Mutagenesis vol.5 no.4 pp.327-331, 1990

Antimutagenic activity of some saponins isolated from Calendula officinaUs L., C.arvensis L. and Hedera helix L.

R.Elias, M.De Meo, E.Vidal-OUivier, M.Laget1, G.Balansard and G.Dumenil1-2 Laboratoire de Pharmacognoste and 'Laboratoire de Microbiologie, Faculty de Pharmacie, 27 Bd Jean Moulin, 13385 Marseille Cedex 5, France 2

To whom correspondence should be addressed

Introduction A variety of compounds exist in nature which exert antimutagenic activity. Ames (1983) reported that some natural substances possess factors which can reduce or inhibit the activity of mutagens from the human diet. Natural antimutagens have been found in plant extracts (Kada et ai, 1978; Morita et al., 1978; Lai et al., 1980; Bala and Grover, 1989). Some plant components have been shown to decrease the mutagenicity of known mutagens. Ellagic acid, a plant phenol, has been found to reduce the mutagenic activity of benzo[a]pyrene (BaP) (Wood et al., 1982). Vitamin C and E decrease the formation of nitroso compounds (Ramel et al., 1986). Catechin, a component of betel quid has been reported to reduce the mutagenic activity of tobacco and masheri extracts (Nagabhushan and Bhide, 1988). Chlorophyll and chlorophyllin, the sodiumcopper salt of chlorophyll, have been shown to reduce the mutagenic activities of known mutagens and complex mixtures (Lai et al., 1980; Terwel and Van der Hoeven, 1985; Ong et al., 1986). During a comparative study, chlorophyllin was found to be more effective than retinol, /3-carotene, and vitamins C and E in inhibiting the mutagenic activities of dietary and environmental complex mixtures (Ong et al., 1989). Chlorophyllin could be antimutagenic by scavenging free radicals (Sato et al., 1984). Smoking plays a prime role in the etiology of lung and bladder cancer. Among the mutagenic environmental complex mixtures, cigarette smoke condensate (CSC) and urine from smokers (SU) have been found to be mutagenic in the Salmonella/microsome assay (S.Y.Sato et al., 1977; Yamasaki and Ames, 1977). © Oxford University Press

Materials and methods Chemicals Chlorophyllin (sodium—copper salt) and BaP were purchased from Sigma (St Louis, MO, USA). Aroclor 1254 was from Analabs (North Heaven, CO, USA). Urine concentrate from one smoker (SU) A 24 h urine sample from a healthy smoker (40 cigarettes per day) was collected. The extraction and concentration of urinary mutagens were carried out by the adsorption technique on Amberlite XAD-2 resin as described by Yamasaki and Ames (1977). Briefly, the urine sample was divided into 100 ml subsamples and filtered through Whatman paper no. 2. The filtered urines were concentrated on glass Econo columns (Bio Rad Laboratories, Richmond, CA, USA) packed with XAD-2 resin (0.9 g/100 ml of urine). The columns were washed with 10 ml of ultra pure water and the mutagens were eluted with 10 ml of acetone. The organic eluate was concentrated to dryness under a stream of nitrogen and resuspended in 400 fii of dimethylsulphoxide (spectro grade). The final concentration was 250-fold. The concentrated urine samples were kept at -80°C and all the assays were performed within 2 weeks. Plant materials The 13 saponins were isolated from the dried leaves of H.helix L., the dried flowers of C.officinalis L. and the dried aerial parts of C.arvensis L. The powered plant materials were extracted with 60% ethanol. All the extracts were concentrated under reduced pressure to eliminate ethanol and the aqueous solutions were lyophilized. The saponins were separated by liquid chromatography (LPC, Jobin Yvon Chromatospac 10, Paris, France) with a water—methanol gradient (methanol from 0 to 100%). Complete purification was achieved on silica columns using as a solvent a mixture of chloroform, methanol and water (60:40:10 for the three saponins of H.helix, 61:32:5 for the 10 saponins of C.officinalis and C.arvensis). Final identification of the saponins was performed by mass spectrometry (MS), FAB, 'H- and I3C-NMR, and two-dimensional NMR studies as previously described (Chemli et al, 1987; Babadjamian et al, 1988). The complete structures of the 13 saponins are described in Figure 1. Mutagenicity and antimulagenicity assays Tester strains. Tester strains of Salmonella typhimurium TA 97, TA 98, TA 100 and TA 102 were generous gifts from Professor B.N.Ames (Berkeley, CA, USA). The strains were stored at -80°C and regularly checked for their genetic markers (Maron and Ames, 1983). Activation mixture (S9 mix). Aroclor 1254 was administered i.p. to five male Sprague-Dawley rats (180-200 g body weight) al a dose of 500 mg/kg. The animals were killed on day 5 by cervical dislocation. Livers were collected under

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Thirteen saponins were isolated and identified from Calendula officinaUs, C.arvensis and Hedera helix. Mutagenic and antimutagenic activities of these products were investigated using a modified liquid incubation technique of the Salmonella/microsomal assay. The Salmonella tester strain TA98 ± S9 mix was used. Screening of the antimutagenic activity was performed with a known promutagen: benzo[a]pyrene (BaP) and a mutagenic urine concentrate from a smoker (SU). Antimutagenic activities were also compared with the activity of chlorophyllin. All the saponins were found to be non-toxic and non-mutagenic for doses of 400 /ig. Chlorophyllin inhibited the mutagenic activities of BaP (1 /*g) and SU (5 /J) in a dose-dependent manner. The four saponins from C.arvensis and the three saponins from H.helix snowed antimutagenic activity against BaP (1 /tg) and SU (5 jil) with a dose—response relationship. The possible mechanism of the antimutagenic activity of saponins is discussed.

The mutagenicity of some constituents of CSC has been evaluated (Florin et al., 1980). The actual metabolic products of SU are still mostly unknown. SU contains trace amounts of 2-aminonaphthalene OS-naphthylamine). However, the main compound is a metabolite of 2-aminonaphthalene: 2-amino-7naphthol (a bladder carcinogen). Some mutagenic metabolites may result from a multistep activation. Thus, SU may contain unique constituents (Connors et al., 1983). The screening of potential antimutagenic compounds could be performed on complex mixtures of biological origin. In the present study, mutagenic and antimutagenic activities of 13 saponins isolated from H.helix L. (common ivy), Calendula officinalis L. (garden marygold) and C.arvensis L. (field marygold) have been investigated using a microsuspension technique of the Salmonella/microsome assay (Ames test). The antimutagenic activity was assayed against a known mutant (BaP) and SU. In addition, antimutagenic activities were compared with the one of chlorophyllin.

R.Ellas et al. 30

XOR,

R1=R2=R3=H

R1=R2=H; R3=OH Plant Calendula arvensis

saponin nane A

o l e a n o l i c acid

hederagenin R2

R3

Gald—^-Glu-

du-

H

ll

H

—>3)-Glu-

B C

- Glu-

Glu-

- Glu->3)Calendula offlcinalis

SI —Glur-

Glu-

S2 —Glur-

Hedera helix

S3

Gal(l—>3)-Glur-

Glu-

H

34

Gal(l~>3)-Glur-

H

H

35

Glur-

Glu-

H

36

Glur-

H

H

aH

Rha»(l—>2)-Ara-

H

OH

BH

Rhaad—>2)-Ara-

H

H

6H

Ara-

H

OH

Glu= glucose; Gal= galactose; Glur= glucuronic acid; Rhaa= Rhannose; Ara= arabinose. Fig. 1. Structures of the saponins isolated from C.officinalis L., C.arvensis L. and H.helix L. sterile conditions. The S9 fraction was prepared as previously described (Ames et aL, 1975; Maron and Ames, 1983). The concentration of proteins in the S9 fraction was 21.0 mg/ml as determined by the technique of Lowry et al. (1951). The S9 mix was a mixture of 10% S9 and a solution of co-factors (NADPH generating system; Maron and Ames, 1983). Tenacity, nwtagenicity and antinwtagenicity assays. All samples were assayed for toxicity, mutagenicity and antimutagenicity by the Ames test (Maron and Ames, 1983). Preliminary screening was performed using the semi-quantitative technique: the Spot test as previously described (Maron and Ames, 1983) with the four tester strains ± S9 mix. The tested doses were 200 and 400 #