidin and naringin show antimutagenicity, that they are active in smaller doses than tangeretin. and even in a molar excess of mutagen; (5) that tangeretin, nobiletin and, although less pronounced, hesperidin, inhibit the mutagenicity of quercetin, a mutagenic flavonoid also occurring in Citrus.

Quercetin itself acts as an antimutagen against 2-aminofluorene in a Salmonella strain (TA 1538) where its mutagenicity is not expressed. Quercetin should not merely be regarded as a genotoxic risk factor in the human diet, since its mutagenicity may be inhibited by accompanying compounds including other flavonoids, and since quercetin itself also exhibits an antimutagenic action against chemical mutagens. Antimutagenicity is a very complex process, highly dependent on nature and concentration of mutagen and antimutagen, and inhibition or enhancement of mutagenicty does not always occur in a dose-related manner. As a general conclusion the Citrus flavonoids tested, and especially tangeretin and hesperidin, may act as chemopreventive agents in the human diet, because of their antimutagenic properties against a range of chemical mutagens, includino the mutagenic flavonoid quercetin. This work was supported by the Dept. of Citrus, Florida (contract No. 92031).

MODULATION OF THE COMPLEMENT SYSTEM BY FLAVONOIDS Aleidis Lasure, Bart Van Poel, Kanyanga Cimanga, Luc Pieters and Arnold Vlietinck, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 8-2610 Antwerp, Belgium

p214 I Flavonoids exhibit a wide range of biological activities including a variety of anti-

inflammatory effects. The complement system plays an important role in the host defence system, inflammations and allergic reactions. However, complement activity can result in a variety of inflammatory and degenerative diseases depending on the site, extent and duration of activation. Therefore modulation of the complement system may prove to be of therapeutic value. In this study the anti-complementary activity of flavonoids was related to their substitution pattern . A hemolytical assay, involving a spectrophotometric measurement at 41 5 nm of the hemoglobin released from erythrocytes. which indicates the degree of lytic action of the complement system. was used to determine the effect of a series of flavonoids on the classical and the alternative activation pathway of the complement system.

For the classical pathway it appeared that the double bond at position 2 and the carbonyl group at position 4 were not essential for the inhibitory activity. On the contrary, the h y d r o d group at position 3 was important to obtain a high inhibitory activity (quercetin I& = 1430 t87 #M, luteolin IC, > 2000 #MI. An increasing number of hydroxyl groups on ring A as well as on ring B also had a beneficial effect on inhibition of the complement system (myricetin 1% - 67 f 1 #M, quercetin IC, = 143Oi.87 ~IM, kaempferol IC, > 2000 #MI. Methodation decreased the inhibitory effect, but the effect of glycosylation was rather ambiguous. Flavonoid C~h/cosides showed no effect, but for 0-glycosides the inhibitory activity depended on the nature of the sugar lquercitrin IC, = 312*6 #M, rutin IC, > 1000 #MI. Anthocyanidins showed e high inhibitory effect lcyanidin chloride ICw = 8 7 i 3 #MI. The most potent compounds for inhibition of the classical activation pathway of the complement system were myricetin, morin, quercetin. quercitin, rutin, taxifolin and the anthocyanidins.

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r215 ] CYTOTOXICITY AND CHEMICAL CONSTlTUENTS OF LAGOTIS BREVITUBA Yu-ying Zong', Jin-hua Jia', Wing-keung Liu' and Chun-tao Che' ' Deparrmenr of Chemisny. Hong Kong University of Science and Technology, ' Deparhnent of Anatomy, Chinese University of Hong Hong, Hong Kong.

Lanotis brevituba is a medicinal herb growing on the Tibetan Plateau. An alcoholic extract of the plant exhibits signifcant cytotoxicity in a battery of test cell cultures. In confirmatory assays, the chloroform-soluble fraction is active in suppressing the growth of S-180 tumour in mice, when given orally to the animals.

From the plant extract, two iridoid glucosides, aucubin and catalpin. have been isolated.

P216 1 ANALYSIS OF GASTRODIA ELATA EXTRACTS BY HPLC Kai-lun Huen. Timothy C.M. Tam and Chun-tao Che Department of Chemisny, Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong.

The tuber of Gastrodia elata ("Tian-ma") is a tonic agent in Chinese medicine for the treatment of arthritic pains, numbness in arms and legs, dizziness and headache. It is also reported to be useful in treating stroke and epilepsy. Major chemical constituents of the drug belong to phenolic glycosides.

We have established an HPLC procedure to record a fingerprint profile of the standard drug material, and to estimate the contents of gastrodin. an active sedative component in Gastrodia extracts. Commercial samples from various sources were analyzed and compared.

(The work wa$ supported partially by an American Society of Pharmacognosy Undergraduate Research Award)

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PENTACYCLIC TRITERPENOIDS FROM MAPROUNEA AFRlCANA AS POTENTIAL INHIBITORS OF CHEMICAL CARCINOGENESIS

',l Monroe E. Swapan K. Chaudhuri.' Fekadu Fu1las.l Dan M. 8rown.l Wall.' Uning Woongchon Mar? Yingde LUO.~ Kyaw h-ong? John

P217 I M. Peuuto.2 and A. Doughs Kinghod Research Triangle Institute, P.O. Box 12194, Research Triangle Park, North Carolina 27709

Pharmacy. Chicago. Illinois 60612-7231 2University of Illinois at Chicago, Department of Medicinal Chemistry and Pharmacognosy. College of

As part of our search for naturally occurring cancer chemopreventive agents, we have re-examined the root of M. afficane Muell. Arg. (Euphorbiiceae). Phorbol dibutyrate (PDBu) receptor binding assay and an omnhine decarboxylase (ODC) cell culture assay-guided hactionation of a 50% MeOWCHC13 extraci of

the root of this plant afforded two new maprounoids, 1 and 2, In addition to the previously Identified triterpenoids. W lh the use of spectroscopic techniques. the structures of 1 and 2 were elucidated as IP,2a-dihydroxymaprounic acid 2,3-bis-phydroxybenzoate and 2a-hydroxymaprounic acid 3-phydroxybenzoate, respectively. Compounds 1 and 2, several other related maprounoids, as well as certain derivatives were evaluated for activity in the PDBu and ODC assays. Spectro- scopic and bioassay data for these compounds will be presented.

,..+

R3 (Supported by PO1 CA48112 from

The National Cancer Institute)

R' R2 1 OH 4'-0HCeH,CO0 #-0)HC6H,C0

2 H OH 4'-0HC6H,C0

p:21 PHMOCHEMICAL AND PHARMACOLOGICAL INVESTIGATIONS OF TRITERPENOIDS f3OM AGLAIA GRANDIS

kira Inada' tiiroko Murata.' Yuka Inatomi.' Tsutomu Nakanishi.' Kentaro lida." Yasuhiro Kohama." and Tsutomu Mimura ,'* 'Faculty of Pharmaceutical Sciences Setsunsn University, Hirakata. Osaka 573-01 (Japan) "Faculty of Pharmaceutical Sciences, Osaka University. Suita. Osaka 565 (Japan)

8J, Aglaia grandis Korth (Meliaceae) is a large tree growing in Kalimanlhan in Indonesia. In our continuing search for bioactive constituents from meliaceous plants. we have investigated the constituents 01 the plant. The MeOH extract of the leaves was dissolved in water, and fractionated with haxane and BuOH, successively .The hexane extracl was subjecled to column chromatography and repeated hplc separation to isolale two new cycloaratane-lype triterpenoid hydroperoxides (1 and 3), related known triterpenoids (2 and 4) and cycloartenol. The structural elucidations of Ihe new compounds were established by chemical (reduction of hydroperoxide moiely) and spectroscopic methods. Next, the cylotoxic activity of 1 and 3 against some cell lines were tested. Compond 3 showed the cytotoxic effects (1C50= 0.14 and 0.40 uglml. respoclively) against mouse monocyle- mauophage J774A.1 and human letal lung W138. while compound 1 did not show any remarkable activities.

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p-220

In v i t r o c u l t u r e o f l i v e r w o r t s : NEW H O P A N E - T Y P E T R I T E R P E N E S F R O M

FOSSOMBRONIA SPEC1 ES er.Guniher Burkhardr and Hans Bccker

ISOLATION AND CHAKA<:TEK17.A'lION OF NOVEI. SECONDAKY METABOI.IWS FKOM MUI'ANlS A N D SIJHSI'KAINS OF

harmakoana r i r und Anoigrtrche P h ~ i o c h e r n i e . U n l v c r ~ ~ I s i der Saarlandes -6612% S a a r b r u c k c n . G e r m a n y

xenic cultures o f l iverworts enable phytochcmical studies on rarely occurring species as F o s r o m b r o n i a p r i l l o r F a r r o m b r o n i a a l o r k a n o ( 1 ) which i s restricted i n habitat to the calcarious silt I n the cent re o f permafros t olygons ( 2 ) L iverwor ts produce essential oil. located in special compartiments within the p lan t cell . t h e so- l l lcd o i l -bodies T h e essential o i l of liverworts contains mono-. sesqui-, d i - and tri lcrpcnoids, some o f them :prcscnting less common skeletons unique to the Hepaticne.Tcrpcno1d patterns o f Iivcr\vorts a rc interesting both )r chemotaxonomica l reasons and a s a new source o f natural products with potential biological ac t iv i ty

(1) G m m a . C . BurUurdl. (i. md H A . H.Tnicrpma horn

0) S1ae .W md 1nouc.H (1974) T k Brvofopii 77,63

ArhwlrdPrmvv The a u l b n wrh 10 thank Pmf Ik D Builc. Nnv Yo* E o m d G.rdm. forrhcimlirl cllliur.ofFarsombmni.dmh

Fasmmbrmir 1iv-n~ (1994) P h y c d m m i i ~ ( m p m r )

P.

hytochcmrcal inves t iga t ion of ether extracts bo th o f the arctic F a l a r k o n o and the european spec ies F. p u s z l l a :vcnled the occurrence o f hopane-type tri tcrpencs. Most o f the compounds could be identified as new na tura l roducts. The hopanoid pa t te rn of the two spcc ics i s very s imi la r but not fully identical . T h e occurrence of spancs i n l iverworts is descrlbed from several genera (I). However informallon about hopanes in l iverwor ts i s :t to scarce to use a s a chemotaxonomical marker. I t i s interesting to note. that hopanes from liverworts a r e a l l :longing Lo the 3-dcsoxyscr ies (corresponding to those isolated f rom lichens and ferns). whereas hopancs f rom ighcr Plants often show oxygen substi tutton a t C-3 of the slclcton

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238 WSTER PRESENTATIONS

P2211 I

HAEMOLYTIC ACTNITIES IN u(TRACl3 OF BARKS FROM TRADITIONAL TANZANIAN DIETS. L.A. Chapman and T. Johns. School of Dietetics and Human Nutrition. McGill University. Montreal H9X 3V9. Canada.

Dietary saponins have been suggested to have therapeutic uses in the prevention and alleviation of hypercholesterolemia. Saponins in uaditional diets of the Maasai of East Africa may help explain the low Occurrence of hypercholesterolemia despite a high fat intake in this population. Saponins were detected in bark extracts of Albizzia anthelrnintica, Myrisine afncana, Acacia goetzii and Croton dictygamous which are used in soup by the Batemi, a tribe living in close proximity to the Maasai and who share the Maasai behaviour of adding non-food plant materials to meat based soups and milk. Haemolytic activity of saponins is thought to be closely related to potential hypocholesterolemic activity, thus haemolytic activity was examined in extracts of A. anthelrninitica and M. africana on the basis of the appearance of haemolyzed spots after thin layer chromatographic separation and by comparison of haemolytic activity determined by an in vitro bovine erythrocyte assay. 4 methanol extract of A. anthelmintica (500 ppm) caused 100% haemolysis, while a methanol extract of M. africana fruit (500 pprn) caused 20 41, haernolysis. both relative to distilled water. These results are the basis for on-going studies examining the in vitro and in vivo interactions of cholesterol and bile salts with saponins from traditional dietary additives.

I

P222 1 THE STRUCTURE OF TAXINE C AND C H E M I C A L STUDIES ON TAXINE A Giovanni Appendino ( l ) , P i e r l u i g i G a r i b o l d i ( 2 ) , Bruno Gabetta ( 3 ) and Ez io Eombarde l l i ( 3 )

(1) D ipa r t imen to d i Scienza e Tecno log ia de l Farmaco. v i a G i u r i a 9, 10125 Tor ino ( 2 ) Dipar t imen to d i Scienze Chimiche, v i a S. Agos t ino I , 62032 Camerino (MC) ( 3 ) Indena S.p.A., v i a Ripamont i 99, 20141 M i lano ( ITALY)

Taxine i s a m i x t u r e o f taxane pseudoa lka lo ids r e s p o n s i b l e f o r t h e poisonous p r o - p e r t i e s o f t he yew t r e e . The major c o n s t i t u e n t o f t a x i n e i s t a x i n e B, a m i x t u r e o f a t l e a s t s i x r e l a t e d compounds, whereas t a x i n e A and t a x i n e C a r e m ino r compounds. Taxine C has mp 221", and i s c l o s e l y r e l a t e d t o t a x i n e A ( I ) . However, t h e s t r u c t u r e o f t a x i n e C i s n o t known. Ne have i s o l a t e d from t a x i n e a compound hav ing the c h a r a c t e r i s t i c s of t a x i n e C,and es tab l i shed i t s s t r u c t u r e as d e a c e t y l t a x i n e A. Taxine A has a rea r ranged 2(3->20)- abeotaxane ske le ton , b u t t he C(7) - C(10) moie ty i s t h e same i n t a x i n e A and dea- c e t y l b a c c a t i n 111. The chemical behav io r o f t h i s m o i e t y i n these two compounds was compared, d i s c l o s i n g remarkable d i f f e r e n c e s . Some unusual spec t roscop ica l f e a t u r e s o f t a x i n e A and t a x i n e C are , r a t i o n a l i z e d . The b iogenes is o f t a x i n e A i s d iscussed.

( 1 ) E. Graf , Angew. Chem. 68, 249 (1956).

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P223 I N S I T U A C Y L A T I O N W I T H T A I I N THE STRUCTURAL E L U C I D A T I O N OF NATURAL PRODUCTS

A NEW DITERPENOID QUINONE WITH ANTITUMOUR ACTIVITY, AGASTAQUINONE FROM AGASTACHE RUCOSA (IABIATAE)

P224 I Hveong-Ky Led, Se-Ryang Oh1 , Jin-Woong Kim2 and Jung-Ok L.ee3

'Genetic Engineering research institute, IUST, Taejon 305-600 ; 2CoUege of Pharmacy, Seoul National University, Seoul 151-742 ; 3Korean Research Institute of Chemical Technology, Taejon 305406, Korea

In the course of searchug for biologically active compounds from natural products, A new diterpene, Agastaquinone, was isolated from the root of Asartache rugosa which has been used for treatment of cholera nostras, vomitting, diarrhea and etc. as a traditional medicine.

Its structure was determined as a highly oxidized norditerpene by means of chemical modification and 'instrumental analysis including COLOC technique. Agastaquinone and its derivative showed antitumour activities against in Gtro

human cancer cell lines (A549, SK-OV-3, SK-MEL-2. XF498 and HCTlS).

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F225 CmCER CHEMOPREVENTIVE ACTIVITY OF NATURAL FOOD c o m w w s : AN EXPERIMENTAL APPROACH A Govind J. Kaoadia', Azanaw Mulaw', Harukuni Tokuda2, Akio

Iwashima2 and Hoyoku Nishino3 'Department of Medicinal Chemistry, College of Pharmacy and Pharmacal Sciences, Howard University, Washington, D. C. 20059, 'Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto 6 0 2 , 'Cancer Prevention Division, National Cancer Center Research Institute, Tokyo 104, Japan

Recently, interest in food, pharmaceutical and cosmetic colorants has been directed to the use of naturally occurring pigments. These plant pigments are safely applicable to processed foods as well as soft drinks as naturally harmless color additives. Commonly used colorants comprise of bixins and other carotenes, anthocyanins, betanin and curcumin. These pigments are available in a variety of formulations as food additives. In search for non-toxic cancer chemopreventive agents, we have evaluated anti-tumor promoting effect of a number of commercial formulations as well as pure /purified pigments. The, products were tested in-vitro for antitumor promoting effect on Epstein-Barr virus early antigen activation induced by tumor provider 12-0-tetradecanoylphorbol-13- acetate. Those found to be potent are candidates for im studies. The results of the bioassays will be presented.

SUPERCRITICAL EXTRACTION, ISOLATION AND CHARACTERIZATION OF ANNONA SOUmOSA SEED CONSTITUENTS Govind J. Kapadia', K. K. Tiwari2,James W. Wheeler' and Feseha Woldul,'

of Medicinal Chemistry, College of Pharmacy and Pharmacal Sciences, Howard University, Washington, D.C. 20059, 'Department of Chemical Technology, University of Bombay, Bombay, India, 'Department of Chemistry, Graduate School of Arts and Sciences, Howard University, Washington, D. C. 20059

nona sauamosa is the most widely grown fruit tree in India and other tropical and subtropical trees of the Bnnona plants. The seeds and unripe fruit have been known to show insecticidal activity as well as vermicidal and abortifacient properties. Leaves are alleged to be anthelmintic, root a purgative and bark astringent and a tonic. Recent investigation on seed and bark of Annona sauamosa have resulted in the isolation and chemical characterization of as many as ten acetogenins. The isolation of the compounds in these studies involved the use of organic solvents which are environmentally unsafe. The new isolation method, known as supercritical fractionation and extraction, utilizes carbon dioxide as the solvent under pressure, which is environmentally safe. We performed the extraction of the seeds at pressure of 300 bar, at temperature 50' C and flow rate of 4 hrs. to yield 13.95% of oil liquid. Fractionation of the oil was carried out to provide non-glyceride product and a fraction containing fats. Chromatographic purification of the non-glyceride product resulted in isolation of acetoqenins and aromatic compounds. Details of the

I solation and CharacteGization studies will ie presented.

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p:228

~

SYNTHESIS OF MEMSATIN C AND OTHER SELECTED HELO CH P227 To ENTOSS A L U M I D S Yogendra E l . Shukla2, James W. Wheeler' and

I L Govind J. Kapadia' 'Department of Chemistry, Graduate School of Arts and Sciences, Howard University, Washington, D.C. 20059, 2Department Medicinal Chemistry, College of Pharmacy and Pharmacal Sciences, Howard University, Washington, D. C. 20059

In our earlier studies, a root extract of pelochia tomentosa (Sterculiaceae) was found to show tumorigenic property in experimental animals. In search of the carcinogens, chemical studies resulted in isolation of several alkaloids, namely, the novel quinolinones, melochinone, melovinone, the cyclopeptides, melonovine A and B and scutianine B, and the novel phenylpentyl- isatins, melosatins A, B, C and D. Our continued interest in these alkaloids has led us to undertake synthesis of melosatin C ( 7 - methoxy-4-(5'-phenylpentyl)isatin. Starting frompanisaldehydethe alkaloid was synthesized in five steps. Synthesis of other selected alkaloids has also been undertaken. Details of the reaction sequence will be preseqted.

'6% I & Melosatin c

CYTOTOXIC ENT-KAURENE DITERPENOIDS FROM THREE /SODON SPECIES

2 Han-Dong Sun', Zhong-Wen Lin', Fang-Di Niu', l=gno-Ze Lin', Heebyung Chai', John M. Pezzuto' and Geoffrey A. Cordell'

'Laboratory of Phytochemistry. Kunming Institute of Botany, Academia Sinica. Kunming 650204, Yunnan, P.R. China;'Program for the Collaborative Research in Pharmaceutical Scincea, Department of Medicinal Chemistw and Pharmacognosy, Collsge of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612, USA.

From lsodon loxothyrss. one new diterpenoid, loxothyrin A (11, together with one known diterpenoid, adenolin B (21, from 1. ~eiophyllus, three known diterpenoids, coetsoidins A (3). B (41 and G (5), and from 1. adsndoma, one known diterpenoid, longikaurin F (61, were isolated. The structure determination of 1, and the unambiguous nmr spectral assignments of the known compounds were made by a combination of one- and two-dimensional NMR techniques and computer modeling calculations. The isolates showed potent cytotoxic activities.

LOzolbyk A ( I ) Mmlb B (1) W Y L . A (3) C-LdL. B (4) COawYh c (9 l a m g i h m r b F(6I

Acknowledgement: This work was ruppported, in part, by a grant from the Division of Cancer Treatment, National Cancer Institute, Bethesda. MD. USA.

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ENT-KAURENE DITERPENOIDS FROM ISODON GESNEROIDES

Han-Dong Sun’, Zhong-Wen Lin’, Fang-Di, Niu‘, Oi-Tai Zhen’, Bin Wu’, Lpno-Ze Lin’, Geoffrey A. Cordell’

‘Laboratory of Phytochemistry, Kunming Institute of Botany, Academia Sinica, Kunming 650204, Yunnan, P.R. China;’lnstitute of Materia Medica. Chinese Academy of Medical Sciences, Beijing 100050, P.R. China; ‘Program for the Collaborative Research in Pharmaceutical Sciences, Department of Medicinal Chemistry and Pharmacognosy, ColleQe of Pharmacy,University of Illinois at Chicago, Chicago, Illinois 6061 2, USA.

From lsodongesneroides. three new diterpenoids, gesneroidins A (1 1, B (21, and C (3). together with one known diterpenoid. dawoensin A (41, were isolated, and their structure determination and unambiguous assignment of the sterochemistry and nmr spectral data were made by a combination of one- and two- dimensional NMR techniques, computer modeling calculation, and X-ray analysis.

Ganemldln A (1) Gammidiu B (2) Gunemidin C (3) Dawcnsiu A (4)

Acknowledgement: This work was suppported, in part, by a grant from the Division of Cancer Treatment, National Cancer Institute, Bethesda, MD, USA.

I P:230 I

REGULATION AND PRODUCTION OF BIOLOGICALLY ACTIVE COXSTITUENTS IN CELI. CULTURES DERIVED FROM AILANTHUS ALTISSIMA AKD BRUCEA JAVANICA M. F. Roberts, C. M. Yeoman, R.M. Romero, R. S. Madina and E. Sommer Department of Pharmacognosy, The School of Pharmacy, University of

/London, 29-39 Brunswick Square, London WClN 1AX

Cell cultures and root cultures developed from Ailanthus alttimsa and Brucea javanica have been investigated for the production of biologically active constituents. namely quassinoids of the ailanthone type and simple indoles, the canthin-6-ones (Roberts 1993). Disorganised cell cultures developed from both these plants produced levels of canthin-6-one alkaloids which were 100 fold greater than the yield from whole plants. However despite variations in media, hormones and light, quassinoids were only produced in shoot cultures grown with giberellic acid added to the media (Yeoman 1993). Signal transduction mechanisms stimulated by the elicitors yeast glucan and methyljasmonate had marked effects on gross alkaloid production and on some of the enzymes involved in their biosynthesis. Experiments with root and shoot cultures and young seedlings suggest that quassinoids are essentially produced in the aerial parts of the plant, whereas alkaloid biosynthesis may occur throughput the plant (Yeoman 1993).

Roberts M . F . (1991), in Biotechnology in agriculture and forestry, Vol. 15, Medicinal and aromatic plants, Vol. 3 , ed. Y.P.S. Bajaj, Springer-Verlag, Berlin, Heidelberg, New York. Yeoman, C.M. (1993) Ph.D. thesis, London University

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ANTIBACTERIAL ACTIVITY OF DIOSOUINONE FROM THE ROOT OF DIOSPYROS MESPILIFORMIS (HOSTCH) (EBENACEAE) P231 1 Bolanie A. Lajubutu(1). R.J. Pinney(2). M.F. Roberts(2). H.A.Odelola(

h a n d B.A. O s o ( 3 ) . Clinical Pharmacy. College of Medicine, University of Ibadan, Ibadan, Nigeria. (2lThe School of Pharmacy, University of London, London WClN 1AX. U.K. (3)Department of Botany, University of Ibadan, Ibadan, Nigeria

(1)Department of Pharmaceutical Microbiology and

Several ethnopharmacological claims have been made in respect of Diospyros mespili formis, including control of leprosy, dysentery and oral infections (Irvine 1961). We report data on the antibacterial activity of diosquinone, isolated from the chloroform extract of plant root. Minimum inhibitory concentrations (MICs) were determined against d range of Gram-positive and -negative organisms for single cel inocula on nutrient agar. determined in nutrient broth or in Davies and Mingioli's buffered salt solution. YICs (mg/l) S. aureus NCTC 6571(3), B. subtilis NCTC 8236, (5). S, pyogenes NCTC 8198 (15). E. coli KL16, (15). p- aeruginosa NCTC 6750 (30). S, typhimurium LTZ(45 Bactericidal activity against S. aureus in nutrient broth increased with concentration up to 6 0 m g / l . >hasic response. moth increased with concentration up to the highest concentration tested. :ompound was much more active pgainst E. coli in DM salt solution than in nutrient xoth: tn nutrient broth.

lowever the first report of its antibacterial activity. igainst a variety of organisms, particularly p- aeruginosa, make it worth further itudy. kvine F.R. (1961) Woody plants of Ghana, OUP, London, UK illie, T.J. et a1 (1973) JCS Chem. Commun. 14: 463-464

Sensitivities to lethal concentrations of drugs were

Above this concentration, activity declined giving a bi-

The This was not observed with E . coli. where activity in nutrient

30 mg/l was highly bactericidal in DM, whereas it was only bacteriostatic Diosquinone, although not previously reported fromLmespili-

has been shown to be present in D. tricolor (Lillie et a1 1973). This is The low H I C s recorded

I OF ESSENTIAL OIL OF STOEBE VULGARIS L

- - J - MOODY JO," ADUEYE S'. GUNDDZA M'

'Mpamnent of Phnnnacognosy. Univcnity of badan. Ibadan. Nigeria

' Faculty of Medicine, Univ. of Zimbabwe. Hmrc ' ddms for coMpondcnce

Stocbe vulnarir Lmyns. (Cornposiuc). o x of over tm Ipecics In Swthcrn Africa (1-3) is much h c h d shrub with smd altunatc l u v s s gmwing up 10 3 metem in height.

Although thc plant m w i n g wild in Zimbabwe hu 1 m g m n t i c smell and k h p O N n t in lad CthnOmedKll ' pncticc. d i g Is known a b u t Ihe essential oil C O ~ S ~ ~ N C ~ ~ S .

5he ascntirl oil of &@be wl~aria w1s obuincd by hydrodiStUtillsrim of rhe laver Using rhc Europun phrrmmpoei. rncrhoda (4). M y s i s of thc oil by GC and GC-MS revalcd Ihc prcsacc of u l u a 100 ampahnu. wt of which 46 were identifed l k ma+ compamds w a found to bs sp.lhulsnol (16.75%). limorrnc (11.32 %) and Wrpincoe 0.19%).

Lkpartmmt of Chemical Engineering, Imperial Collcge. London.

Pcftmm

1. WATTJM, BEER-BRANDWIJK MG.. (1962). Thc Mcdichl md Pohwl Pllnt.5 of Southern ud W r n Africa. znd Ed Vol n Livingaons. London.

2. WILD H (1952s. A S h r n Rhodesian Bdanicd Dictimlry of Native and EngllShPlmt nunu. Govt Rinter. Salisbury p 127.

3. COMPMN RH (19%). lwmd of sanh African B m y Sum No 11. Tnutccs Of National Bwnie Gudsns of Swlh Ahiu p. 637.

4. Eump.n P h m m p o e i r (1975) Ed Md~~nncuvc Vol I& pp 68-71.

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P234

COUMARIN GLYCOSIDES FROM ERIOSTEMON CYMBIFORMIS (RUTACEAE) x v a i i t D Snrker. Alexander L Gray and Peter G Waterman Ph) tochemistry Research Laboratories. Department of Pharmaceutical Sciences IIniversity of Strathclyde. Glasgow. GI IXW, Scotland IJK James A Armstrong Department of Conservation and Land Management, Hackett Drive. Crawley. WA 6009, Australia

p:233 3 wo novel coumarin glycosides esculeun-7.(6-a- rhamnopyranosy1)-P-glucopyranoside (named isobaisreoride) [ I ] and ;culetin-7-(4-poro-coumaroyl-6n-rhamnopyranosyl)-~-glucopyranoside (isobaisseoside-4-p-coumarate) 121 and a known avonoid glycoside, hesperidin. have been isolated from the methanol extract of the aerial parts of En'o~remon

~mbifonnrr, a subshrub of Enosremon sect A'igrompulae, which occurs in Western Australia. An n-hexane extract IS yielded a dihydrocinnamic acid derivative eriostemoic acid which is common in the genus Erioslemon. Final irification of novel compounds was achieved by HPLC using a reversed phase C,, semi-prep silica column Structure ere established by spectroscopic methods. cspcclally by different 2D NMR experiments including COSY 45, COSY-11 OFSY. TOCSY. IIMBC and IIC-COB1 dec The chemolaxonomic significance of these findings will be discussed,

2-SLJBSTITUE3 QUlNOI.INl? AL.KAI.OIIX AS WIl~NI'IAL ANIILEISHMANIAL. DRUGS *, Alcira Angelo Barrios**. Victoria MuAoz". Reynald Hocquemiller t, Franwis Roblot t,

~ M a r i c - I 1 C B n e Munos t. Jean-Charles Gantier tt. Jean Bruneron $ and Pascal Richomme $ 'ORSIOM-PARAGIJAY. C C 97. Asuncion, Paraguay; ** IBBA C P 717. La Paz, Bolivie; t Laboramire

. . .

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NEW NATURAL CYTOTOXIC PRECURSOR OF ACETOGENINS OF ANNONACEAE, ISOLATED FROM THE SEEDS OF A. RETICULATA

VU THI TA 5 , PHAN QUAN CHI mu9. BERTRAND CHAPPEQ, FRANCOIS ROBLOT, O k R LAPRlhOTE5, BRUNO FIGADERE* and AND& CAV6

§lns!ituI dc Chimic dcs Substances Natlmlles. CNRS, avenue de la Tc-. 91198 Gif-sur-Yvem Cedw (Fl=n=) Laboratoh de Pharmacognosie. assaiC au CNRS (URA 1843-BIOCIS). Facult& dc Pharmacie. Universit& Parjs-XI. 922% Chslenay-MfW~ C&X (France).

Since 1982. about 100 teaahydrofuran acetogenins of Annonaceae have now been isolated and characterized. Most of these new natural products have shown interesting biological activities (e.g. cytotoxicity, antiturnoral. antiparasitic, insecticide). Recently, we have isolated new natural compounds bearing 1.2-epoxy functions and/or double bonds in place of THF rings, and shown that they rank among the natural precursors of acetogeninsi In continuation of the investigations of the viemamese plant A. reticulato, we have succeeded in the isolation of an unseparable mixture of two epoxy acetogenins: dieporeticanin-1 and dieporeticanin-2. In addition to these compounds a new epoxy acetogenin bearing one double bond, dieporeticenin, has been isolated and characterized as well as a tris-epoxy acetogenin. meporeticanin. The chemical transformation of these new compounds into known and/or new tetrahydrofuran acetogenins has been performed. The first biological data have shown interesting cytotoxicity against KB cells.

p:236 1 THE SEARCH FOR TllE "HICCUP NUT" TOXIN FROM COMfRETLUFRUIT I - L. VerOaa (a). E. GualIco @), E. Wanke @) and C. B. Rogers (c). (a) DipanimcnU, di Cbimica Organica c Indusbiale, via Veaedan 21. 20133 Milano-Italy. @) Dipartham di Fisiologm e Biochimica Gcnuali, Via CcIoria 26, 20133 Milaoo-Ilaly, (c) Depanment of Chausny. Univasity of Durbao-Westville, Dlnban 4ooo-south Africa

Tbc fiuiruiu of certain Combrefurn spsia arc reported to be highly poisowus (0 humans. One ofthe most important symptoms in all p w n i n g s is a violent and prolonged hiccuping (bena the namc "hirmp nut' given to many spccig). Rnious paprs. dealing with the chemical invcstigatioo of the fiuirit. report the isolation of terpcooid and amiwacid glycosdq wmbmastatins and their glumsides (1.2).

technique. on m a n u d m sensory -nes in culhlre. Only mmbmastah B1 was f d to inhibit (ICm 460 uM), in a fast and completely reverdble manner. a variay of potassium ion channels with ncgllgibk &as on calcium and sodium chaaocls. Aciion potentials elicited in chsc conditions kcam more than 10 times longer We suggesr that thc stroog modification ofthe ocll extilability muM be aso on ably at W base of the reponad symptoms. Tbc m o h h basis ofthe mechanism of action is under investigation

Tbe &'ms of the '"9lated COmpMlads ~exaaallularly applied) wcre studied, with tbe w h d c a l l palch-clamp

c",o@mn~ cn,a

OCHl

COMBRETASTAlN BL

I) I. paDzlni F. Pelizoni L. Verotta and C.B. Rogers (1993) South AJ?. J. sfi. 89, 324. I ) F. Pelinoni, L. Veroaa C.B. Rogers, R Colombo. 8. Pcdmlti. G. Balmni, E. Eha and M D'localci (1993) Nat. p d . Lcn. I. 273.

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P:237 1 CONSTTNENTS OF NOTHAPODY7ES FOETIDA

p:238 1 ENDEMIC PLANT SPECIES OF SARA MOUNTAIN I PHYTOCHEMICAL IWSTIGATION OF ACHlLLEA ATRATA L -~ N KovaEeMcl, D DokWP M FisllC3, N Mmkmt3, S Tad3, D GruhE*C4

'Faculty d Pharmacy. Vopcka Sepe 453, B e l g r A , Yugoslawa 'Faculty of Natural Saences, Sludenta trg 16, Belgrade. Yugoslam 31nstitute lor rrmdlanal plm research "Dr JoaI Pan&e, Tadeuk 41nstitute ICW ~io log i~a~ R~SZWA SINS^ S ~ O V ~ C ' 29 Novedra 14, Belgrade, Yugodawa

1, Belgrade Yugoslavia

Achllea strata L is peremal, shrubty plant, wth aromatic, balsamic odor and biller tasle. It is sub en&& d south par( d Balkan and Alps. The ihlusm d this hefb is used as tonic and lor brorchial and laryngeal troubles as well as lor pulmonary affeaions This art& deals with the investigatm of llavonoids lrom the resin, and the essential oil obtained lrom dry leaves and flowers. Plant material was collected at the dflerent localities of h a mountain, during Ihe summer 1992. The resin was obtained by wastung hem wth c h l o r d m Two llavoroid aglycmes were isolated lrom this resn by Ihe use 01 different c h r a n a r c g r ~ c techmques. The spectral characteristics (UV, MS, NMR) cl these aglymws idcated lhal they bkcged to the hghly methoxyiated group, corrmonly nd so &en distributed withm Achilka genera. The first one was &Milled as 5,8-dihydroxy-6.7.4'-tn~ho~ llawne 4 5.6-dihydroxy- 7,8,4'-tfrnelhoxy I l a w , end Ihe SeCDnd MB as 5 ,7 ,3 ' - t r ihy~koxy-6 ,8 ,4 ' - tn~~~ Ilavaw, or 5.7,B-lrihydroxy- 6,3',4'-lrimethoxy llavone The essemal a1 sanples were isdat& by steam distillation in a Clavenger type apparatus. Analysis d these 011 were p6iformed by GC-FID and GC-MS on fused silica capllary c d u m Supelmwar-10 (60 rn ' 0.32 m i.d., 0.5 pm film thickness). Identification d each indikklual carpound was made by carparism c4 their retention limes wah those d authentic sanples, and by mmplter seardnng. matching mass spectral data with those held in the W e r libraries. The mas( abundarl canpawnts in the bdh oil+ were 1,84nede, a- and p - t h u m , sabmene, and canphor (up to 70% all togelher). These oils mntain only m H quantiIiis d sesquiierpenoids (less then 10%). Oils lrom dinerent localities dnter in their corrposirion espedaliy In the percantage d the m i n conslituerds (1,B-dneole. a- and 8-thuinel.

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p240

p239 I ENDEMIC PLANT SPECIES OF SARA MOUNTAIN.

NEW FINDINGS ON SECONDARY METABOLITES FROM LEAVES OF

Maria del Ravo Carnacho*, J. David Phillipson* and Rachel Mata+. CELAENODENDRUM MEXICANUM.

1 II THE ESSENTIAL OIL OF ACMUEA AGERATIFOLIA VAR AIZOON M RlstiC1, N MenkovlC1. D Dokovi$, S TaslC1. N Km&3\nc3. D GrubSd4

'lnstltute lor r n e d z a n t research ' ~ r JOWI PMW, ~ a d e ~ i a K O X U ~ t Belgrade. Yugodavla 2FacuHy d Natural Saeruas s(udec&sk~ trg 16, Eelgrade. YugoslaMa 'Faculty d Pharmacy. Vofvode Slew 4% Belgrade. Yugoslavia 41nstituie lor Bldagcal Research '?miSa SlsnkmC' 29 Novembra 148 Belgrade Yugoslawa

Spealic lacallon and geological ongin aneded on presence ol great number d abwt 1 3 0 endernc speaes in UU, l l aa d Sara mOuntain Among them, seven speaes d Achrllea genefa were recorded, I e A holosariGea SlbthBSm, Achrysomma Gris Aslarandn-rqs Bor, Aatrafa L, Aabrotano/des Vis A h a b m d s , and A ageralldba var a i z m (Gris Adam As a parl d conprehenslve phflochermcal studies rd endemc speaes, in ths artide the results d A agerathha var dzon essential 011 a d y s s wdl be presented

Plant matenal was collected a1 the thee habtats, located on different altitudes of Sara mwnlain dunng the s u m r 1993 Duvska klisura (500 m), Gradslu karnen (1400 rn) and 051jak (2212 m)

The e558ntnl oil sanples were isolated by steam dstilhtm in a Clavenger type apparatus Analyws d these als were performed by GC FID and GC MS on fused slica capillary d u r n Supelcowax 10 (60 m 0 32 mn i d 0 5 p n film thickness) ldentllicatm d each IndMduaI mmpwnd was made by corrparison d reINKxI times wl(h lbw 01 authentic sarr&s and by maldung mass spadral data Wh MS libraries, uwng a m u t e r search It was lound that all investigated sanples were identical in c h e m d cmpxmm Concen!ralm d mapr msti tumt vaned between 507056 lor camphor, 421% lor 1.8 aneole, w h k all d d b r s were present in much lower concentration (up to 7%) Detailed results of comparative analyws d al samples originated from different habtats will be presented

Celuemdendrwn mexicanwn Stand. (Euphorbiaceae) is the only species of this monotypic genus whicf is endemic to Mexico. It grows in the tropical caducifolia forest on the pacific coast. The tree is knowr commonly as "palo prieto' and is used as an antiseptic medicinal plant. Previous investigations centerec on the phytochemistry of the leaves(1) and the stem bark(2). Because many Euphorbiaceae species art important sources of biologically active compounds and have potential for the development of nove antiprotozoal drugs we decided to investigate this species further. In the present investigation, the leaves of C. mexicanurn were ground and macerated witt ch1oroform:methanol (1: 1). The crude extract was partitioned between chloroform and watecthe aqueou: fraction was separated and partitioned with n-butanol. The chloroformic and butanolic fractions wert chromatographed separately. The compounds which were separated and identified by spectroscopic techniques included three biflavonoids(amentoflavone,ginkgigetin and podocarpusflavone A), i picrotoxane sesquiterpenoid (celaenodendrolide). the pentacyclic mterpenoid friedelin and two tetracyclic triterpenoids which have not been isolated from this plant previously.One of these tetracyclic triterpenoids represents a novel compound and its chemical structure has been established. Thc biflavonoid podocarpusflavone A differs from those flavonoids reported from this plant. The rarc piocrotoxane celaenodendrolide has been isolated from the stem bark of C. mexicanwn but has not beer isolated previously from the leaves. No phorbols were detected in this species. The crude extract and isolated compounds showed weak activities against KB cells, Arfemia salina Plasmodium falciparwn and Leismania donovani in vitro.

1. P. Castaneda. M.R. Garcia et al., J. Chern. Ecol. 18. 1025 (1992) 2. P. Castaneda. A. Baheba et al., J. Nat. Prod. 56. 1575 (1993).

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~

BIOASAY-DIFIECTED ISOLATION OF NIGROSPOROUDE. A NEW MACRWDE FROM N m M m J&&QuB& J. S. liarwood and H. G. Cutler College of Pharmacy, Ohio Northern University, Ada, OH 45810; Dept of Chemistry, WC 111, University of Illinois at Chicago, Chicago, IL 60607; USDA- ARS, Russell Research Center, P. 0. Box 6677, Athens, GA 30613

P2411

A strain of the fungus Nigrospom sphaerica. found on germinating fescue seed, was subjected to bioassaydirected fractlonation using an etiolated wheat coleoptile bioassay. This procedure resulted in the isolation of a new 14-membered lactone, which has been trivially named nigrosporolide. The lactone has plant growth-inhibiting properties, and is structurally related to the known phytotoxin seiricuprolide, from Seiridium cupressi.

F O U R NEW BISRENZYLISOQUINOLINE ALCALOIDS F R O M CYCLEA P242 ATJEHENSIS Clix OVONO ABESSOLO'. Htltne GUNAUDEAU', Bamrung T A h i S E W E * *

des Sciences Medicales er Pharmaceutiques. 49045 Angers Cedex "Depanrnent of Pharmacognosy. Chulalongkom University. 10500 Bangkok. Thailand I n a continuing study of the alkaloids of Cyc/ea arjehensis. collected i n Thailand, four new bisbenzylisoquinolines from the curine goup. have been Isolated. Their structures, related to (-)-

Curicycleacjenine and (-)-lsocuricycieatjenine, were determined by one and two dimendonnal NMR technics ( such as 'H YMR. COSY H-H. COSY C-H. COLOC. HMBC. HMQC and selective W E I T experiments ). Their relationship and derivatives obtained by reduction and N-methylation will be described. The cytotoric activities of rhese compounds will be presented.

( - ) - l s o c u r i c y c l e a t j e h i m i n e 1 R . 1's : (+ ) -2 ' -no rcu r i cyc leah ine l S , 1 ' R : ( - ) -2 ' -no rcu r i cyc leah ine 1 A . 1 ' R : ( - ) - 2 ' - n o r i s o c u r i c y c l e a h i n e

I

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NATURAL DISTRIBUTIOK AND BIOLOGICAL ASPECTS OF p243 I RHUS CORIARIA

Bloshenkn, E. K. Institute of Botany, Dushanbe, Tadjikistan Address for correspondence: YL Begovaya Dom 13 App. 9. 125284 Moscow, RUSSIA

Sumach (Rhus coriaria, L., ) from the Anacardiaceae family, is a perennial shrub used in medicinal preparations, food processing or preservations, veterinary practices and other technical applications due to its high content of tannins. Although there is increasing demand for R. coriariu, there has been no published information about the natural geographical distribution or essential biological characteristics of this species. The main objectives of this study were, therefore, to investigate the extent and nature of its distribution and the influence of complex ecological conditions on certain biological aspects under different geographical regions. This study was carried out in the Central and Eastern Caucasus region of the former Soviet Union. It was found that R. coriaria distribution in the Eastern region, characterized by annual precepitation levels of 500 - 600 mm is disjunctive. The extension of this species North wards as well as vertical distribution in the mountainous regions is limited by lower temperatures. Southern and Eastern distribution is limited by aridity and relatively higher competition from other species. The Pamir-Alai region of Tadjekistan, contained exclusively monodominant phytocenosis of R. coriaria, with relatively large tree-like individual plants of 6-8m height. In contrast to the Eastern Caucasus, in Crimea and other Western areas, i t is a calciphil, having a co-dominant character of distribution, mostly in association with other species. Clear demarcation in zonal distribution of monoecious and dioecious forms of R. coriariu was found. R. coriaria can be multiplied either by seed or vegetative means. However. due to the absence of male plants in the Pamir-Alai region, its propagation is possible only through vegetative means. The cause for the absence of male plants in this area is may be due to ecological problems or the presence of modern development.

GROWING MEDIUM AND SUPPLEMENTAL LIGHTING ALTER ESSENTIAL OIL CONTENT IN Anaelica archanaelica ROOTS p:244 I

gtchamo. W (11.9 J. Holzl (2), B. Desrosches (3) and A. Gosselin (1) 1. Horticultural qesearch Centre, Department of Plant Science, Faculty of Agriculture and Food 3ciences. Lava1 University, Sainte-Foy, Quebec, Canada, G1 K 7P4. 2. lnstitut of 'harmaceutical Biology, Phillips University of Marburg, Germany. 3. Laboratoire de qecherche Bioplant Inc. Varennes, Quebec, Canada.

Angelica archangelica, L. is a biannual herbaceous plant from the Apiaceae family. The extracts from angelica are used in beverage, food, and tobacco processing industries. The objectives of the present work was to elucidate the relationship between active substances content, dry matter production and photosynthesis of angelica plants grown in a greenhouse under two growing substrates and two lighting regimes. Sand culture supplied with standard nutrient solution (SCNS) was compared with peat-based organic substrate (PBOS). Plants were either grown under natural light (NL) or supplemented (SL) with 75 pmol/m2/ s of PPF for a photoperiod of 16 hrs.. Essential oil content was analysed through steam distillation. Higher biomass production for both growing substrates was recorded under.supplementa1 lighting. However, the highest essential oil content (+ 25 %) was recorded only from PBOS grown plants developed under supplemental lighting. In this investigation we found significant differences (200%) in essential oil content between rhizomes and roots. Remarkable differences in the distribution of essential oil in various parts of the plant were also recorded.

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p-245 TAXANES IN TAXUS BACCATA L. CALLUS CULTURES. -, DanaVeselB, Jana MaW, Radka PodllpnB, David Saman Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences,

p246 EXTRACTION AND DETERMINATION OF DIOSGENIN INDIOSCORE4 NIPPONICA BY SUPERCRITICAL FLUID EXTKACTION COUPLED WITH CAPILLARY GAS CHROMATOGRAPHY

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25 1 POSTER PRESENTATIONS

MORPHOLOGICAL AND CHEMICAL PROPERTIES OF FOENICULUM AND

Eva Nkmeth. JenB Bemdth. Ahmd Kattaa University of Horticulture and Food Industry. Department of Medicinal Plant Production. P.O. Box 5 2 . Budapest 1502 (Hungary)

p-247 ACHILLEA TAXA OF DIFFERENT ORIGIN

Species belonging to Foeniculuni and Achillea genera are widely used in pharmacy thorough the word. Knowledge about their intmspecific variability is significant, however, data are often contradictious. Foeniculum vulgar L. is an important crop in Hungary. countinuous improvement of its genetic basis ir necessary. The cultivated types are referred in literature as subsp. capillaceurn (GI.). var. vulgare (Mill.: Thell., var. duke (Mill.) Thell. and var. azoricum (Mill.) Thell. These varieties are characterized mainly b) the amount of specific components (fenchone, estrdgol. anethol) in the essential oil. In o u r investigations 34 genotypes of fennel belonging to the above taxa were tested for their morphologica features as well as essential oil accumulation and composition. The most important connection betwee these properties had been determined. with the aim to stabilize the selected strains both for yiel components as well as active materials’quality.

ones. Although there is a hudge knowledge accumulated ahout mlene compounds in yarrow, taxa wick does not contain these compoun& deslred much less attention. Some result indicate however wide intraspecific chemical variability also in case of them. In our work we investigated individuals of Achilles cridunifolia W. et K. from four spontaneous populations. and as a rcwlt we identified three basic chemotypes, containing camphor, borne01 and 1.8-cineole in their essential oil. The proportion of each chemotype is charactenstic for growing site. and they differ also in some morphological properties(e.g.leaf- form).

. . ’-248 I Survey of Jamu Gendong in Surabaya

ADRIANTA SURJADHANA Research Center of Traditional Medicine

Widya Mandala University Surabaya, Indonesia.

Jamu Gendong (JG) is a traditional medicine in liquid form that is not preserved, and sold without label. It has been used since ancient days and is still being used now in spite of the fast development of modern medicine. JG is sold by the vendors at the markets as well as those who peddle around. Since 1991 several studies has been conducted by the center i.e : - to find the simplicia that are mostly used in the

preparation of the 7 kinds of JG and the formulation of each JG. - to know the antimicrobial activity of the extracts of the simplicia. - to make fingerprints by TLC method of the simplicia.

Besides the center has conducted several upgrading courses for the jarnu gendong vendors to produce better quality JG. Results of the studies will be presented and an outline of future activities are mentioned.

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CHARACTERIZATION OF TAXA OF THE A C H I U M MIIIXFOLICM-GROUP IN CENTRAL EUROPE BY MEANS OF TLC AND OFF-LINE HPLC-MS

?:249 1 Sabine Glasl, Ulrike Kastner, Johann Jurenitsch and Wolfgang Kubelka Institute of Pharmacognosy, University of Vienna, Wahringer StraBe 25, A- 1090 Vienna. Austria.

Aerial parts of Achilleu nrillefdium L. s.1. (Asteraceae) are widely used for the preparation of herbal teas with antiphlogistic and spasmolytic activity. Due to its position in folk medicine (7th range of the most popular plants in Austria [I]) extensive morphological [2-41 and phytochemical [ 5 ] investigations have been started. The morphometric studies resulted in a review of the Achilleu millefolium-group characterizing the different taxa by morphological features and level of ploidy. Based on well defined plant material a characteristic pattern of sesquiterpene lactones was found for the most common indigenous species by means of TLC. In order to distinguish single taxa more properly a HPLC method was developed, offering the advantage of a quick identification of characteristic compounds by mass spectrometric monitoring of the respective HPLC fractions. According to the sensitivity of this procedure (1OOmg dried flower heads per analysis) the single plant analysis can be usehl for chemotaxonomic requirements as well as for the investigation of commercially available drug material.

1 Saukrl J.. Kubdka W (1994) Sci P l m 62. IW 2 a u k e l J.. Langrr R. (1-2) Pliylon32. 47-78 3 . S . u k c l J . U n g n R . ( l W Z ) P h y 1 ~ 3 1 . 185.207

4.Siukcl J..UngaR(I992)Phyion32. 139-172 J S c h r M a H.. K1slrrr U., G~rguh K , nudi3inrky M I

Haslinger E , Jurrmtsch J .Kuklka U’ Ph~kxl~r .m, in press

QUANTIFICATION OF FLAVONOIDS IN HERBA PASSIFLORAE OF DIFFEKENT ORIGIN BY RAPID HPLC-ANALYSIS K. Rahman, B. Kopp, L. Krenn und W. Kubelka institute of Pharmacognosy, University of Vienna, A-1090 Wien, Austria

P250 1 The spectrophotometric quantification of the total flavonoid content of Herba Passiflorac as required by the DAB 10 and the Ph. Helv. VI1 results in too small wnccntrations, because the flavone-C-glywsides cannot be determined by acid hydrolysis1. Quantitative HPLC-analysis2 has proved to be the method of choicc for this purpose. Therefore, a quantitative W-HPLC-method was developed as a gradient elution procedure with internal standardization. Compared to an isocratic elution* the peaks of minor compounds with higher retention time appear more distinct and sharp. The use of an internal standard allows more precise and reproducible quantification of the flavonoid complex. The plant material was extracted with 40% methanol, and after adding the internal standard kacmpferol-7- neohcspcridoside the extract is ready for HPLC-analysis. Baseline separation of flavonoids was achieved in less than IS minutes using aqueous trifluoroacctic acid (pH=3) - acctonitrile (80+20), gradient elution (0,8%dmin; flow rate: 1,2ml/min) on a Nucleosil W 18 column ( 5 pm, 4,O x 250 mm), fitted with a precolumn (Lichrosorb RP 8, 10 pm, 4,O x 40 mm). The detection was performed at 340 nm. The new method was applied to investigate the flavonoid pattern of samples of Hb.Passiflorae from Italy, the USA, Germany and India The chromatograms showed similarities in the quality of the flavonoids but a grcat differencc in the wnccntrations of the individual compounds. The system is uscful as a convenient method for a rapid, reproducible quantification of the single and total content of flavonoids in I-ierba Passiflorac of different origin. This procedure also enables a standardization of alcoholic phytomedicines, which can be injected directly or aftcr dilution.

i U ’ ~ ~ l I . . T i n e l G . B l r d l S . ( 1 9 8 3 ) . ~ l ~ Apah.%Ig 121 J I J - 5 2 1 2 S c h i d l P C.OncgaG G ( 1 9 9 3 ) . b h ApoUi Ztg. 113 44J74466

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abstracts from the international, congress on natural products research

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