This article was downloaded by: [UQ Library]On: 10 November 2014, At: 00:39Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Natural Product LettersPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gnpl19

Steroidal Saponins from the Rhizomesof Paris polyphylla var. chinensis andtheir Cytotoxic Activity on HL-60 CellsYoshihiro Mimaki a , Minpei Kuroda a , Yuusuke Obata a , YutakaSashida a , Mikio Kitahara b , Akira Yasuda c , Noriyuki Naoi c ,Zhen Wen Xu d , Ming Run Li d & Ai Na Lao ea School of Pharmacy, Tokyo University of Pharmacy and LifeScience , 1432-1, Horinouchi, Hachioji, Tokyo, 192-0392, Japanb Takasago Research Laboratories, Life Science RD Center,Kaneka Corporation, 1-8 , Miyamae-machi, Takasago-cho,Takasago, Hyogo, 676-8688, Japanc Medical Devices Division, Kaneka Corporation , 3-2-4,Nakanoshima, Kita-ku, Osaka, 530-8288, Japand Tianjin Institute of Medical and Pharmaceutical Science , 96,Gui Zhou Road, He-ping District, Tianjin, 300070, Chinae Department of Phytochemistry , Shanghai Institute of MateriaMedica Academia Sinica 319 , Yue-yang Road, Shanghai, 200031,ChinaPublished online: 04 Oct 2006.

To cite this article: Yoshihiro Mimaki , Minpei Kuroda , Yuusuke Obata , Yutaka Sashida , MikioKitahara , Akira Yasuda , Noriyuki Naoi , Zhen Wen Xu , Ming Run Li & Ai Na Lao (2000) SteroidalSaponins from the Rhizomes of Paris polyphylla var. chinensis and their Cytotoxic Activity onHL-60 Cells, Natural Product Letters, 14:5, 357-364, DOI: 10.1080/10575630008043768

To link to this article: http://dx.doi.org/10.1080/10575630008043768

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sources

of information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoeveror howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms& Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

Nutural Product Letters Volume 14(5), pp. 3.57-364 Reprints available directly from the Publisher Photocopying permitted by license only

0 2000 OPA (Overseas Publishers Association) N.V. Published by license under

the Harwood Academic Publishers imprint, part of The Gordon and Breach Publishing Group.

Printed in Malaysia.

STEROIDAL SAPONINS FROM THE RHIZOMES OF PARIS POLYPHYLLA VAR. CHINENSIS AND THEIR CYTOTOXIC

ACTIVITY ON HL-60 CELLS

Yoshihiro MIMAKI, * 4 Minpei K U R O D A , ~ Yuusuke O B A T A , ~ Yutaka SASHIDA,* 4 Mikio KITAHARA,~ Akira Y A S U D A , ~

Noriyuki N A O I , ~ Zhen Wen Xu,d Ming Run L I , ~ and Ai Na LAOe

School of Pharmacy, Tokyo University of Pharmacy and Life Science,a 1432-1,

Horinouchi, Hachioji, Tokyo 192-0392, Japan; Takasago Research Laboratories, Life

Science RD Center, Kaneka Corporation,b 1-8, Miyamae-machi, Takasago-cho,

Takasago, Hyogo 676-8688, Japan; Medical Devices Division, Kaneka Corporation: 3-2-4, Nakanoshima, Kita-ku, Osaka 530-8288, Japan; Tianjin Institute of Medical and Pharmaceutical Science,d 96, Gui Zhou Road, He-ping District, Tianjin 300070, China;

Department of Phytochemistry, Shanghai Institute of Materia Medica Academia Sinica: 319, Yue-yang Road, Shanghai 200031, China

(Received 20th June 1999)

Abstract: A new spirostanol steroidal saponin, along with ten known saponins, which

were based upon (25R)-spirost-5-en-3p-o1 (diosgenin) or (25R)-spirost-5-ene-3f3,17a- diol (pennogenin) as the aglycones, were isolated from the rhizomes of Paris polyphylla var. chinensis. Spectral data, including two-dimensional NMR, and the

result of hydrolytic cleavage showed that the structure of the new saponin was

pennogenin 3-O-{O-a-L-rhamnopyranosyl-(l~4)-O-a-L-rhamnopyranosyl-(l-r4)- p-Dglucopyranoside}. The isolated saponins were evaluated for their cytotoxic

activity on human promyelocytic leukemia HL-60 cells.

Key Words: Paris polyphylla var. chinensis, Liliaceae, steroidal saponin, spirostanol saponin, cytotoxic activity, HL-60 cells.

INTRODUCTION

The genus Paris (Liliaceae) with about 20 species is distributed in Europe and Asia.l) There are 15 species in China.2) The two varieties of Paris polyphylla, P.

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

358 YOSHIHIRO MIMAW ef al.

pofyphylfa var. chinensis and P. polyphyfla var. yunnanensis, are important crude

drugs in the traditional Chinese medicine. P. pofyphylla var. chinensis is distributed in the Southwest China and described to have a variety of medicinal uses such as an

antidote for snake bite, antibiotic, antitumor, contraceptive, and sedative.*) Although a few steroidal saponins from P. pofyphylfa var. chinensis have been shown to be

cytotoxic against several tumor cell lines?) detailed investigation paying attention to

the cytotoxic components has not been carried out. In our continuous studies of the

cytotoxic compounds contained in plants of the family Liliaceae?) we have now investigated the rhizomes of P . pofyphylfa var. chinensis.

RESULTS AND DISCUSSION The MeOH extract of P. pofyphylfa var. chinensis rhizomes was passed through a

polystyrene resin (Diaion HP-20) column, successively eluting with 30% MeOH, 50%

MeOH, MeOH, EtOH, and EtOAc. The MeOH eluate fraction exhibited cytotoxic

activity against human promyelocytic leukemia HL-60 cells (99% cell growth

inhibition at the sample concentration of 10 pg/mL; IC50 3.3 pg/mL), and TLC analysis suggested that it contained several saponins. Then, the MeOH fraction was

repeatedly subjected to column chromatography on silica-gel and octadecylsilanized

'Q,,,,, 27 1 8 "'

i a

1 2 3 4 5 6 7 8 9

10 11

RI R2 OH H OH H H Rha

OH Rha H Rha

OH Rha H Rha

OH Rha H Rha

OH Rha OH H

R3 H H H H

Glc Glc H H H H H

R4 H

k a f H H H H

Araf Rha-(l+4)-Rha Rha-(1+4)-Rha Rha-(l-.4)-Rha

k a f

(ODS) silica-gel to give compounds 1-1 1.

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

STEROIDAL SAPONINS FROM THE RHIZOMES OF PARIS POLYPHYLLA VAR. CHINENSIS 359

Compounds 1-10 are known spirostanol steroidal saponins and their structures were

identified as (25R)-spirost-5-ene-3P,17a-diol (pennogenin) 3-0-p-D-glucopyranoside

(1):) pennogenin 3-0-{0-a-L-arabinofuranosyl-(l~4)-~-D-glucopyranoside} (2):) (25R)-spirost-5-en-3f3-01 (diosgenin) 3-0-{0-a-L-rhamnopyranosyl-(l-2)-fi-D- glucopyranoside} (3b6) pennogenin 3-0-{0-a-L-rhamnopyranosyl-(l--2)-~-D- glucopyranoside} (4)$) diosgenin 3-0-{0-a-L-rhamnopyranosyl-(l-2)-0-[~-D- glucopyranosyl-(l--3)]-~-D-glucopyranoside} (5),7) pennogenin 3-0-(0-a-L-

rhamnopyranosyl-(l-.2)-0-[ ~-D-glucopyranosyl-(1-3)]-~-D-glucopyranoside) (6),8) diosgenin 3-0-{0-a-L-rhamnopyranosyl-(l.-+2)-0-[a-L-arabinofuranosyl- (1~4)]-P-D-glucopyranoside} (7),9) pennogenin 3-0-{0-a-L-rhamnopyranosyl-

(1-2)-0-[ a-L-arabinofuranosyl-(1-.4)]-~-D-glucopyranoside} (8):) diosgenin 3-0- (0-a-L-rhamnop yranosyl-( 1 +2)-0-[O-a-L-rhamnopyranosyl-( 1 --4)-a-L-rhamno-

pyranosyl-(l-.4)]-~-D-glucopyranoside (9b9) and pennogenin 3-0-{0-a-L-

rhamnopyranosyl-(l-.2)-0-[0-a-L-rhamnopyranosyl-(l-4)-a-L-rhamnopyranosyl- (1 -.4)]-~-D-glucopyranoside} (10):) respectively.

Compound 11 was obtained as an amovhous solid, [ a ] ~ -101.0" (MeOH), which

was shown to have the molecular formula C45H72017 by the high-resolution (HR) FAB-MS (mh 907.4693 [M+Na]+) and 13C-NMR data (45 carbon signals). The IH- NMR spectrum of 11 in pyridine-dg displayed signals for four steroid methyl groups

at 6 1.23 (3H, d, J = 7.2 Hz), 0.97 (3H, s), 0.95 (3H, s), and 0.69 (d, J = 5.6 Hz).

Furthermore, three anomeric proton signals were identified at 6 6.31 (lH, br s), 5.87

(lH, br s), and 4.94 (d, J = 7.8 Hz). Acid hydrolysis of 11 with 1 M HCl in dioxane-

H20 (1: 1) liberated D-glucose and L-rhamnose, and several degradation products from

the aglycone. The identification of the monosaccharides, including thcir absolute

configurations, was established by direct HPLC analysis of the sugar fraction of the

acid hydrolysate using a combination of the RI and optical rotary (OR) detectors. The

13C-NMR shifts of the aglycone moiety of 11 were in good accordance with those of the reported pennogenin 3 - 0 - g l y ~ o s i d e s . ~ ~ ) The above data were indicative of 11

being a pennogenin 3-0-triglycoside. The exact structure of the triglycoside moiety

was determined by the following two-dimensional NMR analysis. The lH-lH shift

correlation spectroscopy (COSY) experiment allowed the sequential assignment of all

the proton signals for the triglycoside residue, with the easily distinguished anomeric

protons being used as the starting point of analysis. Subsequent inspection of the lH- detected heteronuclear multiple quantum coherence (HMQC) spectrum led to the

correlation of all the proton resonances with those of the corresponding one-bond coupled carbons (Table 1). Comparison of the carbon chemical shifts thus assigned with those of the reference methyl glycosides,ll) taking into account the known effects

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

360 YOSHIHIRO MIMAKI etal.

of 0-glycosylation, indicated that 11 contained a C-4 substituted fLDglucopyranosy1

[ 6 ~ 4 . 9 4 ( d , J = 7.8 Hz); 102.5 ( 0 , 75.6 (CH), 76.5 (CH), 77.7 (CH), 77.2 (CH),

Table 1. 'H- and UC-NMR Data of the Glycoside Moiety of 11'

position 'H @pm) J p z ) l3C Cppm)

1 ' 4.94 d 7.8 102.5 2 ' 3.98 dd 9.3,7.8 75.6 3 ' 4.22 dd 9.3, 9.3 76.5

5 ' 3.68 ddd 9.3,3.1,2.0 77.2 6 'a 4.23 dd 12.1,2.0 61.4

1 " 5.87 br s 102.1 2 " 4.58 brd 3.2 73.4 3 " 4.61 dd 9.0,3.2 73.0 4 " 4.47 dd 9.0, 9.0 80.3 5 " 5.03 dq 9.0,6.2 68.3 6 " 1.69 d 6.2 18.9 1 "I 6.31 br s 103.2

4.90 br d 3.3 72.6 2 ,,t

3 "' 4.53 dd 9.2,3.3 72.9 4 "' 4.31 dd 9.2,9.2 74.0 5 "' 4.39 dq 9.2,6.1 70.4 6 'I' 1.61 d 6.1 18.4

4 ' 4.47 dd 9.3,9.3 77.7

b 4.09 dd 12.1,3.1

a Spectra were measured in pyridine-d5.

and 61.4 (CHz)], a C-4 substituted a-L-rhamnopyranosyl [SH 5.87 @r s); 6~ 102.1

(CH), 73.4 (CH), 73.0 (CH), 80.3 (CH), 68.3 (CH), and 18.9 (Me)], and a terminal a-

L-rhamnopyranosyl [h 6.31 (br s); 6~ 103.2 (CH), 72.6 (0, 72.9 (CH), 74.0 (CH),

€I ,.-

H O ~ " d a , ,

Ho OH

Fig. 1. HMBC Correlations of the Glycoside Moiety of 11

70.4 (CH), and 18.4 (Me)] unit. In the lH-detected heteronuclear multiple-bond

connectivity (HMBC) spectrum, the anomeric proton signals at 6 6.31 (terminal

rhamnosyl), 5.87 (C-4 substituted rhamnosyl), and 4.94 (C-4 substituted glucosyl)

exhibited 3 J c , ~ correlations with the carbon signals at 6 80.3 (C-4 of rhamnosyl), 77.7

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

STEROIDAL SAPONINS FROM THE RHIZOMES OF PARIS POLYPHYLLA VAR. CHhVENSfS 36 I

(C-4 of glucosyl), and 78.2 (C-3 of aglycon), respectively (Fig. 1). Accordingly, the

structure of 11 was elucidated as pennogenin 3-O-{O-a-L-rhamnopyranosyl-( 1 4 4 ) -

O-a-L-rhamnopyranosy1-(1~4)-~-D-glucopyranoside}. Compound 11 is a new

steroidal saponin.

The cytotoxic activity of the isolated compounds on leukemia HL-60 cells was

evaluated. The cells were continuously treated with each sample for 72 h, and the cell growth was measured by using the 3-(4,5-di-methylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay. The saponins, except for 1, 2, and 11,

which have no substituent at C-2 of the glucosyl moiety attached to the aglycone,

showed relatively potent cytotoxic activity with the IC50 values ranging from 0.5

pg/mL to 5.1 pg/mL, whereas that of etoposide used as a positive control was 0.3

pg/mL. Compound 7 was obtained in good yield from the crude drug (0.074% of the

dry weight) and exhibited the most potent cytotoxic activity among the isolated

saponins (IC50 0.5 pghL) . Subsequent evaluation of 7 in the National Cancer

Institute 60 cell line assay12) showed that the mean concentration required to achieve

GI50, TGI, and LC50 levels13) against the panel of cells tested were 0.58, 1.5, and 3.5

@mL, respectively. Compound 7 exhibited no significant differential cellular

sensitivity. However, some cell lines were relatively sensitive to it, namely, the leukemia

MOLT-4 (GI50 0.19 pg/mL; TGI 0.41 pg/mL; LC5o 0.91 pg/mL), non-small cell lung

cancer A549/ATCC (GI50 0.20 pg/mL; TGI 0.36 pg/mL; LC50 0.66 pg/mL), colon

cancer SW-620 (GI50 0.19 pg/mL; TGI 0.42 @mL; LC50 0.91 pg/mL), melanoma

M14 (GI50 0.20 pg/mL; TGI 0.36 pg/mL; LC50 0.65 pg/mL), and renal cancer 786-0

(GI50 0.22 pg/mL; TGI 0.47 pg/mL; LC50 0.98 pg/mL) cell lines. These results are thought to support the medicinal folkloric background of P. polyphylla var. chinensis,

which has long been used as a treatment of a variety of human cancers in China.

EXPERIMENTAL

Optical rotations were measured by using a JASCO DIP-360 automatic digital

polarimeter. IR spectra were recorded on a JASCO A-100 spectrophotometer. NMR spectra were recorded on a Bruker DPX-400 spectrometer (400 MHz for IH-NMR) or on a Bruker DRX-500 spectrometer (500 MHz for lH-NMR) using standard Bruker

pulse programs. Chemical shifts are given as 6 values with reference to

tetramethylsilane (TMS) as internal standard. MS were recorded on a VG AutoSpec E mass spectrometer. Silica-gel (Fuji-Silysia Chemical, Japan), ODS silica-gel (Nacalai

Tesque, Japan), and Diaion HP-20 (Mitsubishi-Kasei) were used for column

chromatography, and precoated Kieselgel 60 F254 (0.25 mm thick, Merck, Germany)

and RP-18 F254 S (0.25 mm thick, Merck) plates, for TLC. The spots on TLC were

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

362 YOSHIHIRO MIMAKI et ul

visualized by spraying the plates with 10% H2SO4 solution, followed by heating.

HPLC was performed by using an LC system comprised of a CCPM pump (Tosoh,

Japan), a CCP PX-8010 controller (Tosoh), an RI-8010 detector (Tosoh), a Shodex

OR-2 detector (Showa-Denko, Japan), and a Rheodyne injection port with a 20 pL

sample loop. A Kaseisorb NH2-60-5 column (4.6 mm i d . x 250 mm, 5 pm, Tokyo-

Kasei, Japan) was employed for HPLC analysis. The following materials and reagents

were used for cell culture and assay of cytotoxic activity: microplate reader, Inter Med

Immuno-M ini NJ-2300 (Japan); 96-well flat-bottom plate, Iwaki Glass (Japan); HL-60 cells, ICN Biomedicals (U.S.A.); RPMI 1640 medium, GIBCO BRL (U.S.A.); MTI',

Sigma (U.S.A.). All other chemicals used were of biochemical reagent grade. Plant Material: Rhizomes of P. polyphylla var. chinensis were collected at Yunnan

Province, China. A vouchcr of the plant is on the file in our laboratory (# PPC-97-2).

Extraction and Isolation: The plant material (dry weight, 2.0 kg) was extracted with

hot MeOH twice (each 2.8 I., 3 h). The MeOH extract was concentrated under reduced

pressure, and the viscous concentrate (702 g) was passed through a Diaion HP-20

column, successively eluting with 30% MeOH, 50% MeOH, MeOH, EtOH, and EtOAc.

The MeOH eluate fraction (60 g) exhibited considerable cytotoxic activity against HL-

60 cells (99% cell growth inhibition at the sample concentration of 10 pg/mL; IC50 3.3

pg/mL), while the other fractions were far less active [30% MeOH eluate fraction (70

g): 47% inhibition at 10 pg/mL; 50% MeOH (25 g): 0% inhibition; EtOH (1 g): 28%

inhibition; EtOAc (1 g): 0% inhibition]. Column chromatography of the MeOH eluate

portion on silica-gel and elution with a stepwise gradient mixture of CHC13-MeOH

(9:l; 4 : l ; 2 : l ; l:l), and finally with MeOH alone, gave six fractions (I-VI). Fraction I1

was chromatographed on silica-gel eluting with CHC13-MeOH-H20 (120:20:1) and

ODS silica-gel with MeOH-H20 (9:2; 4 : l ) to give 1 (96.2 mg). Fraction I11 was

subjected to column chromatography on silica-gel eluting with CHC13-MeOH-H20 (120:20:1) and ODS silica-gel with MeOH-H2O (9:2; 4: l ) and MeCN-H20 (5:3; 1 : l )

to give 2 (739 mg). Fraction IV was subjected to a silica-gel column eluting with

CHC13-MeOH-H2O (120:lO:l) and an ODS silica-gel column with MeOH-H20 (4: l )

and MeCN-H20 (5:3; 1:1) to give 3 (987 mg), 4 (2.15 g), 7 (1.48 g). 8 (1.55 g), 9

(58.0 mg), 10 (203 mg), and 11 (72.3 mg). Fraction V was separated by silica-gel

column chromatography eluting with CHC13-MeOH-H20 (120:20:1; 40:lO:l;

20:lO:l) and ODS silica-gel column chromatography with MeOH-H20 (4:l) to give 5

(59.1 mg) and 6 (626 mg). Compound 11: Amorphous solid, [ a ] ~ 2 5 -101.0" (MeOH, c = 0.21). HR FAB-MS

(positive mode) m/z: 907.4693 [M+Na]+ (C45H72017.Na, Calcd for 907.4667). IR

vmax (KBr) cm-l: 3370 (OH), 2930 (CH), 1450,1375,1240,1030,975,895,885, 820,

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

STEROIDAL SAPONINS FROM THE RHIZOMES OF PARIS POLYPHYLLA VAR. CHINENSIS 363

795. lH-NMR (pyridine-dg) 6: 5.31 (lH, br d, J = 5.0 Hz, H-6), 4.45 (lH, dd, J = 5.6,

5.1 Hz, H-16), 3.85 (lH, m, H-3), 3.51 (2H, H2-26), 2.28 (lH, q, J = 7.2 Hz, H-20),

1.23 (3H, d, J = 7.2 Hz, Me-21), 0.97 (3H, s, Me-18), 0.95 (3H, s, Me-19), 0.69 (3H, d,

J = 5.6 Hz, Me-27). Triglycoside moiety: Table 1. I3C-NMR (pyridine-dg) 6: 37.5 (C-1), 30.2 (C-2), 78.2 (C-3), 39.3 (C-4), 140.8 (C-5), 121.8 (C-6), 32.4 (C-7), 32.3

(C-8), 50.2 (C-9), 37.0 (C-lo), 21.0 (C-ll), 32.1 (C-121, 45.1 (C-13), 53.0 (C-14),

31.8 (C-15), 90.1 (C-16) 90.0 (C-17), 17.2 (C-18), 19.4 (C-19), 44.8 (C-20), 9.7 (C-

21), 109.8 (C-22), 32.1 (C-23), 28.8 (C-24), 30.4 (C-25), 66.7 (C-26), 17.3 (C-27). Triglycoside moiety: Table 1.

Acid Hydrolysis of 11: A solution of 11 (10.1 mg) in 1M HCI (dioxane-Hz0, 1:1,

2 mL) was heated at 100 "C for 2 h under an Ar atmosphere. After cooling, the

reaction mixture was neutralized by passage through an Amberlite IRA-93ZU

(Organo, Japan) column and chromatographed over silica-gel using a discontinuous

gradient of CHClyMeOH (9:l to 1 : l ) to give a mixture of several decomposed

sapogenols and a sugar fraction (4.4 mg). The sugar fraction was dissolved in H20

and passed through a Sep-Pak C18 cartridge (Waters, U.S.A.), which was then analyzed

by HPLC under the following conditions: solvent, MeCN-H20 (3:l); flow rate, 0.8

mL/min; detection, RI and OR. The identification of D-glucose and L-rhamnose present in the sugar fraction was carried out by the comparison of their retention times

and polarities with those of authentic samples. Rt (min) 8.32 (L-rhamnose, negative

optical rotation); 12.86 (D-glucose, positive optical rotation).

Cell Culture Assay: HL-60 cells were maintained in the RPMI 1640 medium

containing 10% fetal bovine serum supplemented with L-glutamine, 100 units/mL

penicillin, and 100 pg/mL streptomycin. The leukemia cells were washed and resuspended in the above medium to 3 x lo4 cells/mL, and 196 pL of this cell

suspension was placed in each well of a 96-well flat-bottom plate. The cells were

incubated in 5% COdair for 24 h at 37 "C. After incubation, 4 pL of EtOH-HzO (1:l)

solution containing the sample was added to give the final concentrations of 0.1-10

pg/mL; 4 pL of EtOH-H20 (1:l) was added into control wells. The cells were further

incubated for 72 h in the presence of each agent, and then cell growth was evaluated

by an MTT assay procedure. The MTT assay was carried out according to a modified method of Sargent and Tayler as f0llows.1~) After termination of the cell culture, 10

p L of 5 mg/mL MTT in phosphate buffered saline was added to every well and the plate was further incubated in 5% Codair for 4 h at 37 "C. The plate was then

centrifuged at 1500 g for 5 min to precipitate cells and formazan. An aliquot of 150 pL of the supernatant was removed from every well, and 175 pL of dimethyl sulfoxide

(DMSO) was added to dissolve the formazan crystals. The plate was mixed on a

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014

364 YOSHIHIRO MIMAKI et al.

microshaker for 10 min, and then read on a microplate reader at 550 nm. A dose

response curve was plotted for the MeOH eluate fraction and compounds 3-10, which

showed more than 50% of cell growth inhibition at the sample concentration of 10

@mL, and the concentration giving 50% inhibition (IC50) was calculated. The IC50 values of 3-10 were 1.8, 5.1, 2.1, 5.0, 0.5, 1.5,0.8, and 1.7 pg/mL, respectively.

Acknowledgments: We wish to thank the Developmental Therapeutics Program,

National Cancer Institute, Bethesda, MD, for performing the cytostatic and cytotoxic

screening studies. We are also grateful to Dr. Y. Shida, Tokyo University of Pharmacy

and Life Science, for the measurements of the mass spectra.

REFERENCES AND NOTES

1) Tsukamoto Y. (ed.), "The Grand Dictionary of Horticulture," Vol. 4, Shogakukan,

Tokyo, 1989, pp. 48-49.

2) Zhou J., Pure & Appl. Chem., 61,457-460 (1989).

3) Mimaki Y., Kuroda M., Fukasawa T., Sashida Y., J. Nut. Prod., 62, 194-197

(1 999).

4) Mahato S. B., Sahu N. P., Ganguly A. N., Phytochemistry, 20,1943-1946 (1981).

5) Miyamura M., Nakano K., Nohara T., Tomimatsu T., Kawasaki T., Chem. Pharm.

Bull.,30,712-718 (1982).

6) Nohara T., Miyahara K, Kawasaki T., Chem. Pharm. Bull., 23,872-885 (1975).

7) Mahato S. B., Sahu N. P., Ganguly A. N., Indian J. Chem., 19B, 817-819 (1980).

8) Chen C., Zhou J., Yunnan Zhiwu Yanjiu,6,111-117 (1984).

9) Nohara T., Yabuta H., Suenobu M., Hida R, Miyahara K, Kawasaki T., Chem.

Pharm. Bull., 21,1240-1247 (1973).

10) Mimaki Y., Nakamura O., Sashida Y., Nikaido T., Ohmoto T., Phytochemistry, 38,

1270-1286 (1995).

11) Agrawal P. K., Jain D. C., Gupta R. K, Thakur R. S. , Phytochemistry, 24, 2479-

2496 (1985); Agrawal P. K., ibid.,31,3307-3330 (1992).

12) Monks A., Scudiero D., Skehan P., Shoemaker R, Paul1 K., Vistica D., Hose C., Langley J., Cronise P., Vaigro-Wolff A, Gray-Goodrich M., Campbell H., Mayo J.,

Boyd M.,J. Natl. Cancer Inst., 83,757-766 (1991).

13) The LC50 is the concentration at which only 50% of the cells are viable, the GI50

value is the concentration that yields 50% cell growth, and the total growth inhibition

(TGI) is the concentration at which no growth is observed.

14) Sargent J. M., Taylor C. G., Br. J. Cancer, 60,206-210 (1989).

Dow

nloa

ded

by [

UQ

Lib

rary

] at

00:

39 1

0 N

ovem

ber

2014