Pharmaceutical Biology 1998, Vol. 36, No. 1, pp.

2932

1388-0209/98/3601-0029$12.00 Swets & Zeitlinger

ANTI-HIV ACTIVITY DIRECTED FRACTIONATION OF THE EXTRACTS OF MARGYRICARPUS SETOSUS

Nunziatina De Tommasi1, Sonia Piacente1, Luca Rastrelli1, Naheed Mahmood2 and Cosimo Pizza1*
1Dipartimento di Scienze Farmaceutiche, Universit degli Studi di Salerno, Piazza Vittorio Emanuele no. 9,

84084 Penta di Fisciano (Salerno), Italy

2MRC Collaborative Centre, 13 Burtonhole Lane, Mill Hill, London, NW71AD, U.K.

ABSTRACT
As part of our screening of anti-HIV agents from medicinal plants, the in vitro anti-HIV activity of the CHCl3, CHCl3:MeOH 9:1 and MeOH extracts of the aerial parts of Margiricarpus setosus (Rosaceae) were tested. Since the MeOH extract showed signicant activity, a bioassay-directed fractionation was carried out. This investigation resulted in the isolation of catechin, epicatechin, quercetin-3-O-(6"-p-coumaroyl)-D-glucoside, -hydroxy-3',4'dimethoxy-phenyl-ethylglucoside, -hydroxy-3',4'-dimethoxyphenylethylrutinoside, -hydroxy-phenylethyl-O--L-rhamnopyranosyl (1 6)--D-glucopyranoside, benzyl -O--L-rhamnopyranosyl (1 6)--D-glucopyranoside, 4methoxybenzyl- O--L-rhamnopyranosyl (1 6)--D-glucopyranoside, tormentic acid 3-O-D-quinovopyranoside, tormentic acid 3- O--D-fuco pyranoside and tormentic acid 3-O--L-rhamnopyranoside. Two of these compounds, catechin and epicatechin, have been identied as the principles responsible for anti-HIV activity. This nding is in good agreement with our previous investigations which demonstrated catechin, epicatechin and their derivatives inhibited the in vitro HIV replication.

INTRODUCTION Margiricarpus setosus R et P. (Rosaceae) is a plant of the Andean area, commonly known as piqui yoyo

(Velasco, 1946). The aerial parts are widely used in folk medicine for their antiinammatory and antiviral properties (Velasco, 1946). However, there are no data in the literature on the possible pharmacological effects of this plant. In a previous paper we described the isolation and structure elucidation of new aryl and triterpenic glycosides from the leaves of M. setosus (De Tommasi et al., 1996). The occurrence of arylglycosides and triterpene derivatives has been demonstrated in a great number of Rosaceae; therefore considerable taxonomic importance seems to emerge from the relatively high frequency of accumulation of such compounds in these species. In the course of our continuing search for novel antiHIV agents from natural sources, we tested the antiHIV activity of extracts, partially puried fractions and pure compounds from M. setosus . The extracts and fractions were bioassayed for in vitro anti-HIV activity. Subsequent bioassay-directed fractionation of the most active fractions has led to the isolation and characterization of catechin and epicatechin as the compounds responsible for the anti-HIV activity elicited by the MeOH extract. These data conrm the role of avans as anti-HIV agents and are in good agreement with our previous investigations which demonstrated catechin, epicatechin and their derivatives inhibit the in vitro HIV replication (Mahmood et al., 1993a,b; Moore & Pizza, 1992; Piacente et al., 1994).

Keywords: Anti-HIV activity, catechin and epicatechin, Margiricarpus setosus, Rosaceae.

MATERIALS AND METHODS General NMR spectra were obtained in CD3OD using Bruker WM-250 or Bruker AMX-500 spectrometers. HPLC

* Author to whom correspondence should be addressed.

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N. DE TOMMASI ET AL.

separations were performed on a Waters 590 series pumping system with a Waters R401 refractive index detector equipped with a Waters -Bondapak C18 column. Plant Material The aerial parts of Margiricarpus setosus were collected at Riobamba, Chimborazo region, in July 1993, and identied by W. Palacios. A voucher sample is deposited at the Herbario Nacional de Quito. Isolation of Compounds The air-dried aerial parts of M. setosus (450 g) were defatted with petrol in a Soxhlet apparatus and then extracted at room temperature with CHCl3, CHCl3:MeOH 9:1 and MeOH in succession to give 6.8, 11.0 and 24 g of residue. The MeOH extract was found to exhibit signicant anti-HIV activity; therefore, a portion (2.5 g) was subjected to Sephadex LH-20 using MeOH as eluent to afford 60 fractions (8 ml each). These fractions were monitored by TLC on silica gel plates using a mixture of chloroform-methanol-H2O (7:3:0.3) as developing solvent. Eluates showing similar TLC proles were pooled to give 6 combined fractions, of which fractions V (fr. 4554, 200 mg) and VI (fr. 5560, 170 mg) were the only ones found to exhibit activity against HIV. Fractionation of V and VI was achieved by RP-HPLC using, respectively, MeOH:H2O (45:55, ow rate 2.5 ml/min) and MeOH:H2O (33:67, ow rate 1.5 ml/min) as the eluents. From fraction V, pure epicatechin (80 mg, Rt 2 min), -hydroxy-3',4'dimethoxyphenylethylglucoside (17.3 mg, Rt 14 min) and -hydroxy-3',4'-dimethoxyphenylethylrutinoside (23.5 mg, Rt 9.5) were obtained; fraction VI yielded catechin (50.3 mg, Rt 10 min), epicatechin (35.2 mg, Rt 12.5 min) and quercetin-3-O-(6"-p-cumaroyl)-D-glucoside (7.8 mg, Rt 23 min). All the isolated compounds were identied by a comparison of their spectroscopic parameters (1H and 13C NMR) with literature values (Andary et al., 1980; Nishibe et al., 1982; Porter et al., 1982; Higuchi & Donelli, 1978; De Tommasi et al., 1996). Antiviral Assays Anti-HIV activity and toxicity of compounds was assessed in C8166 cells infected with HIV-1MN. The cells were cultured in RPMI 1640 with 10% fetal calf serum. Forty-thousand cells per microtiter plate well were mixed with 5-fold dilutions of compounds prior to addition of 10 CCID50 units of virus and incubated for 56 days. Formation of syncytia was examined

from 2 days post-infection. The inhibition of HIVinfection was assessed by examining syncytia, by estimating antigen gp120 by ELISA (Mahmood & Hay, 1992), and by measuring cell viability of virus-infected and uninfected cell controls by the XTT-formazan method (Weislow et al., 1989). Virus Infectivity Assay The total progeny virus was titrated in microtiter plates using double dilutions of freshly collected supernatants and C8166 cells. The end point was determined by examining syncytia formation and by the XTT-formazan method. The virus titer (CCID 50) are expressed as the reciprocal of the dilution which gave a 50% end point. To measure the effects of compounds on virus infectivity, HIV-1IIIB (104105 CCID50) was incubated with compound at 37C for 1 h, the virus was seriallydiluted and the infectivity endpoint determined. In all cases compound was diluted to well below the EC 50, such that residual compound did not interfere with the virus titration.

RESULTS AND DISCUSSION The air-dried aerial parts of M. setosus were extracted successively with CHCl3, CHCl3/MeOH 9:1 and MeOH. The in vitro anti-HIV activity of the three extracts was tested. Table 1 shows the inhibitory effect of the three extracts against HIV-MN in acutely infected C8166 cells. The EC50 values for HIV infection were the same, but signicant differences were seen in terms of toxicity. The lower toxicity of the MeOH extract gave a selectivity index (S.I.) of 50, more than 10-fold that of CHCl3 (S.I. 4) and CHCl3/MeOH 9:1 (S.I. 4) extracts. At the concentration of 200 g/ml, the MeOH extract suppressed completely the formation of syncytia and production of gp120. The above results prompted us to investigate the constituents of the MeOH extract that was subjected to Sephadex LH-20 to afford six main fractions I-VI which were tested for in vitro anti-HIV activity. Since only V and VI exhibited a signicant inhibition of HIV replication, showing EC50 and S.I. comparable to those observed for the MeOH extract (Table 1), the constituents of these two fractions were investigated. By means of reversed-phase HPLC, -hydroxy-3',4'dimethoxyphenylethylglucoside and -hydroxy-3',4'dimethoxyphenylethylrutinoside were isolated from fraction V, and catechin, epicatechin and quercetin-3-O(6"-p-cumaroyl)--D-glucoside from fraction VI. Inves-

MARGYRICARPUS SETOSUS ANTI-HIV ACTIVITY

31

Table 1. Anti-HIV activity of MeOH, CHCl 3 and CHCl3/MeOH (9:1) extracts of M. setosus. Extract Conc. (g/ml) Syncytia () gp120 % of control Estimated cell growth % of control infected MeOH 1000 200 40 8 1.6 100 20 4 100 20 4 10 0.016 0 8 95 79 34 23 18 37 27 20 39 23 100 31 uninfected 5 96 99 10 500 EC50 TC50

18 74 82 18 73 9 71 51 100

CHCl3/MeOH 9:1

16 23 100 19 65 100 101 100

10

40

CHCl3

10

40

AZT*

0.016

> 1000

Control

EC50 represents the concentration which reduces the Ag gp120 by 50% in infected cell cultures. TC50 represents the concentration which reduces cell growth by 50%. *Concentration of AZT is M. Table 2. Anti-HIV activity of catechin and epicatechin. Compound Conc. (g/ml) Syncytia () gp120 % of control Estimated cell growth % of control infected catechin 50 10 2 0.4 100 20 4 0.8 0.16 10 0.016 0 85 64 89 40 90 101 70 42 31 100 31 uninfected 98 100 101 103 92 99 101 102 100 101 100 4 >100 EC50 TC50

25 62

epicatechin

>100

26 64

AZT* Control

0.016

>1000

51 100

EC50 represents the concentration which reduces the Ag gp120 by 50% in infected cell cultures. TC50 represents the concentration which reduces cell growth by 50%. *Concentration of AZT is M.

tigation of inactive fractions of M. setosus afforded three new aryl glycosides, -hydroxy-phenylethyl-O--Lrhamnopyranosyl (1 6)--D-glucopyranoside, benzylO--L-rhamnopyranosyl (1 6)--D-glucopyranoside, 4-methoxybenzyl-O--L-rhamnopyranosyl (1 6)-D-glucopyranoside, and three new triterpene glycosides, tormentic acid 3-O--D-quinovopyranoside, tormentic acid 3-O--D-fucopyranoside and tormentic acid 3O--L-rhamnopyranoside. The structures of the new

compounds were elucidated on the basis of chemical and spectral data including the concerted application of onedimensional NMR and two-dimensional NMR techniques as reported in an our previous work (De Tommasi et al., 1996). Among all the isolated compounds, catechin and epicatechin consistently exhibited the greatest activity, showing an EC50 of 4 g/ml with a selectivity index 25 and an EC50 of 2 g/ml with a selectivity index 50.

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N. DE TOMMASI ET AL.

These data were in good agreement with the results of our previous study on the inhibition of HIV infection by avanoids (Mahmood et al., 1993a). Our previous investigations established that catechin and epicatechin inhibit HIV reverse transcriptase in a non-specic manner (Mahmood et al., 1993b) and that inhibition of virus infection is principally due to their more selective interaction with gp120 (Mahmood et al., 1993a). Studies of their effects on the binding of sCD4 to gp120 indicated that the two compounds interact irreversibly with gp120 to inactivate virus infectivity and block infection. The specity of the interaction with gp120 was apparent from the ability of the compounds to selectively prevent the binding of monoclonal antibody to, for example, the V3 loop of gp120 but not antibodies directed against terminal regions of the molecule. The antiviral action of catechin and epicatechin is not limited to HIV-1 since they exhibited a comparable activity against HIV-2 and SIV infections of C1866 cells, and some effect, although at higher concentrations, also against herpes simplex virus infection of Vero cells (Mahmood et al., 1993a). The identication of catechin and epicatechin as the compounds responsible for the anti-HIV activity elicited by the MeOH extract conrm the role of avans as anti-HIV agents. However, we cannot rule out that the activity of the extracts and fractions could also be due to minor compounds related to catechin and epicatechin, or due to a combination that may act synergistically.

REFERENCES
Andary C, Privat G, Chevallet P, Orzalesi H, Serrano JJ, Bouchard M (1980): Etude chimique et pharmacodinamique desters heterosidiques de lacide cafeique, isoles d Orobanche rapum-genistae . Il Farmaco Ed Sci 35: 330. De Tommasi N, Rastrelli L, Cumanda J, Speranza G, Pizza C

(1996): Aryl and triterpenic glycosides from Margyricarpus setosus. Phytochemistry 42: 163167. Higuchi R, Donelly D (1978): Acylated avonol glucosides of Pinus contorta needles. Phytochemistry 17: 787791. Mahmood N, Hay AJ (1992): An ELISA utilising immobilised snowdrop lectin GNA for the detection of envelope glycoproteins of HIV and SIV. J Immunol Meth 151: 913. Mahmood N, Pizza C, Aquino R, De Tommasi N, Piacente S, Colman S, Burke A, Hay AJ (1993a): Inhibition of HIV infection by avanoids. Antivir Res 22: 189199. Mahmood N, Moore PS, De Tommasi N, De Simone F, Colman S, Hay AJ, Pizza C (1993b): Inhibition of HIV infection by caffeoylquinic acid derivatives. Antiviral Chem Chemother 4: 235240. Moore PS, Pizza C (1992): Observations on the inhibition of HIV-1 reverse transcriptase by catechins. Biochem J 288: 717719. Nishibe S, Okabe K, Tsukamoto H, Sakushima A, Hisada S, Baba H, Akisada T (1982): Studies on the Chinese crude drug Forsythiae Fructus. VI. The structure and antibacterial activity of suspensaside isolated from Forsythia suspensa. Chem Pharm Bull 30: 45484553. Piacente S, Aquino R, De Tommasi N, Pizza C, Lock De Ugaz O, Orellana HC, Mahmood N (1994): Constituents of Werneria ciliolata and their in vitro anti-HIV activity. Phytochemistry 36: 991996. Porter LJ, Newman RH, Foo LY, Wong H (1982): Polymeric proanthocyanidins. J Chem Soc, Perkin Trans I, 12171227. Velasco J (1946): Historia del Rein de Quito, La Historia Natural, Vol. 1. Empresa Editoria El Comercio, Quito. Weislow OS, Kiser R, Fine DL, Bader J, Shoemaker RH, Boyd R (1989): New soluble-formazan assay for HIV-1 cytopathic effects: application to high-ux screening of synthetic and natural products for AIDS-antiviral activity. J Natl Cancer Inst 81 : 577586.

Accepted: October 1, 1997