Fitoterapia 75(2004) 500–504

0367-326X/04/$ - see front matter� 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.fitote.2004.03.009

Short report

Cytotoxic constituents fromPlumbago zeylanica

A.T. Nguyen *, H. Malonne , P. Duez , R. Vanhaelen-Fastre ,a,b, b a a

M. Vanhaelen , J. Fontainea b

Laboratoire de Pharmacognosie, Institut de Pharmacie CP-205y9, Universite Libre de Bruxelles,a ´B-1050 Brussels, Belgium

Laboratoire de Physiologie et de Pharmacologie Fondamentales, Institut de Pharmacie CP-205y7,b

Universite Libre de Bruxelles, B-1050 Brussels, Belgium´

Received 17 October 2003; accepted in revised form 29 March 2004

Abstract

The bioassay-guided fractionation of the dichloromethane extract of aerial parts ofPlumbago zeylanica led to the isolation ofb-sitosterol,b-sitosteryl-3b-glucopyranoside,b-sitosteryl-3b-glucopyranoside-69-O-palmitate (1), lupenone, lupeol acetate, plumbagin andtrilinolein. Compound1 showed cytotoxic activity against MCF7 and Bowes cancer celllines (IC 113 mM and 152mM, respectively), b-sitosterol inhibited Bowes cell growth50

(IC 36.5 mM) and plumbagin was cytotoxic against MCF7 and Bowes cells(IC 1.2850 50

mM and 1.39mM, respectively).� 2004 Elsevier B.V. All rights reserved.

Keywords: Plumbago zeylanica; Cytotoxicity; b-Sitosteryl-3b-glucopyranoside-69-O-palmitate;Plumbagin

Plant. Plumbago zeylanica L. (Plumbaginaceae), aerial parts collected in November2000 and identified by Prof. Vu Van Chuyen, Laboratory of Botany, Hanoi Universityof Pharmacy, where a voucher specimen(N8 501) was preserved. The plant,commonly called ‘Bach hoa xa’ in Vietnam, was selected for study because the

*Corresponding author. Tel.:q32-2-6505273; fax:q32-2-6505430.E-mail address: annhthng@ulb.ac.be(A.T. Nguyen).

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dichloromethane extract of the aerial parts displayed in vitro cytotoxicity againsttwo human cancer cell lines(MCF7 and Bowes).

Uses in traditional medicine. In Vietnamese traditional medicine for the treatmentof rheumatic pain, sprains, scabies, skin diseases, wounds, ulcers, inflammationsand cancerw1,2x.

Previously isolated classes of constituents. Naphthoquinones, steroids, sugars,naphthalenones, alkanes, triterpenes and amino acidsw3,4x.

New-isolated constituents. b-Sitosteryl-3b-glucopyranoside-69-O-palmitate (1)(yield: 0.009%) w5x, plumbagin(0.004%) w6x, lupeol acetate(0.008%), lupenone(0.044%) w7x, trilinolein (0.001%) w8x, b-sitosterol (0.028%) w9–11x and b-sitosteryl-3b-glucopyranoside(0.002%) w12x.

b-sitosteryl-3b-glucopyranoside-69-O-palmitate(1). H-NMR (CDCl , 600 MHz)13

and C-NMR(CDCl , 150 MHz) see Table 1.133

Tested material. b-Sitosterol,b-sitosteryl-3b-glucopyranoside,b-sitosteryl-3b-glu-copyranoside-69-O-palmitate and plumbagin.

Studied activity. Two human cancer cell lines, Bowes,(melanoma cells) and MCF7,(breast cancer cells), were incubated at 378C in sealed 75 cm dishes(Nunc. Life2

Technologies, Inc., Merelbeke, Belgium) containing Eagle’s minimal essentialmedium(MEM, Life Technologies, Inc., Paisley, Scotland) supplemented with 10%and 5% heat-inactivated fetal calf serum for the Bowes and MCF7, respectively. Allthe media were supplemented with 0.6 mgyml glutamine, 200 IUyml penicillin, 200IUyml streptomycin, and 0.1 mgyml gentamicin(all from Life Technologies, Inc.).The cells were incubated for 24 h in 96-microwell plates(at a density of 3=104

cellsyml for MCF7 and 6=10 cellsyml for Bowes) to ensure adequate plating4

before cytotoxicity testing using the MTT assayw13x. The IC and its confidence50

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Table 1H-NMR and C-NMR assignments for compound1 in CDCl1 13

3

N8 C (d) H (d, J (Hz)) N8 C (d) H (d, J (Hz))

b-Sitosterol 25 29.16 1.66(m)1 37.27 a. 1.06(m) 26 19.02 0.82(3H, d, 6.8)

b. 1.85(m)2 29.70 a. 1.61(m) 27 19.82 0.84(3H, d, 6.8)

b. 1.95(m)3 79.65 3.54(1H, m) 28 23.06 1.26(bs)4 38.91 a. 2.27(1H, m) 29 11.97 0.84(3H, t, 7.6)

b. 2.36(1H, m)5 140.30 – Glucopyranoside6 122.14 5.38(1H, m) 19 101.24 4.38(1H, d, 7.7)7 31.94 1.98(2H, m) 29 73.45 3.35(1H, dd, 7.7, 8.7)8 31.88 1.52(m) 39 76.07 3.57(1H, dd, 8.7, 9.9)9 50.15 0.93(m) 49 70.23 3.38(1H, dd, 9.9, 8.6)10 36.70 – 59 73.82 3.45(1H, m)11 21.07 a. 1.02(m) 69 63.41 a. 4.29(1H, dd, 12.1, 1.7)

b. 1.56(m) b. 4.42(1H, dd, 5.3, 12.1)12 39.76 a. 1.18(m)

b. 2.02(m) Palmitic acid13 42.33 – 19 174.56 –14 56.76 1.01(m) 29 34.26 2.34(2H, t, 7.6)15 24.30 a. 1.08(m) 39 24.97 1.61(m)

b. 1.12(m)16 28.26 a. 1.83(m) 49 29.26 1.28(bs)

b. 1.86(m)17 56.11 1.12(m) 59 29.45 1.26(bs)18 11.85 0.68(3H, s) 69 29.68 1.26(bs)19 19.37 1.00(3H, s) 79–129 29.78 1.26(bs)20 36.19 1.36(m) 139 29.40 1.26(bs)21 18.76 0.92(3H, d, 6.4) 149 31.94 1.26(bs)22 33.94 a. 1.00(s) 159 22.70 1.30(bs)

b. 1.34(m)23 26.10 1.18(2H, m) 169 14.13 0.88(3H, t, 7.1)24 45.81 0.95(m)

interval were determined by fitting experimental points to a parametric function bymeans of an original simplex algorithmw14,15x:

NsN8=exp(ykC)

where Csconcentration,Nspercentage of living cells at concentrationC, N8spercentage of living cells at concentration 0 andksparameter.

Results. Reported in Table 2.

Conclusions. b-Sitosterol showed a selective cytotoxic activity on Bowes cells.This common dietary phytosterol has been reported to inhibit 66% of the growth

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Table 2Cytotoxicity of b-sitosterol,b-sitosteryl-3b-glucopyranoside,b-sitosteryl-3b-glucopyranoside-69-O-pal-mitate and plumbagin on MCF7 and Bowes cells

Compound IC (mM)50

MCF7 Bowes

b-Sitosterol 357.51 36.54(335.68"377.43) (32.71"40.69)

b-Sitosteryl-3b-glucopyranoside )500a )500b-Sitosteryl-3b-glucopyranoside- 113.23 151.9869-O-palmitate(1) (109.32"117.21) (144.14"159.80)

Plumbagin 1.28 1.39(1.27"1.29) (1.37"1.41)

Adriamycinb 0.13 0.51(0.12"0.14) (0.47"0.56)

Considered as non-active.a

Cytotoxic reference compound.b

and stimulate the apoptosis of MDA-MB-231 human breast cancer cells in cultureat 16mM. However,b-sitosterol showed no cytotoxic effect on other cancer cells,i.e. PP2A, Colo-205, KB, Hela, HA22T, Hep-2, GBM8401yTSGH and H1477w16,17x. A certain selectivity ofb-sitosterol cytotoxicity against different cancer celllines equally appears in our experiment(Table 2). b-Sitosteryl-3b-glucopyranosideis inactive in the two cell lines tested. By contrast,b-sitosteryl-3b-glucopyranoside-69-O-palmitate(1) showed an activity against both cell lines. Plumbagin showed astrong cytotoxic activity on MCF7 and Bowes cell lines. Our results support thetraditional use ofP. zeylanica in Vietnam as an anticancer remedy.

Acknowledgments

A.T. Nguyen is a PhD scholarship recipient from the Belgian government(BTC-CTB). The authors thank Prof. T.K. Pham and Prof. V.C. Vu, Hanoi University ofPharmacy, for plant collection and identification; Dr Luhmer and Mr Moular(ULB)for the measurements of NMR and MS spectra; Prof. J. Dubois(ULB) for givingus access to the FADHA program and Dr A. Kumps and Mrs J. Genin(ULB) forGC-MS analysis.

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