picrorhiza scrophulariiflora

Picrorhiza scrophulariiflora,from traditional use toimmunomodulatory activity

Friso Smit

Picrorhiza scrophulariiflora,from traditional use toimmunomodulatory activity

Picrorhiza scrophulariiflora,van traditioneel gebruik tot immuunmodulerende activiteit

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de UniversiteitUtrecht op gezag van de Rector Magnificus, Prof. Dr. H.O.Voorma, ingevolge het besluit van het College voor Promotiesin het openbaar te verdedigen op woensdag 7 juni 2000 desnamiddags te 2.30 uur

door

Hobbe Friso Smitgeboren op 7 oktober 1968 te Drachten.Promotores: Prof. Dr. R.P. Labadie

Prof. Dr. H. van Dijk

Co-promotor: Dr. B.H. Kroes

Table of contents

1. General introduction 7

2. Taxonomic notes on Picrorhiza and Neopicrorhiza 11

3. Traditional use of Picrorhiza in Āyurveda 233.1 Āyurveda3.2 Āmavāta3.3 Picrorhiza in Āyurveda

4. Constituents of Picrorhiza with immunomodulatory and related activities 454.1 Cucurbitacins4.2 Iridoid glucosides4.3 Phenylethanoids4.4 Phenolics

5. Immunomodulatory activity of Picrorhiza scrophulariiflora extracts 635.1 Introduction to relevant inflammatory processes5.2 Immunomodulatory activity in vitro5.3 Anti-inflammatory activity in vivo5.4 Exclusion of toxicity in vivo

6. Activity-guided fractionation of constituents modulating complement activation 876.1 Purification of the diethyl-ether extract6.2 Purification of the light-petroleum extract

7. Activity-guided isolation of cucurbitacins inhibiting T-cell proliferation 97

8. Immunomodulatory activity of cucurbitacins from Picrorhiza scrophulariiflora 1118.1 Immunomodulatory effects on T lymphocytes and monocytes in vitro8.2 Immunomodulatory effects in vivo

9. Summarizing discussion and perspective 125

Appendix A. Sanskrit vocabulary 135

Appendix B. Abbreviations 143

Samenvatting 147

Dankwoord 153

Curriculum vitae and 155

Publications 157

General introduction

Chapter 1

Introduction

Natural products have been an important resource for the maintenance of life for ages.Already in the earliest written traditions, e.g. the Rigveda of South Asia (ca. 1500-900 BC), itis evident that plants played an important role in daily life. One of the best-known examples isSoma, a plant that was pressed to yield juice, which was used as a medicine(Mukhopadhyaya, 1922-1929, Mahdihassan and Mehdi, 1989). The interest in medicinalplants has never ceased since. Even today, natural products become increasingly important asa source of pharmacotherapeutics, either directly, for example in the application of herbaldrugs for the treatment of chronic diseases, or as raw material from which more or lesscomplex chemical structures with particular biological activity are isolated. Cragg et al.(1997) reviewed the role of natural products in drug discovery, and concluded that for thedisease indications anticancer and anti-infection, more then 60% of new approved drugs arederived from natural sources. Furthermore, there is a global concern about emerginginfectious diseases, and an urgent need to identify novel means for effective treatment thereof.

The National Cancer Institute has screened randomly a hundred thousand plant extractsfor antineoplastic, anticarcinogenic, and antitumor activity in several model systems. Thisstrategy did not prove very effective (Reynolds, 1991). Traditional systems of medicine,however, provide an extremely vast body of source material for the development of new drugs(Holland, 1994). Evaluation of traditionally used medicines, keeping into account thetraditional principles that are applied in drug therapy, may supply leads towards effective drugdiscovery.

One of the species that emerged from such an inventory is Picrorhiza kurrooa Royle(Labadie et al., 1989). This plant has been the subject of another thesis, which focused on itsmodulatory effects on complement activation and the production of chemiluminescence byactivated neutrophils. Two macromolecules have been isolated that interfered with theclassical pathway and the alternative pathway of complement activation, respectively.Furthermore, several phenols were isolated that inhibited the production ofchemiluminescence by activated neutrophils. One of these, apocynin, also reduced theseverity of symptoms in experimentally induced arthritis (Simons, 1989, ’t Hart et al., 1990).Because oxidative stress due to reactive oxygen species is a major contributor to joint damagein rheumatoid arthritis (Halliwell and Gutteridge, 1999), these data suggest the usefulness ofPicrorhiza in the treatment of this disease. This impelled further research into activeconstituents of Picrorhiza that may be modulating immune responses.

Objective

The objective of this thesis was the further pre-clinical evaluation of theimmunomodulatory activity of Picrorhiza and the activity-guided isolation of other activeconstituents, in order to assess its usefulness in rheumatoid arthritis or related conditions. Thisassessment is presented from a pharmacognostic perspective. The ultimate biomedical goal ofpharmacognosy is “to contribute to the causal and hence rational relationships between thechemical constituents of medicinal plant preparations and the biological and therapeuticeffects they generate” (Labadie et al., 1989). Traditional systems of medicine provide an

extensive source of material, which may demonstrate such relationships. To investigate thenature of these relationships, a multidisciplinary approach is required, ranging fromanthropology, via philology, botany, phytochemistry, pharmacology, to medicine. In thisthesis, several of these fields are taken into consideration in answering questions pertaining tothe usefulness of Picrorhiza in chronic inflammation.

Rhizomes of two Picrorhiza species happen to be available on South-Asian markets: P.kurrooa Royle and P. scrophulariiflora Pennell (Scrophulariaceae). Both species are knownunder the same local name, and used for similar purposes. The material under investigation,which was obtained from the market, was identified as Picrorhiza scrophulariiflora Pennell(Scrophulariaceae). This species is categorized into a separate genus by Hong (1984), whonamed it “Neopicrorhiza scrophulariiflora”. For reasons of clarity, however, described inchapter 2, the name Picrorhiza scrophulariiflora is maintained throughout this thesis.

Outline

Chapter 2 deals with taxonomical and geographical information concerning the properidentity of the plant material used. This chapter is partly based on fieldwork performed in theHimalaya regions of Nepal and India. Chapter 3 elaborates on the traditional use of Picrorhizain Āyurveda, a traditional life science of South Asia, as known from ancient Sanskritliterature, as well as from contemporary use. Two approaches have been followed, one basedon the direct exploitation of empirical knowledge, and the other on the specific application ofgeneralized traditional principles. In chapter 4, an overview is given of metabolites that havebeen isolated from Picrorhiza, and their biological activity is discussed with regard toimmunomodulation. Chapter 5 gives a limited overview of the background necessary tounderstand the assays and models used, and describes the in vitro and in vivoimmunomodulatory activity of Picrorhiza extracts. Chapter 6 describes the activity-guidedfractionation of extracts that interfere with the classical pathway of complement activation.Activity-guided isolation of two cucurbitacins that inhibit proliferation of activated Tlymphocytes is described in chapter 7, while chapter 8 elaborates on the mechanisms of actioninvolved. Finally, chapter 9 provides a summary and a general discussion of the topics thatwere elaborated in this thesis, and extends the discussion to further perspectives. In addition,appendices have been included as reference to Sanskrit words and abbreviations.

References

Cragg, G.M., Newman, D.J., Snader, K.M. (1997) Natural products in drug discovery anddevelopment. J. Nat. Prod. 60, 52-60.

Halliwell, B., Gutteridge, J.M.C. (1999) Free radicals in biology and medicine. Third edition,Oxford University Press, Oxford, pp. 664-677.

’t Hart, B.A., Simons, J.M., Knaan-Shanzer, S., Bakker, N.P., Labadie, R.P. (1990) Antiarthriticactivity of the newly developed neutrophil oxidative burst antagonist apocynin. FreeRadical Bio. Med. 9, 127-131.

Holland, B.K. (1994) Prospecting for drugs in ancient texts. Nature 369, 702.Hong, D.Y. (1984) Taxonomy and evolution of the Veroniceae (Scrophulariaceae) with special

reference to palynology. Opera Bot. 75, 5-60.Labadie, R.P., Van der Nat, J.M., Simons, J.M., Kroes, B.H., Kosasi, S., Van den Berg, A.J.J., ’t

Hart, L.A., Van der Sluis, W.G., Abeysekera, A., Bamunuarachchi, A., De Silva, K.T.D.(1989) An ethnopharmacognostic approach to the search for immunomodulators of plantorigin. Planta Med. 55, 339-348.

Mahdihassan, S., Mehdi, F.S. (1989) Soma of the Rigveda and an attempt to identify it. Am. J.Chinese Med. 17, 1-8.

Mukhopadhyaya, G. (1922-1929) History of Indian medicine. Volume I. Second reprint 1974,Oriental Books Reprint Corporation, New Delhi, pp. 189-203.

Reynolds, T. (1991) Tropical rain forest conservation tied to drug discovery. J. Natl. Cancer I.83, 594-596.

Simons, J.M. (1989) Immunomodulation by Picrorhiza kurroa, basis for its ethnomedical use?Ph.D. Thesis, Universiteit Utrecht, Utrecht.

Taxonomic notes on Picrorhiza andNeopicrorhiza

Chapter 2

12 | Chapter 2

Introduction

The present chapter describes taxonomical aspects of species from the closely relatedgenera Picrorhiza and Neopicrorhiza. Since the plant material used for the experimentsdescribed in this thesis was obtained as simplex, and as there are no discriminative featuresbetween the rhizomes of both species, the identity of the plant material could not directly bedetermined. This chapter provides a basis for the proper identification of the plant materialinvestigated.

Picrorhiza and Neopicrorhiza are small genera belonging to the tribe Veroniceae of thefamily Scrophulariaceae. This family is, according to the taxonomical system of Cronquist,arranged in the order Scrophulariales, subclass Asteridae, class Dicotyledonae, of theAngiospermae. The genus Picrorhiza was considered monotypic, with as only species P.kurrooa, until Pennell (1943) distinguished a second species: P. scrophulariiflora (originallywritten as “P. scrophulariaeflora”). This new species is brought under a separate genus byHong (1984), who named it Neopicrorhiza scrophulariiflora. This name has been accepted asthe official name of this species (Brummit, 1992). However, several arguments are in favor ofthe use of the basionym Picrorhiza scrophulariiflora within the scope of this thesis. Firstly,most literature on this species published after 1984 sticks to this synonym. Secondly, becauseof their similarity, rhizomes of both species are comparable with regard to their morphology,histology, accumulated metabolites, bitterness, activity, and traditional use (Zhang et al.,1965, Wang et al., 1993). Thirdly, because distinguishing criteria between rhizomes of bothspecies have not been developed so far, plant material described in publications on biologicalactivity or constituents as referred to P. kurrooa and obtained from raw material suppliersmight erroneously be mixed up with P. scrophulariiflora. Therefore, the name Picrorhizascrophulariiflora is used throughout this thesis.

Discussion

PicrorhizaThe genus Picrorhiza and the species P. kurrooa appeared for the first time on a drawing

published by Royle on August 24, 18351 in his “Illustrations of Botany” (figure 1) (Royle,1835-1840a). Bentham described the genus and the species in his “Scrophularineae Indicae”,which was published a few months later, on November 17, 1835 (Bentham, 1835). However,he wrote the species name as “P. kurroa”, probably correcting or misspelling the name ofRoyle’s publication. In May 1836, Royle published the text on Picrorhiza, and retained thespecies name “P. kurrooa” (Royle, 1835-1940b). Because Bentham was the first to give awritten description of the species, the accepted species name has been “Picrorhiza kurrooaBentham”, or alternatively “Picrorhiza kurrooa Royle ex Bentham”. However, according tothe International Code of Botanical Nomenclature article 42, an illustration with analysispublished before January 1, 1908 is acceptable as valid publication, instead of a writtendescription or diagnosis (Greuter et al., 1994). Therefore, the correct name of the species is“Picrorhiza kurrooa Royle”.

1 For publication dates see: Stafleu and Cowan, 1983.

Taxonomic notes on Picrorhiza and Neopicrorhiza | 13

Figure 1. First publication on Picrorhiza kurrooa by Royle in 1835.

14 | Chapter 2

Royle states that the generic name is derived from the bitter root, which is used in nativemedicine (Royle, 1835-1940b). In Greek, “picros” means bitter, while “rhiza” means root.The specific name is derived from “Karu”, the Punjabi name of the plant, which means bitteras well (Coventry, 1927).

In 1876, Bentham and Hooker described the genus Picrorhiza in their “GeneraPlantarum”, mentioning the existence of only one species. The genus was consideredmonotypic until Pennell distinguished another species, based on information in Hooker’s“Flora of British India” (Pennell, 1943). Hooker considered the flowers of P. kurrooa asdimorphic, which was so far not known in the Scrophulariaceae. He distinguished a form withlong stamens and a short corolla with five sub-equal lobes, and a form with short stamens anda bilabiate corolla, of which the upper lip is longer and the lower lip consists of three shorterlobes (Hooker, 1885).

Smith and Cave collected a Picrorhiza species at the base of the Zemu glacier in Sikkimat 4300 meter, and identified it as P. kurrooa (Smith and Cave, 1911). There is a specimen atthe Herbarium of the Academy of Natural Sciences of Philadelphia (PH), which wasidentified by Pennell as the short-stamened form described by Hooker. Furthermore, henoticed that all collections seen from the Western Himalaya are in agreement with the formhaving long stamens, while those from the eastern Himalaya and Yunnan correspond to theform with short stamens. Therefore, he characterized two species, one of the dry western (P.kurrooa), and the other of the moist eastern Himalaya (P. scrophulariiflora) (Pennel, 1943).

NeopicrorhizaBased on the differences described above, and additional differences between the pollen

of both species, Hong (1984) suggested to place both taxa into two distinct genera: Picrorhizaand Neopicrorhiza. Both genera are monotypic, including P. kurrooa and P. scrophularii-flora, respectively. In the former, the corolla is almost actinomorphic with a short corolla tubeand five equal lobes, and with stamens about 3 times longer than the corolla (Hong, 1984).The name “Neopicrorhiza scrophulariiflora (Pennell) Hong” has been reserved as the officialname of this species (Brummitt, 1992).

Description and distributionA. Family Scrophulariaceae, tribe Veroniceae, genus Picrorhiza:

Perennial herb, usually with a long rhizome. Leaves basal and alternate. Spikes terminal.Calyx nearly equally 5-partite. Corolla 4- or 5-lobed, bilabiate with lobes more or lessspreading or nearly actinomorphic. Stamens 4, inserted on corolla tube, slightlydidynamous, as long as corolla or strongly exserted. Stigma capitate. Capsule turgid,tapered at top, dehiscing first septicidally and then loculicidally into 4 valves. Seedsnumerous, ellipsoid; seed coat very thick, transparent and alveolate. Pollen grainsspheroidal, 3-colpate, with partial or perforate tectum, the partial tectum microreticulate,colpus membrane smooth or sparsely granular.1 Corolla 4-5 mm long, 5-lobed, and nearly actinomorphic; stamens many times

longer than corolla. – Picrorhiza Royle2. Corolla 9-10 mm long, 4-lobed, and bilabiate; stamens slightly didynamous,

equalling corolla. – Neopicrorhiza Hong


1. Picrorhiza kurrooa Royle, Ill. Bot.: t. 71. 1835; Benth., Scroph. Ind.: 47. 1835; Benth. etHook.f. Gen. Plant. 2: 962. 1876; Hook.f. Fl. Brit. Ind. 4: 290. 1885.Distribution: Kashmir to Kumaon, 3000-4300 m (Pennell, 1943), Pakistan to Uttar

Pradesh, 3300-4300 m (Polunin and Stainton, 1990). Jammu & Kashmir: Apharwat,Kashmir (Rau, 1975), Burzil Pass, Stewart 18856, 19084, 19224 (Pennell, 1943), Gumri, VirJee 133 (Jee et al., 1987), Kamri Pass (Naigund), Inayat 25732 (Pennell, 1943), Kolohoi;Zojpal; Sonsa Nag, 10,000-13,000 ft (Coventry, 1927), Lipper Valley, northwest of KashmirValley (Singh and Kachroo, 1976), Mir Panzil Pass, Deosai Road, Stewart 19945a (Pennell,1943), Nafran, Stewart 12537 (Pennell, 1943), Pahlgam, Stewart 5839, 7823, 9278 (Pennell,1943), Pir Panjal range; Krishan Ganga valley; upper Lidder valley (Kapahi et al., 1993),Pirpanjal range; Krishnaganga Valley; Upper Lidder Valley (Kapil, 1995), Simthan, Jammu(Sharma and Kachroo, 1981), Sonamarg, Stewart 6417, 6597½ (Pennell, 1943), Tragbal,Stewart 4618 (Pennell, 1943), Zojibal Pass, Stewart 18234 (Pennell, 1943; HimachalPradesh: Basharh: Chhit Kul, Parmanand 1032; Hangla Pass, Parmanand 688; Pieri Pass,Kinnawar, Parmanand 521 (Pennell, 1943), Chafuwa, Talla Johar, Eastern Kumaon, 3500 m2126 (Mehta et al., 1994), Chamba and Kangra (Gammie, 1898), Chamba: Kukti Pass, Koelz8600 (Pennell, 1943), Chamba; Kinnaur; Kulu; Lahul; Spiti (Chowdhery and Wadhwa,1984), Chhakinal watershed, District Kullu (Dobriyal et al., 1997), Kangra (Kulu):Chandrokani, Koelz 61; Sumdo, Koelz 5011 (Pennell, 1943), Lahaul; Kinnaur; Kulu; Rohru;Kangra; Pangi-Bharmour above 3500 m (Chauhan, 1988), Lahul: Dilburig, Chenab Valley,Koelz 9917; Kyelang, Koelz 538 (Pennell, 1943), Manimahesh, 3300 m; Rotang Pass, 3800m, Lahaul Spiti Forest Division (Uniyal et al., 1982), Pangi valley; Birmor valley; Lahaulvalley; Dauladhar valley (Kapahi et al., 1993), Pangi; Bharmour; Lahaul; Kalpa; Dauladhar;Barabhangal ranges (Kapil, 1995), Rohtang slope, Lahaul-Spiti Aswal 6517 (Aswal andMehrotra, 1994), Rudranath Bugyal, Chamoli, Garhwal (Joshi, 1989), Satrundi, Chamba(Rau, 1975); Uttar Pradesh: around Agora-Dodital, Uttarkashi district (Chandra and Pandey,1983), Bhillangna Valley, Sahasru Tal, Tehri-Garhwal 5,300 m, Gupta RK 330 (Gupta, 1957,1989), Buhna, Nar Parbat, Garhwal, 3000-4500 m, Rau MA 10271 (Rau, 1961, 1975),Damdar Valley, Tehri-Garhwal 3700 m, Duthie 229 (Gupta, 1989), Dayara, Devkund 3300m, Uniyal 3994 (Uniyal, 1968), Deodi Ramani, Garhwal, Anon 73386 (Gupta, 1989), EnrouteRupkund, Garhwal, Bhattacharya and Mehrotra (Gupta, 1989), Gangotri, Tehri-Garhwal2400-5000 m, Keshwanand 30 (Gupta, 1989), Harsil, Raithal, Sukhi, Sayara, Tehri-Garhwal3000 m in Bhagirathi Valley, Uniyal 3994 (Gupta, 1989), Jumnotri, Tehri-Garhwal (Rau,1963, 1975), on the way to Yumnotri, Tehri-Garhwal 2500-3500 m, Rau MA (Gupta, 1989),Kedar Konta, east of Sutlej gorge (Royle, 1835-1940b), Kedar Nath; Herker Dun; Ponwali;Tali; Harsil, Garhwal Himalaya 3000-4000 m (Uniyal, 1989), Kedarkanta, Tehri-Garhwal4300 m, Drummond 22760 (Gupta, 1989), Kedarnath, Har-ki-dun, Ponwati, Tali, Harshil andGangotri in Garhwal Hills (Kapil, 1995), Khaljhuni, Garhwal 2200-2500 m, cultivated (Raoand Saxena, 1994), Kidarnath, Garhwal; Gangotri, Garhwal; Kumaon hills (Kapahi et al.,1993), Kumaon, alpine regions (Rawat and Pangtey, 1987), Kumaon and Garhwal, alpineregions (Joshi et al., 1995), Madhari Pass, Kumaun Strachey & Winterbottom (Duthie, 1906,Pennell, 1943), Milam, Kumaun Schlagintweit 9647 (Pennell, 1943), Milam, Pindari,Kumaon (Rau, 1975), Narparbat, Garhwal, 3000 m Rau MA 10271, 10506, Falconer 778(Gupta, 1989), Pithoragarh District of Kumaon Hills (Kapil, 1995), Ponwali, Tehri-Garhwal3300-3600 m, Uniyal 1667, 3843 (Gupta, 1989), Rupkund, Garhwal (Bhattacharyya, 1984),Tungnath, Garhwal, 3600 m (Nautiyal, 1988), Tungnath, Garhwal, 3300 m, Kala and Gaur

16 | Chapter 2

200 (Gupta, 1989), Valley of Flowers, Glacial Valley, Garhwal, 3800-4200 m, Malhotra BM4601, 4645 (Gupta, 1989).

2. Neopicrorhiza scrophulariiflora Hong, Opera Bot. 75: 56. 1984 – Picrorhiza kurrooaauct. non Royle: Hook. Fl. Brit. Ind. 4: 290. 1885; Picrorhiza auct. non Royle: Pennell,Monogr. Acad. Nat. Sci. Philad. 5: 65. 1943 (as ‘scrophulariaeflora’).Distribution: Eastern Himalaya to mountains of Yunnan, 4300-5200 m (Pennell, 1943),

Uttar Pradesh to Southwest China, 3600-4800 m (Polunin and Stainton, 1990). UttarPradesh: Munsiari tehsil, Pithoragarh District, Kumaon (Malhotra and Balodi, 1984),Pithoragarh District, Garhwal (Balodi, 1995), Thungnath, Uttarakhand, Garhwal, 3600 m,cultivated, Smit 9602; Nepal: Bagmati zone, Rasuwa district, Langtang (Suwal, 1993),Central Nepal (Bhattarai, 1993), Gorkha district, Central Nepal Olsen 303 (Olsen, 1998),Gosaikunda (lake Gosainkund, lo: 08524E, la: 2802N), Langtang 4270 m Kanai & Malla16257 (Malla, 1976), Jaljale Himal, East Nepal: Jaljale–Tin Pokhari, 4030-4130 m,TI9110180; around Banduke, 4150 m, TI9110217 (Ohba and Akiyama, 1992), Jumla district,Karnali, Western Nepal (Manandhar, 1986), Laurivinayak, Langtang 3900 m Malla 9246(Malla et al., 1976), Murkhagari, Gorkha, Central Nepal, 4000 m, cultivated, Smit 9601C,Thaple Himal, 4700 m (Kihara, 1955), West Nepal: PSW 4215; Central Nepal: SSW 1190;East Nepal: Williams 309; TI720508, 3500-4800 m (Hara et al., 1982); Sikkim: Naku Chhu,Smith & Cave 1965* (Pennell, 1943), North Sikkim; East Sikkim* (Kapahi et al., 1993),Zemu valley, Llonakh, 13,000-16,000 ft, Smith 1343, Smith 1965, Smith 2251* (Smith andCave, 1911; P. scrophulariiflora Pennell, 1943), Choktsering Chhu, north of Jongri, 4000-4200 m (Hara, 1966), Gamothang (Gopethang), 3800 m (Hara, 1966), Jongri (Dzongri), 4000m (Hara, 1963), Jongri–Olothang (Onglakthang), 3900 m (Hara, 1966), Preig Chu–Jongri(Prek Chhu), 2200-4000 m (Hara, 1966); Bhutan: Bhutan, 1971 Ramesh bedi 87, 121* (Bedi,1971).

Distribution of both species is restricted to the Himalayan region and China. While P.kurrooa occurs mainly in the Western Himalaya at an altitude of 3000-4300 meters, P.scrophulariiflora is found mainly in the Eastern Himalaya at an altitude of 4300-5200 meters(Pennell, 1943). These findings are confirmed by most of the botanical references, whichdescribe the occurrence of Picrorhiza species, as listed above. Marked locations (*) indicateinaccurate identification. The nomenclature of the collections by Smith and Cave has beencorrected by Pennell (1943), while Kapahi et al. (1993) have not made any distinctionbetween the two species (Kapahi, personal communication). Bedi (1971) does not include adescription of the plant, and probably unaware of the existence of P. scrophulariiflora, hemay have identified his material erroneously.


Figure 2. Distribution of Picrorhiza species in the Himalaya region: ▬ ▬ Picrorhizakurrooa Royle; ▬▬ Picrorhiza scrophulariiflora Pennell.

FieldworkThe plant material that was used for the experiments described in this thesis was

purchased from Gorkha Ayurved Company, Gorkha, Nepal. To establish the identity ofPicrorhiza species in this area, we performed fieldwork into Gorkha District, at Murkhaghari(Centre for Community Development and Research; alt. 4000 m). At this location, theexclusive presence of P. scrophulariiflora was observed (1996). Other observations are inagreement with the exclusive occurrence of P. scrophulariiflora in Gorkha District, CentralNepal (Olsen, 1998). Additional fieldwork was carried out at Thungnath, Garhwal in UttarPradesh, India (G.B. Pant Institute of Himalayan Environment and Development; alt. 4000m). At this location, the presence of P. scrophulariiflora was observed, as identified based onthe inflorescence (1996).

Except for fresh samples, market samples were obtained from different origin. Becausethe suppliers were generally unaware of the proper scientific names, they were asked for theorigin of the material, and this information was used to establish a preliminary identity (table1). Microscopical analysis of the material obtained from the market could not discriminatebetween the two species.

All identifications were done by H.F. Smit, Department of Medicinal Chemistry,Universiteit Utrecht, The Netherlands. Herbarium specimens are deposited at the NationalHerbarium of The Netherlands, Utrecht University Branch (U).

18 | Chapter 2

Table 1. Market samples obtained during fieldwork or by mail order.Supplier Acquired Origin Proposed identityRatna Enterprises, Colombo,Sri Lanka

1995 South India, Nepala P. scrophulariiflora

Nardevi, Kathmandu, Nepal Aug. 1996 Nepal P. scrophulariifloraLaurabir, Varanasi, UP, India Sept. 1996 Nepal P. scrophulariifloraArya Vastu Bhandar, DehraDun, Garhwal, UP, India

Oct. 1996 Garhwalb P. kurrooa

Arya Vastu Bhandar, DehraDun, Garhwal, UP, India

Spring 1997 Garhwalb P. kurrooa

Jammu, JK, India Nov. 1998 Himachal Pradesh P. scrophulariifloraHwa To Centre, Utrecht,Netherlands

July 1999 China, Nepalc P. scrophulariiflora

a According to the supplier, the material is obtained from South India, and probably originatesfrom Nepal; b According to the supplier, the material originates from Garhwal, and is P.kurrooa; c Olsen (1998), in describing the trade of medicinal plants from Gorkha, notices thatfor several years, China purchased all Picrorhiza stocks from the Delhi market, being mostlyPicrorhiza from Nepali origin. Therefore, the material probably belongs to P.scrophulariiflora.

Conclusion

In the present chapter, we described taxonomic aspects of the closely related speciesPicrorhiza kurrooa and P. scrophulariiflora. Because the plant material that was used for theexperiments described in this thesis was obtained as simplex, and as there are nodiscriminative features between the rhizomes of both species, the plant material could notdirectly be identified. However, an inventory of the geographical distribution of bothPicrorhiza species provided sufficient circumstantial evidence to establish its proper identity.The material was obtained from Gorkha Ayurved Company, Gorkha, Nepal, and originatedfrom Gorkha District. Literature review revealed that the distribution of P. kurrooa isrestricted to the Western Himalaya, from Kashmir to Kumaon, while P. scrophulariiflora isfound in the Eastern Himalaya, from Garhwal to Sikkim. This observation is a strongargument to consider the identity of the Gorkha material to be P. scrophulariiflora.Observations by Olsen (1998) confirm the exclusive occurrence of P. scrophulariiflora inGorkha District of Central Nepal. Fieldwork into Gorkha District at Murkhaghari (4000meter) supports this conclusion, showing only the presence of P. scrophulariiflora in thisarea. In flowering season, both species can readily be distinguished, on basis of theremarkable differences in the inflorescence.


References

Aswal, B.S., Mehrotra, B.N. (1994) Flora of Lahaul-Spiti. Bishen Singh Mahendra PalSingh, Dehra Dun, pp. 484-485, pl. 22.

Balodi, B. (1995) Studies on the floristics of Pithoragarh district in N.W. Himalaya withparticular reference to rare and endangered species and their conservation. In: Gupta,B.K. Higher plants of Indian subcontinent. Bishen Singh Mahendra Pal Singh, DehraDun, pp. 273-283.

Bedi, R. (1971) Herbal wealth of Bhutan. Publisher unknown, p. 9.Bentham, G. (1835) Scrophularineae Indicae. James Ridgway and Sons, London, p. 47.Bentham, G., Hooker, J.D. (1876) Genera Plantarum. Volume 2. Reprint 1979, Periodical

Expert Book Agency, Delhi, pp. 962-963.Bhattacharyya, U.C., Malhotra, C.L. (1984) A botanical exploration enroute Rupkund lake

(North-East Garhwal). In: Paliwal, G.S. (Ed.) The vegetational wealth of the Himalayas.Puja Publishers, Delhi, pp. 161-174.

Bhattarai, N.K. (1993) Folk medicinal use of plants for respiratory complaints in centralNepal. Fitoterapia 64, 163-170.

Brummitt, R.K. (1992) Vascular plant families and genera. Royal Botanical Gardens, Kew,p. 667.

Chandra, K., Pandey, H.C. (1983) Collection of plants around Agora-Dodital in Uttarkashidistrict of Uttar Pradesh, with medicinal values and folklore claims. Int. J. Crude DrugRes. 21, 21-28.

Chauhan, N.S. (1988) Endangered Ayurvedic pharmacopoeial plant resources of HimachalPradesh In: Kaushik, P. (Ed.), Indigenous medicinal plants – including microbes andfungi. Today and Tomorrow’s Printers and Publishers, New Delhi, pp.199-205.

Chowdhery, H.J., Wadhwa, B.M. (1984) Flora of Himachal Pradesh. Vol. 2. BotanicalSurvey of India, New Delhi, p. 530.

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20 | Chapter 2

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22 | Chapter 2

Traditional use of Picrorhiza inĀyurveda

3.1 Āyurveda

3.2 Āmavāta

3.3 Picrorhiza in Āyurveda

Chapter 3

24 | Chapter 3

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Traditional use of Picrorhiza in Āyurveda | 25

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26 | Chapter 3

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28 | Chapter 3

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32 | Chapter 3

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34 | Chapter 3

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A'J7'?&(/7&*<G

36 | Chapter 3

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M,A':6,C,J#)"'"P2,A':%6,,w,A':.,X

38 | Chapter 3

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40 | Chapter 3

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42 | Chapter 3

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44 | Chapter 3

@#&A#&+7*'-'+'C,rXNX,?DEMDG,@'P&<#'S*&<7'*(."7.+'*'SX,r7$S'#''O,@#&A#&+7*'+'+#$+7(&*'CH)S"'2X

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Constituents of Picrorhiza withimmunomodulatory and relatedactivities

4.1 Cucurbitacins

4.2 Iridoid glucosides

4.3 Phenylethanoids

4.4 Phenolics

Chapter 4

46 | Chapter 4

Introduction

Most of the publications on the biological activities and constituents of Picrorhiza dealwith the species P. kurrooa, while P. scrophulariiflora is scarcely described. However, theidentification of the raw material has usually not been elaborated on in these publications.Quite often, plant material is obtained through a chain of buyers and sellers, and ultimatelythere is no information on the origin of the material. Generally, no distinction is madebetween samples from Nepal (P. scrophulariiflora) and India (P. kurrooa). A properidentification can only be made when selective criteria are found to distinguish betweenrhizomes from both species, e.g. microscopically, chemically, or by DNA analysis.However, these criteria have not been met so far. The difficulty in distinguishing bothspecies from each other based on rhizomes only makes interpretation of experimental resultson Picrorhiza species ambiguous. This uncertainty has to be taken into consideration whenreferring to publications on P. kurrooa. In cases where the origin of the material in not clearlydescribed, there is a possibility that the material investigated is erroneously substituted for P.scrophulariiflora. These observations provide ample arguments to introduce publicationson both species in this chapter.

4.1 Cucurbitacins

IntroductionThe cucurbitacins constitute a group of triterpenic substances, especially known by

their bitterness and toxicity. They are characterized by the cucurbitane skeleton, are mainlytetracyclic, and oxygenated at different sites. The basic structure is 9β-methyl-19-norlanosta-5-ene (figure 1), also known as 10α-cucurbit-5-ene (Lavie and Glotter, 1971).Cucurbitacins are usually present within plants as β-glucosides and have wide-rangingpharmacological activities. Several authors have reviewed this group of compounds, froma chemical (Lavie and Glotter, 1971), biological (Rehm, 1960, Guha and Sen, 1975), orpharmacological perspective (Miró, 1995).

2

3 4

1

10

56

7

9

8

1211

19

H 13

14

15

16

17

20

22

23

24

25

26

27

21

18

30

H

H

28

H

29

Figure 1. Basic structure of the 10α-cucurbit-5-ene skeleton (9β-methyl-19-norlanosta-5-ene)

Constituents of Picrorhiza with immunomodulatory and related activities | 47

Cucurbitacins from PicrorhizaSo far, 23 different cucurbitacin glucosides and 1 aglucone have been isolated from

Picrorhiza species, mainly from P. kurrooa (table 1).

Table 1. Cucurbitacins isolated from P. kurrooa and P. scrophulariiflora.

OOHOHOH

OH O

OH

R

OH

R =

OH20

O

Stuppner et al., 1991†

Li et al., 1998‡

OH20

OH

Wang et al., 1993‡

20

O


OH20

OHO

Stuppner and Wagner, 1989a†

Li et al., 1998‡

OH20

OO

OStuppner et al., 1991†

OH20

OHO


OH20

OO

OLaurie et al., 1985†,Wang et al., 1993‡

OH20

OO

O


H20

OO

OStuppner et al., 1991†

R

OH

OH

OHO

OH

R =

HO


OOHOHOH

O O

O


48 | Chapter 4

Table 1. Continued.

OOHOHOH

OH O

OH

R

OH

O

R =

OH20

OHStuppner and Müller, 1993†

OH20

OHO


OH20

OO

OStuppner and Wagner, 1989a†

20

OOH

Stuppner and Müller, 1993†

20

OOH

OH


OOHOHOH

OH O

R

OH

O

O

R =

OH20

O


OH20

OH


OH20

OHStuppner and Müller, 1993†

OH20

OHO

arvenin IIIStuppner et al., 1990†

OH20

OO

Oarvenin IStuppner and Wagner, 1989a†

OH20

OO

Oarvenin IIStuppner and Wagner, 1989a†

20

OOH


20

OOH

OH


† Constituent of P. kurrooa; ‡ constituent of P. scrophulariiflora.

CytotoxicityStrong cytotoxic effects have been described for several cucurbitacins, using KB cell

cultures, derived from human nasopharyngeal carcinoma, or HeLa cells (Miró, 1995). Itwas suggested that the cytotoxic mechanism involves selective interference with DNAsynthesis, as seen by the inhibition of [3H]-thymidine incorporation in cultured


lymphocytes incubated for 30 min with cucurbitacin B, D, or dihydrocucurbitacin B. RNAand protein synthesis were not affected (Whitehouse and Doskotch, 1969). Laterexperiments showed that cucurbitacin I inhibits the incorporation of radioactive precursorsinto DNA, RNA, as well as protein in HeLa S3 cells. ID50 values of several cucurbitacinswere similar to the ED50 values of cucurbitacin-induced growth inhibition, suggesting arelationship between inhibition of intracellular metabolic activity and growth-inhibitoryactivity of cucurbitacins. Cell lysis due to exposure to cucurbitacins was excluded(Witkowski et al., 1984).

Table 2. Schematic presentations of some relevant cucurbitacins.

OH

O

OH

OHO

O

H HO O

cucurbitacin B

OH

O

OH

OHO

OH

H HO

cucurbitacin D

OH

O

OH

OHO

O

H HO O

cucurbitacin E

OH

OH

OH

OHO

OH

H HO

cucurbitacin F

OH

O

OH

OHO

OH

H HO

cucurbitacin I

OH

O

OH

OHO

OH

H HO

cucurbitacin R

Cucurbitacins were also shown to inhibit the binding of cortisol to glucocorticoidreceptor (GCR) in HeLa cells at 37 °C in a dose-dependent manner. At 0-4 °C and in cell-free HeLa systems, however, inhibition was decreased or completely abrogated, suggestinga metabolic transformation of cucurbitacins in functionally HeLa cells into derivatives witha higher affinity to GRC than the parent compounds. Moreover, the inhibition of cortisolbinding to GCR by cucurbitacins in intact cells at 37 °C showed a strong correlation withcytotoxic activity, suggesting that cytotoxicity of cucurbitacins involves binding to theglucocorticoid receptor (Witkowski and Konopa, 1981). Incorporation experimentsconfirmed the suggestion of an active metabolite, because cucurbitacin I suppressed the

50 | Chapter 4

incorporation of radio-labeled precursors when cells were permeabilized upon exposure,but did not suppress incorporation when cells were exposed to cucurbitacin I afterpermeabilization (Witkowski et al., 1984). Later experiments, however, have shown thatglucocorticoids alone do not affect the incorporation of radio-labeled precursors into HeLaS3 cells and, in combination with cucurbitacin I, could not reverse its inhibitory activity,while the concentrations of steroids used were large enough to induce complete inhibition.These data suggest that the inhibition of macromolecule synthesis is not mediated by theGCR, but instead might involve immediate extranuclear effects of glucocorticoids, whichare not necessarily receptor-mediated (Witkowski et al., 1984).

Effects on cytoskeletonCucurbitacins D, I, E, and 2-methoxy-cucurbitacin E were shown to induce

morphological changes to Ehrlich ascites tumor cells at low concentrations. Only at higherconcentrations, respiration, permeability, and viability of these cells were affected, withoutan increase in morphological changes. Similar changes were observed in samples that weretaken 1 hour after i.p. injection of cucurbitacin D into ascites-bearing mice. In samplestaken 24 h after injection, however, no deformed or necrotized tumor cells were found(Gitter et al., 1961). Furthermore, it was shown that lymphocytes of chronic lymphaticleukemia patients as well as those of leukocytotic lymphosarcoma patients were muchmore sensitive to cucurbitacin D (elatericin A) than those isolated from normal individuals(Shohat et al., 1967). Exposure of prostrate carcinoma cells to cucurbitacin E also led tothe development of morphologically abnormal, multinucleated cells, which presented achange in vimentin distribution. Furthermore, cucurbitacin E induced cell growthinhibition of prostrate carcinoma cells, even after drug removal. It was shown to disrupt theF-actin cytoskeleton, which correlated with antiproliferative effects (Duncan et al., 1996).Moreover, cucurbitacin E was shown to interfere with the actin cytoskeleton of humanendothelial cells, preferentially targeting proliferating versus quiescent cells. It wassuggested that the condensation of actin microfilaments in quiescent cells, which does notoccur in proliferating cells, renders the molecular targets for cucurbitacin E unavailable forinteraction (Duncan and Duncan, 1997).

Antitumor activityCytotoxic and antiproliferative effects, as described above, make cucurbitacins

interesting agents in the treatment of tumors. The National Cancer Institute (NCI) hasscreened several plants containing cucurbitacins and has isolated their active constituents(Fuller et al., 1994). Cucurbitacins have shown in vivo inhibitory activity in severalcarcinoma, sarcoma, and leukemia models (Gitter et al., 1961, Gallily et al., 1962,Kupchan et al., 1967, Lavie and Glotter, 1971, Kupchan et al., 1972, Konopa et al., 1974,Fang et al., 1984, Reddy et al., 1988).

Immunological and related activitiesCucurbitacin B, isolated from Ecballium elaterium (Cucurbitaceae), exhibited anti-

inflammatory activity in acetic acid-induced vascular permeability, serotinin-induced pawedema, and bradykinin-induced paw edema in mice (Yesilada et al., 1988, 1989).Furthermore, cucurbitacins B and E, isolated from Wilbrandia ebracteata (Cucurbitaceae),showed anti-inflammatory activity in carrageenan-induced paw edema in rats (Peters et al.,


1997), while cucurbitacin B reduced PGE2 levels in exudates of carrageenan-inducedpleurisy in mice (Peters et al., 1999). Cucurbitacin E, isolated from Conobea scoparioides(Scrophulariaceae), and several related cucurbitacins were demonstrated to inhibit LFA-1-and ICAM-1-mediated JY/HeLa cell adhesion. These results were in line with theinhibition of actin polymerization in fMLP-stimulated neutrophils, suggesting that themechanism of inhibiting cell adhesion is by interference with the cytoskeleton (Musza etal., 1994).

In addition to modulation of cell-mediated immunity, interference with humoralimmune responses has also been described. Picfeltarraenins, cucurbitacin glycosidesisolated from Picria fel-terrae (Scrophulariaceae), were shown to inhibit the classical andalternative pathways of the complement system (Huang et al., 1998). Cucurbitacin Ishowed only minor inhibition of the classical pathway (Knaus and Wagner, 1996).

In contrast to cucurbitacin B, cucurbitacin D was shown to enhance capillarypermeability. The activity was in a similar order of magnitude as that of histamine, whilethe activity could not be reversed with antihistamines. The enhanced permeability wasassociated with a persistent fall in blood pressure and the accumulation of fluid in thoracicand abdominal cavities in mice. The long latent period before the appearance ofpharmacological effects, suggests that the compound had to be converted into an activemetabolite (Edery et al., 1961).

Adaptogenic activitiesCucurbitacin R-diglucoside was shown to moderately stimulate the secretion of

corticosterone by the adrenal cortex, and to inhibit stress- or ACTH-induced corticosteroneformation in vitro and in vivo, suggesting an adaptogenic effect. Furthermore, preliminaryadministration of cucurbitacin R reversed stress-induced alterations in eicosanoid levels,increasing PGE2 and decreasing 5-HETE, which supports the suggestion of adaptogenicactivity (Panossian et al., 1997, 1999). Cucurbitacin B, isolated from Ecballium elaterium,was shown to have antihepatotoxic activities. It reduced CCl4-induced liver damage asshown by a decrease in sGPT levels, hepatic collagen and β-lipoproteins, and a reductionof pathological liver changes (Han et al., 1979, Miró, 1995, Agil et al., 1999).

In vivo toxicityDue to severe toxicity, the medicinal usage of plants that produce high amounts of

cucurbitacins has become obsolete. LD50 values of several cucurbitacins are described inliterature, ranging from 0.5 mg/kg for cucurbitacin B (rabbits, i.v.) to 340 mg/kg forcucurbitacin E (mice, p.o.) (David and Vallance, 1955, Edery et al., 1961, Le Men et al.,1969). Particularly, the absence of ∆1, and the presence of ∆23 and 25-acetoxy functionalitiesseem to enhance toxicity (Le Men et al., 1969).

4.2 Iridoid glucosides

IntroductionIridoids represent a large group of monoterpenes and accumulate in a number of plant

families, mainly concentrated in the subclass Asteridae (Cronquist system). They consist of

52 | Chapter 4

a basic cyclopentano-[c]-pyran skeleton, which is formed during ring closure of 8-epi-iridodial (figure 2) and usually occur as glucosides. Several authors have reviewed theiroccurrence (El-Nagar and Beal, 1980, Boros and Stermitz, 1990), distribution (Hegnauerand Kooiman, 1978), biosynthesis (Inouye, 1978), and biological activities (Ghisalberti,1998, Mandal et al., 1998).

1O

34

9

5

6

8

7

H

HOH10

11

1O

34

9

5

6

8

7

H

HO10

11

Figure 2. Diagram of 8-epi-iridodial, precursor in the biosynthesis of catalpol.

Iridoids from PicrorhizaSeveral iridoid glucosides have been isolated from Picrorhiza species (table 3).

Extensive research has been devoted to standardized iridoid fractions of P. kurrooa, e.g.kutkin and Picroliv. Kutkin is obtained by crystallization and consists of the glucosidespicroside I and kutkoside in a ratio of 1: 2 and other minor glycosides (Singh and Rastogi,1972, Ansari et al., 1988). Picroliv is a similar but less purified fraction, containing about60 % of an equal mixture of picroside I and kutkoside (Dwivedi et al., 1989). More recentpublications describe Picroliv as a standardized iridoid fraction containing 60 % of thismixture in a 1: 1.5 ratio (Dhawan, 1995).

Table 3. Iridoid glucosides isolated from P. kurrooa and P. scrophulariiflora.

O

R1

O

R2O

OOHOHOH

R3

R1 = OH, R2 = OH, R3 = OHcatalpolWang et al., 1993‡

R2 = OH, R3 = OH, R1 = R2 = OH, R3 = OH, R1 = R1 = OH, R3 = OH, R2 =

O

O

veronicosideStuppner and Wagner, 1989b†

O

O

OH

OMe

picroside IIWeinges et al., 1972†


O

O

OH

OMe

kutkosideSingh and Rastogi, 1972†


Table 3. Continued.R2 = OH, R3 = OH, R1 =

O

O

OH

speciosideLi et al., 1998‡

O

O

OH

OH

verminosideLi et al., 1998‡

O

O

OH

OMe

6-feruloylcatalpolStuppner and Wagner, 1989b†

Simons, 1989†

OH

OMe

OO

picroside VSimons, 1989†

O

O

OMe

OH

minecosideStuppner and Wagner, 1989b†

Simons, 1989†

R1 = OH, R2 = OH, R3 =

O

O

picroside IKitagawa et al., 1971†

O

O

OH

picroside IVLi et al., 1998‡

O

O

OH

OMe

picroside IIIWeinges and Künstler, 1977†

O

OH

OHO

OOHOHOH

OH

aucubinWang et al., 1993‡

O

O

O

HOOH

OH

OOHOH OH

OH

O

OH

MeO

pikurosideJia et al., 1999†


Biological activitiesA wide range of biological activities have been attributed to iridoids, such as

cardiovascular, antihepatotoxic, choleretic, hypoglycemic, hypolipidemic, anti-inflammatory, antispasmodic, antitumor, antiviral, purgative, immunomodulatory,antioxidant, anti-phosphodiesterase, neuritogenic, molluscicidal, and leishmanicidalactivities (reviewed by Ghisalberti, 1998).

In a number of publications referring to the antihepatotoxic, hepatoregenerative,choleretic, and hypolipidemic effects of Picroliv, the preparation was shown to havesimilar or more potent activities than silymarin, a purified fraction of Silybum marianum(Asteraceae), commonly used in the treatment of liver disorders (reviewed by Dhawan,1995). Hepatoregenerative effects were associated with an increased recovery of the liveranti-oxidant system after partial hepatectomy or carbon tetrachloride-induced liver damageof Picroliv-treated rats (Rastogi et al., 1995, 1997). Hypolipidemic effects were observed

54 | Chapter 4

in rats with triton- and cholesterol-induced hyperlipidemia (Khanna et al., 1994). Oraladministration of Picroliv to mice prior immunized with sheep erythrocytes resulted in anincrease in hemagglutinating antibody titers, antibody-forming cells, and DTH responses tosheep erythrocytes. Furthermore, it enhanced non-specific immune responses of peritonealmacrophages and the [3H]-thymidine uptake by lymphocytes isolated from mice treatedwith Picroliv (Puri et al., 1992).

Anti-inflammatory activity was observed for kutkin in experimentally inducedarthritis, edema, vascular permeability, and leukocyte migration in rodents (Singh et al.,1993). Aucubin was also shown to potently inhibit phorbolester-induced edema in miceears, while catalpol and picroside II were not active (100 mg/kg, p.o.). The latter iridoidsshowed only minor anti-inflammatory effects upon topical administration (Recio et al.,1994). Moderate anti-inflammatory activity of picroside II, when administered topically,was confirmed later, while pikuroside was ineffective (Jia et al., 1999). Picrosides II, III,V, 6-feruloylcatalpol, and minecoside moderately inhibited chemiluminescence generatedby activated polymorphonuclear neutrophils (PMNs), while picroside I was not active;scavenging effects of these compounds were excluded (Simons, 1989, p. 102). Picrolivhowever, as well as picroside I, were shown to be moderate superoxide scavengers, whilekutkoside showed only weak activity (Chander et al., 1992). Furthermore, Picrolivprotected cells against hypoxia, enhanced the expression of VEGF and HIF-1, selectivelyinhibited protein tyrosine kinase activity, and reduced PKC (Gaddipati et al., 1999).

4.3 Phenylethanoids

Only recently, another group of compounds have been isolated from Picrorhizaspecies. P. scrophulariiflora was shown to constitute the phenylethanoid glucosidesscroside A-C and plantamajoside (Li et al., 1998). Plantamajoside, isolated from Plantagomajor (Plantaginaceae), is a potent inhibitor of phosphodiesterase and 5-lipoxygenase(Ravn et al., 1990), as is reported for similar compounds (Zhou et al., 1998). Moreover,phenylethanoids were shown to be antibacterial (Didry et al., 1999) and scavengers ofreactive oxygen species (ROS; Çalis et al., 1999).

Table 4. Phenylethanoids isolated from P. scrophulariiflora.

OR3OH

OHOH

OO

OHR2R1

O

OH

OMe

R2 = OH, R3 = glucose, R1 = R2 = OH, R3 = OH, R1 = R1 = OH, R3 = glucose, R2 =O

OH

MeOO

scroside A; Li et al., 1998‡

O

OH

MeOO

scroside B; Li et al., 1998‡

O

OH

MeOO

scroside C; Li et al., 1998‡


Table 4. Continued.

OO

OHOOH

OH

OH

OH

O

OH

OH

plantamajosideLi et al., 1998‡

‡ Constituent of P. scrophulariiflora.

4.4 Phenolics

IntroductionMonocyclic phenolic compounds are very common in the plant kingdom. They are

precursors and degradation products of lignin, which provides sturdiness to the plant and aphysical defense barrier against parasites (Steinegger and Hänsel, 1988, p. 366).Furthermore, they usually act as antifungal agents, providing direct protection to the plant(Kokubun and Harborne, 1995, Aoyama et al., 1997).

Phenolics from PicrorhizaVanillic acid, apocynin, and their respective glucosides have been isolated from

Picrorhiza (table 5).

Table 5. Phenolic compounds isolated from P. kurrooa and P. scrophulariiflora.R = OH R = glucoseOHO

ROMe vanillic acid

Rastogi et al., 1949†piceinStuppner and Wagner, 1989b†

R = OH R = glucoseO

ROMe

apocyninBasu et al., 1971†

androsinStuppner and Wagner, 1989b†



Antioxidant activitiesApocynin, and to a minor extent vanillic acid, were isolated guided by their inhibition

of chemiluminescence by activated PMNs (Simons et al., 1989). Apocynin completelyinhibited oxygen uptake by activated neutrophils and human eosinophils, but not by humanalveolar macrophages. This effect was mediated by an inhibition of NADPH oxidaseactivity, which was associated with the translocation of the cytosolic oxidase componentsp47phox and p67phox to the membrane fraction of neutrophils (Simons et al., 1990, Stolk et

56 | Chapter 4

al., 1994). In order to exhibit these effects, apocynin needs conversion into an activemetabolite by MPO and hydrogen peroxide, but so far, the structure of this compound hasnot been elucidated (’t Hart and Simons, 1992, Stolk et al., 1994, Müller et al., 1999).Furthermore, apocynin, upon prior activation, revealed inhibitory activity towards LTB4-,fMLP-, and PAF-induced chemotaxis of PMNs, which was associated with an inhibition ofcytoskeletal rearrangement after stimulation with fMLP (Müller et al., 1999). Anotheroxygen-related effect was discovered in the form of enhanced intracellular GSH synthesis,which appeared to be mediated by increased expression of γ-GCS mRNA and enzymeactivity through activation of transcription factor AP-1 (Lapperre et al., 1999). Theseeffects have led to the testing of apocynin in several experimental models pertaining to awide range of diseases, especially those in which NADPH oxidase plays a role.

Anti-inflammatory activityApocynin reduced joint swelling in collagen-induced arthritis in rats (’t Hart et al.,

1990). Furthermore, it reversed the inhibition of cartilage matrix proteoglycan synthesisinduced by mononuclear cells isolated from rheumatoid arthritis (RA) patients. Theseeffects were accompanied by a reduced production of pro-inflammatory cytokines frommonocytes and T cells, without affecting T-cell proliferation. Moreover, apocynindiminished the release of proteoglycan from cartilage matrix (Lafeber et al., 1999).Continuous administration of apocynin prevented the formation of ulcerative skin lesionsinduced by complete Freund’s adjuvant, without affecting humoral and cellular immuneresponses, as determined by serum antibodies and DTH responses (’t Hart et al., 1992).Moreover, apocynin concentration-dependently inhibited TXA2 formation, which effectwas associated with an inhibition of arachidonic acid-induced aggregation of bovineplatelets, whereas the release of PGE2 and PGF2α was stimulated (Engels et al., 1992, Linet al., 1999).

Other activitiesIn relation to the inhibitory effects of apocynin on NADPH oxidase activity,

implications for several diseases have been investigated. It has been shown to be effectivein mechanisms underlying, among others, sepsis (Wang et al., 1994, Mattson et al., 1996),atherosclerosis (Aviram et al., 1996, Holland et al., 1997, 1998, Chen et al., 1998), andasthma (Zhou and Lai, 1994, Tsukagoshi et al., 1994, 1995, Iwamae et al., 1998).Apocynin did not demonstrate genotoxic effects, as determined by the Ames test and thesister chromatid exchange assay (Pfuhler et al., 1995).

Conclusion

Picrorhiza species accumulate cucurbitacin glucosides, iridoid glucosides,phenylethanoid glucosides, and acetophenone glucosides. These compounds have a widerange of biological activities. Although hardly any information on the effects of thecucurbitacins from Picrorhiza is available, it seems reasonable that they exhibit effectssimilar to other cucurbitacins. In chapters 8 and 9, we confirm that some of the activities


described here specifically relate to particular cucurbitacins isolated from Picrorhiza aswell.

Regarding the activities described for constituents of Picrorhiza, anti-inflammatoryeffects seem to prevail. As far as iridoid glucosides are concerned, the effects describedmainly relate to a protection of the liver against damage and subsequent inflammation. Incontrast, phenylethanoid glucosides exhibit anti-inflammatory effects by inhibiting pro-inflammatory responses, e.g. by diminishing phosphodiesterase and 5-lipoxygenaseactivities. Acetophenones like apocynin contribute to the anti-inflammatory effects byinterfering with the formation of reactive oxygen species and enhancing the physiologicalantioxidant system, which lead to a wide range of secondary effects.

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Immunomodulatory activity ofPicrorhiza scrophulariiflora extracts

5.1 Introduction to relevant inflammatory processes

5.2 Immunomodulatory activity in vitro

5.3 Anti-inflammatory activity in vivo

5.4 Exclusion of toxicity in vivo

Chapter 5

64 | Chapter 5

5.1 Introduction to relevant inflammatory processes

Introduction

As described in chapter 3, Picrorhiza species have been used extensively for centuriesin the traditional medicine of India for the treatment of various immune-related diseases. Theprevious chapter gave an overview of the literature published on immunomodulatoryactivity of constituents from Picrorhiza species. The current chapter describesimmunomodulatory activity of P. scrophulariiflora Soxhlet extracts. To evaluate thisactivity, a series of in-vitro bioassays was performed as a reflection of nonspecific humoral(complement) and cellular (neutrophil leukocytes) defense mechanisms, and specificimmune responses (T-cell activation). This section provides a background on severalinflammatory processes that are relevant to the assays described.

The complement systemThe complement system provides an important nonspecific defense mechanism, which

involves a multitude of immune responses. Its primary goal is to recognize and eliminatepotentially harmful exogenous and endogenous structures from the human body. Thecomplement cascade is activated by complexes of antigens and their correspondingantibodies, which activate the classical pathway (CP), or by a variety of other initiators, asmicrobes or microbe- or plant-derived polysaccharides (zymosan, LPS, or inulin), whichinduce the lectin pathway (figure 1).

The classical activation pathway involves the complement components C1, C4, andC2, whereas the lectin pathway utilizes mannose-binding lectin (MBL) instead of C1, andfurther uses C4 and C2 as well. As stipulated, C1 binds to antigen-bound IgM or IgGantibodies, whereas MBL binds to sugar residues on their substrates, with as mostprominent specificities mannose, N-acetylglucosamine, and fucose. Activated C1 andMBL-associated serine protease (MASP) lead to the formation of a surface-bound C4/C2convertase, which converts C4 and C2 into a C3 convertase (C4bC2a), which is surface-bound as well. This enzyme splits the most important complement component, C3, intoC3b and C3a. Subsequently, the alternative pathway, which provides a kind ofamplification loop, becomes activated by binding factor B to surface-bound C3b. Theresulting complex is activated by factor D, which is present in serum, producing thealternative pathway C3 convertase (C3bBb). Binding of another C3b molecule to thiscomplex results in the formation of a C5 convertase (C3bBbC3b). Likewise, the classicaland lectin pathway generate another C5 convertase (C4bBbC3b). Both enzymes give rise tothe cleavage of C5 into C5b and C5a. Together with C6 and C7, C5b forms a lipophiliccomplex, which intrudes the bilayers of cells. In concert with C8, a C9 polymerase isformed which generates amphiphilic cylinders of about 13 C9 molecules. This complex(membrane-attack complex, MAC) destabilizes the membrane and causes cell deaththrough lysis of the cell (Liszewski and Atkinson, 1993, Law and Reid, 1995, Matsushitaand Fujita, 1996).

The smaller subunits, which are deposited into plasma, act as granulocyte attractants(C3a and C5a), anaphylatoxins (histamine releasers; C3a and C4a), or as kinins (C2b).

Immunomodulatory activity of Picrorhiza scrophulariiflora | 65

They ultimately lead to a vast array of immune responses, such as the migration anddegranulation of granulocytes, contraction of smooth-muscle cells, increased vascularpermeability, and vasodilatation, which are instrumental in eliminating the harmful factor(Frank and Fries, 1991, Law and Reid, 1995).

Figure 1. The activation steps of the complement system. Light shaded componentsindicate enzymatic activity, dark shaded components are subunits, released in plasma.Bold components are bound to cell-surfaces.

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Because of its potent pro-inflammatory and destructive capabilities, the complementsystem is provided with a very extensive regulatory system (Liszewski et al., 1996). Ifcomplement activation is not adequately controlled, it might cause tissue damage. Sublyticamounts of MACs were shown to induce non-lethal stimulatory effects in synovial cells,inducing the production of reactive-oxygen species and arachidonic-acid metabolites, therelease of IL-6, and increased expression of collagenase mRNA (Daniels et al., 1990, Jahnet al., 1993). Furthermore, C3 metabolites and C9 were detected in synovia of patients withacute arthritides and rheumatoid arthritis (RA), suggesting that the complement system isinvolved in acute-transient inflammatory reactions in the arthritic joints. Normally,complement-inhibiting proteins are present that protect against complement-mediatedtissue damage. However, the expression of the cell-associated complement inhibitorprotectin (CD59) was not seen in synovia of RA patients, making them targets for MAC-mediated effects (Konttinen et al., 1996). In such cases, it would be vital to enhance theregulation of complement activation in order to diminish tissue damage during flare-ups ofsynovitis.

Polymorphonuclear leukocytes (PMNs)A further role of complement proteins is opsonization of antigens, allowing them to

bind to receptors on PMNs. This causes activation of these neutrophils, ultimately leadingto a respiratory burst and degranulation of these cells. The products of this activationprocess consist of reactive oxygen species, peroxidases, proteinases, proteases, hydrolases,cytokines, and several other activator proteins (Root and Cohen, 1981, Cassatella, 1995).These mediators of inflammation activate other immune cells, such as lymphocytes andmonocytes, or directly target the potentially pathogenic substance to the liver in order toeliminate it from the body. However, due to lack of control of these processes, as is thecase in autoimmune diseases, such an immune response leads to a massive local andultimately systemic inflammation. Tissues may be damaged, mutilated, and destroyed,releasing even more mediators that sustain the inflammation and lead to degenerativechronic diseases, like rheumatoid arthritis (Bird, 1989).

T lymphocytesIn a later stage of the disease, T lymphocytes take over the leading role in the

inflammatory process. T cells are key players in cell-mediated immunity. In order togenerate an effective immune response, activation and subsequent expansion of antigen-specific lymphocytes is required. Activation of T cells initiates when T-cell receptors(TCRs) bind to specific antigens, in association with major histocompatibility complex(MHC) molecules on the membrane of antigen presenting cells (APC). Several otherproteins and receptors are associated with the TCR and play a cooperative role in acontrolled manner of signal transduction (Abraham et al., 1992, Chambers and Allison,1997, 1999). A persistent stimulatory signal ultimately leads to expansion anddifferentiation of the specific T cells, under the influence of an array of cytokines andgrowth factors (Swain, 1999).


Proliferation versus apoptosisActivation of T-cells induces signal transduction pathways that lead to the release of

IL-2 and the expression of IL-2 receptor (IL-2R) during the transformation of T-cells fromthe G0 to the G1 phase (Swain, 1999). The autocrine interaction of IL-2 with its receptorinduces T-cell progression from the G1 to the S phase, ultimately leading to proliferation(Cantrell and Smith, 1984, Stern and Smith, 1986). In normal situations, cell proliferationand cell death are balanced, leading to homeostasis. Several autoimmune diseases,however, are associated with an increased survival of potential autoreactive T lymphocytes.Cell accumulation can result from either increased proliferation or the failure of cells toundergo apoptosis in response to appropriate stimuli (Thompson, 1995). In contrast tonecrotic cell death, apoptosis follows a specific pattern of membrane rearrangement,cytoplasmic and nuclear condensation, and DNA cleavage. This process leads to theformation of encapsulated cellular fragments, which are ingested by surroundingphagocytes, without leading to a massive immune response, as is the case in cell necrosis(Kerr et al., 1972, Savill et al., 1993).

Rheumatoid arthritisActivated T lymphocytes that are differentiated into pro-inflammatory effector cells

release cytokines, which further induce cell-mediated immune responses (Swain, 1999).They have been implicated as important mediators in inflammation and in the pathogenesisof autoimmune diseases such as rheumatoid arthritis (Maini et al., 1995, Perkins, 1998).Within the articular synovium, they activate synovial macrophages that produce a range ofpro-inflammatory cytokines such as IL-1β, TNFα, IL-6, and IL-15. These cytokines, whichare abundantly present in articular synovium, induce proliferation of synoviocytes,enhancing local inflammatory processes. These affect lymphocytes, chondrocytes,fibroblasts, and osteoclasts, inducing damage of bone, cartilage, and other connective tissuecomponents (Feldmann et al., 1996, Vaishnaw, et al., 1997, Starkebaum, 1998).

Furthermore, T cells that predominantly belong to the CD4+ subpopulation showpartial cross-reacting with stress protein, including heat-shock protein 60. Since similarproteins are generated by bacteria, but also the human body, the inflammatory response isat first instance heteroimmune, and later on predominantly autoimmune.

Therapeutic measures of RA often focus on immunomodulatory activity, by means ofnonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, or disease-modifyingantirheumatic drugs (DMARDs; ACR, 1996a). While NSAIDs only afford symptomaticrelief, without altering the course of the disease or preventing joint damage,glucocorticoids and DMARDs interfere with the release of pro-inflammatory cytokines(e.g. TNFα, IL-1, IL-2). As such, they have the potential to reduce and prevent jointdamage and to preserve joint integrity and function (Shevach, 1995, Cronstein, 1996). Amajor disadvantage of these drugs, however, is their toxicity, which is usually proportionalto their efficacy (ACR, 1996b, Jackson and Williams, 1998). These side effects imply anongoing search for lead compounds that are able to selectively interfere with thepathological processes underlying chronic inflammation.

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5.2 Immunomodulation in vitro

Introduction

Hence, our search for compounds that interfere with the activation of complement, therelease of chemiluminescence by neutrophil leukocytes, and the proliferation of Tlymphocytes, as a reflection of nonspecific humoral and cellular defense mechanisms, andspecific immune responses.

Materials and methods

MaterialsHuman pooled serum (HPS) was obtained from healthy volunteers (Bloedbank

Midden-Nederland, Utrecht, Netherlands), veronal buffers, and antigen-coated sheep andrabbit erythrocytes were prepared according to Klerx et al. (1983). Veronal-buffered saline(VBS0) contained 5 mM veronal and 150 mM sodium chloride in bidistilled water,adjusted to pH 7.35. VBS0 with additional 0.15 mM Ca2+ and 0.5 mM Mg2+ is referred toas VBS2+, whereas VBS0 containing 2.5 mM Mg2+ and 0.8 mM EGTA is called EGTA-VBS. Erythrocytes were obtained from arterial blood supplied by Charles River (Someren,Netherlands) and were washed thrice before use. Sheep erythrocytes were sensitized byincubation with 1/800 diluted amboceptor (IgG antibody; Behring Diagnostics, Marburg,Germany) for 10 min, washed, and resuspended in VBS2+ (4⋅108 cells/ml). Rabbiterythrocytes were suspended in EGTA-VBS (3⋅108 cells/ml).

A suspension of serum-treated cell walls of Saccharomyces cerevisiae (zymosan) wasprepared by incubation of zymosan (Sigma Chemicals, St. Louis, MO, USA) with HPS at37 °C for 30 min and subsequent washing and suspension in Hank’s balanced salt solution(HBSS; Gibco BRL, Life Technologies, Pailsey, Scotland).

Phythaemagglutinin (PHA) was obtained from Difco (Detroit, MI, USA); RPMI 1640medium and fetal calf serum (FCS) from Gibco BRL and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) from Sigma Chemicals. Sodium dodecyl sulphate(SDS) solution (ICN Biomedicals, Aurora, Ohio, USA) was prepared by dissolving 20 gSDS in a mixture of 50 ml distilled water and 50 ml dimethyl formide and adjusting the pHto 4.7 with 80 % (v/v) acetic acid.

Plant materialDried rhizomes of P. scrophulariiflora were purchased from Gorkha Ayurved

Company (Gorkha, Nepal). These samples had been collected in the Gorkha District. Olsen(1998) described the exclusive occurrence of P. scrophulariiflora Pennell in this area.Fieldwork confirmed the presence of P. scrophulariiflora in Gorkha District, at an altitudeof 4000 meter onwards, as identified by H.F. Smit, Department of Pharmacy, UniversiteitUtrecht, The Netherlands (Smit 9601), and described in chapter 2. Herbarium specimensare deposited at the National Herbarium of The Netherlands, Utrecht University Branch(U).


Extraction procedureFive hundred grams of powdered rhizomes were subjected to sequential Soxhlet

extraction, using light petroleum, diethyl ether, ethyl acetate, and methanol as extractionfluids, respectively. The residual plant material was extracted with water under reflux for 2hours. Organic solvents were removed under reduced pressure, the residue was mixed withwater to get a homogenous suspension, and all aqueous samples were lyophilized. Theextracts yielded 1 %, 2 %, 6 %, 17 %, and 10 % (w/w), respectively.

Analytical methodHPLC separation was performed on a modular Gilson system (dual 306 pumps, 234

auto injector, 170 diode array detector) provided with UniPoint software (v. 1.65) forautomated sample handling, and an Alltech Alltima C18 column (5 µm, 250 x 4.6 mm),using a gradient of water and acetonitrile (0-5 min: 90 %; 5-10 min: 90 % → 80 %; 10-25min: 80 %; 25-40 min: 80 % → 20 %).

Complement assaysInhibitory activities of the extracts towards the classical (CP) and the alternative (AP)

pathways of the complement system were determined by a modified hemolytic microassay(Klerx et al., 1983). In 96-wells round-bottom microtiter plates, HPS or heat-inactivatedserum as a control (CP: 50 µl; AP: 25 µl) was pre-incubated for half an hour at 37 °C withan appropriate dilution series of the extracts in buffer (CP: VSB2+; AP: EGTA-VB).Subsequently, erythrocytes (CP: 50 µl antibody-coated sheep erythrocytes; AP: 25 µl rabbiterythrocytes) were added and the plates were incubated for one hour at 37 °C. Finally, theplates were centrifuged (5 min, 2500 rpm), and 50 µls of the supernatants were mixed with200 µl water per well in flat-bottom microtiter plates. The amount of hemoglobin releasedwas measured at 405 nm by means of an ELISA reader (SLT Labinstruments). Controlsconsisted of erythrocytes incubated with water (100 % hemolysis) or buffer (0 %hemolysis).

Chemiluminescence assayPolymorphonuclear leukocytes (PMNs) were prepared from buffycoat residues, kindly

provided by the Bloedbank Midden-Nederland (Utrecht, The Netherlands), aftercentrifugation in Ficoll-Hypaque, according to manufacturer’s instructions (AmershamPharmacia, Uppsala, Sweden). Cells were diluted to 1×107 PMNs/ml HBSS and dispensedin white 96-wells flat-bottom microtiter plates in 50-µl amounts. Subsequently, 50 µl of anappropriate dilution range of extract and 50 µl luminol (0.1 mM) were added to each well.The cells were activated with 50 µl serum-treated zymosan (0.8 mg/ml) after which theluminescence of each well was monitored every 2 min during a 30-min period, in a TitertekLuminoscan luminometer (TechGen International). Maximum peak levels were used tocalculate the inhibitory activity of extracts compared with a control. Controls consisted ofcells with luminol and buffer.

70 | Chapter 5

T-cell proliferation assayPeripheral blood mononuclear cells (PBMC) were prepared from buffycoat residues

(Bloedbank Midden-Nederland) using Ficoll-Hypaque centrifugation, according tomanufacturer’s instructions (Amersham Pharmacia). Under sterile conditions, the cells werediluted to 2×106 cells/ml RPMI 1640 (supplemented with 10 % FCS) and dispensed in 96-wells microtiter plates (50 µl/well). Proliferation of cells was induced by 50 µl PHA (0.1mg/ml), and 50 µl test samples were added in appropriate dilution ranges. After 4-dayincubation at 37 °C, T-cell proliferation was determined using a modified colorimetric MTTassay (Hansen et al., 1989). To each well, 25 µl MTT (1 mg/ml) was added and the platewas incubated for 4 hours at 37 °C. The formazan product formed was dissolved by adding50 µl SDS-solution and by shaking for 1 h on a microtiter plate shaker. Absorbance valueswere measured by an ELISA reader (SLT Labinstruments) operating at 550 nm. Controlsconsisted of PBL with PHA (100 % activity), PBL with medium (0 % activity), or sampleswith non-stimulated PBL.

Results

ComplementStrong inhibitory activity of P. scrophulariiflora towards the classical pathway of

complement was observed for the diethyl ether extract (IC50 = 1.9 ± 0.3 µg/ml). Theaqueous, light petroleum, methanol, and ethyl acetate extracts showed only moderateactivity, with IC50 values of 13 ± 2 µg/ml, 17 ± 5 µg/ml, 38 ± 5 µg/ml, and 55 ± 9 µg/mlrespectively (figure 2). None of the extracts tested inhibited the alternative pathway ofcomplement activation up to 200 µg/ml, the highest concentration tested.

��

��

��

��

PE DE EA Me Aq0

25

50

75

extract

IC50

(µ µµµg/

ml)

Figure 2. Inhibitory activity of Picrorhiza Soxhlet extracts on the classical pathway of thecomplement system. Data are expressed as means ± SEM of at least six experiments.

ChemiluminescenceModerate inhibitory activity against chemiluminescence produced by activated PMNs

was observed for the diethyl ether (IC50 = 53 ± 5 µg/ml), ethyl acetate (IC50 = 50 ± 5µg/ml), methanol (IC50 = 80 ± 2 µg/ml), and aqueous extracts (IC50 = 157 ± 34 µg/ml),


respectively. Light petroleum extracts were inactive up to the highest concentration tested(IC50 > 250 µg/ml; figure 3).

��

��

��

��

��

PE DE EA Me Aq0

100

200

300

extract

IC50

(µ µµµg/

ml)

Figure 3. Inhibitory activity of Picrorhiza Soxhlet extracts on chemiluminescence ofactivated neutrophils. Data are expressed as means ± SEM of at least six experiments.

T-cell proliferationActivity against mitogen-induced proliferation of T lymphocytes was only seen for the

diethyl ether extract (IC50 = 13 ± 2 µg/ml), while the other extracts were inactive up to thehighest concentration tested (IC50 > 200 µg/ml; figure 4).

��

��

��

��

PE DE EA Me Aq0

100

200

300

400

extract

IC50

(µ µµµg/

ml)

Figure 4. Inhibitory activity of Picrorhiza Soxhlet extracts on T-cell proliferation. Data areexpressed as means ± SEM of at least six experiments.

5.3 Anti-inflammatory activity in vivo

Introduction

Carrageenan-induced paw edema is a well-known model for studying the activity ofanti-inflammatory agents. Although the mechanisms underlying this model are not fullyunderstood yet, several inflammatory processes have been suggested to play a role, e.g.:activation of complement, release of reactive oxygen or nitrogen species by activated

72 | Chapter 5

phagocytes, and the release of pro-inflammatory cytokines by monocytes (Di Rosa, 1972,Hirschelmann and Bekemeier, 1981, Vinegar et al., 1987). Although these processes aremainly described for carrageenan-induced paw edema in rats, they may also apply toexperimentally induced immune responses in mice.

Although no experimental animal model completely resembles the features ofrheumatoid arthritis in humans, collagen-induced arthritis (CIA) in mice mimicsconsiderably the processes that play a role here (Trentham, 1982). Several authors havedemonstrated the role of T cells in the development of CIA, by the isolation of collagentype II (CII)-reactive T cells (Dallman and Fathman, 1985, Holmdahl et al., 1985), theresistance to CIA of anti-CD4 treated mice (Ranges et al., 1985), and amelioration of CIAby restricted TCR-specific antibodies (Moder et al., 1992, Osman et al., 1993).Furthermore, the development and progression of CIA was inhibited by IL-10, whichsuppresses Th1 cells and monocytes/macrophages (Tanaka et al. 1996, Walmsley et al.,1996). Direct evidence demonstrated that, except for a cellular response, an additionalhumoral factor is necessary in order to maintain an arthritic response (Seki et al., 1988).Additional observations suggest an important role for IL-12 to enhance the cellular andhumoral anti-CII responses in DBA/1 mice (Szeliga et al., 1996). This is supported by theobservation that administration of CII along with Freund’s adjuvant supplemented withMycobacterium (CFA) is strongly enhancing the susceptibility to CIA (Courtenay et al.,1980). Other predisposing factors that are required in order to induce CIA in mice are themouse strain: only mice with the MHC haplotype H-2q and with a certain Vα/β-TCRrepertoire are found susceptible to CIA (Wooley et al., 1981, Moder et al., 1992, Osman etal., 1993); sex: female sex hormones suppress the development of CIA (Holmdahl et al.1986); and age: highest susceptibility was found in mice ≥ 8 weeks of age (Wooley et al.,1983). In addition, especially native bovine collagen type II was able to elicit an arthriticresponse (Courtenay et al., 1980), and a booster immunization of CII enhanced thesusceptibility to CIA (Kato et al., 1996). These predisposing factors are all taken intoconsideration in applying this model to assess the effects of P. scrophulariiflora extracts inchronic inflammation.


Materialsλ-Carrageenan was obtained from Fluka (Buchs, Switzerland), and sodium

carboxymethylcellulose (CMC), highly viscous, from Bufa (Uitgeest, The Netherlands).Native bovine collagen type II from the metacarpal joint of the front paws was a kind giftfrom the Department of Rheumatology, Nijmegen, Netherlands. Complete Freund’sadjuvant (CFA) with Mycobacterium butyricum was obtained from Difco (Detroit, US).Cucurbitacin E was obtained from Extrasynthese (Genay, France), dexamethasone andprednisone from Bufa, and apocynin from Roth (Karlsruhe, Germany). Androsin wasobtained by glucosylation of apocynin using α-acetobromoglucose (Mauthner, 1918). Itsspectral data were found to be identical with those reported in literature (Junior, 1986).


AnimalsSpecific-pathogen-free male BALB/c mice were obtained from the Central Animal

Laboratory (Utrecht, The Netherlands) and female DBA/1 mice from Bomholtgard (Rye,Denmark). Both strains were maintained under standard housing conditions and providedwith food and water ad libitum. At the start of the experiments, the mice were 8-10 weeksold.

Carrageenan-induced paw edemaFor the determination of effects on acute inflammation, a carrageenan-induced paw

edema model was used. Male BALB/c mice (6 per group) were orally given extracts,suspended in 0.5 % CMC, either in three consecutive daily doses or in a single dose, onehour before the induction of paw edema. The control group was given dexamethasone (30mg/kg p.o.) one hour before induction, while the blank groups received vehicle only, eitherin three consecutive daily doses or in one dose. Inflammation was induced by subplantarinjection of a homogenous suspension of 1 % λ-carrageenan in physiological saline (25 µl).Paw edema was measured in every mouse hourly during seven hours after induction with acomputerized micrometer device. Mean values of treated groups were compared with meanvalues of a control group. The differences between the experimental groups and the controlgroup were statistically analyzed using the Student’s t-test.

Collagen-induced arthritisTo assess any effect on chronic inflammation, extracts were tested in a modified

collagen-induced arthritis model in mice (Joosten et al., 1996). A solution of CII in 0,05 Macetic acid (2 mg/ml) was suspended in an equal volume of CFA solution containing 4mg/ml Mycobacterium butyricum. At the start of the experiment, the mice wereconditioned to the handling procedures for one week to decrease stress. The mice wereimmunized by injecting 100 µl FCA emulsified in CII solution intradermally at the tail baseunder a mild anesthesia with ether. After three weeks, a booster injection of 200 µl (100µg) CII in phosphate-buffered saline was given intraperitoneally. In the course of the nextthree weeks, the clinical severity of arthritis was graded every other day, based on thechanges in swelling and redness of toes, foot pad, and ankle, with a maximum score of 2per paw (table 1).

74 | Chapter 5

Table 1. Scoring table of arthritis assessment.symptoms scoreswelling and redness of 1 toe 0.25swelling and redness of at least 2 toes 0.50swelling of foot pad 0.75swelling and redness of toes and swollen foot pad 1.00swelling and redness of toes and foot pad 1.25swelling and redness of toes and minor swelling of foot pad and ankle 1.50swelling and redness of toes and major swelling of foot pad and ankle 1.75swelling and redness of toes, foot pad, and ankle 2.00

Two persons independently performed the scoring, and the mean cumulative value forall paws was taken as the arthritic index (AI). Mice were considered to have arthritis whenAI was at least 1. Three endpoints were chosen for the evaluation of the experiment:incidence of arthritis (only including mice with AI ≥ 1), day of onset of arthritis (AI ≥ 1),and severity of arthritis (including all mice).

Extracts were suspended in 0.5 % CMC and given orally in one daily dose of 200mg/kg for 36 consecutive days, starting on the day of immunization. Prednisone (3 mg/kgp.o.) served as a positive control. Mean AI values of treated groups (n = 10) werecompared with mean values of the control group. The differences between the experimentalgroups and the control group were statistically analyzed using the Wilcoxon rank testunless indicated otherwise.

Results

Carrageenan-induced paw edemaTreatment of mice with light petroleum, diethyl ether, ethyl acetate, methanol, or

aqueous extracts (500 mg/kg) of P. scrophulariiflora one hour before the induction of pawedema did not inhibit paw swelling. The aqueous extract even enhanced the edema forabout 25 %, but this was only significant at t = 4 h. Treatment with dexamethasone (30mg/kg p.o.) inhibited paw edema significantly from two hours after induction onward, witha maximum effect (76 %) at t = 5 h (figure 5).


0 1 2 3 4 5 6 7 80

1

2PEblank

time (h)

swel

ling

(mm

)

0 1 2 3 4 5 6 7 80

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2DEblank

time (h)

swel

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(mm

)0 1 2 3 4 5 6 7 8

0

1

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time (h)

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(mm

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0 1 2 3 4 5 6 7 80

1

2

* * ** **

dexblank

time (h)

swel

ling

(mm

)

*** ***

Figure 5. Effects of single treatment with Picrorhiza extracts (500 mg/kg) anddexamethasone (30 mg/kg) on carrageenan-induced paw edema (PE = light petroleumextract, DE = diethyl ether extract, EA = ethyl acetate extract, Me = methanol extract, Aq= aqueous extract, dex = dexamethasone). Data were expressed as means ± SEM (n = 6per group) and statistically analyzed by the Student’s t-test. P-values are < 0.05 (*), <0.01 (**), or < 0.001 (***).

Administration of 500 mg/kg/day diethyl ether extract for three days induced asignificant inhibition of paw edema from 3 hours after induction onward, with a maximumeffect (41 %) at t = 3 h. Other extracts were not active. Prednisone (3 doses of 5mg/kg/day) inhibited paw edema for 51 % at t = 3 h, while a maximum effect (61 %) wasreached 5 hours after induction (figure 6).

76 | Chapter 5

0 1 2 3 4 5 6 7 80

1

2PEblank

time (h)

swel

ling

(mm

)

0 1 2 3 4 5 6 7 80

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2DEblank

time (h)

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(mm

)

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*

0 1 2 3 4 5 6 7 80

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time (h)

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(mm

)

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2Meblank

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)

0 1 2 3 4 5 6 7 80

1

2blankpred

**

*

** ** ***

time (h)

swel

ling

(mm

)

Figure 6. Effects of prolonged oral treatment with Picrorhiza extracts (500 mg/kg) andsingle oral treatment of prednisone (5 mg/kg) on carrageenan-induced paw edema (PE =light petroleum extract, DE = diethyl ether extract, EA = ethyl acetate extract, Me =methanol extract, Aq = aqueous extract, pred = prednisone). Data were expressed asmeans ± SEM (n = 6 per group) and statistically analyzed by the Student’s t-test. P-valuesare < 0.05 (*), < 0.01 (**), or < 0.001 (***).

To assess whether the inhibitory effects of the diethyl ether extract were dose-dependent, a second experiment was performed in which 500 and 1000 mg/kg/day,respectively, were administered orally for three days. Based on pilot experiments, it wasassumed that three doses of 250 mg/kg did not significantly inhibit carrageenan-inducedpaw edema. A significant inhibition of paw edema was observed upon administration of500 and 1000 mg/kg/day, starting two hours or one hour after induction, respectively, up tot = 6 h. A maximum effect was observed for 1000 mg/kg (64 %) at t = 3 h, and for 500mg/kg (48 %) at t = 4 h (figure 7).


0 1 2 3 4 5 6 7 80.0

0.5

1.0

1.5

*

****

**** **

***** ***

******

DE 1000 mg/kgDE 500 mg/kgblank

time (h)

swel

ling

(mm

)

Figure 7. Effect of prolonged treatment with diethyl ether Picrorhiza extract (DE; 1000and 500 mg/kg p.o.) on carrageenan-induced paw edema. Data were expressed as means ±SEM (n = 6 per group) and statistically analyzed by the Student’s t-test. P-values are <0.05 (*), < 0.01 (**), or < 0.001 (***).

In addition, apocynin, an inhibitor of NADPH oxidase and responsible for several anti-inflammatory effects (chapter 4), was tested to assess whether it was responsible for theeffects observed. Quantitative analysis was performed to determine the content of thismetabolite and its glucoside, androsin, in Soxhlet extracts of P. scrophulariiflora. HPLCanalysis revealed the presence of 1.4 % apocynin and 0.9 % androsin in the diethyl etherextract (table 2). Therefore, apocynin was tested in three doses (1, 10, and 100 mg/kg),given orally for three days before induction of paw edema. Measurement of paw swelling,however, did not show any inhibitory effect by apocynin in this model (figure 8).

Table 2. Contents of apocynin and androsin in Soxhlet extracts of P. scrophulariiflora.Extract Apocynin Androsinlight petroleum 0.35 ± 0.00 % nddiethyl ether 1.4 ± 0.1 % 0.86 ± 0.26 %ethyl acetate 1.0 ± 0.0 % 1.9 ± 0.01 %methanol nd 0.47 ± 0.02 %aqueous nd ndnd = not detectable

0 1 2 3 4 5 6 70.0

0.5

1.0

1.5100 mg/kg10 mg/kg1 mg/kgblank

time (h)

swel

ling

(mm

)

Figure 8. Effect of prolonged treatment with apocynin (100, 10, and 1 mg/kg p.o.) oncarrageenan-induced paw edema. Data were expressed as means ± SEM (n = 6 per group)and statistically analyzed by the Student’s t-test.

78 | Chapter 5

Collagen-induced arthritisIn addition to acute inflammation, we also examined the effects of extracts of P.

scrophulariiflora in a model of chronic inflammation. Preliminary experiments showedthat sub-chronic administration of high doses of plant material (1 g/kg) resulted in severegastro-intestinal symptoms (accumulation of undigested material in the stomach,obstruction in intestine), while administration of the diethyl ether extract (200 mg/kg) for 6weeks did not reveal these effects, as confirmed by toxicity studies (section 5.4). Ethylacetate and methanol extracts of P. scrophulariiflora (200 mg/kg) given from t = 0 onwardshowed only a minor decrease, while diethyl ether extract significantly (χ2; P < 0.05)inhibited the incidence of arthritis (AI ≥ 1). Furthermore, treatment with apocynin alsosignificantly inhibited incidence of arthritis (χ2; P < 0.01), while prednisone completelyinhibited development of collagen-induced arthritis (figure 9).

23 25 27 29 31 33 35 370

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100DEEAMeblank

time (d)

inci

denc

e (%

)

23 25 27 29 31 33 35 370

25

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100apopredblank

time (d)

inci

denc

e (%

)

Figure 9. Effects of oral treatments with Picrorhiza extracts (200 mg/kg), apocynin (20mg/kg), or prednisone (3 mg/kg) on the incidence of arthritis (DE = diethyl ether extract,EA = ethyl acetate extract, Me = methanol extract, apo = apocynin, pred = prednisone).Data were expressed as percentages of animals with AI ≥ 1.

The mean arthritic scores of mice treated with Picrorhiza extracts or controls,expressed as the arthritic index, did not differ significantly. Treatment with apocynin,however, moderately diminished the severity of arthritis, although not significantly. Themean arthritic index of mice treated with prednisone, however, remained zero throughoutthe experiment. The day of onset of arthritis shifted from 27 to 29 days for the extracts andto 31days for apocynin (figure 10).


23 25 27 29 31 33 35 370

1

2

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EADE

Meblank

time (d)

arth

ritic

inde

x

23 25 27 29 31 33 35 370

1

2

3 apopredblank

time (d)

arth

ritic

inde

x

Figure 10. Effects of oral treatment with Picrorhiza extracts (200 mg/kg), apocynin (20mg/kg), or prednisone (3 mg/kg) on the development of arthritis (DE = diethyl etherextract, EA = ethyl acetate extract, Me = methanol extract, apo = apocynin, pred =prednisone). Data were expressed as means ± SEM of all animals.

5.4 Exclusion of toxicity in vivo

Introduction

As indicated in section 5.3, in combination with carboxymethylcellulose (CMC),higher doses of P. scrophulariiflora extracts incidentally gave rise to gastro-intestinalobstructive reactions, sometimes leading to death of the animals. To exclude that thesetoxic effects had to be ascribed to metabolites probably enriched in the form of extracts,rather than to the whole plant product, or to a combination of extract with CMC, thetoxicity of extracts was tested in the absence of vehiculum.

Material and methods

AnimalsRandomly bred Swiss mice, aged 8-12 weeks, were obtained from Harlan (Zeist, The

Netherlands). The mice were randomly divided in groups of 7, maintained under standardhousing conditions, and provided with food and water ad libitum throughout theexperiment.

Toxicity assayDiethyl ether Soxhlet extract of P. scrophulariiflora (200 mg/kg) was administered by

gastrointestinal catheter three times a week (Mon., Wed., Fri.). After 3 weeks and 6 weeks,respectively, mice were weighed, killed, and their thymus, spleen, liver, and mesentericlymph nodes were removed. All organs were weighed individually, except for themesenteric lymph nodes, which were weighed together. Means of the treated groups werecompared with means of a control group. The differences between the experimental groupsand a control group were statistically analyzed using the Student’s t-test.

80 | Chapter 5

Results

Diethyl ether Soxhlet extract and ethanol reflux extract of P. scrophulariiflora wereadministered orally thrice a week for 3 or 6 weeks. Although the weights of the spleen andthe liver were somewhat increased after administration of diethyl-ether extract for 6 weeks,these differences were not statistically significant, as determined using the Students t-test(figure 12B). Moreover, no significant differences were found in total weight or in weightof thymus, spleen, liver, or mesenterial lymph nodes between mice treated with diethylether extract (200 mg/kg p.o.) and blanks or between mice treated with ethanol extract (200mg/kg p.o.) and blanks (figures 11 and 12).

3 bl 6 bl 3 bl 6 bl0

10

20

30

40DE extractEt extractblank

treatment (weeks)

wei

ght (

g)

Figure 11. Weights of mice after 3 or 6 weeks of treatment with diethyl ether Soxhletextract (DE) or ethanol reflux extract (Et) of P. scrophulariiflora. Data were expressed asmeans ± SEM (n = 6-7 per group) and statistically analyzed by the Student’s t-test.

2DE extractA

B

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thymus spleen liver lymph nodes0

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.
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igure 12. Weights of organs isolated from mice treated with diethyl ether Soxhlet extractDE) or ethanol reflux extract (Et) of P. scrophulariiflora. A: After 3 weeks of treatmentith DE extract. B: After 6 weeks of treatment with DE extract. C: After 3 weeks of

reatment with Et extract. D: After 6 weeks of treatment with Et extract. Data werexpressed as means ± SEM (n = 6-7 per group) and statistically analyzed by the Student’s-test.

iscussion

The diethyl ether Soxhlet extract of P. scrophulariiflora was the most anti-nflammatory of all extracts prepared. The activity concerned both nonspecificcomplement and respiratory burst) and specific (T-cell proliferation) immune responses.urthermore, significant inhibition of acute inflammation in vivo was observed for thisxtract after prolonged treatment. The light petroleum and aqueous extracts wereoderately active in inhibiting the classical complement pathway, while the ethyl acetate

nd methanol extracts moderately inhibited chemiluminescence by activated PMNs. Inivo, however, these extracts were completely inactive.

The inhibition of the classical complement pathway can be due to several effects.ctive compounds in the extract can bind or alter particular components of the

omplement system, or inactivate converting enzymes, eliminating their involvement in theascade. Alternatively, certain compounds can activate the complement cascade, with asesult a depletion of complement activity. Further research has to reveal the nature of theffects observed.

82 | Chapter 5

The in vivo anti-inflammatory activity of the diethyl ether extract is in line with theeffects observed in vitro for this extract. Although not all mechanisms underlyingcarrageenan-induced paw edema have been identified yet, it is a very useful model for thescreening of anti-inflammatory agents. In rats, the model has been used since 1962 for thispurpose (Winter et al., 1962). Regarding the possible mechanisms involved, severalinflammatory processes have been suggested to play a role, as e.g. activation ofcomplement, release of mediators by activated mast cells, release of kinins, the release ofreactive oxygen or nitrogen species by activated phagocytes, arachidonic acid metabolites,and pro-inflammatory cytokines (Di Rosa, 1972, Hirschelmann and Bekemeier, 1981,Vinegar et al., 1987). We assume that at least a part of these processes is subject ofinhibition by P. scrophulariiflora extracts.

Moderate inhibitory activity on activated neutrophils in vitro, as shown for the diethylether and the ethyl-acetate extracts, may be ascribed to acetophenones. Similar effects havebeen ascribed to P. kurrooa extracts, in which such an activity is mainly attributed toapocynin (Simons, 1989). The presence of apocynin has been reported in P.scrophulariiflora as well (Wang et al., 1993). The presence of apocynin was established byHPLC in diethyl ether and ethyl acetate extracts, while androsin, its O-β-D-glucoside, wasidentified in diethyl ether, ethyl acetate, and methanol extracts of P. scrophulariiflora.However, androsin did not show any inhibitory effect on the respiratory burst of activatedneutrophils in vitro (data not shown). Therefore, apocynin may be largely responsible forthe moderate in vitro anti-oxidative activity of the diethyl ether and the ethyl acetateextracts.

Furthermore was assessed whether apocynin was responsible for the anti-inflammatoryactivity of the diethyl ether extract in the carrageenan-induced paw edema model. Althoughandrosin was not active in vitro, it may play a similar role as apocynin in vivo, because thesugar moiety of O-β-D-glucosides is generally hydrolyzed by β-glucosidase, produced bythe intestinal flora, following oral administration (Rowland et al., 1986). The content ofapocynin and androsin in the diethyl ether extract was determined at 1.4 % and 0.9 %(w/w), respectively. Therefore, apocynin was tested in three consecutive daily doses of 1,10, or 100 mg/kg in carrageenan-induced paw edema. However, apocynin did not show anyanti-inflammatory effect in this model, suggesting that compounds other than apocynin areresponsible for the anti-inflammatory effects observed.

Collagen-induced arthritis (CIA) was used as a model to determine the effects of P.scrophulariiflora extracts on chronic inflammation. Treatment with 200 mg/kg diethylether extract significantly reduced the incidence of arthritis (AI ≥ 1), while ethyl acetateand methanol extracts did not affect this parameter. However, neither extract affected theseverity of arthritis. In addition, the effect of apocynin (20 mg/kg p.o.) on the developmentof CIA was assessed. Apocynin significantly reduced arthritis incidence, while it onlymoderately decreased arthritis severity. These results are partially in line with the anti-arthritic activity of apocynin in collagen-induced arthritis in rats, obtained when thesubstance was added to the drinking water of rats (’t Hart et al., 1990). One possibleexplanation for the discrepancy observed with our experiments is the short half-life ofapocynin, which might require continuous exposure of animals to apocynin in order tomaintain body concentrations that are sufficient to reach the desired effect. Moreover,


apocynin might interfere with processes in CIA in rats, which are of lower importance inthe mouse model. At least, our results suggest that the in-vitro inhibitory effects of thediethyl ether extract on complement activation, chemiluminescence of activated PMNs,and T-cell proliferation, are not sufficient to inhibit collagen-induced arthritis in mice. Thismight be due to underdosage, low biological availability, an insufficient pharmacokineticprofile, or to metabolic inactivation of active compounds in vivo. It is also possible thatpathological processes, other than those referred to and insufficiently inhibited by thediethyl ether extract, lead to the development of collagen-induced arthritis in mice.

Because sub-chronic administration of powdered rhizomes of P. scrophulariifloraresulted in severe gastro-intestinal symptoms, the toxicity of the active diethyl ether extractand an ethanol reflux extract were determined. Administration of diethyl ether or ethanolextract of P. scrophulariiflora for 3 and 6 weeks thrice a week to Swiss mice did not revealany significant toxicity involving body weigh and weights of critical lymphoid organsincluding thymus, spleen, liver, and mesenterial lymph nodes.

In conclusion, of the extracts of P. scrophulariiflora tested, the diethyl ether extractwas the most effective in inhibiting the classical pathway of complement, the release ofreactive oxygen species by activated neutrophil leukocytes, and the mitogen-inducedproliferation of T lymphocytes. Furthermore, this extract exposed anti-inflammatoryactivity in vivo, as demonstrated in an acute carrageenan-induced paw inflammation model.This effect may be due to the presence of compounds, which inhibit activation ofcomplement. The contribution of compounds that interfere with the release of reactiveoxygen species by activated PMNs to the anti-inflammatory effect observed seems lesspronounced, since apocynin, a potent anti-oxidant and present in the diethyl ether extract,was not effective in inhibiting carrageenan-induced paw edema. A combination ofimmunomodulating compounds with different mechanisms of action might be necessary toproduce anti-inflammatory activity in vivo. Several known, but also as yet unidentifiedimmunomodulatory compounds may contribute to this activity. To this end, a search forbiological active compounds was undertaken by means of activity-guided isolation, asdescribed in chapters 6 and 7.

The in vivo effects were not associated with significant toxicity, as tested in mice by asemi-chronic assay involving body weight and weights of critical lymphoid organs. Even indoses up to 100 × the intended dose of application in humans, the extracts were devoid ofsignificant toxicity.

References

Abraham, R.T., Karnitz, L.M., Secrist, J.P., Leibson, P.J. (1992) Signal transduction throughthe T-cell antigen receptor. Trends Biochem. Sci. 17, 434-8.

ACR - American College of Rheumatology (1996a) Guidelines for the management ofrheumatoid arthritis. Arthritis Rheum. 39, 713-722.

ACR - American College of Rheumatology (1996b) Guidelines for monitoring drug therapyin rheumatoid arthritis. Arthritis Rheum. 39, 723-731.

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Bird, I.N. (1989). Role of polymorphonuclear leukocytes in the pathogenesis of infectivearthritis. In: Infections and arthritis. Calabro, J.J., Dick, W.C. (Eds.) Kluwer AcademicPublishers, Dordrecht, pp. 135-152.

Cantrell, D.A., Smith, K.A. (1984) The interleukin-2 T-cell system: a new cell growth model.Science 224, 1312-6.

Cassatella, M.A. (1995) The production of cytokines by polymorphonuclear neutrophils.Immunol. Today 16, 21-26

Chambers, C.A., Allison, J.P. (1997) Co-stimulation in T cell responses. Curr. Opin.Immunol. 9, 396-404.

Chambers, C.A., Allison, J.P. (1999) Costimulatory regulation of T cell function. Curr. Opin.Cell Biol. 11, 203-10.

Courtenay, J.S., Dallman, M.J., Dayan, A.D., Martin, A., Mosedale, B. (1980) Immunisationagainst heterologous type II collagen induces arthritis in mice. Nature 283, 666-668.

Cronstein, B.N. (1996) Molecular therapeutics: methotrexate and its mechanism of action.Arthritis Rheum. 39, 1951-1960.

Dallman, M., Fathman, C.G. (1985) Type II collagen-reactive T cell clones from mice withcollagen-induced arthritis. J. Immunol. 135, 1113-1118.

Daniels, R.H., Houston, W.A.J., Petersen, M.M., Williams, J.D., Williams, B.D., Morgan,B.P. (1990) Stimulation of human rheumatoid synovial cells by non-lethal complementmembrane attack. Immunology 69, 237-242.

Di Rosa, M. (1972) Biological properties of carrageenan. J. Pharm. Pharmacol. 24, 89-102.Feldmann, M., Brennan, F.M., Maini, R.N. (1996) Role of cytokines in rheumatoid arthritis.

Annu. Rev. Immunol. 14, 397-440.Frank, M.M., Fries, L.F. (1991) The role of complement in inflammation and phagocytosis.

Immunol. Today 12, 322-326.Hansen, M.B., Nielsen, S.E., Berg, K.J. (1989) Re-examination and further development of a

precise and rapid dye method for measuring cell growth/cell kill. J. Immunol. Methods119, 203-210.

’t Hart, B.A., Simons, J.M., Knaan-Shanzer, S., Bakker, N.P., Labadie, R.P. (1990)Antiarthritic activity of the newly developed neutrophil oxidative burst antagonistapocynin. Free Radical Bio. Med. 9, 127-131.

Hirschelmann, R., Bekemeier, H. (1981) Effects of catalase, peroxidase, superoxidedismutase and 10 scavengers of oxygen radicals in carrageenin edema and in adjuvantarthritis of rats. Experientia 37, 1313-1314.

Holmdahl, R., Jansson, L., Andersson, M. (1986) Female sex hormones suppressdevelopment of collagen-induced arthritis in mice. Arthritis Rheum. 29, 1501-1509.

Holmdahl, R., Klareskog, L., Rubin, K., Larsson, E., Wigzell, H. (1985) T Lymphocytes incollagen II-induced arthritis in mice. Characterization of arthritogenic collagen II-specificT-cell lines and clones. Scand. J. Immunol. 22, 295-306.

Jackson, C.G., Williams, H.J. (1998) Disease-modifying antirheumatic drugs. Drugs 56, 337-344.

Jahn, B., Von Kempis, J., Kramer, K.L., Filsinger, S., Hänsch, G.M. (1993) Interaction of theterminal complement components C5b-9 with synovial fibroblasts – binding to the


membrane surface leads to increased levels in collagenase-specific messenger RNA.Immunology 78, 329-334.

Joosten, L.A.B., Helsen, M.M.A., Van de Loo, F.A.J., Van den Berg, W.B. (1996)Anticytokine treatment of established type II collagen-induced arthritis in DBA/1 mice.Arthritis Rheum. 39, 797-809.

Junior, P. (1986) Acetophenonglucoside aus Penstemon pinifolius. Planta Med. 52, 218-220.Kato, F., Nomura, M., Nakamura, K. (1996) Arthritis in mice induced by a single

immunization with collagen. Ann. Rheum. Dis. 55, 535-539.Kerr, J.F.R., Wyllie, A.H., Currie, A.R. (1972) Apoptosis: a basic biological phenomenon

with wide-ranging implications in tissue kinetics. Brit. J. Cancer 26, 239-257.Klerx, J.P.A.M., Beukelman, C.J., Van Dijk, H. and Willers, J.M.N. (1983) Microassay for

colorimetric estimation of complement activity in Guinea pig, human and mouse serum.J. Immunol. Methods 63, 215-220.

Konttinen, Y.T., Čeponis, A., Meri, S., Vuorikoski, A., Kortekangas, P., Sorsa, T., Sukura,A., Santavirta, S. (1996) Complement in acute and chronic arthritides: assessment ofC3c, C9, and protein (CD59) in synovial membrane. Ann. Rheum. Dis. 55, 888-894.

Law, S.K.A., Reid, K.B.M. (1995) Complement. Second edition, Oxford University Press,Oxford.

Liszewski, M.K., Atkinson, J.P. (1993) The complement system. In: Fundamentalimmunology. Paul, W.E. (Ed.). Third edition, Raven Press, New York, pp. 917-939.

Liszewski, M.K., Farries, T.C., Lublin, D.M., Rooney, I.A., Atkinson, J.P. (1996) Control ofthe complement system. Adv. Immunol. 61, 201-283.

Maini, R.N., Chu, C.Q., Feldmann, M. (1995) Aetiopathogenesis of rheumatoid arthritis. In:Mechanisms and models in rheumatoid arthritis. Henderson, B., Edwards, J.C.W.,Pettipher, E.R. (Eds.). Academic Press, London, pp. 25-46.

Matsushita, M., Fujita, T. (1996) The lectin pathway. Res. Immunol. 147, 115-118.Mauthner, F. (1918) Mitteilung aus dem II. chemischen Institut der Universität Budapest.

Über neue synthetische Glucoside. J. Prakt. Chem. 97, 217-224.Moder, K.G. Luthra, H.S., Kubo, R., Griffiths, M., David, C.S. (1992) Prevetnion of collagen

induced arthritis in mice by treatment with an antibody against the T cell receptor αβframework. Autoimmunity 11, 219-224.

Olsen, C.S. (1998) The trade in medicinal and aromatic plants from Central Nepal toNorthern India. Econ. Bot. 52, 279-292.

Osman, G.E., Toda, M., Kanagawa, O., Hood, L.E. (1993) Characterization of the T cellreceptor repertoire causing collagen arthritis in mice. J. Exp. Med. 177, 387-395.

Perkins, D.L. (1998) T-cell activation in autoimmune and inflammatory diseases. Curr. Opin.Nephrol. Hy. 7, 297-303.

Ranges, G.E., Sriram, S., Cooper, S.M. (1985) Prevention of type II collagen-induced arthritisby in vivo treatment with anti-L3T4. J. Exp. Med. 162, 1105-1110.

Root, R.K., Cohen, M.S. (1981) The microbial mechanisms of human neutrophils andeosinophils. Rev. Infect. Dis. 3, 565-598.

Rowland, I.R., Mallett, A.K., Bearne, C.A., Farthing, M.J. (1986) Enzyme activities of thehindgut microflora of laboratory animals and man. Xenobiotica 16, 519-523.

86 | Chapter 5

Savill, J.S., Fadok, V., Henson, P., Haslett, C. (1993) Phagocyte recognition of cellsundergoing apoptosis. Immunol. Today 14, 131-136.

Seki, N., Sudo, Y., Yoshioka, T., Sugihara, S., Fujitsu, T., Sakuma, S., Ogawa, T., Hamaoka,T., Senoh, H., Fujiwara, H. (1988) Type II collagen-induced murine arthritis. I. Inductionand perpetuation of arthritis require synergy between humoral and cell-mediatedimmunity. J. Immunol. 140, 1477-1484.

Shevach, E.M. (1995) The effects of cyclosporin A on the immune system. Annu. Rev.Immunol. 3, 397-423.

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Starkebaum, G. (1998) Role of cytokines in rheumatoid arthritis. Sci. Med., 6-15.Stern, J.B., Smith, K.A. (1986) Interleukin-2 induction of T-cell G1 progression and c-myb

expression. Science 233, 203-6.Swain, S.L. (1999) Helper T cell differentiation. Curr. Opin. Immunol. 11, 180-185.Szeliga, J., Hess, H., Rüde, E., Schmitt, E., Germann, T. (1996) IL-12 promotes cellular but

not humoral type II collagen-specific Th1 responses in C57BL/6 and B10.Q mice andfails to induce arthritis. Int. Immunol. 8, 1221-1227.

Tanaka, Y., Otsuka, T., Hotokebuchi, T., Miyahara, H., Nakashima, H., Kuga, S., Nemoto,Y., Niiro, H., Niho, Y. (1996) Effect of IL-10 on collagen-induced arthritis in mice.Inflamm. Res. 45, 283-288.

Thompson, C.B. (1995) Apoptosis in the pathogenesis and treatment of disease. Science 267,1456-1462.

Trentham, D.E. (1982) Collagen arthritis as a relevant model for rheumatoid arthritis:evidence pro and con. Arthritis Rheum. 25, 911-916.

Vaishnaw, A.K., McNally, J.D., Elkon, K.B. (1997). Apoptosis in the rheumatic diseases.Arthritis Rheum. 40, 1917-1927.

Vinegar, R., Truax, J.F., Selph, J.L., Johnston, P.R., Venable, A.L., McKenzie, K.K. (1987)Pathway to carrageenan-induced inflammation in the hind limb of the rat. FederationProc. 46, 118-126.

Walmsley, M., Katsikis, P.D., Adney, E., Parry, S., Williams, R.O., Maini, R.N., Feldmann,M. (1996) Interleukin-10 inhibition of the progression of established collagen-inducedarthritis. Arthritis Rheum. 39, 495-503.

Wang, D.-Q., He, Z.-D., Feng, B.-S. and Yang, C.-R. (1993) Chemical constituents fromPicrorhiza scrophulariiflora. Acta Bot. Yunnan. 15, 83-88.

Winter, C.A., Risley, E.A., Nuss, G.W. (1962) Carrageenin-induced edema in hind paw ofthe rat as an assay for anti-inflammatory drugs. P. Soc. Exp. Biol. Med. 111, 544-547.

Wooley, P.H., Dillon, A.M., Luthra, H.S., Stuart, J.M., David, C.S. (1983) Genetic control oftype II collagen-induced arthritis in mice: factors influencing disease susceptibility andevidence for multiple MHC-asociated gene control. Transplant. P. 15, 180-185.

Wooley, P.H., Luthra, H.S., Stuart, J.M., David, C.S. (1981) Type II collagen-inducedarthritis in mice. I. Major histocompatibility complex (I region) linkage and antibodycorrelates. J. Exp. Med. 154, 688-700.

Activity-guided fractionation ofconstituents modulatingcomplement activation

6.1 Purification of the diethyl-ether extract

6.2 Purification of the light-petroleum extract

Chapter 6

88 | Chapter 6

6.1 Purification of the diethyl-ether extract

Introduction

As described in chapter 5, the effects of sequential Soxhlet extracts of P.scrophulariiflora in the classical complement pathway were determined. The diethyl etherextract showed the highest inhibitory activity, with an IC50 value of 1.9 µg/ml.

The complement system provides an important nonspecific defense mechanism, whichinvolves a multitude of immune responses. Its primary goal is to recognize and eliminatepotentially harmful exogenous and endogenous structures from the human body.Furthermore, split products of complement activation (anaphylatoxins) accumulate inplasma, and ultimately lead to a vast array of immune responses (Liszewski and Atkinson,1993; chapter 5). If complement activation is not adequately controlled, it might causeserious tissue damage. Deranged complement activation has been implicated in thedegeneration of synovial structures in rheumatoid arthritis patients (Konttinen et al., 1996).In such cases, regulation of the complement activation should be enhanced in order todiminish tissue damage during flare-ups of synovitis. In search for new compoundsinhibiting complement activation, the diethyl ether extract of P. scrophulariiflora wassubjected to activity-guided fractionation, based on its effect on the classical complementpathway, leading to complement depletion.


Materials and analytical methodsSilica 60 and Silica LowBar columns were obtained from Merck (Darmstadt,

Germany) and Sephadex LH20 from Pharmacia (Uppsala, Sweden).TLC analysis was carried out on precoated Si60 F254 Silica plates (Merck). Typically,

10-µl solutions were applied to the plate and dried. The plate was developed in a saturatedchamber with a mixture of ethyl acetate, methanol, and water in a ratio of 20: 3: 2, andsprayed with a 1: 1 mixture of vanillin (1 % w/v in ethanol) and sulfuric acid (5 % v/v inethanol). Colored spots were detected after heating for 5 min at 110 °C.

HPLC separation was performed on a modular Gilson (Middleton, USA) system (dual306 pumps, 234 auto injector, 170 diode array detector, FC 204 fraction collector)provided with UniPoint software (v. 1.65) for automated sample handling, and an AlltechAdsorbosphere Silica column (Deerfield, USA; 5 µm, 250 x 10 mm). Preparative HPLCwas performed using a gradient with cyclohexane and ethanol (0-2 min: 100 %; 2-10 min:100 % → 93 %; 10-20 min: 93 %; 20-30 min: 93 % → 85 %).

Complement assayThe fractions were tested in the classical complement pathway, as described

previously in chapter 5.

Activity-guided fractionation of complement activation inhibitors | 89

Silica column chromatographySoxhlet extracts from the rhizomes of P. scrophulariiflora were obtained and tested in

the complement assay as described in chapter 5. The diethyl-ether extract (0.5 g) wassubjected to Silica column chromatography (50 cm × 2 cm diameter) using a diethyl ether:ethyl acetate mixture in a ratio of 1: 1 as eluent. The column was eluted and 6 fractions of5 ml and 4 fractions of 10 ml were collected. Pilot trials showed that further elution with100 % ethyl acetate did not enhance the separation at this stage. Therefore, the remainingmaterial on the column was eluted with 500 ml dried methanol. The separation wasmonitored with TLC and all fractions were tested in the complement assay.

Sephadex LH20 and preparative HPLCA similar Silica column separation as described above was performed on the diethyl

ether extract (3 g) using a larger column (50 cm × 5 cm diameter) to obtain more material(0.9 g methanol fraction). The methanol fraction (170 mg) was subjected to SephadexLH20 chromatography with methanol as eluent. Sixty fractions of 7 ml were collected,analyzed with TLC, and tested on volume base in the complement assay. Fractions 19-37were pooled (120 mg) and further purified with preparative HPLC (figure 4), as describedabove. The separation was performed 7 times and corresponding fractions were pooled.Ten fractions were collected, dried, and tested.

Results and discussion

Powdered rhizomes of P. scrophulariiflora were subjected to sequential Soxhletextraction by light petroleum and diethyl ether, respectively, as described in chapter 5. Thefirst step of the purification process of the diethyl ether extract consisted of a separationwith Silica column chromatography (figure 1).

The total yield of the fractions (117 %) exceeded the initial amount due to Silica,which partly dissolved in the eluent. Fraction 10 showed the strongest inhibitory activity(IC50 = 0.5 ± 0.0 µg/ml). Fractions 3 and 4 were similar in both TLC profile and activity(IC50 = 2.4 ± 0.3 µg/ml and 3.1 ± 0.5 µg/ml, respectively), while other fractions were lessactive (figure 2).

90 | Chapter 6

Figure 1. Purification scheme of fractions inhibiting activation of the classical pathway ofthe complement system.

0

10

20

30

40

50

1 2 3 4 5 6 7 8 9 10

fraction

IC50

(g/

ml)

0%

60%Yi

eld CPYield

Figure 2. Effects of Silica column chromatography fractions on the classical pathway ofcomplement activation. Data of fractions were expressed as means ± SEM of 2measurements. Results were reproduced in 2 independent separation experiments.

A similar purification of the diethyl ether extract (3 g) as described above wasperformed on a larger Silica column, yielding a methanol fraction with a similar TLCprofile and activity (IC50 = 1.6 ± 0.6 µg/ml; 30 % yield over one step) as the methanolfraction of the previous separation. A part of this fraction (170 mg) was further separatedwith Sephadex LH20 using methanol as eluent. Fractions 19-37, which showed highestactivity on volume base (figure 3), were pooled yielding 124 mg (73 % yield over one step;IC50 = 0.9 ± 0.1 µg/ml). This fraction was further purified using preparative HPLC (figure4), yielding 10 fractions, which were dried and tested (figure 5). Fractions 3 (8 % yieldover one step) and 4 (11 % yield over one step) showed highest activity (IC50 = 0.8 µg/mland 0.5 µg/ml, respectively). However, these fractions revealed at least five spots on TLC


analysis, while HPLC separation did not enhance the purity. Further purification of theactive compounds is in progress.

0

100

200

300

400

12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58

fraction

abso

rban

ce (4

05 n

m)

Figure 3. Volume-based inhibition of the classical route of complement activation byfractions obtained by Sephadex LH20 separation of active Silica fractions.

0

50

100

% M

obile

Pha

se

0

1

AU

0 20 40 Minutes

fraction 1 2 3 4 5 6 7 8 9 10

Figure 4. Chromatogram with absorbance units (AU) at 220 nm obtained by preparative-HPLC separation of active Sephadex LH20 fractions.

92 | Chapter 6

0

20

40

60

80

1 2 3 4 5 6 7 8 9 10

fraction

IC50

(µg/

ml)

0%

10%

20%

yiel

d (%

)

CPyield

Figure 5. Inhibition of the classical pathway of complement activation by preparative-HPLC fractions, obtained by separation of active Sephadex LH20 fractions.

6.2 Purification of the light-petroleum extract

Introduction

As described above, relatively apolar fractions of the Silica column separation of thediethyl ether extract showed complement inhibition as well. TLC analysis revealed thepresence of similar compounds in the light petroleum extract. Therefore, this extract wasused to purify relatively apolar compounds that inhibited the classical complementpathway.


Analytical methodsTLC analysis was carried out on precoated Si60 F254 Silica plates (Merck). Typically,

10 µl solution was applied to the plate and dried. The plate was developed in a saturatedchamber with a mixture of cyclohexane and acetone in a ratio of 3: 1 and sprayed with a 1:1 mixture of vanillin (1 % w/v in ethanol) and sulfuric acid (5 % v/v in ethanol). Coloredspots were detected after heating for 5 min at 110 °C.

Silica column chromatography and Sephadex LH20 chromatographyA Silica column separation was performed (column size 50 cm × 5 cm diameter),

similar to the one described above, but with light petroleum extract as the starting material.The extract (1 g) was eluted with diethyl ether: ethyl acetate in a ratio of 1: 1, and 19fractions of 100 ml were collected. The remaining material on the column was eluted with


1000 ml dried methanol, yielding fraction 20. All fractions were dried and tested on weightbase in the complement assay. Active fractions (4-6) were pooled (130 mg) and furtherpurified by Silica LowBar chromatography.

Silica LowBar chromatography, and preparative TLCActive fractions of the Silica separation described previously (130 mg) were subjected

to Silica LowBar column chromatography using cyclohexane: acetone in a ratio of 8: 2 aseluent. Fourty fractions of 8 ml were collected, analyzed by TLC, and tested in thecomplement assay. Based on similarity in TLC profile and activity, fractions 11-15 werepooled as well as fractions 17-23. These fractions were applied to preparative TLC platesand developed using cyclohexane: methanol in a ration of 3: 1 as eluent. Fluorescent bands(360 nm) were scraped off and extracted three times with cyclohexane: acetone in a ratioof 8: 2. The respective extracts were pooled and filtrated. Filtrates were dried, analyzed byTLC, and tested on weight base for complement depletion.


Light-petroleum Soxhlet extract of rhizomes of P. scrophulariiflora was obtained asdescribed in chapter 5. Separation of this extract using Silica column chromatography withdiethyl ether: ethyl acetate as eluent yielded active fractions with a similar TLC profile andactivity to fraction 3-4 of the previously described Silica column separation of the diethyl-ether extract (figures 2 and 7). Fractions 4-6 were pooled (13 % yield over one step) andfurther separated on a Silica LowBar column with cyclohexane: acetone in a ratio of 8: 2 aseluent (figure 6).

Figure 6. Purification scheme of fractions from the light petroleum extract inhibiting theclassical pathway of complement activation.

All fractions were tested and showed two separate groups of active fractions, whichseemed to consist of at least two active compounds each (figure 8). Fractions 11-15 werepooled, as well as fractions 17-23, and these were separated by preparative TLC.Fluorescent bands were scraped off, extracted, and tested for anticomplementary activity

94 | Chapter 6

(figures 9 and 10). Band 3 of fractions 11-15 showed highest activity (IC50 = 0.5 ± 0.2µg/ml). This fraction revealed at least 8 spots on TLC analysis, some of them coincidingwith phytosterols, according to color and Rf value after detection. Further research is beingundertaken to isolate the active compounds and establish their identity.

0

5

10

15

20

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

fraction

IC50

(µg/

ml)

0%

25%

50%

yiel

d (%

)

CPYield

Figure 7. Inhibition of the classical pathway of complement activation by fractionsobtained by Silica-column separation of the light-petroleum extract of P. scrophulariiflora.

0

100

200

300

400

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35

fraction

abso

rban

ce (4

05 n

m)

Figure 8. Volume-based inhibition of complement activation by Silica LowBar fractionsobtained by Silica-column chromatography.


0

4

8

12

1 2 3 4 5

fraction

IC50

( µg

/ml)

0%

30%

60%

yiel

d CPYield

Figure 9. Inhibition of the classical pathway of complement activation by fractionsobtained by preparative TLC of active LowBar fractions 11-15. Data were expressed asmeans ± SEM of two duplicate experiments.

0

20

40

60

80

1 2 3 4

band

IC50

( µg

/ml)

0%

30%

60%

yiel

d CPYield

Figure 10. Inhibition of the classical pathway of complement activation by fractionsobtained by preparative TLC of active LowBar fractions 17-23. Data were expressed asmeans ± SEM of two duplicate experiments.

Several methods have been followed in order to isolate compounds from P.scrophulariiflora that inhibit complement activation, and a representative overview ispresented in this chapter. It has become clear that in both the light petroleum and thediethyl ether extracts several compounds are present, which potently inhibit the classicalpathway of complement activation. It seems, however, that the respective amounts of thesecompounds in the extracts are rather low. Furthermore, the difficulty to purify activecompounds suggests that many compounds are present with similar physical properties, atleast with respect to their solubility and absorption in the separation systems used. This hasmade isolation very tedious and incomplete within the time span available. However, TLCanalysis suggested the presence of phytosterols in the light petroleum extract. Althoughthese compounds have not been demonstrated in Picrorhiza species before, stigmasterol,campesterol, and β-sitosterol are ubiquitous in higher plants and are known for their potentcomplement-depleting activity (Yamada et al., 1987, Oh et al., 1998).

96 | Chapter 6

In conclusion, P. scrophulariiflora light petroleum and diethyl ether extracts containseveral compounds that inhibit the classical pathway of complement activation. Moreresearch will be required to isolate these compounds and to elucidate their structure.

References

Konttinen, Y.T., Čeponis, A., Meri, S., Vuorikoski, A., Kortekangas, P., Sorsa, T., Sukura,A., Santavirta, S. (1996) Complement in acute and chronic arthritides: assessment ofC3c, C9, and protein (CD59) in synovial membrane. Ann. Rheum. Dis. 55, 888-894.

Liszewski, M.K., Atkinson, J.P. (1993) The complement system. In: Fundamentalimmunology. Paul, W.E. (Ed.). Third edition, Raven Press, New York, pp. 917-939.

Oh, S.R., Kim, D.S., Lee, I.S., Jung, K.Y., Lee, J.J., Lee, H.-K. (1998) Anticomplementaryactivity of constituents from the heartwood of Caesalpinia sappan. Planta Med. 64,456-458.

Yamada, H., Yoshino, M., Matsumoto, T., Nagai, T., Kiyohara, H., Cyong, J.-C., Nakagawa,A., Tanaka, H., Ōmura, S. (1987) Effects of phytosterols on anti-complementaryactivity. Chem. Pharm. Bull. 35, 4851-4855.

Activity-guided isolation ofcucurbitacins inhibiting T-cellproliferation

Chapter 7

98 | Chapter 7

Introduction

T-lymphocytes are among the key players in cell-mediated immunity. In order togenerate an effective immune response, activation and subsequent expansion of antigen-specific lymphocytes is required. Activation of T cells initiates when T-cell receptors(TCRs) bind to specific antigens, in association with major histocompatibility complex(MHC) molecules on the membrane of antigen-presenting cells (APC). Several otherproteins and receptors are associated with the TCR and play a cooperative role in acontrolled manner of signal transduction (Abraham et al., 1992, Chambers and Allison,1997, 1999). A persistent stimulatory signal ultimately leads to the expansion anddifferentiation of the specific T cells, under the influence of an array of cytokines andgrowth factors (Swain, 1999). Activated T-cells have been implicated as importantmediators in inflammation and in the pathogenesis of autoimmune diseases such asrheumatoid arthritis (Maini et al., 1995, Perkins, 1998). Several compounds that suppresspro-inflammatory T cells have been successfully used in the treatment of rheumatoidarthritis, e.g. azathioprine (Berry et al., 1976), chloroquine (Landewé et al., 1995),methotrexate (Boerbooms et al., 1988), cyclosporin (Van Rijthoven et al., 1986), andglucocorticoids (Kirwan, 1995). However, toxicity induced by these drugs stresses the needfor new therapeutics in the treatment of autoimmune diseases.

Along this line, our research is aiming at the disclosure of compounds that interferewith the proliferation of lymphocytes. As described in the previous chapter, a diethyl etherSoxhlet extract of Picrorhiza scrophulariiflora Pennell strongly inhibited T-cellproliferation, while other extracts were not active. This chapter describes the isolation andidentification of two cucurbitacins, which strongly inhibit mitogen-induced T-cellproliferation.


MaterialsRPMI 1640 medium, fetal calf serum (FCS), and M199 medium were obtained from

Gibco BRL, Life Technologies (Paisley, Scotland); Phytohemagglutinin (PHA) fromMurex Diagnostics (Dartford, GB); Concanavalin A type IV, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-carboxyfluorescein diacetate (CFDA), andpropidium iodide (PI) from Sigma Chemicals (St. Louis MO, USA). Lactatedehydrogenase (LDH) substrate mixture consisted of 1 mg NAD (β-nicotinamide adeninedinucleotide ⋅ 4H2O; Janssen Chimica, Geel, Belgium), 80 µg phenazine methosulphate(Janssen Chimica), 4.8 mg calcium lactate, and 0.5 mg MTT per ml 0.2 M Tris buffer(tris(hydroxymethyl)aminomethane; Merck, Darmstadt, Germany), adjusted to pH 7.2.

Silica 60 was obtained from Merck, and Sephadex LH20 from Pharmacia (Uppsala,Sweden).

Activity-guided isolation of cucurbitacins inhibiting T-cell proliferation | 99

Analytical methodsTLC analysis was carried out on precoated Si60 F254 Silica plates (Merck). Typically,

10-µl solutions were applied to the plate and dried. The plate was developed in a saturatedchamber with a mixture of ethyl acetate, methanol, and water in a ratio of 20: 3: 2, andsprayed with a 1: 1 mixture of vanillin (1 % w/v in ethanol) and sulfuric acid (5 % v/v inethanol). Colored spots were detected after heating for 5 min at 110 °C.

HPLC separation was performed on a modular Gilson (Middleton, USA) system (dual306 pumps, 234 auto injector, 170 diode array detector, FC 204 fraction collector) providedwith UniPoint software (v. 1.65) for automated sample handling. For preparative HPLC, anAlltech Adsorbosphere Silica column (Deerfield, USA; 5 µm, 250 x 10 mm) was used, anda gradient of cyclohexane and ethanol (0-2 min: 100 %; 2-10 min: 100 % → 93 %; 10-24min: 93 %; 24-30 min: 93 % → 100 %). For analytical HPLC, an Alltech Alltima C18column (5 µm, 250 x 4.6 mm) was used, and a gradient of water and acetonitrile (0-5 min:90 %; 5-10 min: 90 % → 80 %; 10-25 min: 80 %; 25-40 min: 80 % → 20 %).

Pilot purification procedureDiethyl-ether Soxhlet extract of rhizomes of P. scrophulariiflora was obtained as

described in chapter 6. Dried extract (190 mg) was dissolved in 10 ml of a mixture ofdiethyl ether and ethyl acetate in a ratio of 1: 1 and subjected to Silica columnchromatography (50 cm × 2 cm diameter). Diethyl ether: ethyl acetate (1: 1) served aseluent and 6 fractions of 50 ml and 4 fractions of 100 ml were collected. All fractions weredried, analyzed by TLC, and tested for antiproliferative activity.

Isolation procedure of compound 1Diethyl-ether extract (2.5 g) was subjected to Silica column chromatography (50 cm ×

5 cm diameter) with diethyl ether: ethyl acetate (1: 1) as eluent. Thirty fractions of 100 mlwere collected, three times concentrated, analyzed by TLC, and tested on volume base forantiproliferative activity. Compound 1 was directly obtained from fractions 18-30 (300 mg)by crystallization (50 mg; 0.04 % of dried rhizomes).

Isolation procedure of compound 2Active fractions 10-13 of the previous described Silica column separation were pooled

(200 mg) and pooled with the corresponding fractions of a similar separation. Thesefractions (370 mg) were separated by Sephadex LH20 (50 cm × 2 cm diameter) withacetone as eluent. Sixty fractions of 400 drops (ca. 8 ml) were collected and concentratedthree times. Even fractions were analyzed by TLC and odd fractions were tested forantiproliferative activity. Fractions corresponding in TLC profile and activity (18-26) werepooled (150 mg) and further purified by preparative HPLC using a gradient of cyclohexaneand ethanol, yielding compound 2 (40 mg; 0.02 % of dried rhizomes).

Purification of unknown compoundThe active fractions 1-9 of the previously described Silica separation of the diethyl-

ether extract were pooled (1.3 g) and further purified by Sephadex LH20 with acetone aseluent. Fifty fractions of about 10 ml were collected, three times concentrated, analyzed by

100 | Chapter 7

TLC, and tested on volume base in the T-cell proliferation assay. Fractions 6-11 werepooled (100 mg) because they showed antiproliferative activity and a similar TLC profile.The pooled fractions were subjected to Sephadex LH20 separation using dichloromethane(DCM) as eluent, yielding 40 fractions. The fractions were three times concentrated, oddfractions being analyzed by TLC, and the even fractions being tested on volume base in theT-cell proliferation assay.

Structure elucidationThe structures of compounds 1 and 2 were determined using EI-MS, FAB-MS, IR, 1H

NMR, and 13C NMR spectroscopy (including APT, COSY and HETCOR).

T-cell proliferation assayThe T-cell proliferation assay was performed as described in chapter 6.

T-cell toxicity assaysFor the assessment of cytotoxic effects of picracin and deacetylpicracin towards T-

lymphocytes, LDH-release (Babson and Phillips, 1965), and CFDA/PI-labeling (Bruning etal., 1980) assays were performed. Cell suspensions (50 µl) were incubated at 37 °C in U-well microtiter plates with 50 µl medium and appropriate dilution ranges of test samples.To determine the amount of LDH released after 24 h, plates were spun (5 min, 300 × g),supernatants (50 µl) were transferred to flat-bottom microtiter plates, and 25 µls of LDHsubstrate mixture were added. After incubation (10 min, 37 °C), the optical densities at 550nm were measured. Controls consisted of cells incubated with test samples and 50 µlTriton X-100 (0.1 % v/v), cells incubated with 50 µl Triton X-100 (0.1 % v/v), or cellsincubated with medium only.

To assess cytotoxicity after 4 days, stimulated lymphocytes were labeled withcarboxyfluorescein (CFDA) and propidium iodide (PI) to distinguish viable from deadcells. After 4 days of incubation (37 °C), 50 µl of a 1 µl/ml CFDA dilution in RPMImedium was added. After incubation in the dark (15 min, room temperature), 50 µl PI inink was added. The plates were inspected under an upside-down fluorescence microscopeand the ratio of viable/dead cells was assessed. Controls consisted of cells incubated withmedium only.


Two cucurbitacins were isolated from the rhizomes of P. scrophulariiflora, guided byinhibition in a mitogen-induced human T-lymphocyte proliferation assay. The diethyl-etherextract, which potently inhibited proliferation (IC50 = 13 ± 2 µg/ml), was subjected toSilica column chromatography using a mixture of diethyl ether and ethyl acetate in a ratioof 1: 1 as eluent. TLC analysis of the fractions showed a good separation with major bluespots at Rf = 0.70 and Rf = 0.65 in fractions 4-5 and 6-7, respectively. The fractions weretested on a weight base in the T-cell proliferation assay, revealing high activity for thefractions 4-7 (figure 1).


-

10

20

30

40

50

DE 1 2 3 4 5 6 7 9 10

fraction

IC50

( µ µµµg/

ml)

0%

3%

6%

9%

12%

15%

Yiel

d TCPYield

Figure 1. Inhibition of proliferation of T cells by Silica fractions of the diethyl ether (DE)extract of P. scrophulariiflora. Data were expressed as means ± SEM of 2 experiments.

In order to obtain higher yields, a similar separation as described earlier was used witha larger Silica column. The fractions were tested in the T-cell proliferation assay, in whichtwo groups of fractions revealed inhibitory activity (figure 2). Fractions 1-9 were pooledsince they showed a similar TLC profile. The pooled fractions were subjected to SephadexLH20 chromatography using acetone and dichloromethane as eluents, respectively (figures4 and 5). However, this method did not result in a good separation, and revealed at leasteight different compounds in the active fractions 13-24 of the dichloromethane separation,as shown by TLC analysis. Because more material was needed to isolate the activecompound, priority was given to the other fractions (figure 3).

Fractions 10-13 of the Silica column separation (figure 2) were pooled and subjectedto Sephadex LH20 column chromatography using acetone as eluent. Fractions 18-26 werepooled, based on their TLC profile and activity (figure 6), and subjected to HPLC using agradient of cyclohexane and ethanol. The major peak yielded compound 2, which potentlyinhibited T-cell proliferation.

Fractions 18-30 of the Silica column separation (figure 3) showed a similar TLCprofile, revealing a blue spot at Rf = 0.66, which corresponded to the spot observed withfractions 6-7 of the pilot separation (figure 1). Repetitive crystallization from the pooledfractions yielded a pure compound (1), which also potently inhibited T-cell proliferation.

102 | Chapter 7

-

100

200

300

400

500

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

fraction

abso

rban

ce (5

50 n

m)

Figure 2. Volume-based inhibition of T-cell proliferation by Silica fractions of the diethylether extract.

Figure 3. Isolation scheme of picracin and deacetylpicracin from rhizomes of P.scrophulariiflora.


-

200

400

600

800

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49

fraction

abso

rban

ce (5

50 n

m)

Figure 4. Volume-based inhibition of fractions obtained by separation of Silica fractions 1-9 on LH20 using acetone as eluent.

0

200

400

600

800

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40fraction

abso

rban

ce (5

50 n

m)

Figure 5. Volume-based inhibition of fractions obtained by separation of LH20-acetonefractions 6-11 on LH20 using DCM as eluent.

104 | Chapter 7

0

200

400

600

1 5 9 13 17 21 25 29 33 37 41 45 49 53 57

fraction

abso

rban

ce (5

50 n

m)

Figure 6. Volume-based inhibition of Sephadex LH20 fractions of Silica fractions 10-13,containing compound 2.

Both compounds were identified as aglycones of known 11-deoxycucurbitacinglucosides (Laurie et al., 1985, Stuppner and Wagner, 1989) and named deacetylpicracin(1) and picracin (2). Using TLC, they revealed blue spots with Rf values of 0.65 and 7.0,respectively. Their structures were determined by EI-MS, FAB-MS, IR, 1H NMR, and 13CNMR spectroscopy (including APT, COSY and HETCOR). Deacetylpicracin (1) wasobtained as colorless needles (ethyl acetate): mp 224-228 °C (decomposition); IR (KBr)νmax 3400, 2939, 1682, 1633, 1282 cm-1; FAB-MS m/z 505 [M+H]+. Picracin (2) wasobtained as colorless amorphous powder: mp 181-182 °C; IR (KBr) νmax 3575, 2947, 1716,1672, 1618, 1258 cm-1; EI-MS m/z 486 [M-CH3COOH]+ (1.5), 96 (100).

Picracin was shown to be 25-acetoxy-2,3,16,20-tetrahydroxy-9-methyl-19-norlanosta-5,23-diene-22-one (2, figure 2) which was reported previously being an aglucone obtainedby hydrolysis of the corresponding 2-O-β-D-glucoside isolated from P. kurrooa (Laurie etal., 1985).

The 1H NMR spectrum of deacetylpicracin (2,3,16,20,25-pentahydroxy-9-methyl-19-norlanosta-5,23-diene-22-one; 1, figure 2) showed the characteristic signals of an 11-deoxycucurbitacin skeleton with signals for eight methyl groups between 0.85 and 1.29ppm and three olefinic protons at δ 5.49 (H-6, d, J = 5.5 Hz), 6.69 and 6.87, the latter twosignals being an AB system (H-24, H-23, d, J = 15.4 Hz) attributable to the α,β-unsaturated ketone function of the side chain. In addition, signals at δ 3.29, 3.88, and 4.32were assigned to three single protons being attached to hydroxylated carbons. 1H-1H COSYexperiments showed the coupling of a broad doublet at δ 3.88 (H-2, J = 10.7 Hz) to adoublet at δ 3.29 (H-3, J = 2.2 Hz). The triplet at δ 4.32 was assigned to H-16 (J = 7.5 Hz).Compared with the 1H NMR spectrum of picracin, the methyl groups at C-25 (26-Me and27-Me) were shifted upfield to δ 1.22 (- 0.2 ppm), whereas the signal for OCOMe at δ 1.90was missing, indicating the absence of the C-25 acetyl group.


In addition, it was shown by TLC analysis that methanolysis of picracin with sodiummethoxide resulted in the formation of deacetylpicracin.

Table 1. 1H NMR data (ppm) of 1 and 2 in CD3OD and CDCl3 (300 MHz); J is expressedin Hz; s = singlet, d = doublet, t = triplet, br. = broad.

δ (1111) CD3OD δ (2) CD3OD δ (2) CDCl3*

0.85 (3H, s) 0.84 (3H, s) 0.90 (3H, s)0.93 (3H, s) 0.92 (3H, s) 1.00 (3H, s)0.96 (3H, s) 0.92 (3H, s) 1.04 (3H, s)1.02 (3H, s) 1.00 (3H, s) 1.04 (3H, s)1.07 (3H, s) 1.06 (3H, s) 1.16 (3H, s)1.29 (3H, s) 1.29 (3H, s) 1.39 (3H, s)

H-26 1.22 (3H, s) 1.46 (3H, s) 1.51 (3H, s)H-27 1.22 (3H, s) 1.44 (3H, s) 1.51 (3H, s)OCOMe 1.90 (3H, s) 1.97 (3H, s)

H-17 2.25 (d, J = 7.0)H-3 3.29 (1H, d, J = 2.2) 3.29 (1H, s) 3.43 (1H, br.s)H-2 3.88 (1H, br.d, J = 10.7) 3.87 (1H, br.d, J = 11.7) 3.96 (1H, br.d, J = 11.7)H-16 4.32 (1H, t, J = 7.5) 4.38 (1H, t, J = 7.9) 4.30 (1H, br.)H-6 5.49 (1H, d, J = 5.5) 5.48 (1H, d, J = 5.1) 5.62 (1H, d, J = 5.5)H-23 6.69 (1H, d, J23-24 = 15.4) 6.68 (1H, d, J23-24 = 15.8) 6.44 (1H, d, J23-24 = 15.7)H-24 6.87 (1H, d) 6.87 (1H, d) 7.00 (1H, d)* Values are in agreement with Laurie et al., 1985.

The 13C NMR spectrum of deacetylpicracin was very similar to that of picracin exceptfor some signals of the side chain. In contrast to picracin, only one carbonyl carbon signalwas found (C-22 at δ 204.8), the signal for C-24 being shifted downfield to 155.3 (+ 5.7ppm), whereas the signal for C-25 showed a strong upfield shift to 70.1 (- 9.6 ppm). Inaddition, the absence of the acetyl group at C-25 was confirmed by the mass difference of42 between picracin and its deacetylated derivative. Compared with its 2-O-β-D-glucoside(Stuppner and Wagner, 1989), the signal of C-2 in the 13C NMR spectrum ofdeacetylpicracin in CD3OD showed a strong upfield shift (about - 8 ppm), whereas signalsof C-1 and C-3 shifted to a lower field (about + 2-3 ppm). These observations are in goodagreement with picracin and its 2-O-β-D-glucoside (Stuppner and Wagner, 1989) and withliterature data reported for the 23,24-dihydro derivative of deacetylpicracin and a series of2-O-β-D-glucosides of related aglucones (Stuppner et al., 1991).

Concerning the stereochemistry of deacetylpicracin, the coupling constant of 2.2 Hzbetween H-2 and H-3 indicated a 2β,3β-diol configuration since 2α,3α-diol systems havenot been found in natural cucurbitacins so far (Rao et al., 1974, Che et al., 1985, Laurie etal., 1985). In analogy with other cucurbitacins, it was assumed that the 20-OH ofdeacetylpicracin is β-oriented, whereas the 16-OH is in the α-position (Vande Velde andLavie, 1983, Stuppner et al., 1991). The α-orientation of 16-OH and the β-orientation of20-OH have been confirmed by X-ray crystallographic analysis of (20R)-16α-acetoxy-20-

106 | Chapter 7

hydroxy-24,25,26,27-tetranor-2β-(β-D-tetraacetylglucopyranosyloxy)-10α-cucurbit-5-en-3,11,22-trion, a derivative of a cucurbitacin obtained from P. kurrooa, and the di-p-iodobenzoate of datiscoside, a cucurbitacin isolated from Datisca glomerata(Cucurbitaceae) (Kupchan et al., 1972, Weinges et al., 1989).

Table 2. 13C NMR (APT) data (ppm) of 1 and 2 in CD3OD and pyridine-d5 (75 MHz).APT δ (1)

CD3ODδ (1)pyridine-d5

δ (2)CD3OD

δ (2)pyridine-d5

C-1 CH2 30.5 31.9 30.5 31.9C-2 CH 69.9 68.9 69.9 68.9C-3 CH 80.4 79.8 80.4 79.8C-4 C 42.6 41.9 42.6 41.9C-5 C 141.9 142.0 141.9 142.0C-6 CH 121.5 119.8 121.7 119.8C-7 CH2 25.2 24.5 25.3 24.5C-8 CH 44.1 42.7 44.1 42.8C-9 C 35.3 34.4 35.3 34.5C-10 CH 38.3 37.2 38.2 37.3C-11 CH2 31.5 30.5 31.3 30.6C-12 CH2 32.9 30.7 32.8 30.6C-13 C † 46.7 49.5 46.6C-14 C 49.9 48.9 49.7 48.9C-15 CH2 47.0 48.9 46.9 48.8C-16 CH 72.4 70.9 72.6 71.2C-17 CH 60.0 59.6 60.5 60.1C-18 CH3 18.6 18.4 18.7 18.4C-19 CH3 29.2 29.8‡ 28.4 27.9‡

C-20 C 80.5 79.6 81.1 80.1C-21 CH3 25.1 25.2‡ 24.9 25.2‡

C-22 C 205.4 204.8 205.6 204.7C-23 CH 121.7 121.0 123.0 122.7C-24 CH 155.3 155.3 151.4 149.6C-25 C 71.6 70.1 80.9 79.7C-26 CH3 29.2 29.5! 26.5 26.0‡

C-27 CH3 28.5 27.9‡ 26.8 26.5‡

C-28 CH3 27.5 27.2‡ 27.5 27.2‡

C-29 CH3 26.1 26.2‡ 26.1 26.2‡

C-30 CH3 19.2 18.4 19.2 18.5OCOMe, C 172.0 169.8OCOMe, CH3 21.8 21.5

† Signal obscured by the solvent signal; ‡ tentatively assigned.


OH

OH

OH

OHO

R

1: R = OH2: R = OAc

Figure 7. Cucurbitacins isolated from P. scrophulariiflora with T-cell antiproliferativeactivity (1: deacetylpicracin; 2: picracin).

Table 3. Contents of picracin and deacetylpicracin in Soxhlet extracts of P.scrophulariiflora.

Picracin DeacetylpicracinPs.PE 0.69 ± 0.20 % ndPs.DE 4.5 ± 1.3 % 3.2 ± 0.7 %Ps.EA nd ndPs.Me 0.19 ± 0.00 % ndPs.Aq nd ndPk = P. kurrooa; Ps = P. scrophulariiflora; nd = not detectable.

The contents of picracin and deacetylpicracin were determined in the extracts prepared,and were found to be 4.5 % and 3.2 % (v/v), respectively. Furthermore, picracin was detectedin the light-petroleum extract and the methanol extract (table 3).

Both compounds exhibited a dose-dependent inhibition of PHA-induced T-cellproliferation. The IC50 values were determined to be 1.0 ± 0.2 µM for both picracin anddeacetylpicracin.

DE extract picracin DAP0

10

IC50

(µ µµµg/

ml)

Figure 8. Effects of diethyl ether (DE) extract, picracin, and deacetylpicracin (DAP) onPHA-induced proliferation of T cells. Data were expressed as means ± SEM of at least 9experiments.

108 | Chapter 7

Because cucurbitacins are well known for their cytotoxic behavior (Fuller et al., 1994),cytotoxic properties of the two compounds were determined. Labeling the PHA-stimulatedlymphocytes after 4-day incubation with carboxyfluorescein diacetate and propidiumiodide to distinguish between viable and dead cells, showed only minor cell death (< 10 %)for concentrations of picracin and deacetylpicracin below 60 µM. Another cytotoxicityexperiment, using the lactate dehydrogenase release assay, did not show any toxic effect upto 50 µM for both cucurbitacins after 24 h. As these concentrations are much higher thanthe IC50 values of both compounds (1 µM), it can be concluded that the inhibitory activityof picracin and deacetylpicracin is most probably not to be ascribed to cytotoxic effects.

Several cucurbitacins have been shown to inhibit incorporation of radioactiveprecursors into DNA, RNA, and protein of HeLa S3 cells at ID50 values that were similarto the ED50 values of cucurbitacin-induced growth inhibition of these cells. This suggests arelationship between the inhibition of intracellular metabolic activity and growth-inhibitoryactivity (Witkowski et al., 1984). Furthermore, cucurbitacin E was shown to inducemorphological changes and inhibit cell growth of prostrate carcinoma cells and humanendothelial cells, which were associated with the disruption of the filamentous (F)-actincytoskeleton (Duncan et al., 1996, Duncan and Duncan, 1997). The cytoskeleton has beenimplicated as a critical intermediate in signal transduction pathways controlling celldivision (Luna and Hitt, 1992, Penninger and Crabtree, 1999). Therefore, the mechanism ofpicracin and deacetylpicracin may also include interference with the cytoskeleton andsubsequent abrogation of proliferative signal transduction, leading to inhibition of T-cellproliferation.

In conclusion, picracin and deacetylpicracin, two cucurbitacins isolated from P.scrophulariiflora, are potent inhibitors of mitogen-induced proliferation of T lymphocytes.These findings may explain the usefulness of P. scrophulariiflora in the treatment ofdiseases such as asthma, jaundice, and arthritis (chapter 3), in which T cells play a pivotalrole (Walker et al., 1991, Löhr et al., 1996, Klimiuk et al., 1999). In addition, picracin anddeacetylpicracin may serve as lead compounds for the development of future anti-inflammatory drugs.

References

Abraham, R.T., Karnitz, L.M., Secrist, J.P., Leibson, P.J. (1992) Signal transduction throughthe T-cell antigen receptor. Trends Biochem. Sci. 17, 434-438.

Babson, A.L., Phillips, G.E. (1965) A rapid colorimetric assay for serum lacticdehydrogenase. Clin. Chim. Acta 12, 210-215.

Berry, H., Liyange, S.P., Durance, R.A., Barnes, C.G., Berger, L.A., Evans, S. (1976)Azathioprine and penicillamine in treatment of rheumatoid arthritis: a controlled trial.Brit. Med. J. 1, 1052-1054.

Boerbooms, A.M.T., Jeurissen, M.E.C., Westgeest, A.A.A., Theunisse, H., Van de Putte,L.B.A. (1988) Methotrexate in refractory rheumatoid arthritis. Clin. Rheumatol. 7, 249-253.


Bruning, J.W., Kardol, M.J., Arentzen, R. (1980) Carboxyfluorescein fluorochromasiaassays. I. Non-radioactively labeled cell-mediated lympholysis. J. Immunol. Methods 33,33-44.

Chambers, C.A., Allison, J.P. (1997) Co-stimulation in T-cell responses. Curr. Opin.Immunol. 9, 396-404.

Chambers, C.A., Allison, J.P. (1999) Costimulatory regulation of T-cell function. Curr. Opin.Immunol. 11, 203-210.

Che, C.-T., Fang, X., Phoebe Jr. C.H., Kinghorn, A.D., Farnsworth, N.R. (1985) High-field1H-NMR spectral analysis of some cucurbitacins. J. Nat. Prod. 48, 429-434.

Duncan, K.L., Duncan, M.D., Alley, M.C., Sausville, E.A. (1996) Cucurbitacin E-induceddisruption of the actin and vimentin cytoskeleton in prostate carcinoma cells. Biochem.Pharmacol. 52, 1553-1560.

Duncan, M.D., Duncan, K.L. (1997) Cucurbitacin E targets proliferating endothelia. J. Surg.Res. 69, 55-60.

Fuller, R.W., Cardellina II, J.H., Cragg,G.M., Boyd, M.R. (1994) Cucurbitacins: differentialcytotoxicity, dereplication and first isolation from Gonystylus keithii. J. Nat. Prod. 57,1442-1445.

Kirwan, J.R. (1995) The effect of glucocorticoids on joint destruction in rheumatoid arthritis.New Engl. J. Med. 333, 142-146.

Klimiuk, P.A., Yang, H., Goronzy, J.J., Weyland, C.M. (1999) Production of cytokines andmetalloproteinases in rheumatoid synovitis is T cell dependent. Clin. Immunol. 90, 65-78.

Kupchan, S.M., Sigel, C.W., Guttman, L.J., Restivo, R.J., Bryan, R.F. (1972) Datiscoside, anovel antileukemic cucurbitacin glycoside from Datisca glomerata. J. Am. Chem. Soc.94, 1353-1354.

Landewé, R.B., Miltenburg, A.M., Verdonk, M.J., Verweij, C.L., Breedveld, F.C., Daha,M.R., Dijkmans, B.A. (1995) Chloroquine inhibits T cell proliferation by interferingwith IL-2 production and responsiveness. Clin. Exp. Immunol. 102, 144-151.

Laurie, W.A., MacHale, D., Sheridan, J.B. (1985) A cucurbitacin glycoside from Picrorhizakurroa. Phytochemistry 24, 2659-2661.

Löhr, H.F., Schlaak, J.F., Kollmannsperger, S., Dienes, H.P., Meyer zum Büschenfelde, K.H.,Gerken, G. (1996) Liver-infiltrating and circulating CD4+ T cells in chronic hepatitis C:immunodominant epitopes, HLA-restriction and functional significance. Liver 16, 174-182.

Luna, E.J., Hitt, A.L. (1992) Cytoskeleton – plasma membrane interactions. Science 258,955-964.

Maini, R.N., Chu, C.Q., Feldmann, M. (1995) Aetiopathogenesis of rheumatoid arthritis. In:Mechanisms and models in rheumatoid arthritis. Henderson, B., Edwards, J.C.W.,Pettipher, E.R. (Eds.). Academic Press, London, pp. 25-46.

Penninger, J.M., Crabtree, G.R. (1999) The actin cytoskeleton and lymphocyte activation.Cell 96, 9-12.

Perkins, D.L. (1998) T-cell activation in autoimmune and inflammatory diseases. Curr. Opin.Nephrol. Hy. 7, 297-303.

110 | Chapter 7

Rao, M.M., Meshulam, H., Lavie, D. (1974) The constituents of Ecballium elaterium L. PartXXIII. Cucurbitacins and hexanorcucurbitacins. J. Chem. Soc. - Perkin Trans. I 2552-2556.


Stuppner, H., Wagner, H. (1989) New cucurbitacin glycosides from Picrohiza kurrooa.Planta Med. 55, 559-563.

Swain, S.L. (1999) Helper T cell differentiation. Curr. Opin. Immunol. 11, 180-185.Van Rijthoven, A.W.A.M., Dijkmans, B.C., Goei Thé, H.S., Herman, J., Montnor-Beckers,

Z.L.B.M., Jacobs, P.C.J., Cats, A. (1986) Cyclosporin treatment for rheumatoid arthritis:a placebo controlled, double blind, multicentre study. Annals Rheum. Dis. 45, 726-731.

Vande Velde, V., Lavie, D. (1983) 13C NMR spectroscopy of cucurbitacins. Tetrahedron 39,317-321.

Walker, C., Kaegi, M.K., Braun, P., Blaser, K. (1991) Activated T cells and eosinophilia inbronchoalveolar lavages from subjects with asthma correlated with disease severity. J.Allergy Clin. Immunol. 88, 935-942.

Weinges, K., Schick, H., Neuberger K., Ziegler, H.J., Lichtenthäler, J., Irngartinger, H.(1989) Isolierung und Strukturaufklärung neuer Inhaltstoffe aus dem sodaalkalischenExtract von Picrorhiza kurroa. Liebigs Ann. Chem. 1113-1122.

Witkowski, A., Woynarowska, B., Konopa, J. (1984) Inhibition of the biosynthesis ofdeoxyribonucleic acid, ribonucleic acid and protein in HeLa S3 cells by cucurbitacins,glucocorticoid-like cytotoxic triterpenes. Biochem. Pharmacol. 33, 995-1004.

Immunomodulatory activity ofcucurbitacins from Picrorhizascrophulariiflora

8.1 Immunomodulatory effects on T lymphocytes andmonocytes in vitro

8.2 Immunomodulatory effects in vivo

Chapter 8

112 | Chapter 8

Introduction

In the previous chapter, the isolation of two bioactive cucurbitacins from the rhizomesof Picrorhiza scrophulariiflora was described. Both compounds potently inhibitedmitogen-induced proliferation of T cells. In this chapter, experiments are delineated thatpertain to the underlying mechanisms of this immunomodulatory effect.

As described in chapter 5, the activation of T cells requires signal transduction thatleads to the release of IL-2 and the expression of IL-2 receptor (IL-2R), ultimately leadingto proliferation. In normal situations, cell proliferation and cell death are in balance.However, autoimmune diseases are associated with an increased survival of potentiallyautoreactive T lymphocytes. Activated T lymphocytes that are differentiated into pro-inflammatory effector cells release cytokines, which sustain cell-mediated immuneresponses (Swain, 1999). Within the articular synovium, these T cells activate synovialmacrophages that produce a range of pro-inflammatory cytokines, which induceproliferation of synoviocytes, enhancing the local inflammatory process. These involvelymphocytes, chondrocytes, fibroblasts, and osteoclasts, inducing damage of bone,cartilage, and other connective tissue components (Feldmann et al., 1996, Vaishnaw, et al.,1997, Starkebaum, 1998).

Therapeutic measures of rheumatoid arthritis (RA) often focus on immunomodulatoryactivity, by means of nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, ordisease-modifying antirheumatic drugs (DMARDs; AMR, 1996a). While NSAIDs onlyafford symptomatic relief, without altering the course of the disease or preventing jointdamage, glucocorticoids and DMARDs interfere with the release of pro-inflammatorycytokines. As such, they have the potential to reduce and prevent joint damage and topreserve joint integrity and function (Shevach, 1995, Cronstein, 1996). A majordisadvantage of these drugs, however, is their toxicity, which is usually proportional totheir efficacy (AMR, 1996b, Jackson and Williams, 1998). These side effects imply anongoing search for lead compounds that are able to selectively interfere with pathologicalprocesses underlying chronic inflammation.

8.1 In vitro immunomodulatory effects on T lymphocytes and monocytes


MaterialsPicracin and deacetylpicracin were isolated from P. scrophulariiflora Pennell as

described earlier (chapter 7). Cucurbitacin E was obtained from Extrasynthese (Genay,France). M199 medium, RPMI 1640 medium, and heat-inactivated fetal calf serum (FCS)were obtained from Gibco BRL, Life technologies (Pailsey, Scotland);Phytohemagglutinin (PHA) from Murex Diagnostics (Dartford, GB); 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5-carboxyfluoresceindiacetate (CFDA), propidium iodide (PI), and cyclophosphamide from Sigma Chemicals(St. Louis MO, USA); and sodium dodecyl sulphate (SDS) from ICN Biomedicals. Sheep

Immunomodulatory activity of cucurbitacins from Picrorhiza scrophulariiflora | 113

red blood cells (SRBC) were obtained from citrated arterial blood supplied by CharlesRiver (Someren, The Netherlands) and were washed thrice before use.

IL-2 releasePeripheral blood lymphocytes (PBLs) were prepared from buffycoat residues, kindly

provided by the Blood bank Midden-Nederland (Utrecht, Netherlands), using Ficoll-Hypaque centrifugation, according to the supplier’s intructions (Amersham Pharmacia).Cells were diluted to 2⋅106 cells/ml RPMI 1640 supplemented with 10 % FCS anddispensed in 96-wells microtiter plates (50 µl/well). Proliferation was induced using 50 µlPHA (0.1 mg/ml), and cells were incubated at 37 °C with 50 µl of test samples inappropriate dilution ranges. After incubation, 50-µl samples of supernatants weretransferred to flatbottom plates and IL-2 levels were determined using an ELISA-kit (CLB,Amsterdam, Netherlands). Controls consisted of stimulated PBLs without sample (100%activity), non-stimulated PBLs without sample (0% activity), and non-stimulated PBLswith samples.

Pro-inflammatory cytokine releaseMononuclear cells were obtained from buffycoat residues (Bloedbank Midden-

Nederland) using Ficoll-Hypaque centrifugation, according to the manufacturer’sinstructions (Amersham Pharmacia). Under sterile conditions, 100-µl samples of cellsuspension were dispensed in 96-wells flatbottom microtiter plates, and monocytes wereallowed to adhere overnight at 37 °C under 5 % CO2-atmosphere in a humidifiedincubator, at a concentration of 106 cells/ml M199. After removal of non-adhering cells,50-µl sample solutions in appropriate dilution ranges were added. Cells were stimulated byadding 50 µl LPS (0.5 µg/ml M199 medium), and incubated overnight at 37 °C under 5 %CO2 atmosphere in a humidified incubator. After incubation, 50-µl samples of supernatantswere transferred to flatbottom microtiter plates, and used for the detection of IL-1β andTNFα, using ELISA-kits (CLB, Amsterdam, Netherlands). IC50-values of the testcompounds were calculated in µg/ml. Controls consisted of non-stimulated cells incubatedwith sample solutions.

Toxicity assaysFor the assessment of cytotoxic effects of picracin and deacetylpicracin, cells were

labeled with CFDA and PI to distinguish between viable and dead cells. After 4 days ofincubation (37 °C), 50 µl of a 1 µl/ml CFDA dilution in RPMI medium was added. Afterincubation in the dark (15 min, room temperature), 50 µl PI in ink was added. The plateswere inspected under an upside-down fluorescence microscope and the ratio of viable/deadcells was assessed. Controls consisted of cells incubated with medium only.

ApoptosisPBLs (2 × 106 /ml), obtained as described above, were added to equal volumes of

picracin or deacetylpicracin in different concentrations and incubated in a humidifiedincubator (36 °C). After incubation, cells were washed with phosphate-buffered saline(PBS) and incubated shed from light for 15 min in binding buffer containing FITC-conjugated annexin V (1:100). Propidium iodide (PI; 10 µg/ml) was added just before

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analysis to detect dead cells. T cells were detected using FITC-conjugated anti-CD3antibodies (1:100). Fluorescence of stained cells were measured by flow cytometry on aFACScan apparatus (Becton Dickinson) and analyzed with WinMDI (vs. 2.8; ScrippsInstitute, La Jolla, USA).T cells that were annexin V positive and PI negative were regarded as apoptotic cells.

Results

IL-2 releaseControl experiments revealed maximum IL-2 release by PHA-activated T cells about

24 h after activation. This incubation time was used in determining the effects of picracinand deacetylpicracin on IL-2 release of activated PBLs. Picracin, deacetylpicracin, andcucurbitacin E inhibited IL-2 release by activated T cells after 24 h at IC50 values of 5 ± 2µM, 16 ± 7 µM, and 5 ± 1 µM, respectively (figure 1). Activated T cells that were stainedwith CFDA and PI, following exposure to either of the compounds, revealed a direct toxiceffect of cucurbitacin E, while cells treated with picracin or deacetylpicracin did not differsubstantially from controls, as estimated by with fluorescent microscopy.

picracin DAP cuc E0

10

20

30

IC50

(µ µµµM

)

Figure 1. Effects of picracin, deacetylpicracin (DAP), and cucurbitacin E (cuc E) on IL-2release by PHA-activated T lymphocytes after 24 h incubation, as determined by ELISA.Data were expressed as means ± SEM of 6 (picracin) or 3 (DAP and cuc E) separateexperiments.

IL-1ββββ, TNFαααα releaseTo investigate additional immunomodulatory activities of picracin and DAP on the

development of chronic inflammation, we assessed the effect of both compounds on therelease of pro-inflammatory cytokines by monocytes. After stimulation of monocytes withLPS, cells were incubated for 24 h with different concentrations of picracin or DAP. Uponanalysis of the supernatants of these cells, using ELISA, we found that picracin inhibitedthe release of both IL-1β and TNFα at IC50-values of 7 ± 2 µM and 10 ± 4 µM,respectively. Cucurbitacin E inhibited TNFα release more potently (IC50 = 3 ± 2 µM),while IL-1β-inhibiting activity was a factor 5 lower (IC50 = 16 ± 3 µM). Contradictory,deacetylpicracin did not show inhibitory activity on the release of IL-1β up to 200 µM, the


highest concentration tested, while it moderately inhibited TNFα release (IC50 = 120 ± 36µM). Dexamethasone showed similar activity as deacetylpicracin (figure 3). Staining ofactivated monocytes, exposed to either of the compounds, with CFDA and PI, did notreveal any direct toxic effect after 24 h, as determined by analysis with fluorescentmicroscopy.

0

100

200

300picracinDAPcucurbitacin Edexamethasone

IC50

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)

IL-1β TNFα

Figure 2. Effects of picracin, deacetylpicracin (DAP), cucurbitacin E, and dexamethasoneon the release of pro-inflammatory cytokines by LPS-activated monocytes. Data wereexpressed as means ± SEM of 6 (picracin and DAP) or 3 (cucurbitacin E anddexamethasone) separate experiments.

Apoptosis of T cellsTo assess whether picracin and deacetylpicracin were able to induce apoptosis in non-

stimulated T cells, PBLs were exposed to the two compounds. After incubation for 2, 4, 6,or 24 h, cells were stained with FITC-conjugated annexin V and propidium iodide (PI) andanalyzed by flow cytometry. Stained cells that were annexin V-positive and PI-negativewere regarded as apoptotic. Flow cytometric analysis of stained T cells that were exposedto picracin, showed a significant induction of apoptosis (70-85 % of gated T cells) after 4,6, and 24 h, at concentrations of 20 µM and 200 µM (figure 2A). Deacetylpicracin,however, only slightly induced apoptosis (30-35 %) after 4, 6, and 24 h, at 200 µM, thehighest concentration tested (figure 2).

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A. t = 2 h t = 4 h t = 6 h t = 24 h

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Figure 3. Flow-cytometric analysis of picracin- (A) and deacetylpicracin (DAP; B)-induced apoptosis in non-stimulated T-cells. After incubation, cells were stained withFITC-conjugated annexin V (FL1; x-axis) and propidium iodide (FL2; y-axis). Results arerepresentative of two separate experiments. C. Percentage of T cells treated with picracinor deacetylpicracin (DAP) undergoing apoptosis (annexin-V positive and propidium iodidenegative). Data were expressed as means ± SEM of two separate experiments andstatistically analyzed by Student’s t-test. P-values are < 0.05 (*).

118 | Chapter 8

Discussion

In the previous chapter, the isolation of two cucurbitacins that inhibit T-cellproliferation from rhizomes of Picrorhiza scrophulariiflora was described. In this chapter,possible underlying mechanisms are investigated. As described earlier, proliferation ofactivated T-cells is mediated by the release of IL-2 and the subsequent interaction with theIL-2 receptor (IL-2R), which is only expressed in T cells that are activated. Controlexperiments show that IL-2 release by mitogen-activated peripheral blood T lymphocytesis optimal at 24 h after induction, which is in agreement with literature (Swain, 1999).Incubation of activated T cells with picracin or deacetylpicracin showed a markedinhibition of IL-2 release after 24 h, at IC50 values of 5 and 16 µM, respectively, whichcould not be ascribed to a direct toxic effect. Furthermore, cucurbitacin E, a cucurbitacinknown to inhibit the proliferation of endothelial cells (Duncan and Duncan, 1997),inhibited IL-2 release at an IC50 value of 5 µM. However, fluorescence microscopicalanalysis following staining of cells with CFDA and PI demonstrated that in this case adirect cytotoxic effect was involved. These data may explain the antiproliferative activityof picracin and cucurbitacin, since IL-2 is responsible for signaling T-cell proliferation(Smith, 1988). The difference observed between picracin and deacetylpicracin regardinginhibition of IL-2 release might be ascribed to differences in lipophility between the twocompounds.

In addition to the effects on T lymphocytes, the effects of picracin anddeacetylpicracin on the release of the pro-inflammatory cytokines IL-1β and TNFα byactivated monocytes was investigated. These cytokines play an important role in thepathogenesis of RA, because they enhance the release of metalloproteinases bychondrocytes and fibroblasts, inducing cartilage destruction (Dayer et al., 1986) andstimulating bone erosion by osteoclasts (Thomas et al., 1987). Incubation of humanmonocytes with picracin for 24 h inhibited the release of IL-1β and TNFα at IC50-values of7 µM and 10 µM, respectively. Deacetylpicracin, however, did not inhibit the release ofIL-1β up to 200 µM, the highest concentration tested, and slightly inhibited TNFα release(IC50 = 120 µM). Cucurbitacin E, which differs from picracin in the presence of carbonylfunctionalities at C3 and C11, and a C1-2 double bond (chapter 4), inhibited the release ofIL-1β at IC50 = 16 µM and TNFα release at IC50 = 3 µM. This suggests the involvement ofat least the acetyl group in the side chain in the inhibition of IL-1β and TNFα release,although other functional differences between the two compounds were not taken inconsideration.

The interference of picracin and deacetylpicracin with IL-2 release by activated Tlymphocytes could be the result of a specific interaction with signal transduction pathwaysleading to the release of IL-2, or due to the induction of apoptosis, which renders the T cellincapable of releasing IL-2. To assess the apoptotic effect of picracin and deacetylpicracin,PBL were stained with FITC-conjugated annexin V, a phospholipid-binding protein withhigh affinity to phosphatidylserine, a marker of early apoptosis (Martin et al., 1995,Vermes et al., 1995). Because phosphatidylserine is exposed in necrotic cells as well, cellswere double stained with propidium iodine (PI). Cells that were annexin V-positive and PI-negative were regarded as apoptotic. Cytometric analysis of T cells, exposed to picracin,and stained with annexin V and PI, showed a significant induction of apoptosis (70-85 %)


after 4, 6, and 24 h, at concentrations of 20 µM and 200 µM. Deacetylpicracin, however,was about three times less effective, and induced apoptosis for 30-35 % after 4, 6, and 24h, at 200 µM, the highest concentration tested, which was not significantly different fromuntreated cells. Nevertheless, preliminary experiments suggest the induction of apoptosisin T cells, two days after incubation with deacetylpicracin. The effects observed might bedue to the lower lipophilicity of deacetylpicracin, which could result in a less pronouncedinteraction with cell membranes, and, consequently, a delayed capacity to induceapoptosis. The observation that deacetylpicracin was about three times less effective indecreasing the release of IL-2 by T cells after 24 h of incubation, while its capacity toinhibit T-cell proliferation after 4 days of incubation was not inferior to picracin, are insupport of this hypothesis.

8.2 Immunomodulatory effects in vivo

Material, animals, and methods

AnimalsSpecific-pathogen-free female BALB/c mice of 7-9 weeks were obtained from the

Central Animal Laboratory (Utrecht, The Netherlands), maintained under standard housingconditions, and provided with food and water ad libitum. At the start of the experiments,the mice were 8-10 weeks old.

DTH responseDelayed-type hypersensitivity was induced in BALB/c mice according to Kerckhaert

et al. (1974). Before priming, the mice were pretreated with 5 mg cyclophosphamide inphosphate buffered saline (PBS) intraperitoneally to suppress a B-cell mediated response.Eight hours after pretreatment, the mice were primed by intraperitoneal injection of 108

SRBC in 200 µl PBS. After 10 days, the mice were challenged by injecting 108 SRBC in25 µl PBS subplantar in the left paw. Picracin or deacetylpicracin (10, 30, or 100 mg/kg)was administered, either intraperitoneally 1 hour before challenge (200 µl), orsubcutaneously along with the challenge (25 µl). Dexamethasone served as a positivecontrol. The locally administered samples, including the controls, contained 20 % DMSOin order to dissolve the drug properly. A separate control experiment was performed withprimed mice, which were not challenged with antigen, and treated with subcutaneousinjection of picracin or deacetylpicracin (0.1, 1, 10 µg/ml PBS), subplantar in the left paw.Swelling of the paws was measured 24 h after challenge using a computerized micrometerdevice (University Medical Center, Utrecht, The Netherlands), and compared to the controlgroup. The differences between the experimental groups and the control group werestatistically analyzed using the Students t-test.

120 | Chapter 8

Results

A delayed-type hypersensitivity (DTH) model was used to assess theimmunomodulatory activity of picracin and deacetylpicracin in vivo. Intraperitonealinjection of sensitized mice, one hour before challenge with either picracin or DAP,showed significant inhibition of DTH responses for 100 mg/kg picracin (45 %) and for 30mg/kg DAP (33 %). Dexamethasone (30 mg/kg i.p.) served as a control and inhibited DTHfor 99 % (figure 4).

0 30 1000.0

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Figure 4. Effects of picracin, deacetylpicracin (DAP), and dexamethasone on DTHresponses in mice after intraperitoneal administration. Results are representative of twoindependent experiments. Data were expressed as means ± SEM (n = 6 per group) andstatistically analyzed by the Student’s t-test. P values were < 0.05 (*), 0.01 (**), or <0.001 (***).

While these results were obtained by intraperitoneal injection of picracin or DAP,another experiment was performed to determine whether the inhibitory activity wasenhanced after local administration of either of the two compounds. For this, a mixture ofsheep erythrocytes with picracin, deacetylpicracin, or dexamethasone was given by meansof subplantar injection in the hind paw. In this experiment, however, an increase inswelling rather than a decrease was observed in groups treated with picracin or DAP,suggesting an irritating or toxic response for both compounds at all concentrations tested(figure 5).


0 10 300

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picracinDAPdexamethasoneblank

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swel

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)

Figure 5. Effects of picracin, deacetylpicracin (DAP), and dexamethasone on DTH-response in mice after subplantar administration. Results are representative of twoindependent experiments. Data were expressed as means ± SEM (n = 6 per group) andstatistically analyzed by the Student’s t-test. P values are < 0.01 (**) or < 0.001 (***).

To confirm the direct irritating or toxic effect of picracin or DAP, sensitized micewere given a subplantar injection of picracin or DAP (10, 1.0, or 0.1 µg/ml), suspended inPBS, in the absence of antigen. After 24 h, the paw swelling was measured. Picracin (10µg/ml and 1.0 µg/ml), and to a lesser extend DAP (10 µg/ml), showed a significantirritating effect (figure 6).

0 0.1 1 100.00

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swel

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Figure 6. Direct irritant effects of picracin and deacetylpicracin (DAP) after subplantaradministration. Data were expressed as means ± SEM (n = 5 per group) and statisticallyanalyzed by the Student’s t-test. P values are < 0.05 (*), < 0.01 (**), or < 0.001 (***).

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Discussion

Delayed-type hypersensitivity (DTH) is a valuable model to demonstrate cell-mediated immune responses, which are mediated mainly by T cells. Kerckhaert et al.(1974) showed that a single injection of cyclophosphamide (300 mg/kg) 8 h beforeimmunization resulted in an enhanced DTH-response 10 days after immunization, and adecreased B-cell response during the whole experiment. Later research demonstrated theselective inactivation of suppressor T cells or their precursors by cyclophosphamide(Kaufmann et al., 1980, Bovbjerg et al., 1986), and the selective induction of apoptosis indifferentiating B cells as compared with T cells in vivo (Hemendinger and Bloom, 1996).Therefore, this model was chosen to determine the effects of picracin or deacetylpicracin invivo, with dexamethasone serving as a positive control (Schrier et al., 1985). Thesignificant decrease in paw edema upon systemic administration of picracin ordeacetylpicracin suggests that these substances are immunosuppressive. However, theireffects were less prominent than the effects of dexamethasone, which is considered an anti-inflammatory rather than an immunosuppressive drug. To determine whether localadministration would enhance the effects observed, the compounds were injected directlywithin the area of inflammation. The inhibitory effects of both cucurbitacins, however,could not be enhanced by local, subcutaneous administration. Instead, an increasedswelling was observed. Control experiments revealed a concentration-dependent directinflammatory response of picracin, which was significant at low concentrations (1 µg/mland 10 µg/ml), and a minor but significant effect of deacetylpicracin at 10 µg/ml, thehighest concentration tested.

The inflammatory and immunosuppressive effects observed for picracin anddeacetylpicracin suggest a dualistic effect of these compounds. Upon local administration,they might induce a direct inflammatory response, while upon systemic administration(i.p.) they might be metabolically converted into compounds that exhibitimmunosuppressive effects instead. Alternatively, the effects might be related to theinduction of T-cell apoptosis in the paw. Apoptosis is usually not associated withinflammation, because the apoptotic bodies, which are the result of apoptosis, are ingestedby surrounding phagocytes and any leakage of their contents is thus avoided (Savill et al.,1993). However, Uchimura et al. (1997) demonstrated that apoptotic CTL-L2 cells, co-cultured with peritoneal exudate cells, resulted in the production of both anti-inflammatoryand pro-inflammatory cytokines. Culture supernatants induced the accumulation ofneutrophils in vivo (Uchimura et al., 1997). These findings suggest that apoptosis isassociated with leukocyte infiltration in vivo. Whether this effect is associated with edemaand inflammation remains unclear. However, the intraperitoneal administration of highdoses of picracin or deacetylpicracin, e.g. 30 and 100 mg/kg, might induce a strong influxof leukocytes secondary to T-cell apoptosis. Leukocytes might as well be withheld fromthe paw, leaving behind a reduced number of inflammatory cells to elicit a local immuneresponse after subcutaneous injection of antigen. A similar phenomenon has beendescribed before as counter-irritation, e.g. inflammation in progress at one site decreasesedema formation at a second and separate inflammatory focus (Normann et al., 1985).

In conclusion, picracin and deacetylpicracin, two cucurbitacins isolated fromPicrorhiza scrophulariiflora, which inhibit mitogen-induced proliferation of T cells, are


potent inhibitors of IL-2 release. Furthermore, picracin potently induced apoptosis of non-stimulated T cells, while deacetylpicracin was much less active. The induction of apoptosisby picracin might explain its inhibitory effects on the proliferation of T cells and itsinhibition of IL-2 release. The inferior effects of deacetylpicracin in inhibiting IL-2 releaseand inducing apoptosis might be due to the lower lipophilicity of deacetylpicracin. Theinactivity of deacetylpicracin and the activity of picracin to inhibit the release of IL-1β andTNFα in LPS-stimulated monocytes upon 24-h co-incubation, suggest that these effectsmight be ascribed to the induction of apoptosis as well, although further research has toconfirm any apoptosis-inducing effect on monocytes. Furthermore, picracin anddeacetylpicracin moderately inhibited delayed-type hypersensitivity responses in miceupon intraperitoneal administration, while subplantar injection in the paw elicited a localinflammatory response. These observations suggest the attraction of phagocytes, secondaryto T-lymphocyte apoptosis in vivo, and producing a counter-irritant effect uponintraperitoneal administration of these compounds.

Although our findings do not favor the usefulness of picracin and deacetylpicracin inautoimmune disorders such as rheumatoid arthritis, these compounds might be useful toolsin studying the effects of apoptosis induction in vivo. Furthermore, the effect of picracin onT-cell proliferative disorders needs study, as well as its effect on cell arrest extended toother cell types and relevant diseases. Picracin might serve as a lead compound for thedevelopment of pharmacotherapeutics, effective in such diseases.

References

AMR - American College of Rheumatology (1996a) Guidelines for the management ofrheumatoid arthritis. Arthritis Rheum. 39, 713-722.

AMR - American College of Rheumatology (1996b) Guidelines for monitoring drug therapyin rheumatoid arthritis. Arthritis Rheum. 39, 723-731.

Bovbjerg, D.H., Ader, R., Cohen, N. (1986) Long-lasting enhancement of the delayed-typehypersensitivity response to heterologous erythrocytes in mice after a single injection ofcyclophosphamide. Clin. Exp. Immunol. 66, 539-550.

Cronstein, B.N. (1996) Molecular therapeutics: methotrexate and its mechanism of action.Arthritis Rheum. 39, 1951-1960.

Dayer, J.-M., De Rochemonteix, B., Burrus, B., Semczuk, S., Dinerello, C.A. (1986) Humanrecombinant interleukin 1 stimulates collagenase and prostaglandin E2 production byhuman synovial cells and dermal fibroblasts. J. Exp. Med. 77, 645-648.

Duncan, M.D., Duncan, K.L. (1997) Cucurbitacin E targets proliferating endothelia. J. Surg.Res. 69, 55-60.

Feldmann, M., Brennan, F.M., Maini, R.N. (1996) Role of cytokines in rheumatoid arthritis.Annu. Rev. Immunol. 14, 397-440.

Hemendinger, R.A., Bloom, S.E. (1996) Selective mitomycin C and cyclophosphamideinduction of apoptosis in differentiating B lymphocytes compared to T lymphocytes invivo. Immunopharmacology 35, 71-82.

Jackson, C.G., Williams, H.J. (1998) Disease-modifying antirheumatic drugs. Drugs 56,337-344.

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Kaufmann, S.H., Hahn, H., Diamantstein, T. (1980) Relative susceptibilities of T cell subsetsinvolved in delayed-type hypersensitivity to sheep red blood cells to the in vitro actionof 4-hydroperoxycyclophosphamide. J. Immunol. 125, 1104-1108.

Kerckhaert, J.A.M., Van den Berg, G.J., Willers, J.M.N. (1974) Influence ofcyclophosphamide on the delayed hypersensitivity of the mouse. Ann. Immunol. (Inst.Pasteur) 125C, 415-426.

Martin, S.J., Reutelingsperger, C.P., McGahon, A.J., Rader, J.A., van Schie, R.C., LaFace,D.M., Green, D.R. (1995) Early redistribution of plasma membrane phosphatidylserineis a general feature of apoptosis regardless of the initiating stimulus: inhibition byoverexpression of Bcl-2 and Abl. J. Exp. Med. 182, 1545-1556.

Normann, S.J., Schardt, M., Sorkin, E. (1985) Antileukocyte activity I. Systemic inhibitionof cellular emigration following local inflammation. J. Leukocyte Biol. 37, 319-330.

Savill, J.S., Fadok, V., Henson, P., Haslett, C. (1993) Phagocyte recognition of cellsundergoing apoptosis. Immunol. Today 14, 131-136.

Schrier, D.J. (1985) Effects of anthiarthritic compounds on sheep erythrocyte-induceddelayed-type hypersensitivity. J. Immunol. 7, 313-323.

Shevach, E.M. (1995) The effects of cyclosporin A on the immune system. Annu. Rev.Immunol. 3, 397-423.

Smith, K.A. (1988) Interleukin-2: inception, impact, and implications. Science 240, 1169-1176.

Starkebaum, G. (1998) Role of cytokines in rheumatoid arthritis. Sci. Med. 6-15.Swain, S.L. (1999) Helper T cell differentiation. Curr. Opin. Immunol. 11, 180-185.Thomas, B.M., Mundy, G.R., Chambers, T.J. (1987) Tumour necrosis factor α and β induce

osteoblastic cells to stimulate osteoclast bone resorption. J. Immunol. 138, 775-780.Uchimura, E., Kodaira, T., Kurosaka, K., Yang, D., Watanabe, N., Kobayashi, Y. (1997)

Interaction of phagocytes with apoptotic cells leads to production of pro-inflammatorycytokines. Biochem. Biophys. Res. C. 239, 799-803.

Vaishnaw, A.K., McNally, J.D., Elkon, K.B. (1997). Apoptosis in the rheumatic diseases.Arthritis Rheum. 40, 1917-1927.

Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. (1995) A novel assay forapoptosis. Flow cytometric detection of phosphatidylserine expression on earlyapoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39-51.

Summarizing discussion andperspective

Chapter 9

126 | Chapter 9

Summarizing discussion

Natural products have been an important resource for the maintenance of life for ages.The interest in medicinal plants has never ceased since. Even today, natural productsbecome increasingly important as a source of pharmacotherapeutics, either directly, e.g. inthe application of herbal drugs for the treatment of chronic diseases, or as a source of moreor less complex chemical structures are isolated, with a particular biological activity.Instead of random screening of a large number of plant extracts, traditional systems ofmedicine provide an extremely vast body of source material for the development of newdrugs (Holland, 1994). Evaluation of traditionally used medicines, keeping into account thetraditional principles that are applied in drug therapy, may supply leads towards effectivedrug discovery.

Previously, based on such an approach, P. kurrooa Royle was identified as a potentialsource of immunomodulating compounds (Labadie et al., 1989), and its modulatory effectson complement activation and the production of chemiluminescence by activatedneutrophils were investigated (Simons, 1989). Because oxidative stress due to reactiveoxygen species is a major contributor to joint damage in rheumatoid arthritis (Halliwelland Gutteridge, 1999), these data suggested the usefulness of Picrorhiza in the treatment ofthis disease. This thesis deals with the further pre-clinical evaluation of theimmunomodulatory activity of Picrorhiza and the activity-guided isolation of activeconstituents, in order to assess its usefulness in rheumatoid arthritis or related conditions.

Rhizomes of two Picrorhiza species happen to be available on South-Asian markets,e.g. P. kurrooa Royle and P. scrophulariiflora Pennell (Scrophulariaceae). Both speciesare known under the same local name, and used for similar purposes. Because the plantmaterial under investigation was obtained as a simplex, and there are no discriminativefeatures between the rhizomes of both species, the identity of the plant material could notdirectly be determined. However, an inventory of the geographical distribution of bothPicrorhiza species provided sufficient circumstantial evidence to establish the properidentity, as described in chapter 2. The material was obtained from Gorkha District, Nepal.Literature review revealed that the distribution of P. kurrooa is restricted to the WesternHimalaya, from Kashmir to Kumaon, while P. scrophulariiflora is found in the EasternHimalaya, from Garhwal to Sikkim. This observation is a strong argument to consider theidentity of the Gorkha material to be P. scrophulariiflora. Observations by Olsen (1998)confirm the exclusive occurrence of P. scrophulariiflora in Gorkha District of CentralNepal. Fieldwork into Gorkha District supports this conclusion, showing the exclusivepresence of P. scrophulariiflora in this area. Although Hong categorized this species into aseparate genus and named it “Neopicrorhiza scrophulariiflora” (Hong, 1984), for reasonsof clarity, described in chapter 2, the name Picrorhiza scrophulariiflora is maintainedthroughout this thesis.

Chapter 3 deals with the uses of Picrorhiza in Āyurveda, the traditional medicine ofSouth Asia. The applicability of a plant for the treatment of a particular disease can bederived from generalized traditional principles, or obtained directly from traditional uses.When using the first approach, in which the properties of Picrorhiza according toĀyurveda were considered, it became clear that Picrorhiza increases vāta (wind), and assuch, causes or aggravates vātika disorders. Vāta plays an important role in thedevelopment of āmavāta (rheumatoid arthritis), although this disease is usually not

Summarizing discussion and perspective | 127

classified among the vātika diseases, but described as a separate disease entity. Its primarycause is not derangement of vāta, but the formation of āma (undigested food materials).Picrorhiza has the properties of inducing digestion and removal of āma. This suggests that,according to Āyurvedic principles, it will be useful in the prevention of āmavāta(rheumatoid arthritis). For the treatment of established āmavāta, however, it is notrecommended, because of its vāta-enhancing activity. Regarding the inductive approach,e.g. direct exploitation of traditional claims, references to the treatment of arthritis withPicrorhiza as a single drug are not found in Āyurvedic literature. Upon investigating theuse of Āyurvedic preparations of Picrorhiza, a similar result was obtained. Moreover,fieldwork in northern India and Nepal yielded thirty-three different commercially availablepreparations that contain Picrorhiza, while four of them were prescribed for the treatmentof rheumatic conditions. Seventeen of the products obtained were classical Āyurvedicpreparations, while the others were proprietary products, developed by Āyurvediccompanies. However, according to the original sources, only ten of these classicalpreparations contained Picrorhiza, and only one is sometimes used for the treatment ofāmavāta, although this is not indicated in the original source. Furthermore, this preparationcontains just a fraction of Picrorhiza, if any. Therefore, based on both deductive andinductive approaches, it can be concluded that Picrorhiza does not play an important rolein the treatment of āmavāta. However, according to Āyurvedic principles, Picrorhizaremains a potent therapy in the prevention of āmavāta (rheumatoid arthritis).

Review of the experimental literature on Picrorhiza species, as described in chapter 4,revealed the accumulation of cucurbitacin glucosides, iridoid glucosides, phenylethanoidglucosides, and acetophenone glucosides in the rhizomes of Picrorhiza. These compoundshave a wide range of biological activities, among which anti-inflammatory effects seem toprevail. Hardly any information on the effects of the cucurbitacins from Picrorhiza isavailable. Other cucurbitacins, however, are described to have cytotoxic and antitumoreffects, immunomodulating and adaptogenic activities. Some particular cucurbitacins arehighly toxic. As far as iridoid glucosides concerned, the effects described relate mainly to aprotection of the liver against experimentally induced damage. In contrast, phenylethanoidglucosides exhibit anti-inflammatory effects by inhibiting pro-inflammatory responses, e.g.by diminishing phosphodiesterase and 5-lipoxygenase activities. Acetophenones likeapocynin contribute to the anti-inflammatory effects by interfering with the formation ofreactive oxygen species and enhancing the physiological antioxidant system, which lead toa wide range of secondary effects.

Chapter 5 describes the modulating activities of Soxhlet extracts of P.scrophulariiflora on the classical complement pathway, the release of chemiluminescenceby neutrophil leukocytes, and the proliferation of T lymphocytes, as a reflection ofnonspecific humoral and cellular defense mechanisms, and specific cellular immuneresponses. The diethyl-ether Soxhlet extract was the most anti-inflammatory of all extractsprepared, concerning both nonspecific (complement and respiratory burst) and specific (T-cell proliferation) immune responses. Furthermore, prolonged administration of this extractshowed inhibition of carrageenan-induced paw edema in mice. The light petroleum andaqueous extracts were less active in inhibiting the classical complement pathway, while theethyl acetate and methanol extracts moderately inhibited chemiluminescence by activatedPMNs. In vivo, however, these extracts were completely inactive in the doses tested. The invivo anti-inflammatory activity of the diethyl-ether extract is in line with the effects

128 | Chapter 9

observed in vitro for this extract. We assume that at least a part of the processes suggestedto be involved in carrageenan-induced paw edema (activation of complement;degranulation of mast cells; release of kinins; release of reactive oxygen or nitrogenspecies by activated phagocytes; arachidonic acid metabolites; and pro-inflammatorycytokines) is subject of inhibition by P. scrophulariiflora extracts. Moderate inhibitoryactivity on activated neutrophils in vitro, as shown for the diethyl ether and the ethyl-acetate extracts, was mainly attributed to apocynin. However, apocynin did not show anyanti-inflammatory effect in the acute inflammation model, suggesting that compoundsother than apocynin are responsible for the anti-inflammatory effects observed. This effectmay be due to the presence of compounds, which inhibit activation of complement.Alternatively, a combination of immunomodulating compounds with different mechanismsof action might be necessary to produce anti-inflammatory activity in vivo.

Collagen-induced arthritis in mice was used as a model to determine the effects of P.scrophulariiflora extracts on chronic inflammation. Treatment with 200 mg/kg diethyl-ether extract or 20 mg/kg apocynin reduced the incidence of arthritis, while ethyl acetateand methanol extracts were not active. However, neither of the samples tested,significantly affected the severity of arthritis. These results suggest that the inhibitoryactivity of the diethyl-ether extract can be attributed to apocynin. Furthermore, it seemsthat the in-vitro inhibitory effects of the diethyl ether extract on complement activation,chemiluminescence of activated PMNs, and T-cell proliferation, do not predict inhibitoryactivity in collagen-induced arthritis in mice. This might be due to under-dosage, lowbiological availability, an insufficient pharmacokinetic profile, or to metabolic inactivationof active compounds in vivo. It is also possible that pathological processes, other than thosereferred to and not or insufficiently inhibited by the diethyl ether extract, lead to thedevelopment of collagen-induced arthritis in mice. The in vivo effects were not associatedwith significant toxicity, as tested in mice by a semi-chronic assay involving body weightand weights of critical lymphoid organs. Even in doses up to 100 × the intended dose ofapplication in humans, the extracts were devoid of significant toxicity.

Several activity-guided purification methods have been followed in order to isolatecompounds from P. scrophulariiflora that inhibit complement activation. A representativeoverview of these methods is presented in chapter 6. It has become clear that in both thelight petroleum and the diethyl ether extracts several compounds are present, whichpotently inhibit the classical pathway of complement activation. It seems, however, that therespective amounts of these compounds in the extracts are rather low, making isolation andidentification very tedious and incomplete within the time span available. However, TLCanalysis suggested the presence of phytosterols in the light petroleum extract. Althoughthese compounds have not been demonstrated in Picrorhiza species before, phytosterolsare ubiquitous in higher plants and are described to exhibit potent complement-depletingactivity (Yamada et al., 1987, Oh et al., 1998). Furthermore, cucurbitacins may also play arole in the interference of the diethyl-ether extract with the complement system. Huang etal. (1998) demonstrated the presence of complement-inhibiting cucurbitacin glycosidesfrom Picria fel-terrae Lour. (Scrophulariaceae). Cucurbitacin glucosides are abundantlypresent in Picrorhiza species (Stuppner et al., 1991; Wang et al., 1993). While cucurbitacinglucosides might not be present in the apolar diethyl-ether extract, it is possible that theircorresponding aglucones are responsible for the activity observed.


Chapter 7 describes the isolation of two cucurbitacins from the rhizomes of P.scrophulariiflora, guided by inhibitory activity in a mitogen-induced human T-lymphocyteproliferation assay. The diethyl-ether extract, which potently inhibited proliferation, wassubjected to Silica column chromatography, followed by crystallization of active fractions,yielding deacetylpicracin. Other fractions were subjected to Sephadex LH20 columnchromatography and preparative HPLC, yielding picracin. A third active compound couldnot be isolated yet. The structures of both isolated compounds were determined by EIMS,FABMS, IR, 1H NMR, and 13C NMR spectroscopy, revealing their identity as aglycones ofknown 11-deoxycucurbitacin glucosides (Laurie et al., 1985, Stuppner and Wagner, 1989).Both compounds exhibited a potent inhibition of PHA-induced T-cell proliferation.

Because cucurbitacins are well known for their cytotoxic behavior, direct cytotoxicproperties of the two compounds were determined. Labeling the PHA-stimulatedlymphocytes after 4-day incubation with carboxyfluorescein diacetate and propidiumiodide to distinguish between viable and dead cells, showed only minor cell death (< 10 %)for concentrations of picracin and deacetylpicracin below 60 µM. Another cytotoxicityexperiment, using the lactate dehydrogenase release assay, did not show any toxic effect upto 50 µM for both cucurbitacins after 24 h. As these concentrations are much higher thanthe IC50 values of both compounds (1 µM), it can be concluded that the inhibitory activityof picracin and deacetylpicracin is most probably not to be ascribed to direct cytotoxiceffects.

Following the isolation of compounds that inhibit T-cell proliferation, possibleunderlying mechanisms were investigated, as described in chapter 8. Since proliferation ofactivated T cells is mediated by the release of IL-2 and the subsequent interaction with theIL-2 receptor, the effects of these compounds on the release of IL-2 by activated Tlymphocytes were determined. Incubation of activated T cells with picracin ordeacetylpicracin for 24 h showed a marked inhibition of IL-2 release after 24 h, whichcould not be ascribed to a direct toxic effect. Furthermore, cucurbitacin E, a cucurbitacinknown to inhibit the proliferation of endothelial cells (Duncan and Duncan, 1997),inhibited IL-2 release as well. However, fluorescence microscopical analysis followingstaining of cells with CFDA and PI demonstrated that in this case a direct cytotoxic effectmight be involved. These data explain the antiproliferative activity of picracin andcucurbitacin, since IL-2 is responsible for signaling T-cell proliferation (Smith, 1988).

The interference of picracin and deacetylpicracin with IL-2 release by activated Tlymphocytes could be the result of a specific interaction with signal transduction pathwaysleading to the release of IL-2, or due to the induction of apoptosis, which renders the T cellincapable of releasing IL-2. To assess the apoptotic effect of picracin and deacetylpicracin,PBL were stained with FITC-conjugated annexin V and propidium iodine. Cytometricanalysis showed a significant induction of apoptosis by picracin, while deacetylpicracinwas about three times less effective, and not significantly different from untreated cells(chapter 8). The inferior effects observed for deacetylpicracin in pro-inflammatorycytokine release and the induction of apoptosis may be due to its lower lipophilicity, whichmight result in a less pronounced interaction with cell membranes, and a delayed capacityto induce apoptosis.

In addition to the effects on T lymphocytes, the effects of picracin anddeacetylpicracin on the release of the pro-inflammatory cytokines IL-1β and TNFα by

130 | Chapter 9

activated monocytes was investigated. Incubation of human monocytes with picracin for24 h inhibited the release of IL-1β and TNFα, while deacetylpicracin was not active. Theinhibitory activity of cucurbitacin E was found to be in the same order of magnitude as thatof picracin. As both compounds differ only in the presence of carbonyl functionalities atC3 and C11, and a C1-2 double bond, the acetyl functionality in the side chain might beessential in the inhibiting of IL-1β and TNFα release.

Delayed-type hypersensitivity, a valuable model to demonstrate cell-mediated immuneresponses, was used to determine the effects of picracin and deacetylpicracin in vivo. Bothpicracin and deacetylpicracin significantly inhibited paw edema, at doses of 100 mg/kg and30 mg/kg, respectively, upon intraperitoneal administration. To determine whether localadministration would enhance the effects observed, the compounds were injected directlywithin the area of inflammation. The inhibitory effects of both cucurbitacins, however,could not be enhanced by local, subcutaneous administration. Instead, an increasedswelling being observed. Control experiments revealed a concentration-dependent directinflammatory response, which was significant even at low concentrations.

The inflammatory and immunosuppressive effects observed for picracin anddeacetylpicracin suggest a dualistic effect of these compounds. Upon local administration,they might induce a direct inflammatory response, while upon systemic administration(i.p.) they might be metabolically converted into compounds that exhibitimmunosuppressive effects instead. Alternatively, the effects might be due to the inductionof T-cell apoptosis in the paw. Although apoptosis is usually not associated withinflammation, because the apoptotic cells are ingested by surrounding phagocytes beforeany leakage of their contents can occur, they can induce the production of cytokines byperitoneal exudate cells in vitro, which was related to the accumulation of neutrophils invivo (Uchimura et al., 1997). These findings suggest that apoptosis is associated withleukocyte infiltration in vivo. Whether this effect is associated with edema andinflammation remains unclear. However, the intraperitoneal administration of high dosesof picracin or deacetylpicracin might induce a strong influx of leukocytes secondary to T-cell apoptosis. Leukocytes might as well be withheld from the paw, leaving behind areduced number of inflammatory cells to elicit a local immune response after subcutaneousinjection of antigen.

Summarizing, the diethyl-ether Soxhlet extract of dried rhizomes of Picrorhizascrophulariiflora potently inhibited the classical complement pathway, the release ofchemiluminescence by activated neutrophils, and the mitogen-induced proliferation of Tlymphocytes. Prolonged oral administration of this extract inhibited carrageenan-inducedpaw edema in mice and delayed the incidence of collagen-induced arthritis. The activity inacute inflammation may involve compounds interfering with the classical complementpathway, while the activity in chronic inflammation could be ascribed to apocynin.Activity-guided isolation revealed the presence of several complement-modulatingcompounds, although these compounds were not isolated yet. Furthermore, based onactivity in the T-cell proliferation assay, two cucurbitacins were isolated from this extractand their structures were elucidated. In addition to the inhibitory effects of thesecompounds on the proliferation of T lymphocytes, they also inhibited the release of IL-2.Moreover, picracin, but not deacetylpicracin, inhibited the release of IL-1β and TNFα byactivated monocytes. These effects might be due to the induction of apoptosis, which was


demonstrated for T cells after incubation with picracin. Induction of apoptosis might play arole in the local immunostimulatory effects of picracin and deacetylpicracin in vivo, whilea similar effect might be responsible for the inhibitory effects on delayed typehypersensitivity, mediated via a counter-irritation phenomenon. Alternatively, upon localadministration these compounds might induce a direct inflammatory response, while uponsystemic administration (i.p.) they might be metabolically converted into compounds thatexhibit immunosuppressive effects.

In conclusion, several compounds play a role in immunomodulatory effects inducedby Picrorhiza extracts. Picracin and deacetylpicracin induce a local immune response uponlocal administration, while they tend to decrease a local inflammatory response aftersystemic (i.p.) administration. Although the latter results are promising with regard to theanti-inflammatory activity of Picrorhiza, it cannot be excluded that these compoundsenhance local inflammation and for example aggravate rheumatoid arthritis. Testing ofpicracin and deacetylpicracin in experimentally induced arthritis should be performed inorder to evaluate such effects. Both compounds are present in the diethyl-ether extract,which decreased the onset of arthritis in mice. However, apocynin was shown to exhibitsimilar effects, therefore, this compound might be responsible for the effects observed forthe diethyl-ether extract in collagen-induced arthritis. Neither the diethyl-ether extract, norapocynin decreased the severity of joint inflammation in the dosages used. These findingsare in agreement with the contraindication of Picrorhiza for the treatment of rheumatoidarthritis, both according to contemporary use and according to Āyurvedic principles.However, the experimental results as well as the conclusions drawn from the Āyurvedicproperties of Picrorhiza suggest that it can play an important role in the prevention ofrheumatoid arthritis.

Perspectives

A possible application of the cellular effects induced by picracin might be in theinterference with the cell skeleton. Several cucurbitacins (cucurbitacin D, I, E, and 2-methoxy-cucurbitacin E) are shown to induce morphological changes to Ehrlich ascitestumor cells at low concentrations. Only at higher concentrations, viability, respiration, andpermeability of these cells were affected, without an increase of the morphologicalchanges. Similar changes were observed in vivo, 1 hour after i.p. injection of cucurbitacinD into ascites-bearing mice. In samples drawn 24 h after injection, however, no deformedor necrotized tumor cells were found (Gitter et al., 1961).

These observations are confirmed by Duncan et al. (1996), who demonstratedcucurbitacin E-induced cell growth inhibition of prostrate carcinoma cells, even after drugremoval. Furthermore, exposure to cucurbitacin E led to the development ofmorphologically abnormal, multinucleated cells, associated with a change in vimentindistribution. Cucurbitacin E was shown to disrupt the filamentous (F)-actin cytoskeleton,which correlated with its antiproliferative effect (Duncan et al., 1996). Moreover,cucurbitacin E interfered with the actin cytoskeleton of human endothelial cells, showingpreferential sequestration in proliferating cells (Duncan and Duncan, 1997). The actincytoskeleton has been implicated as critical intermediates in several signal transductionpathways controlling cell division (Aderem, 1992, Luna and Hitt, 1992). TCR-mediated

132 | Chapter 9

stimulation of T cells leads to the organization of Caps, assemblies of receptors andsignaling molecules at the surface of T lymphocytes following activation (Monks et al.,1998, Penninger and Crabtree, 1999). The forces driving the formation of these structuresrequire actin polymerization in T cells (Wülfing et al., 1998). This cytoskeletonreorganization plays an important role in downstream signal transduction (Valitutti et al.,1995).

Although interference of picracin and deacetylpicracin with the cytoskeleton ofactivated T cells has not been reported, the similarity of picracin and cucurbitacin E as faras structure and biological effects are concerned could indicate a similar mechanism inblocking signal transduction pathways. Furthermore, although the induction of T-cellapoptosis by cucurbitacin E has not been demonstrated so far, the effects of cucurbitacin Eon the cytoskeleton, in association with its antiproliferative effect towards tumor cells andproliferating endothelial cells, might be explained by the induction of apoptosis in thesecells. A similar mechanism might explain the induction of apoptosis by picracin anddeacetylpicracin upon exposure to activated T cells.

Several compounds, mainly derived from natural products, are known to interfere withthe cytoskeleton, some of them being very effective in cancer chemotherapy (Jordan andWilson, 1998). Cytochalasins are fungal metabolites and among the best-studied agentsacting on actin-filament organization and the subsequent effect on cell functioning(Cooper, 1987). Cytochalasin D abrogates actin polymerization, Cap formation, andsubsequent downstream signal transduction leading to IL-2 transcription upon antigenreceptor activation of T lymphocytes (Holsinger et al., 1998). Furthermore, cytochalasin Dinduces apoptosis of CD4+ T cells, which correlates with its affinity for the polymerizationends of actin filaments. Moreover, the cytochalasin-D-induced cytoskeletal disruption wasassociated with the up-regulation of caspase-3 protease activity and an increasedaccessibility of active caspase-3 to its substrate (Suria et al., 1999). Caspases are proteases,involved in the intracellular proteolytic machinery ultimately leading to apoptosis. Theyinactivate proteins that protect living cells from apoptosis, disassemble cell structures, andas such contribute to chromatin condensation, and inactivate proteins involved incytoskeletal regulation (Thornberry and Lazebnik, 1998).

Although several gaps have yet to be filled in, the mechanism of picracin-mediatedcell arrest might be of similar nature. Its effects on T lymphocytes might be due to adisturbance of the cytoskeletal organization, leading to a downstream activation ofcaspases, and a subsequent induction of apoptosis. Future research in order to substantiatethis hypothesis should be focusing on the effect of picracin on the actin-cytoskeleton andsubsequent effects on caspase activity. Furthermore, the effect of picracin on T-cellproliferative disorders needs study, as well as its effect on cell viability extended to othercell types and relevant diseases. Finally, picracin and deacetylpicracin may serve as leadcompounds for the development of novel anticancer drugs.


References

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Cooper, J.A. (1987) Effects of cytochalasin and phalloidin on actin. J. Cell. Biol. 105, 1473-1478.

Duncan, K.L.K., Duncan, M.D., Alley, M.C., Sausville, E.A. (1996) Cucurbitacin E-induceddisruption of the actin and vimentin cytoskeleton in prostrate carcinoma cells. Biochem.Pharmacol. 52, 1553-1560.

Duncan, M.D., Duncan, K.L.K. (1997) Cucurbitacin E targets proliferating endothelia. J.Surg. Res. 69, 55-60.

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Holland, B.K. (1994) Prospecting for drugs in ancient texts. Nature 369, 702.Holsinger, L.J., Graef, I.A., Swat, W., Chi, T., Bautista, D.M., Davidson, L., Lewis, R.S.,

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Hong, D.Y. (1984) Taxonomy and evolution of the Veroniceae (Scrophulariaceae) withspecial reference to palynology. Opera Bot. 75, 5-60.

Huang, Y., De Bruyne, T., Apers, S., Ma, Y., Claeys, M., Vanden Berghe, D., Pieters, L.,Vlietinck, A. (1998) Complement-inhibiting cucurbitacin glycosides from Picria fel-terrae. J. Nat. Prod. 61, 757-761.

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Labadie, R.P., Van der Nat, J.M., Simons, J.M., Kroes, B.H., Kosasi, S., Van den Berg,A.J.J., ’t Hart, L.A., Van der Sluis, W.G., Abeysekera, A., Bamunuarachchi, A., DeSilva, K.T.D. (1989) An ethnopharmacognostic approach to the search forimmunomodulators of plant origin. Planta Med. 55, 339-348.

Laurie, W.A., McHalle, D., Sheridan, J.B. (1985) A cucurbitacin glycoside from Picrorhizakurroa. Phytochemistry 24, 2659-2661.

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Stuppner, H., Wagner, H. (1989) New cucurbitacin glycosides from Picrorhiza kurrooa.Planta Med. 55, 559-563.

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Thornberry, N.A., Lazebnik, Y. (1998) Caspases: enemies within. Science 281, 1312-1316.Uchimura, E., Kodaira, T., Kurosaka, K., Yang, D., Watanabe, N., Kobayashi, Y. (1997)

Interaction of phagocytes with apoptotic cells leads to production of pro-inflammatorycytokines. Biochem. Biophys. Res. C. 239, 799-803.

Valitutti, S., Dessing, M., Aktories, K., Gallati, H., Lanzavecchia, A. (1995) Sustainedsignaling leading to T cell activation results from prolonged T cell receptor occupancy.Role of T cell actin cytoskeleton. J. Exp. Med. 181, 577-584.

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Sanskrit vocabulary

Appendix A

136 | Appendices

Introduction

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138 | Appendices

3(&-(".-N*( '5,(.)5*"#:-$/$#3(&-(#&/$+54)*(.$"g#$@>@#.-$#]5)*."3(/4(*# 459$4$*.#5/#(,.)5*3(eN6(## ("./)*>$*.3)``(## :(".$#&/5+7,."37`3O## \),/5/-)f(37e.-(# "3)*#+)"$("$g#'$&/5"63S4)# :5/4"3S4)>-*(# (*.-$'4)*.),3$d(## -()/#5;#.-$#-$(+3(74N/(<-S.6(# &$+)(./),"3/)6N3N'(# ".(>$"#5;#&(.-5>$*$")"3/5e`73(dO/e(# ,5*+).)5*#)*#:-),-#.-$#3*$$#)"#&()*;7''6#":5''$*#+7$#.5#9N.(#(*+

d5b).(3'$+(3( 45)".$*"#.-$#;55+#)*#.-$#".54(,-3e(6( :(".)*>#+)"$("$3-(4('(## $%,/$.)5*"3-(/(## /57>->7b(# &/5&$/.6>7/7## -$(96]('( :(.$/]9(/( ;$9$/]9(/N^](6$.# /$459)*>#;$9$/.(/&(3( *57/)"-$"#.-$#"$*"$#5/>(*".)3.(# <)..$/.)3.(/("( <)..$/#)*#.(".$.)3.(9(/>( >/57#<)..$/".)3.("3(*+-(# >/57#<)..$/".O3eb(## "-(/&.$](" ;)/$.9(3"*$-(## "4$(/#5;#.-$#"3)*+N-(# <7/*)*>#"$*"(.)5*+O&(*(# +)>$".)9$1#(&&$.)f$/+5e( &-6")5'5>),('#;(,.5/+/(9(## ')?7)++/(96(# "7<".(*,$1#+/7>+/(96(>7b(## &-(/4(,5'5>6+-(4(*O# (/.$/6+-N.7 .)""7$+-N/(b(# .5#"7&&5/.*(3-(## *()'"*)>-(b`7 >'5""(/6#5;#4$+),)*('#&'(*."*)+N*(# &(.-5'5>61#,(7"(.)9$#;(,.5/"*6(>/5+-N+)>(b(# >/57#+/7>"#/$&/$"$*.$+#<6#*6(>/5+-(##HD),7"#<$*>('$*")"#c@L&(^,(35'(# >/57#;)9$#&7*>$*.#+/7>"&(^,(4(-N<-R.(#"# ;)9$#<("),#$'$4$*."&(`5'N+)>(b(# >/57#+/7>"#/$&/$"$*.$+#<6#&(`5'(#HP/),-5"(*.-$"#+)5),(#_5%<@L&(+N/.-(#" ,(.$>5/)$"#5;#/$(')(#5;#.-$#:5/'+

Appendices | 139

&N3( ".(>$#5;#+)>$".)5*&N,(3( +)>$"."#.-$#;55+&N,(*(## +)>$".)9$&),,-)'(## "')46&)..(## .5#<$#5/#4(3$#-5.g#;)/$1#&/)*,)&'$#5;#H4$.(<5'),L#./(*";5/4(.)5*&)..(]). ,5*?7$/"#&)..(&)..(9)/$,(*(## $%&$'')*>#.6&$#5;#&7/>(.)9$"&)..("N/(3(# ,-5'(>5>7$&)&&('6N+)>(b(# >/57#+/7>"#/$&/$"$*.$+#<6#&)&&('O##H\)&$/#'5*>74#c@L&7/Oe(# ;$,$"&R/9(/R&(# &/5+/54$"&S.-)9O# $(/.-&5e(b(# .5#*57/)"-&/(3S.)# ,5*".).7.)5*&/(35&( $%,).(.)5*#5;#.-$#+5e(#"g#"$,5*+#+)"$("$#".(>$&/(3eN'6(# .5#<$#:("-$+#5/#&7/);)$+g#-(9)*>#:("-$+#5/#/)*"$+&/(]N".-N&(*( (*.)F(<5/.);(,)$*.&/(&N3(## &/$')4)*(/6#+)>$".)5*&/(<-N9(## "&$,);),#&/5&$/.6#5;#(#+/7>&/(4$-( #7/)*(/6#+)"$("$&/("(/(# +$'5,(')f(.)5*#(*+#"&/$(+)*>#5;#.-$#+5e(#"g#.-)/+#+)"$("$#".(>$&/Nb( ;)''"#.-$#'7*>"#(*+#.-$#".54(,-1#$.,@<('N"N 3(&-(<('N"N/5,(3N*#-(*.) +$"./56"#3(&-(#(*+#+)">7".#;5/#;55+<('6( .5*),<N'(.(*./( &$+)(./),"<5+-(3( "(')9(.$"#.-$#;55+#;5/#.(".$#&$/,$&.)5*1#&/545.$"#-$(')*><-R.(9)+6N# +$45*5'5>6#(*+#&"6,-)(./6<-$+(# +$9$'5&4$*.#5;#,54&'),(.)5*"g#")%.-#+)"$("$#".(>$<-$+(*O6(# </$(3)*><-$+)3(# </$(3)*><-$+)*# &7/>(.)9$<-/N](3( 4()*.()*"#.-$#.$4&$/(.7/$1#,5'5/"#.-$#"3)*4(]](* <5*$#4(//5:4(]]N## <5*$#4(//5:4(+-7/(## ":$$.4(*+(## "'7>>)"-4(*+N>*)# )*+)>$".)5*4('(" :(".$#&/5+7,."4(-N3(eN6N >/57&1#,(.$>5/64NW"( 47",7'(/#.)""7$47".N+)>(b(# >/57#+/7>"#/$&/$"$*.$+#<6#47".N#HY6&$/7"#/5.7*+7"##c@L4R./(## 7/)*$4S+7## "5;.4$+("## (+)&5"$#.)""7$6(3S. ')9$/6(3S+7..$](3(# ')9$/#".)47'(*.

140 | Appendices

/(3.( <'55+/(3.(/7]N&(-(# /$459)*>#<'55+#+)"$("$/(^](3( ,5'5/"#.-$#<'55+1#,5*9$/."#.-$#*7./)$*."#)*.5#<'55+/("(## *7./)$*.#;'7)+1#&'("4(/("N6(*(# ",)$*,$#5;#'5*>$9).6/7]N&(-(# /$459)*>#"),3*$""/R3e(# +/61#/57>-/R3e(*(## +/6)*>/R&(# "64&.54"/$,(*(# '(%(.)9$/5>(# +)"$("$/5,(*( (&&$.)f)*>/5-)bO## \),/5/-)f('(>-7# ')>-.'(9(b(## "57/'$3-(*( $4(,)(.)*>'$3-(*O6(# $4(,)(.)*>'54(*## <5+6#-()/9N]O3(/(b(# ",)$*,$#5;#(&-/5+)")(,"9N.(## .5#459$g#:)*+1#&/)*,)&'$#5;#459$4$*.9N.(/(3.(# >57.9N67 ()/1 wind9)`"*$-(## "4$(/#)*#;$,$"9)+(>+-( )4&/5&$/'6#&/5,$""$+1#"&5)'$+9)&N3( .(".$#5;#.-$#)*>$".$+#4(.$/)('#(;.$/#+)>$".)5*9)d(+(## ,'$(/9)d$e(# &(/.),7'(/).6#5/#+)"")4)'(/).69)e(# &5)"5*1#9$*541#<(*$1#(*6.-)*>#(,.)9$'6#&$/*),)57"9)e(4(]9(/(# )//$>7'(/1#/$4)..$*.#;$9$/9)e(4(]9(/(*Nd)*O# /$459$"#/$4)..$*.#;$9$/ ###9O/6(## &5.$*,6#5;#(#+/7>9S++-) (>>/(9(.)5*96(3.)# 4(*);$".(.)5*#5;#(#+)"$("$g#;);.-#+)"$("$#".(>$96N*( 459$"#.-$#<5+61#)*+7,$"#<'55+#;'5:d(37'N+(*O## \),/5/-)f(d(/O/(## <5+61#(*(.546d('6(.(*./(# "7/>$/6dO.(# ,5'+1#,55'dO.(&)..(# 7/.),(/)(dO.('(# ,55')*>dO.(9O/6( ,5'+#&5.$*,6dN'N36(.(*./(# +)"$("$"#5;#-$(+#(*+#.-/5(.dO.N"/(+N-(]). ,5*?7$/"#,5'+1#<'55+1#(*+#<7/*)*>#"$*"(.)5*d73/( "$4$*d5b).(# <'55+d5+-(*(## $')4)*(.)5*#.-$/(&6

Appendices | 141

d'(3eb(## "455.-d'$e(3( '7</),(.$"#.-$#]5)*."d'$e4(*# .5#(+-$/$d9N"(# /$"&)/(.5/6#+)"$("$d9N"(# "-5/.*$""#5;#</$(.-"(W&/N&.)# &(.-5>$*$")""(Wd(4(*(# &(,);6)*>"(Wd5+-(*(# &7/);6)*>"(W-).N ./$(.)"$g#(*6#4$.-5+),(''6#(//(*>$+#,5''$,.)5*#5;#.$%."#5/#9$/"$""(^,(6(# (,,747'(.)5*#5;#+5e(#"g#;)/".#+)"$("$#".(>$"(..9(# 4)*+"(*+-)>(.(9N.(## 5".$5(/.-/).)""(*+-)9N.( 5".$5(/.-/).)""(**(4# "7*3#+5:*1#+$&/$""$+1#:$(3"(4(9N6(# )*-$/$*,$"(4N*( "$&(/(.$"#.-$#*7./)$*."#(*+#.-$#:(".$1#/$459$"#.-$#:(".$"(/(# ,(.-(/.),1#&7/>(.)9$1#'(%(.)9$g#;'5:)*>"N+-(3( >/("&"1#)*+7,$"#)*")>-."N*+/(## 9)",)+"N4(+5e(## N4(##,54<)*$+#:).-#+5e("N4N*6(# >$*$/(').6#5/#")4)'(/).6")/N#"# 9$)*""R3e4(## "7<.'$"*$-(*(## 5'$(.)5*#.-$/(&6"*)>+-(## 7*,.757"".(*6(d5+-(*( &7/);6)*>#</$(".#4)'31#>('(,.5+$&7/(*.".-N*("(Wd/(6(# '5,(')f(.)5*#5;#9).)(.$+#+5e(#"g#;57/.-#+)"$("$#".(>$".-)/(## )445<)'$".-R'(## >/5"""/5.(" ,-(**$'"/5.NW")# ,-(**$'""/5.5/5+-( 5<"./7,.)5*#5;#.-$#,-(**$'""9$+(## ":$(."9$+(*(## "7+(.)5*#.-$/(&6-)4(# ,5'+-S+# -$(/.1#,(/+)(,#/$>)5*-S+6(# &'$(")*>#.5#.-$#-$(/.g#,(/+)(,-S+/5>(# +)"$("$"#5;#.-$#,(/+)(,#/$>)5*g#-$(/.#+)"$("$-S+/5>(*Nd)*)# /$459)*>#-$(/.#+)"$("$

_$;$/$*,$"_$;$/$*,$"_$;$/$*,$"_$;$/$*,$"

E5*)$/FG)'')(4"1#E@#HIJKKL#!#2(*"3/).F=*>')"-#+),.)5*(/6@#_$&/)*.#IKKh1#E5.)'('i(*(/")+(""1#M$'-)@

Appendices | 143

Abbreviations

Appendix B

144 | Appendices

Abbreviations

dN dN/O/(".-N*(ACTH adrenocorticotropic hormone!j (e`N[>(-S+(6N"(W-).NAI arthritic indexAP alternative pathwayAPC antigen-presenting cellapo apocynin!\P (..(,-$+#&/5.5*#.$".Aq aqueous (extract)!2 (e`N[>("([>/(-(iZ# <-N9(&/(3Nd(*)>-(b`7i_# <-()e(]6(/(.*N9('Obr. broadCCl4 carbon tetrachlorideYM ,(3/(+(..(CFA complete Freund’s adjuvantCFDA 5-carboxyflourescein diacetate,) ,)3)."N".-N*(CIA collagen-induced arthritisCII collagen type IICMC carboxymethylcelluloseYk2l ,5//$'(.$+#"&$,./5",5&6CP classical pathwayY2 ,(/(3("(W-).Ncuc E cucurbitacin Ed doubletDAP deacetylpicracinDCM dichloromethaneDE diethyl ether (extract)dex dexamethasoneDMARD disease-modifying antirheumatic drugDMSO dimethylsulphoxideMZ +-(*9(*.(/O*)>-(b`7DNA deoxyribonucleic acidDTH delayed-type hypersensitivityEA ethyl acetate (extract)ED50 dose at 50 % effectEGTA ethylene glycol-bis-(β-aminoethyl ether)=AFE2 $'$,./5*#)4&(,.F4(""#"&$,./54$./6ELISA enzyme-linked immunosorbent assayEtOH ethanol (extract)D!iFE2 ;(".#(.54#<54<(/+4$*.F4(""#"&$,./54$./6FACScan fluorescence activated cell scanF-actin filamentous actin

Appendices | 145

FCS fetal calf serumFITC fluorescein isothiocyanatefMLP N-formyl-methionyl-leucyl-phenylalanineGCR glucocorticoid receptorGCS glutamylcysteine synthetaseGSH glutathioneHBSS Hank’s buffered salt solutionj=PYk_ -$.$/5*7,'$(/#,5//$'(.)5*HETE hydroperoxy eicosatetraenoic acidHIF-1 hypoxia inducible factor-1HPLC high performance liquid chromatographyHPS human pooled serumi.p. intraperitoneallyi.v. intravenouslyIC50 concentration at 50 % inhibitionICAM-1 intercellular adhesion molecule-1ID50 dose at 50 % inhibitionIgG immunoglobulin GIgM immunoglobulin MIL interleukinIL-2R interleukin-2 receptorIR infrared spectroscopy3( 3('&(".-N*(LD50 dose at 50 % lethalityLDH lactate dehydrogenaseLFA lymphocyte function associated moleculeLPS lipopolysaccharideLTB leukotrieneMAC major attack complexMASP mannose-binding lectin-associated serine proteaseMBL mannose-binding lectinMCP monocyte chemoattractant proteinMe methanol (extract)MHC major histocompatibility complexEZ 4N+-(9(*)+N*(MPO myeloperoxidasemRNA messenger RNAMTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideNAD β-nicotinamide adenine dinucleotideNADPH nicotinamide adenine dinucleotide phosphatend not detectable*) *)+N*(".-N*(NMR nuclear magnetic resonance spectrometryNSAID non-steroid anti-inflammatory drugp.o. per os; orallyPAF platelet activating factor

146 | Appendices

PBMC peripheral blood mononuclear cellsPBS phosphate-buffered salinePE light petroleum (extract)PG peptidoglycanPGE2 prostaglandin E2

PGF2α prostaglandin F2α

PHA phythemagglutininPI propidium iodidePKC protein kinase CPMA phorbol myristate acetatePMN polymorphonuclear neutrophilpred prednisoneRA rheumatoid arthritisRNA ribonucleic acidROS reactive oxygen species__2# /("(/(.*("(47,,(6(s singlets.c. subcutaneously"R "R./(".-N*(2(2 dN/[>(+-(/("(W-).NSDS sodium dodecyl sulphateSEM standard error of meansGPT serum glutamic-pyruvic transaminase2Z# dN')>/N4(*)>-(b`7SRBC sheep red blood cell22 "7d/7.("(W-).N2l# "(-("/(65>(t tripletTCR T-cell receptorTh1 cell T-helper-1 cellTLC thin-layer chromatographyTNFα tumor necrosis factor αTXA2 thromboxane A2

v/v volume per volumeVBS veronal-buffered salineVEGF vascular endothelial growth factor9) 9)4N*(".-N*(w/w weight per weightl_ 65>(/(.*N3(/(

Appendices | 147

Samenvatting .

Het gebruik van medicinale planten ter behoud en verhoging van de kwaliteit vanleven is eeuwenoud en nog steeds actueel. De laatste jaren is er zelfs een sterke toenamewaar te nemen van de interesse voor en het gebruik van natuurlijke produkten in degezondheidszorg. Dit gebeurt aan de ene kant door het rechtstreeks toepassen van kruidenof afgeleide bereidingen, bijvoorbeeld bij de behandeling van chronische aandoeningen;aan de andere kant dienen planten als grondstof voor de isolatie van biologisch actieveverbindingen. Deze verbindingen kunnen worden ontsloten door het willekeurigonderzoeken van grote aantallen planten extracten, maar ook een voorselectie wordengemaakt op grond van traditionele kennis van het gebruik van kruiden. De beoordeling vantraditioneel gebruikte kruidenpreparaten, waarbij recht wordt gedaan aan de traditioneleprincipes van gezondheid en ziekte, kan leiden tot het ontwikkelen van nieuwegeneesmiddelen.

Voorheen werd via een dergelijke benadering Picrorhiza kurrooa Royle (letterlijk:“Bitterwortel”) geselecteerd als een mogelijk interessante middel voorimmuunmodulerende verbindingen. Resultaten van onderzoek naar de effecten van dezeplant op het complement systeem en op de vorming van zuurstof radicalen doorgeactiveerde witte bloedcellen (leukocyten) suggereerde een mogelijke toepassing bijreumatoïde gewrichtsontsteking. Dit proefschrift handelt over aanvullend onderzoek naarde immuun modulerende eigenschappen van Picrorhiza en de isolatie van biologischactieve verbindingen, om een beter beeld te krijgen van de toepasbaarheid van deze plantin chronische gewrichtsontsteking.

Wortelstokken van twee verschillende Picrorhiza soorten zijn verkrijgbaar op Zuid-Aziatische kruidenmarkten, te weten P. kurrooa Royle en P. scrophulariiflora Pennell. Erwordt geen onderscheid gemaakt tussen de wortelstokken van beide soorten. Aangezien hetplantenmateriaal dat voor dit onderzoek werd gebruikt bestond uit gedroogdewortelstokken, en er geen methoden bekend zijn om wortelstokken van beide soorten teonderscheiden, kon de identiteit van het plantenmateriaal niet rechtsreeks wordenvastgesteld. Echter, een inventarisatie van de geografische verspreiding van beide soorten,beschreven in hoofdstuk 2, leverde voldoende aanwijzingen om de identiteit van hetgebruikte plantenmateriaal vast te kunnen stellen. Het materiaal werd verkregen uit hetGorkha district in Nepal. Een inventarisatie van de locaties waar Picrorhiza soorten zijngevonden wees uit dat de verspreiding van P. kurrooa zich beperkt tot de WestelijkeHimalaya, terwijl P. scrophulariiflora voorkomt in de Oostelijke Himalaya. Voorts bleekuit onderzoek dat in het Gorkha district slechts P. scrophulariiflora voorkomt, hetgeenwerd bevestigd door veldwerk in dit gebied. Op basis van deze informatie werdverondersteld dat het gebruikte plantenmateriaal vastgesteld als P. scrophulariiflora.

148 | Appendices

Hoewel deze soort in 1984 werd ondergebracht in een nieuw geslacht, namelijkNeopicrorhiza, is het gebruik van de synoniem Picrorhiza scrophulariiflora gehandhaafdin dit proefschrift. De argumenten die ten grondslag liggen aan deze keuze wordenbeschreven in hoofdstuk 2.

Hoofdstuk 3 beschrijft het gebruik van Picrorhiza in de Āyurveda, de traditionelegeneeskunde van Zuid Azië. De toepasbaarheid een medicinale plant voor de behandelingvan een bepaalde aandoening kan worden afgeleid aan de hand van gegeneraliseerdetraditionele principes, of rechtstreeks worden ontleend aan het traditionele gebruik.Wanneer voor de eerste benadering wordt gekozen, waarbij de Āyurvedischeeigenschappen van Picrorhiza in aanmerking worden genomen, wordt het duidelijk datdeze plant de vāta (vgl. wind) verhoogd en als zodanig ziekten die zijn veroorzaakt doorvāta kunnen verergeren. Hoewel vāta een belangrijke rol speelt in het ontstaan vanāmavāta (reumatoïde artritis), is dit toch niet de hoofdoorzaak. Deze wordt veeleergevormd door het verschijnsel āma (onvolledig verteerde nutriënten). Picrorhiza heeft alseigenschappen het bevorderen van de spijsvertering en het verwijderen van āma. Ditsuggereert dat Picrorhiza, volgens de Āyurvedische principes, een belangrijke rol kanspelen in het voorkómen van reumatoïde gewrichtsontsteking. Voor de behandelinghiervan wordt het echter niet aangeraden vanwege het stimulerende effect of vāta.Eenzelfde conclusie werd getrokken na onderzoek van de toepassing van Āyurvedischebereidingen van Picrorhiza. Verwijzingen uit de klassieke literatuur naar het gebruik vanPicrorhiza bij gewrichtsontsteking werden niet gevonden. Bovendien bleek uitveldonderzoek in Noord India en Nepal dat complexe bereidingen met een substantiëlehoeveelheid Picrorhiza niet gebruikt worden voor de behandeling van chronischegewrichtsontsteking.

Uit onderzoek van Picrorhiza soorten, zoals beschreven in hoofdstuk 4, blijkt dat metname cucurbitacin glucosiden, iridoid glucosiden, phenylethanoid glucosiden enacetophenon glucosiden worden geaccumuleerd in de wortelstokken van Picrorhizasoorten. Deze componenten hebben een wijd uiteenlopende biologische activiteit, waarinechter ontstekingsremmende effecten de overhand hebben. Hoewel vrijwel geen informatiebeschikbaar is over de biologische activiteit van cucurbitacinen uit Picrorhiza, worden vanandere vergelijkbare verbindingen cytotoxische, antitumor, immuun modulerende enadaptogene effecten beschreven. Tevens is bekend dat sommige cucurbitacinen sterk giftigzijn. De effecten die beschreven zijn voor iridoid glucosiden houden meestal verband metde bescherming van de lever tegen experimenteel geïnduceerde schade. De werking vanphenylethanoid glucosiden wordt meestal in verband gebracht met de remming vanenzymen die een rol spelen bij het ontstaan van ontstekingsreacties, zoalsphosphodiesterase en 5-lipoxygenase. Acetophenonen zoals apocynin dragen bij aan deontstekingsremmende werking door de remming van de vorming van zuurstofradicalen ende verhoging van het cellulaire antioxidant systeem.

Hoofdstuk 5 beschrijft het onderzoek naar de modulerende eigenschappen vanextracten van P. scrophulariiflora op de klassieke route van het complement systeem, deproduktie van chemiluminescentie door leukocyten en de proliferatie van T lymfocyten. Deonderzochte processen weerspiegelen aspecifieke humorale en cellulaireverdedigingsmechanismen en tevens specifieke immuunreacties. Van de geteste extractenvertoonde het diethylether Soxhlet extract de sterkste effecten, zowel in aspecifieke alsspecifieke immuunprocessen. Bovendien vertoonde dit extract een remming van

Appendices | 149

carrageenan-geïnduceerde pootzwelling in muizen na meerdere toedieningen. Depetroleumether en water extracten vertoonden minder sterke remmende effecten op deklassieke route van het complement systeem, terwijl de ethylacetaat en methanol extractende chemiluminescentie van leukocyten matig remden. Deze extracten vertoonden echtergeen remmend effect op carrageenan-geïnduceerde pootzwelling in de onderzochtedoseringen. De remming van chemiluminescentie door de diethylether en ethylacetaatextracten kan voor een groot deel worden toegeschreven aan apocynin. Echter, apocyninvertoonde geen remmend effect in bovengenoemd model, hetgeen suggereert dat anderestoffen verantwoordelijk zijn voor het ontstekingsremmende effect van het diethyletherextract. Het is mogelijk dat verbindingen die het complement systeem remmen hiervoorverantwoordelijk zijn. Ook is het mogelijk dat een combinatie van verbindingen met eenverschillende werking nodig is om een onstekingsremmend effect in vivo te verkrijgen.

Kraakbeen geïnduceerde gewrichtsontsteking in muizen werd gebruikt als model omeen beeld te krijgen van de effecten van P. scrophulariiflora extracten op chronischeontsteking. De behandeling met 200 mg/kg diethylether extract of 20 mg/kg apocyninvertraagde de ontwikkeling van gewrichtsontsteking, hoewel geen van de geteste monstersde ernst van de ontsteking verminderden. Deze resultaten suggereren dat apocyninverantwoordelijk is voor de effecten van het diethylether extract op chronischegewrichtsontsteking. Bovendien kan gesteld worden dat de remming van het diethyletherextract op het complement systeem, de chemiluminescentie en T cel proliferatie geenvoorspellende waarde hebben voor de remming van kraakbeen geïnduceerdegewrichtsontsteking in muizen onder de onderzochte omstandigheden. De oorzaak hiervankan liggen in een te lage dosering, een slechte biologische beschikbaarheid, een ongunstigfarmacokinetisch profiel, of metabolische inactivering van werkzame componenten in vivo.Het is ook mogelijk dat pathologische processen een rol spelen in het ontstaan vanchronische gewrichtsontsteking die niet zijn onderzocht of die onvoldoende wordengeremd door het diethylether extract. De in vivo effecten gingen niet gepaard metbelangrijke toxische effecten, zoals bleek uit de analyse van belangrijke immunologischeorganen van muizen die gedurende 3 of 6 weken extracten van P. scrophulariiflora kregentoegediend.

Verschillende methoden zijn gebruikt om op geleide van activiteit stoffen te isolerenuit P. scrophulariiflora die interfereren met de klassieke route van het complementsysteem remmen. Een representatief overzicht van enkele methoden is weergeven inhoofdstuk 6. Gedurende het onderzoek werd duidelijk dat zowel in het petroleumether alshet diethylether extract meerdere componenten aanwezig zijn die een effect hebben opcomponenten van het complementsysteem. Het gehalte aan actieve verbindingen bleekechter zeer laag wat isolatie en structuuropheldering onmogelijk maakte binnen hetbeschikbare tijdsbestek. DLC analyse suggereerde echter de aanwezigheid van fytosterolenin het petroleumether extract. Hoewel deze verbindingen niet eerder zijn aangetoond inPicrorhiza soorten, komen fytosterolen algemeen voor in hogere planten en is hunremmende werking op het complement systeem beschreven.

Hoofdstuk 7 beschrijft de isolatie van 2 cucurbitacinen uit de wortelstokken van P.scrophulariiflora op geleide van groeiremming van humane T lymfocyten. Hetdiethylether extract, dat een sterke remming vertoonde van celproliferatie, werd gescheidenmet behulp van Silica kolomchromatografie. Uit actieve fracties werd door kristallisatieeen zuivere actieve verbinding verkregen, deacetylpicracin. Andere actieve fracties werden

150 | Appendices

vervolgens gescheiden met behulp van Sephadex kolomchromatografie, gevolgd doorHPLC, waardoor een tweede zuivere actieve verbinding werd verkregen, picracin. Eenderde actieve verbinding kon nog niet worden geïsoleerd. De structuur van beideverbindingen werd opgehelderd door middel van meerdere massaspectrometrische enNMR spectroscopische methoden. Beide componenten vertoonden een sterke remming vanT cel proliferatie. Nader onderzoek toonde aan dat deze effecten niet het gevolg waren vandirect toxische stoffen.

Mogelijke mechanismen werden onderzocht die aanleiding geven tot de waargenomeneffecten. Aangezien bij de proliferatie van T lymfocyten interleukine 2 (IL-2) eenbelangrijke rol speelt, werd het effect van de geïsoleerde stoffen op de afgifte van IL-2onderzocht. Incubatie van geactiveerde T cellen met picracin of deacetylpicracin vertoondeeen duidelijke remming van IL-2 afgifte. Dit effect kon eveneens niet wordentoegeschreven aan een direct cytotoxisch effect. Vervolgens werd onderzocht of deremming van IL-2 afgifte het gevolg was van apoptose, ofwel geprogrammeerde celdood.Flow cytometrische analyse van gemerkte cellen toonden aan dat picracin apoptoseinduceerde in T lymfocyten, terwijl deacetylpicracin niet significant werkzaam was.Bovendien bleek dat beide verbindingen een remmende werking hadden op de afgifte vande ontstekingsmediatoren IL-1 en TNFα door geactiveerde monocyten. Deze stoffenspelen een belangrijke rol in het ontstaan van chronische ontstekingen.

Beide verbindingen werden onderzocht in een diermodel voor vertraagd typeovergevoeligheid, een model voor onderzoek naar cellulaire immuunreacties. Zowelpicracin als deacetylpicracin vertoonden remming van de ontsteking na intraperitonealetoediening. Lokale toediening veroorzaakte echter toename van de ontsteking. Controleexperimenten toonden aan dat met name picracin en in mindere mate deacetylpicracin eendosis afhankelijke onstekingsreactie induceren in lage doseringen. Deze resultatensuggereren een dualistisch effect van picracin en deacetylpicracin. Bij toediening in depoot vertonen ze een plaatselijke immuunreactie, terwijl het mogelijk is dat ze nasystemische (i.p.) toediening worden omgezet in metabolieten die systemisch juist eenontstekingsremmend effect hebben. Het is ook mogelijk dat de plaats van toedieningrelevant is voor het waargenomen effect: wanneer er in de buikholte een depletie vanimmuuncellen ontstaat als gevolg van apoptose, is het mogelijk dat vervolgens op eenandere plaats minder cellen beschikbaar zijn wanneer ter plekke een immuunreactie wordtopgewekt.

Samengevat, het diethylether Soxhlet extract van gedroogde wortelstokken vanPicrorhiza scrophulariiflora een remmende werking heeft op de klassieke route van hetcomplement systeem, de afgifte van zuurstofradicalen of afgeleide verbindingen en deproliferatie van T lymfocyten. Meervoudige toediening van dit extract aan muizen waarineen acute lokale ontsteking was opgewekt resulteerde in een vermindering van de zwelling.Bovendien vertraagde het extract de ontwikkeling van gewrichtsontsteking in muizen,hoewel de ernst van de ontsteking niet werd beïnvloed. Activiteitsgeleide isolatie van hetdiethylether extract resulteerde in 2 zuivere verbindingen die een remmend effectvertoonden op de proliferatie van geactiveerde T lymfocyten. Beide verbindingen remdende afgifte van IL-2 door deze cellen, en de afgifte van IL-1 en TNFα door geactiveerdemonocyten. Deze effecten zijn vermoedelijk het gevolg van het induceren van apoptose,wat werd aangetoond door T lymfocyten te incuberen met picracin. De inductie vanapoptose speelt mogelijk ook een rol in de immuunmodulerende effecten in vivo.

Appendices | 153

Dankwoord .

Tot slot van dit proefschrift een woord van dank. Niet omdat het protocol dit vereist,maar omdat de totstandkoming van een proefschrift over het algemeen niet te danken isaan één persoon. De inzet van een hele groep mensen is bepalend voor het welslagen vaneen promotieonderzoek en het resultaat: een proefschrift.

Allereerst wil ik graag mijn promotoren en co-promotor bedanken, Prof. Dr. R.P.Labadie, Prof. Dr. H. van Dijk en Dr. B.H. Kroes. Beste Rudi, bedankt voor jeenthousiasme. Altijd was er wel tijd voor een filosofisch of meer inhoudelijk gesprek overde wetenschap, oftewel de farmacognosie. Je inzichten in dit vakgebied hebben me vaakverwonderd en werkten bijzonder aanstekelijk. Beste Hans, bedankt voor je medewerkingen de vele praktische tips die je me gedurende het onderzoek en het schrijven hebtgegeven. Ze hebben een belangrijke bijdrage geleverd aan het onderzoek en detotstandkoming van het resultaat. Burt jonge, ik vond het erg plezierig om met je samen tewerken. Ik moet toegeven dat ik wel eens eigenwijs kon zijn en je geduld op de proef hebgesteld, maar dat belette je niet om tot de kern van de zaak door te dringen.

Verder een woord voor mijn paranimfen, Edwin van den Worm en Bart Halkes.Bedankt voor jullie bemoediging en vriendschap van de afgelopen jaren en jullieondersteuning tot het laatste toe. Er zijn veel leuke momenten om aan terug te denken.Laten we het nog eens dunnetjes overdoen.

Ook de andere collega’s van de sectie farmacognosie wil ik graag bedanken voor hunhulp en enthousiasme. Velen van hen hebben een substantiële bijdrage geleverd aanverschillende aspecten van het onderzoek, zoals de bioassays (Linda), destructuuropheldering (Bert) en de gehaltebepalingen (Dick). Een woord van dank ook voorde studenten die ik gedurende het onderzoek mocht begeleiden: Sigyn, Jan, Frank, Anil,Heleen en Rosalie. Mijn vrienden bij de vakgroep medicinal chemistry en daarbuiten wil ikgraag bedanken voor hun vriendschap en de hulp die ik van hen mocht krijgen.

Tijdens het veldwerk in Nepal en India kon ik altijd rekenen op mijn buitenlandsevrienden die voor me klaar stonden. Met name Prof. Dr. L.M. Singh en zijn familie ben ikzeer erkentelijk. Dear Guruji, thanks for your hospitality, your warm friendship, and yourwisdom. I’ll always remember you as a great human, full of love and compassion.

Dr. G.J. Meulenbeld wil ik graag bedanken voor zijn inspiratie en zijn bijdrage aanmijn verkenning van de Āyurveda. Beste Jan, bedankt voor de tijd en moeite die jegespendeerd hebt om me de weg te wijzen in het doolhof van de Āyurveda. Ik heb mealtijd bijzonder gelukkig geprezen om terug te kunnen vallen op dé Āyurveda deskundige.

Prof, Paul Maas en Gea Zijlstra wil graag bedanken voor hun bereidwilligheid hethoofdstuk over de taxonomie kritisch door te nemen en van waardevol commentaar tevoorzien.

154 | Appendices

Graag wil ik alle “nieuwe” collega’s en vrienden van Numico Research bedanken voorhun begrip voor de situatie waarin in nog niet volledig tot hun beschikking stond. Ook alleoverige vrienden, binnen de universiteit en daarbuiten, familie en allen die me dierbaar zijnwil ik graag bedanken voor hun vriendschap en al het andere dat het leven te bieden heeft.

Lieve Harma, jou ga ik persoonlijk bedanken.

Sanskrit vocabulary

Appendix A

136 | Appendices

Introduction

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138 | Appendices

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Appendices | 139

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140 | Appendices

/(3.( <'55+/(3.(/7]N&(-(# /$459)*>#<'55+#+)"$("$/(^](3( ,5'5/"#.-$#<'55+1#,5*9$/."#.-$#*7./)$*."#)*.5#<'55+/("(## *7./)$*.#;'7)+1#&'("4(/("N6(*(# ",)$*,$#5;#'5*>$9).6/7]N&(-(# /$459)*>#"),3*$""/R3e(# +/61#/57>-/R3e(*(## +/6)*>/R&(# "64&.54"/$,(*(# '(%(.)9$/5>(# +)"$("$/5,(*( (&&$.)f)*>/5-)bO## \),/5/-)f('(>-7# ')>-.'(9(b(## "57/'$3-(*( $4(,)(.)*>'$3-(*O6(# $4(,)(.)*>'54(*## <5+6#-()/9N]O3(/(b(# ",)$*,$#5;#(&-/5+)")(,"9N.(## .5#459$g#:)*+1#&/)*,)&'$#5;#459$4$*.9N.(/(3.(# >57.9N67 ()/1 wind9)`"*$-(## "4$(/#)*#;$,$"9)+(>+-( )4&/5&$/'6#&/5,$""$+1#"&5)'$+9)&N3( .(".$#5;#.-$#)*>$".$+#4(.$/)('#(;.$/#+)>$".)5*9)d(+(## ,'$(/9)d$e(# &(/.),7'(/).6#5/#+)"")4)'(/).69)e(# &5)"5*1#9$*541#<(*$1#(*6.-)*>#(,.)9$'6#&$/*),)57"9)e(4(]9(/(# )//$>7'(/1#/$4)..$*.#;$9$/9)e(4(]9(/(*Nd)*O# /$459$"#/$4)..$*.#;$9$/ ###9O/6(## &5.$*,6#5;#(#+/7>9S++-) (>>/(9(.)5*96(3.)# 4(*);$".(.)5*#5;#(#+)"$("$g#;);.-#+)"$("$#".(>$96N*( 459$"#.-$#<5+61#)*+7,$"#<'55+#;'5:d(37'N+(*O## \),/5/-)f(d(/O/(## <5+61#(*(.546d('6(.(*./(# "7/>$/6dO.(# ,5'+1#,55'dO.(&)..(# 7/.),(/)(dO.('(# ,55')*>dO.(9O/6( ,5'+#&5.$*,6dN'N36(.(*./(# +)"$("$"#5;#-$(+#(*+#.-/5(.dO.N"/(+N-(]). ,5*?7$/"#,5'+1#<'55+1#(*+#<7/*)*>#"$*"(.)5*d73/( "$4$*d5b).(# <'55+d5+-(*(## $')4)*(.)5*#.-$/(&6

Appendices | 141

d'(3eb(## "455.-d'$e(3( '7</),(.$"#.-$#]5)*."d'$e4(*# .5#(+-$/$d9N"(# /$"&)/(.5/6#+)"$("$d9N"(# "-5/.*$""#5;#</$(.-"(W&/N&.)# &(.-5>$*$")""(Wd(4(*(# &(,);6)*>"(Wd5+-(*(# &7/);6)*>"(W-).N ./$(.)"$g#(*6#4$.-5+),(''6#(//(*>$+#,5''$,.)5*#5;#.$%."#5/#9$/"$""(^,(6(# (,,747'(.)5*#5;#+5e(#"g#;)/".#+)"$("$#".(>$"(..9(# 4)*+"(*+-)>(.(9N.(## 5".$5(/.-/).)""(*+-)9N.( 5".$5(/.-/).)""(**(4# "7*3#+5:*1#+$&/$""$+1#:$(3"(4(9N6(# )*-$/$*,$"(4N*( "$&(/(.$"#.-$#*7./)$*."#(*+#.-$#:(".$1#/$459$"#.-$#:(".$"(/(# ,(.-(/.),1#&7/>(.)9$1#'(%(.)9$g#;'5:)*>"N+-(3( >/("&"1#)*+7,$"#)*")>-."N*+/(## 9)",)+"N4(+5e(## N4(##,54<)*$+#:).-#+5e("N4N*6(# >$*$/(').6#5/#")4)'(/).6")/N#"# 9$)*""R3e4(## "7<.'$"*$-(*(## 5'$(.)5*#.-$/(&6"*)>+-(## 7*,.757"".(*6(d5+-(*( &7/);6)*>#</$(".#4)'31#>('(,.5+$&7/(*.".-N*("(Wd/(6(# '5,(')f(.)5*#5;#9).)(.$+#+5e(#"g#;57/.-#+)"$("$#".(>$".-)/(## )445<)'$".-R'(## >/5"""/5.(" ,-(**$'"/5.NW")# ,-(**$'""/5.5/5+-( 5<"./7,.)5*#5;#.-$#,-(**$'""9$+(## ":$(."9$+(*(## "7+(.)5*#.-$/(&6-)4(# ,5'+-S+# -$(/.1#,(/+)(,#/$>)5*-S+6(# &'$(")*>#.5#.-$#-$(/.g#,(/+)(,-S+/5>(# +)"$("$"#5;#.-$#,(/+)(,#/$>)5*g#-$(/.#+)"$("$-S+/5>(*Nd)*)# /$459)*>#-$(/.#+)"$("$

_$;$/$*,$"_$;$/$*,$"_$;$/$*,$"_$;$/$*,$"

E5*)$/FG)'')(4"1#E@#HIJKKL#!#2(*"3/).F=*>')"-#+),.)5*(/6@#_$&/)*.#IKKh1#E5.)'('i(*(/")+(""1#M$'-)@

142 | Appendices

Appendices | 143

Abbreviations

Appendix B

144 | Appendices

Abbreviations

dN dN/O/(".-N*(ACTH adrenocorticotropic hormone!j (e`N[>(-S+(6N"(W-).NAI arthritic indexAP alternative pathwayAPC antigen-presenting cellapo apocynin!\P (..(,-$+#&/5.5*#.$".Aq aqueous (extract)!2 (e`N[>("([>/(-(iZ# <-N9(&/(3Nd(*)>-(b`7i_# <-()e(]6(/(.*N9('Obr. broadCCl4 carbon tetrachlorideYM ,(3/(+(..(CFA complete Freund’s adjuvantCFDA 5-carboxyflourescein diacetate,) ,)3)."N".-N*(CIA collagen-induced arthritisCII collagen type IICMC carboxymethylcelluloseYk2l ,5//$'(.$+#"&$,./5",5&6CP classical pathwayY2 ,(/(3("(W-).Ncuc E cucurbitacin Ed doubletDAP deacetylpicracinDCM dichloromethaneDE diethyl ether (extract)dex dexamethasoneDMARD disease-modifying antirheumatic drugDMSO dimethylsulphoxideMZ +-(*9(*.(/O*)>-(b`7DNA deoxyribonucleic acidDTH delayed-type hypersensitivityEA ethyl acetate (extract)ED50 dose at 50 % effectEGTA ethylene glycol-bis-(β-aminoethyl ether)=AFE2 $'$,./5*#)4&(,.F4(""#"&$,./54$./6ELISA enzyme-linked immunosorbent assayEtOH ethanol (extract)D!iFE2 ;(".#(.54#<54<(/+4$*.F4(""#"&$,./54$./6FACScan fluorescence activated cell scanF-actin filamentous actin

Appendices | 145

FCS fetal calf serumFITC fluorescein isothiocyanatefMLP N-formyl-methionyl-leucyl-phenylalanineGCR glucocorticoid receptorGCS glutamylcysteine synthetaseGSH glutathioneHBSS Hank’s buffered salt solutionj=PYk_ -$.$/5*7,'$(/#,5//$'(.)5*HETE hydroperoxy eicosatetraenoic acidHIF-1 hypoxia inducible factor-1HPLC high performance liquid chromatographyHPS human pooled serumi.p. intraperitoneallyi.v. intravenouslyIC50 concentration at 50 % inhibitionICAM-1 intercellular adhesion molecule-1ID50 dose at 50 % inhibitionIgG immunoglobulin GIgM immunoglobulin MIL interleukinIL-2R interleukin-2 receptorIR infrared spectroscopy3( 3('&(".-N*(LD50 dose at 50 % lethalityLDH lactate dehydrogenaseLFA lymphocyte function associated moleculeLPS lipopolysaccharideLTB leukotrieneMAC major attack complexMASP mannose-binding lectin-associated serine proteaseMBL mannose-binding lectinMCP monocyte chemoattractant proteinMe methanol (extract)MHC major histocompatibility complexEZ 4N+-(9(*)+N*(MPO myeloperoxidasemRNA messenger RNAMTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromideNAD β-nicotinamide adenine dinucleotideNADPH nicotinamide adenine dinucleotide phosphatend not detectable*) *)+N*(".-N*(NMR nuclear magnetic resonance spectrometryNSAID non-steroid anti-inflammatory drugp.o. per os; orallyPAF platelet activating factor

146 | Appendices

PBMC peripheral blood mononuclear cellsPBS phosphate-buffered salinePE light petroleum (extract)PG peptidoglycanPGE2 prostaglandin E2

PGF2α prostaglandin F2α

PHA phythemagglutininPI propidium iodidePKC protein kinase CPMA phorbol myristate acetatePMN polymorphonuclear neutrophilpred prednisoneRA rheumatoid arthritisRNA ribonucleic acidROS reactive oxygen species__2# /("(/(.*("(47,,(6(s singlets.c. subcutaneously"R "R./(".-N*(2(2 dN/[>(+-(/("(W-).NSDS sodium dodecyl sulphateSEM standard error of meansGPT serum glutamic-pyruvic transaminase2Z# dN')>/N4(*)>-(b`7SRBC sheep red blood cell22 "7d/7.("(W-).N2l# "(-("/(65>(t tripletTCR T-cell receptorTh1 cell T-helper-1 cellTLC thin-layer chromatographyTNFα tumor necrosis factor αTXA2 thromboxane A2

v/v volume per volumeVBS veronal-buffered salineVEGF vascular endothelial growth factor9) 9)4N*(".-N*(w/w weight per weightl_ 65>(/(.*N3(/(

Appendices | 147

Samenvatting .

Het gebruik van medicinale planten ter behoud en verhoging van de kwaliteit vanleven is eeuwenoud en nog steeds actueel. De laatste jaren is er zelfs een sterke toenamewaar te nemen van de interesse voor en het gebruik van natuurlijke produkten in degezondheidszorg. Dit gebeurt aan de ene kant door het rechtstreeks toepassen van kruidenof afgeleide bereidingen, bijvoorbeeld bij de behandeling van chronische aandoeningen;aan de andere kant dienen planten als grondstof voor de isolatie van biologisch actieveverbindingen. Deze verbindingen kunnen worden ontsloten door het willekeurigonderzoeken van grote aantallen planten extracten, maar ook een voorselectie wordengemaakt op grond van traditionele kennis van het gebruik van kruiden. De beoordeling vantraditioneel gebruikte kruidenpreparaten, waarbij recht wordt gedaan aan de traditioneleprincipes van gezondheid en ziekte, kan leiden tot het ontwikkelen van nieuwegeneesmiddelen.

Voorheen werd via een dergelijke benadering Picrorhiza kurrooa Royle (letterlijk:“Bitterwortel”) geselecteerd als een mogelijk interessante middel voorimmuunmodulerende verbindingen. Resultaten van onderzoek naar de effecten van dezeplant op het complement systeem en op de vorming van zuurstof radicalen doorgeactiveerde witte bloedcellen (leukocyten) suggereerde een mogelijke toepassing bijreumatoïde gewrichtsontsteking. Dit proefschrift handelt over aanvullend onderzoek naarde immuun modulerende eigenschappen van Picrorhiza en de isolatie van biologischactieve verbindingen, om een beter beeld te krijgen van de toepasbaarheid van deze plantin chronische gewrichtsontsteking.

Wortelstokken van twee verschillende Picrorhiza soorten zijn verkrijgbaar op Zuid-Aziatische kruidenmarkten, te weten P. kurrooa Royle en P. scrophulariiflora Pennell. Erwordt geen onderscheid gemaakt tussen de wortelstokken van beide soorten. Aangezien hetplantenmateriaal dat voor dit onderzoek werd gebruikt bestond uit gedroogdewortelstokken, en er geen methoden bekend zijn om wortelstokken van beide soorten teonderscheiden, kon de identiteit van het plantenmateriaal niet rechtsreeks wordenvastgesteld. Echter, een inventarisatie van de geografische verspreiding van beide soorten,beschreven in hoofdstuk 2, leverde voldoende aanwijzingen om de identiteit van hetgebruikte plantenmateriaal vast te kunnen stellen. Het materiaal werd verkregen uit hetGorkha district in Nepal. Een inventarisatie van de locaties waar Picrorhiza soorten zijngevonden wees uit dat de verspreiding van P. kurrooa zich beperkt tot de WestelijkeHimalaya, terwijl P. scrophulariiflora voorkomt in de Oostelijke Himalaya. Voorts bleekuit onderzoek dat in het Gorkha district slechts P. scrophulariiflora voorkomt, hetgeenwerd bevestigd door veldwerk in dit gebied. Op basis van deze informatie werdverondersteld dat het gebruikte plantenmateriaal vastgesteld als P. scrophulariiflora.

148 | Appendices

Hoewel deze soort in 1984 werd ondergebracht in een nieuw geslacht, namelijkNeopicrorhiza, is het gebruik van de synoniem Picrorhiza scrophulariiflora gehandhaafdin dit proefschrift. De argumenten die ten grondslag liggen aan deze keuze wordenbeschreven in hoofdstuk 2.

Hoofdstuk 3 beschrijft het gebruik van Picrorhiza in de Āyurveda, de traditionelegeneeskunde van Zuid Azië. De toepasbaarheid een medicinale plant voor de behandelingvan een bepaalde aandoening kan worden afgeleid aan de hand van gegeneraliseerdetraditionele principes, of rechtstreeks worden ontleend aan het traditionele gebruik.Wanneer voor de eerste benadering wordt gekozen, waarbij de Āyurvedischeeigenschappen van Picrorhiza in aanmerking worden genomen, wordt het duidelijk datdeze plant de vāta (vgl. wind) verhoogd en als zodanig ziekten die zijn veroorzaakt doorvāta kunnen verergeren. Hoewel vāta een belangrijke rol speelt in het ontstaan vanāmavāta (reumatoïde artritis), is dit toch niet de hoofdoorzaak. Deze wordt veeleergevormd door het verschijnsel āma (onvolledig verteerde nutriënten). Picrorhiza heeft alseigenschappen het bevorderen van de spijsvertering en het verwijderen van āma. Ditsuggereert dat Picrorhiza, volgens de Āyurvedische principes, een belangrijke rol kanspelen in het voorkómen van reumatoïde gewrichtsontsteking. Voor de behandelinghiervan wordt het echter niet aangeraden vanwege het stimulerende effect of vāta.Eenzelfde conclusie werd getrokken na onderzoek van de toepassing van Āyurvedischebereidingen van Picrorhiza. Verwijzingen uit de klassieke literatuur naar het gebruik vanPicrorhiza bij gewrichtsontsteking werden niet gevonden. Bovendien bleek uitveldonderzoek in Noord India en Nepal dat complexe bereidingen met een substantiëlehoeveelheid Picrorhiza niet gebruikt worden voor de behandeling van chronischegewrichtsontsteking.

Uit onderzoek van Picrorhiza soorten, zoals beschreven in hoofdstuk 4, blijkt dat metname cucurbitacin glucosiden, iridoid glucosiden, phenylethanoid glucosiden enacetophenon glucosiden worden geaccumuleerd in de wortelstokken van Picrorhizasoorten. Deze componenten hebben een wijd uiteenlopende biologische activiteit, waarinechter ontstekingsremmende effecten de overhand hebben. Hoewel vrijwel geen informatiebeschikbaar is over de biologische activiteit van cucurbitacinen uit Picrorhiza, worden vanandere vergelijkbare verbindingen cytotoxische, antitumor, immuun modulerende enadaptogene effecten beschreven. Tevens is bekend dat sommige cucurbitacinen sterk giftigzijn. De effecten die beschreven zijn voor iridoid glucosiden houden meestal verband metde bescherming van de lever tegen experimenteel geïnduceerde schade. De werking vanphenylethanoid glucosiden wordt meestal in verband gebracht met de remming vanenzymen die een rol spelen bij het ontstaan van ontstekingsreacties, zoalsphosphodiesterase en 5-lipoxygenase. Acetophenonen zoals apocynin dragen bij aan deontstekingsremmende werking door de remming van de vorming van zuurstofradicalen ende verhoging van het cellulaire antioxidant systeem.

Hoofdstuk 5 beschrijft het onderzoek naar de modulerende eigenschappen vanextracten van P. scrophulariiflora op de klassieke route van het complement systeem, deproduktie van chemiluminescentie door leukocyten en de proliferatie van T lymfocyten. Deonderzochte processen weerspiegelen aspecifieke humorale en cellulaireverdedigingsmechanismen en tevens specifieke immuunreacties. Van de geteste extractenvertoonde het diethylether Soxhlet extract de sterkste effecten, zowel in aspecifieke alsspecifieke immuunprocessen. Bovendien vertoonde dit extract een remming van

Appendices | 149

carrageenan-geïnduceerde pootzwelling in muizen na meerdere toedieningen. Depetroleumether en water extracten vertoonden minder sterke remmende effecten op deklassieke route van het complement systeem, terwijl de ethylacetaat en methanol extractende chemiluminescentie van leukocyten matig remden. Deze extracten vertoonden echtergeen remmend effect op carrageenan-geïnduceerde pootzwelling in de onderzochtedoseringen. De remming van chemiluminescentie door de diethylether en ethylacetaatextracten kan voor een groot deel worden toegeschreven aan apocynin. Echter, apocyninvertoonde geen remmend effect in bovengenoemd model, hetgeen suggereert dat anderestoffen verantwoordelijk zijn voor het ontstekingsremmende effect van het diethyletherextract. Het is mogelijk dat verbindingen die het complement systeem remmen hiervoorverantwoordelijk zijn. Ook is het mogelijk dat een combinatie van verbindingen met eenverschillende werking nodig is om een onstekingsremmend effect in vivo te verkrijgen.

Kraakbeen geïnduceerde gewrichtsontsteking in muizen werd gebruikt als model omeen beeld te krijgen van de effecten van P. scrophulariiflora extracten op chronischeontsteking. De behandeling met 200 mg/kg diethylether extract of 20 mg/kg apocyninvertraagde de ontwikkeling van gewrichtsontsteking, hoewel geen van de geteste monstersde ernst van de ontsteking verminderden. Deze resultaten suggereren dat apocyninverantwoordelijk is voor de effecten van het diethylether extract op chronischegewrichtsontsteking. Bovendien kan gesteld worden dat de remming van het diethyletherextract op het complement systeem, de chemiluminescentie en T cel proliferatie geenvoorspellende waarde hebben voor de remming van kraakbeen geïnduceerdegewrichtsontsteking in muizen onder de onderzochte omstandigheden. De oorzaak hiervankan liggen in een te lage dosering, een slechte biologische beschikbaarheid, een ongunstigfarmacokinetisch profiel, of metabolische inactivering van werkzame componenten in vivo.Het is ook mogelijk dat pathologische processen een rol spelen in het ontstaan vanchronische gewrichtsontsteking die niet zijn onderzocht of die onvoldoende wordengeremd door het diethylether extract. De in vivo effecten gingen niet gepaard metbelangrijke toxische effecten, zoals bleek uit de analyse van belangrijke immunologischeorganen van muizen die gedurende 3 of 6 weken extracten van P. scrophulariiflora kregentoegediend.

Verschillende methoden zijn gebruikt om op geleide van activiteit stoffen te isolerenuit P. scrophulariiflora die interfereren met de klassieke route van het complementsysteem remmen. Een representatief overzicht van enkele methoden is weergeven inhoofdstuk 6. Gedurende het onderzoek werd duidelijk dat zowel in het petroleumether alshet diethylether extract meerdere componenten aanwezig zijn die een effect hebben opcomponenten van het complementsysteem. Het gehalte aan actieve verbindingen bleekechter zeer laag wat isolatie en structuuropheldering onmogelijk maakte binnen hetbeschikbare tijdsbestek. DLC analyse suggereerde echter de aanwezigheid van fytosterolenin het petroleumether extract. Hoewel deze verbindingen niet eerder zijn aangetoond inPicrorhiza soorten, komen fytosterolen algemeen voor in hogere planten en is hunremmende werking op het complement systeem beschreven.

Hoofdstuk 7 beschrijft de isolatie van 2 cucurbitacinen uit de wortelstokken van P.scrophulariiflora op geleide van groeiremming van humane T lymfocyten. Hetdiethylether extract, dat een sterke remming vertoonde van celproliferatie, werd gescheidenmet behulp van Silica kolomchromatografie. Uit actieve fracties werd door kristallisatieeen zuivere actieve verbinding verkregen, deacetylpicracin. Andere actieve fracties werden

150 | Appendices

vervolgens gescheiden met behulp van Sephadex kolomchromatografie, gevolgd doorHPLC, waardoor een tweede zuivere actieve verbinding werd verkregen, picracin. Eenderde actieve verbinding kon nog niet worden geïsoleerd. De structuur van beideverbindingen werd opgehelderd door middel van meerdere massaspectrometrische enNMR spectroscopische methoden. Beide componenten vertoonden een sterke remming vanT cel proliferatie. Nader onderzoek toonde aan dat deze effecten niet het gevolg waren vandirect toxische stoffen.

Mogelijke mechanismen werden onderzocht die aanleiding geven tot de waargenomeneffecten. Aangezien bij de proliferatie van T lymfocyten interleukine 2 (IL-2) eenbelangrijke rol speelt, werd het effect van de geïsoleerde stoffen op de afgifte van IL-2onderzocht. Incubatie van geactiveerde T cellen met picracin of deacetylpicracin vertoondeeen duidelijke remming van IL-2 afgifte. Dit effect kon eveneens niet wordentoegeschreven aan een direct cytotoxisch effect. Vervolgens werd onderzocht of deremming van IL-2 afgifte het gevolg was van apoptose, ofwel geprogrammeerde celdood.Flow cytometrische analyse van gemerkte cellen toonden aan dat picracin apoptoseinduceerde in T lymfocyten, terwijl deacetylpicracin niet significant werkzaam was.Bovendien bleek dat beide verbindingen een remmende werking hadden op de afgifte vande ontstekingsmediatoren IL-1 en TNFα door geactiveerde monocyten. Deze stoffenspelen een belangrijke rol in het ontstaan van chronische ontstekingen.

Beide verbindingen werden onderzocht in een diermodel voor vertraagd typeovergevoeligheid, een model voor onderzoek naar cellulaire immuunreacties. Zowelpicracin als deacetylpicracin vertoonden remming van de ontsteking na intraperitonealetoediening. Lokale toediening veroorzaakte echter toename van de ontsteking. Controleexperimenten toonden aan dat met name picracin en in mindere mate deacetylpicracin eendosis afhankelijke onstekingsreactie induceren in lage doseringen. Deze resultatensuggereren een dualistisch effect van picracin en deacetylpicracin. Bij toediening in depoot vertonen ze een plaatselijke immuunreactie, terwijl het mogelijk is dat ze nasystemische (i.p.) toediening worden omgezet in metabolieten die systemisch juist eenontstekingsremmend effect hebben. Het is ook mogelijk dat de plaats van toedieningrelevant is voor het waargenomen effect: wanneer er in de buikholte een depletie vanimmuuncellen ontstaat als gevolg van apoptose, is het mogelijk dat vervolgens op eenandere plaats minder cellen beschikbaar zijn wanneer ter plekke een immuunreactie wordtopgewekt.

Samengevat, het diethylether Soxhlet extract van gedroogde wortelstokken vanPicrorhiza scrophulariiflora een remmende werking heeft op de klassieke route van hetcomplement systeem, de afgifte van zuurstofradicalen of afgeleide verbindingen en deproliferatie van T lymfocyten. Meervoudige toediening van dit extract aan muizen waarineen acute lokale ontsteking was opgewekt resulteerde in een vermindering van de zwelling.Bovendien vertraagde het extract de ontwikkeling van gewrichtsontsteking in muizen,hoewel de ernst van de ontsteking niet werd beïnvloed. Activiteitsgeleide isolatie van hetdiethylether extract resulteerde in 2 zuivere verbindingen die een remmend effectvertoonden op de proliferatie van geactiveerde T lymfocyten. Beide verbindingen remdende afgifte van IL-2 door deze cellen, en de afgifte van IL-1 en TNFα door geactiveerdemonocyten. Deze effecten zijn vermoedelijk het gevolg van het induceren van apoptose,wat werd aangetoond door T lymfocyten te incuberen met picracin. De inductie vanapoptose speelt mogelijk ook een rol in de immuunmodulerende effecten in vivo.

Appendices | 151

Geconcludeerd kan worden dat meerdere verbindingen een rol spelen inimmuunmodulerende effecten van Picrorhiza extracten. Picracin en deacetylpicracininduceren een plaatselijke immuunreactie na lokale toediening, terwijl ze een remmendeffect hebben op een lokale onstekingsreactie na systemische (i.p.) toediening. Hoeweldeze laatste waarnemingen interessant zijn met het oog op de ontstekingsremmendewerking van Picrorhiza, kan niet worden uitgesloten dat deze verbindingen een lokaleontsteking in de gewrichten verergeren. Het testen van picracin en deacetylpicracin inexperimenteel geïnduceerde gewrichtsontsteking kan hierover uitsluitsel geven. Hoewelhet diethylether extract waarin beide verbindingen aanwezig zijn het ontstaan vangewrichtsontsteking in muizen vertraagt, zijn deze verbindingen niet noodzakelijkverantwoordelijk voor dit effect. Aangezien een dergelijk effect werd aangetoond voorapocynin, kan worden gesuggereerd dat apocynin verantwoordelijk is voor hetwaargenomen effect van het diethylether extract op gewrichtsontsteking. Zowel hetdiethylether extract als apocynin vertoonden echter geen effect op de ernst van degewrichtsontsteking in de onderzochte doseringen. Deze waarnemingen zijn inovereenstemming met het feit dat in de traditionele geneeskunde het gebruik vanPicrorhiza bij de behandeling van gewrichtsontsteking niet is geïndiceerd en dat eendergelijk gebruik volgens Āyurvedische principes juist moet worden afgeraden. Echter,zowel de experimentele gegevens als de conclusies die getrokken zijn op basis vanonderzoek van de Āyurvedische principes suggereren dat Picrorhiza een belangrijke rolkan spelen in het voorkómen van chronische gewrichtsontsteking.

152 | Appendices

Appendices | 153

Dankwoord .

Tot slot van dit proefschrift een woord van dank. Niet omdat het protocol dit vereist,maar omdat de totstandkoming van een proefschrift over het algemeen niet te danken isaan één persoon. De inzet van een hele groep mensen is bepalend voor het welslagen vaneen promotieonderzoek en het resultaat: een proefschrift.

Allereerst wil ik graag mijn promotoren en co-promotor bedanken, Prof. Dr. R.P.Labadie, Prof. Dr. H. van Dijk en Dr. B.H. Kroes. Beste Rudi, bedankt voor jeenthousiasme. Altijd was er wel tijd voor een filosofisch of meer inhoudelijk gesprek overde wetenschap, oftewel de farmacognosie. Je inzichten in dit vakgebied hebben me vaakverwonderd en werkten bijzonder aanstekelijk. Beste Hans, bedankt voor je medewerkingen de vele praktische tips die je me gedurende het onderzoek en het schrijven hebtgegeven. Ze hebben een belangrijke bijdrage geleverd aan het onderzoek en detotstandkoming van het resultaat. Burt jonge, ik vond het erg plezierig om met je samen tewerken. Ik moet toegeven dat ik wel eens eigenwijs kon zijn en je geduld op de proef hebgesteld, maar dat belette je niet om tot de kern van de zaak door te dringen.

Verder een woord voor mijn paranimfen, Edwin van den Worm en Bart Halkes.Bedankt voor jullie bemoediging en vriendschap van de afgelopen jaren en jullieondersteuning tot het laatste toe. Er zijn veel leuke momenten om aan terug te denken.Laten we het nog eens dunnetjes overdoen.

Ook de andere collega’s van de sectie farmacognosie wil ik graag bedanken voor hunhulp en enthousiasme. Velen van hen hebben een substantiële bijdrage geleverd aanverschillende aspecten van het onderzoek, zoals de bioassays (Linda), destructuuropheldering (Bert) en de gehaltebepalingen (Dick). Een woord van dank ook voorde studenten die ik gedurende het onderzoek mocht begeleiden: Sigyn, Jan, Frank, Anil,Heleen en Rosalie. Mijn vrienden bij de vakgroep medicinal chemistry en daarbuiten wil ikgraag bedanken voor hun vriendschap en de hulp die ik van hen mocht krijgen.

Tijdens het veldwerk in Nepal en India kon ik altijd rekenen op mijn buitenlandsevrienden die voor me klaar stonden. Met name Prof. Dr. L.M. Singh en zijn familie ben ikzeer erkentelijk. Dear Guruji, thanks for your hospitality, your warm friendship, and yourwisdom. I’ll always remember you as a great human, full of love and compassion.

Dr. G.J. Meulenbeld wil ik graag bedanken voor zijn inspiratie en zijn bijdrage aanmijn verkenning van de Āyurveda. Beste Jan, bedankt voor de tijd en moeite die jegespendeerd hebt om me de weg te wijzen in het doolhof van de Āyurveda. Ik heb mealtijd bijzonder gelukkig geprezen om terug te kunnen vallen op dé Āyurveda deskundige.

Prof, Paul Maas en Gea Zijlstra wil graag bedanken voor hun bereidwilligheid hethoofdstuk over de taxonomie kritisch door te nemen en van waardevol commentaar tevoorzien.

154 | Appendices

Graag wil ik alle “nieuwe” collega’s en vrienden van Numico Research bedanken voorhun begrip voor de situatie waarin in nog niet volledig tot hun beschikking stond. Ook alleoverige vrienden, binnen de universiteit en daarbuiten, familie en allen die me dierbaar zijnwil ik graag bedanken voor hun vriendschap en al het andere dat het leven te bieden heeft.

Lieve Harma, jou ga ik persoonlijk bedanken.

Appendices | 155

Curriculum vitae .

Friso Smit werd geboren op 7 oktober 1968 te Drachten. Aldaar doorliep hij deMAVO, waarna in Groningen in 1988 het VWO diploma werd behaald. Vervolgendstudeerde hij Farmacie aan de Rijksuniversiteit Groningen waar hij zich onder leiding vanwijlen Prof. T.M. Malingré specialiseerde in de Farmacognosie. Zijn bijvakonderzoekspeelde zich grotendeels af in India en Nepal, waar hij onderzoek deed naar traditioneelgebruikte planten in de Āyurveda met een mogelijke cytostatische werking. In 1994vertrok hij naar Utrecht, alwaar hij gedurende anderhalf jaar onderzoek deed naarplantaardige adjuvantia die aangrijpen op het mucosale immuunsysteem. Van 1996 tot2000 werkte hij als assistent in opleiding onder leiding van Prof. R.P. Labadie bij de sectieFarmacognosie, vakgroep Medicinal Chemistry, van de Faculteit Farmacie van deUniversiteit Utrecht. Gedurende deze periode werd het onderzoek dat onderwerp is van ditproefschrift uitgevoerd. Sinds januari 2000 werkt hij bij de afdeling NutritionalSupplements van Numico Research te Wageningen.

156 | Appendices

Appendices | 157

Publications .

Smit, H.F. Plantaardige cytostatica in de Ayurveda. (1994) Nederlands Tijdschrift voorFytotherapie 7 (1), 1-8.

Smit, H.F., Woerdenbag, H.J., Singh, R.H., Meulenbeld, G.J., Labadie, R.P., Zwaving, J.H.(1995) Ayurvedic herbal drugs with possible cytostatic activity. Journal ofEthnopharmacology 47 (2), 75-84.

Bos, R., Woerdenbag, H.J., Hendriks, H., Smit, H.F., Vikström, H.V., Scheffer, J.J.C. (1997)Composition of the essential oil from roots and rhizomes of Valeriana wallichii DC.Flavour and Fragrance Journal 12, 123-131.

Smit, H.F., Woerdenbag, H.J., Zwaving, J.H., Singh, R.H., Labadie, R.P. (1997) Selectionand evaluation of Ayurvedic herbal drugs which might be useful in the treatment ofmalignant swellings. Journal of the European Āyurvedic Society 5, 113-128.

Smit, H.F. (1997) The universal interest of traditional medicine. In: Harvesting herbs –2000; medicinal and aromatic plants – an action plan for Uttarakhand. Nautiyal, A.R.,Nautiyal, M.C., Purohit, A.N. (Eds.), Bishen Singh Mahendra Pal Singh, Dehra Dun,India, pp. 1-11.

Van Dijk, H., Beukelman, C.J., Kroes, B.H., Halkes, S.B.A., Smit, H.F., Quarles van Ufford,L.C., Van den Worm, E., Tinbergen-de Boer, T.L., Van Meer, J.H., Van den Berg,A.J.J., Labadie, R.P. (1999) In-vitro assays for activity-guided enrichment ofimmunomodulatory plant constituents. In: Bioassay methods in natural productsresearch and drug development. Bohlin, L., Bruhn, J.G. (Eds.), Kluwer AcademicPublishers, Dordrecht/Boston/London, pp. 101-111.

Smit, H.F., Kroes, B.H., Van den Berg, A.J.J., Van der Wal, D., Van den Worm, E.,Beukelman, C.J., Van Dijk, H., Labadie, R.P. (2000) Immunomodulatory and anti-inflammatory activity of Picrorhiza scrophulariiflora. Journal of Ethnopharmacology.Submitted.

Smit, H.F., Van den Berg, A.J.J., Kroes, B.H., Beukelman, C.J., Quarles van Ufford, H.C.,Van Dijk, H., Labadie, R.P. (2000) Inhibition of T-lymphocyte proliferation bycucurbitacins from Picrorhiza scrophulariiflora Pennell. Journal of Natural Products.Submitted.

158 | Appendices

Appendices | 157

Publications .

Smit, H.F. Plantaardige cytostatica in de Ayurveda. (1994) Nederlands Tijdschrift voorFytotherapie 7 (1), 1-8.

Smit, H.F., Woerdenbag, H.J., Singh, R.H., Meulenbeld, G.J., Labadie, R.P., Zwaving, J.H.(1995) Ayurvedic herbal drugs with possible cytostatic activity. Journal ofEthnopharmacology 47 (2), 75-84.

Bos, R., Woerdenbag, H.J., Hendriks, H., Smit, H.F., Vikström, H.V., Scheffer, J.J.C. (1997)Composition of the essential oil from roots and rhizomes of Valeriana wallichii DC.Flavour and Fragrance Journal 12, 123-131.

Smit, H.F., Woerdenbag, H.J., Zwaving, J.H., Singh, R.H., Labadie, R.P. (1997) Selectionand evaluation of Ayurvedic herbal drugs which might be useful in the treatment ofmalignant swellings. Journal of the European Āyurvedic Society 5, 113-128.

Smit, H.F. (1997) The universal interest of traditional medicine. In: Harvesting herbs –2000; medicinal and aromatic plants – an action plan for Uttarakhand. Nautiyal, A.R.,Nautiyal, M.C., Purohit, A.N. (Eds.), Bishen Singh Mahendra Pal Singh, Dehra Dun,India, pp. 1-11.

Van Dijk, H., Beukelman, C.J., Kroes, B.H., Halkes, S.B.A., Smit, H.F., Quarles van Ufford,L.C., Van den Worm, E., Tinbergen-de Boer, T.L., Van Meer, J.H., Van den Berg,A.J.J., Labadie, R.P. (1999) In-vitro assays for activity-guided enrichment ofimmunomodulatory plant constituents. In: Bioassay methods in natural productsresearch and drug development. Bohlin, L., Bruhn, J.G. (Eds.), Kluwer AcademicPublishers, Dordrecht/Boston/London, pp. 101-111.

Smit, H.F., Kroes, B.H., Van den Berg, A.J.J., Van der Wal, D., Van den Worm, E.,Beukelman, C.J., Van Dijk, H., Labadie, R.P. (2000) Immunomodulatory and anti-inflammatory activity of Picrorhiza scrophulariiflora. Journal of Ethnopharmacology.Submitted.

Smit, H.F., Van den Berg, A.J.J., Kroes, B.H., Beukelman, C.J., Quarles van Ufford, H.C.,Van Dijk, H., Labadie, R.P. (2000) Inhibition of T-lymphocyte proliferation bycucurbitacins from Picrorhiza scrophulariiflora Pennell. Journal of Natural Products.Submitted.

picrorhiza scrophulariiflora

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