Antiviral activities of medicinal plants of southern Nepal

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Journal of

ETHNOPHARMACOLOG~ ELSEVIER

Journal of Ethnopharmacology 53 (1996) 105-110

Investigation of antifungal and analgesic activities of extracts from Sium nodiflorum M. Larhsini a, H.B. L a z r e k *a, H. A m a r o u c h b, M. J a n a a aLaboratory of Phytochemistry, Department of Biology, Faculty of Sciences-Semlalia, P.O.B:SI5, Marrakesh, Morocco bLaboratory of Microbiology, Faculty of Sciences Ain Choc, Route d'El Jadida, Casablanca, Morocco Received 23 September 1995; revised 13 May 1996; accepted 13 May 1996

Abstract

Different extracts of aerial parts of Sium nodiflorum were examined for their antifungal activity against two groups of fungi: the growth of both yeast and mold was significantly inhibited. Analgesic activity study of these extracts was also carried out but showed no significant effect in mice on the writhing induced by acetic acid. Keywords: Antifungal activity; Analgesic; Sium nodiflorum; Apiaceae; Yeast; Mold

1. Introduction

In recent years, interest in new, safer and more effective antifungal agents has grown with the increasing incidence of fungal infections. In the field of natural products, the antimycotic activity of higher plants remains largely unexplored compared with that of microorganisms (Debono and Gordee, 1990). The systematic search in higher plants for antifungal activity has shown that plant extracts have the ability to inhibit spore germination in many fungal species (Guerin and Reveillere, 1984, 1985). Several compounds with antifungal activity have been isolated which are grouped by Stoessl (1970) according to their chemical family. In order to verify the biological properties of * Corresponding author.

Moroccan medicinal plants and within a valorization programme of natural regional resources, we have been interested to study Sium nodiflorum L. (Apiaceae): a plant widely used in Moroccan folkmedicine against abscess, stomatitis, gumboil and mouth ulcer (Jana, 1996). In a previous study, we have shown an antibacterial effect of extracts from this plant (Jana et al., 1992). The purpose of the present study was to examine the effects of Sium nodiflorum extracts on the growth of some pathogenic fungi and on analgesic activity. 2. Material and methods

2.1. Plant material Slum nodiflorum L. (syn. Helosciadium nodiflorum Koch) (Apiaceae) is a herbaceous plant

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widely distributed in ditches and streams (Jahandiez and Maire, 1932). The aerial parts were collected from Marrakesh in June 1988 and the identity of the plant was confirmed in the botany laboratory of the Faculty of Sciences, Semlalia, Marrakesh. A voucher specimen (MARK 1298) is deposited in the herbarium of the Faculty of Sciences, Semla!ia, Marrakesh.

2.2. Preparation of extracts The dried powdered aerial parts (800 g) of the plant were extracted first with anhydrous chloroform to remove fats. The defatted powder was extracted in Soxhlet with ethanol (70%) and ethanol-water (V/V). The hydroalcoholic solution was concentrated to dryness under vacum. The residue obtained was dissolved in hot water and kept at +4°C for 24 h. Then, the aqueous solution was filtered and extracted with solvents of increasing polarity as described by Netien and Lebretron (1964). Three extracts were obtained: ethyl ether extract (E.E) = 2.8 g, ethyl acetate extract (E.AC) = 3.36 g, butanol extract (E.But) = 4.5 g. The n-butanol residue (4.5 g) was adsorbed on 10 g of silica gel (230-400 mesh) and chromatographed over 60 g of the same adsorbent: eight fractions have been collected using a gradient of CH2CI2-MeOH. All these fractions were tested for their antifungal activity.

2.3. In vitro experiments The fungal species used in this study were the following: Candida albicans and Candida AFNOR (ATCC 2094) (26 strains), Candida parapsilosis (8 strains), Candida tropicalis (8 strains), Candida krusei (8 strains), Torulopsis glabrata (8 strains), Aspergillus fumigatus (8 strains), Aspergillus niger (8 strains), Aspergillus flavus (8 strains), and Scopurialopsis brevicaulis (8 strains) originally isolated from the Hospital center of Toulouse Rangueil (France). All cultures were maintained on Sabouraud glucose agar medium. The strains were cultivated on this medimn for about 5-8 days at 37°C until they were well sporulated and then

the spores were suspended in sterile distilled water and adjusted to give a final spore concentration of approximately 106 spores/ml for mold and 107 blastospores/ml for yeast. Minimum inhibitory concentration (MIC) was determined by the agar dilution method. Two media were used comparatively for this sensitivity test: yeast nitrogen base glucose 2% (YNBG) and Casitone (CAS). These media were sterilized at 110°C for 30 min. Plant extracts were solubilized in 5% DMSO and passed through a membrane filter (pore size 0.2/Lm). The extracts were assayed at 128, 64, 32, 16, 8, 4 and 2/~g/mi. Spore inoculum was spread over the surface of each plate with an alcohol flamed glass rod. Fungal growth was evaluated after incubation for 48 h at 36°C and compared with an appropriate control receiving a similar volume of 5% DMSO. Each test culture was compared with an uncontrolled culture, which showed uninhibited growth of the inoculated fungus.

2.4. In vivo experiments 2.4.1. Animals Male albino Swiss mice (18-22 g) were deprived of food for 14 h before the experiments. Experimental animals were housed in groups of 6 in plastic cages and the experiments were carried out at a laboratory temperature of 20-24°C.

2.4.2. Analgesic activity The test extracts and reference drug (acetylsalicylic acid) were administrated orally to mice caged at a constant temperature of 22-24°C. After 1 h, 0.6% acetic acid was injected intraperitoneally (15 ml/kg) (Bentley et al., 1983). Nociception was evaluated by counting the number of abdominal constrictions 15 min after acetic acid injection for a period of 5 min.

2.4.3. Acute toxicity The plant extracts were tested for their toxicity in mice when given by gavage (10 ml/kg). The mice were fed ad libitum during the 7 day trial and observed for 7 days for toxic effects.

M. Larhsini et al. / Journal of Ethnopharmacology 53 (1996) 105-110

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Table 1 Minimum inhibitory concentration (MIC) ~g/ml) affecting the growth of yeast group Extracts tested

Culture medium

Candida albicans

Candida prapsilosis

Candida tropicalis

Candida krusei

Torulopsis glabrata

Ethyl ether

YNBG CAS YNBG CAS YNBG CAS

27 38 26 29 22 46

47 72 50 98 44 76

74 86 54 78 > 128 >128

38 48 41 53 24 42

39 46 48 59 54 60

Ethyl acetate n-Butanol

MIC is an average of 8 strains.

nodiflorum extracts were more effective against Candida than against Aspergillus. The n-butanol extract inhibited growth of C. albicans and C. krusei, respectively at 22 and 24 #g/ml in the

3. Results

3.1. Antifungal activity The antifungal activity of the plant extracts on spore germination in this investigation revealed that the effects varied among the three extracts and the nine fungal species tested. In general, Sium

YNBG medium (Table 1), but showed no inhibitory effect on germination of Aspergillus strains (Table 2). The ethyl ether and ethyl acetate extracts also

Table 2 Minimum inhibitory concentration (MIC) ~g/ml) affecting the growth of mold group Extracts tested

Culture medium

Eth~ ether

YNBG CAS YNBG CAS YNBG CAS

Ethyl acetate n-Butanol

Aspergillus fumigatus 53 56 72 102 >128 >128

.4spergillus niger

Aspergillus flavus

Scopurialopsis brevicaulis

72 82 > 128 >128 > 128 >128

48 54 > 128 >128 > 128 >128

78 94 73 92 >128 >128

MIC is an average of 8 strains. Table 3 Minimum inhibitory concentration (MIC) (#g/ml) obtained for n-butanol fractions on Candida species Fractions tested

Candida albicans

Candida parapsilosis

Candida tropicalis

Candida krusei

Torulopsis glabrata

F1 F2 F3 F4 F5 F6 F7 F8

24 36 20 21 25 54 76 78

24 29 36 28 29 54 74 68

48 44 55 48 46 >128 92 86

28 33 22 24 28 58 62 69

26 36 38 44 34 68 72 69

MIC is an average of 8 strains.

M. Larhsiniet al./ Journal of Ethnopharmacology53 (1996) 105-110

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Table 4 Effects of oral administration of extracts on abdominal writhing induced in mice by i.p. injectionof acetic acid Treatment

Dose N (mg/kg)

Vehicle control -Ethyl ether extract 100 Ethyl acetate extract 100 n-Butanol extract 100 Acetyi salicylicacid 100

Mean ± InhibiS.E.M. tion (%) abdominal constrictions in 5 rain (n)

6 6 6 6

6

30.6 ± 4.8 13.3 ± 6.1 17.1 ± 2.0 14.3 ± 3A 8.0 ± 4.0

-57 44 53 74*

Significant from control: *P < 0.001. were active inhibitors of growth of C albicans, with MIC of 27 and 26/~g/mi, respectively. Among the yeast group, C albicans appears to be the most sensitive. In contrast, C tropicalis had growth inhibition responses to Slum nodiflorum extracts only at high concentrations. The ethyl ether extract was the most active on the growth of Aspergellus species (Table 2), and was the only extract showing inhibitory activity for all species tested. A. niger and A. flavus were only inhibited by the ethyl ether extract. The n-butanol extract effective on Candida afforded, after silica gel column chromatography, eight fractions. The F3 and F4 fractions were the most active on Candida albicans, with MIC values of 20 and 21 #g/mi (Table 3). F3 was also very active on Candida krusei at a low MIC (MIC = 22 mg/ml). Table 5 Acute toxicityof Slum nodiflorum aerial parts extracts Oral dose (g/kg) Quantal incidence of symptoms

Quantalincidence of mortality

E.E. E.Ac. E.B.

E.E. E.Ac. E.B.

0/6 0/6 0/6 0/6 1/6 2/6

0/6 0/6 0/6 0/6 0/6 1/6

Vehiclecontrol (10 ml/kg) 1

2 3 4 5

0/6 0/6 0/6 0/6 0/6 0/6

0/6 0/6 0/6 0/6 0/6 0/6

0/6 0/6 0/6 0/6 0/6 0/6

0/6 0/6 0/6 0/6 0/6 0/6

3.2. Analgesic activity Administration of ethyl ether, ethyl acetate, and n-butanol extracts (100 m~/kg) to mice elicited a weak but not significant inhibitory effect on writhing induced by injection of 0.6% acetic acid. In contrast, treatment with acetyl salicylic acid (100 mg/kg) significantly inhibited the writhing induced by acetic acid (Table 4).

3.3. Acute toxicity From Table 5 it is evident that the plant extracts were well tolerated orally in mice up to a dose of 5 g/kg with no mortality or serious side effects except for the ethyl ether extract, which caused 16% mortality at this dose. All mice died from mixed convulsions within 48 h post-administration. 4. Discussion The antifungal properties of the aerial pazts of Slum nodiflorum are well evidenced from the results obtained here. The growth response to ethyl ether and ethyl acetate extracts suggests that these extracts have a broad spectrum of antifungal activity. The aqueous solubility of the ethyl ether extract is very low, and therefore the lipophilicity of this extract may play an important role in membrane permeability and thus affect the organisms. The ethyl ether and ethyl acetate extracts consist mostly of phenolics and flavonoids, so the activity observed is probably due to these compounds. The literature reports that, in the Umbelliferae, flavonoids and coumarins are characteristic chemical constituents (Heywood, 1971; Hegnauer, 1973). Phenolics from other vegetable sources have been found to be inhibitory toward the growth of fungi (Swaminathan and Koeler, 1976; Tomas-Barberan et al., 1988). The n-butanol extract has no activity on Aspergillus strains but has strong activity against Candida species, especially on Candida albicans, one of the most important fungi responsible for human systemic infections (Fukuzawa and Kagawa, 1985). According to Guerin and Reveillere (1985), an inhibitory effect on spore germination of C. albicans and A. niger is an important criterion in deter-

M. Larhsini et al./ Journal of Ethnopharmacology 53 (1996) 105-110

mining the antifungal activity of a compound. In our case, the ethyl ether extract is the only extract which has a simultaneous effect on both these organisms. The activity of one extract varied within a genus, for instance, the ethyl acetate extract exhibited antifungal activity against A. fumigatus but no activity on the growth of A. niger and A. flavus. Results in Table 1 and 2 indicate that the different extracts have varied inhibitory potencies; this suggests that different compounds with inhibitory activity may be isolated from these extracts. The literature reports several naturally occurring compounds which exhibit antifungal activity such as flavonoids (Tomas-Barberan et al., 1988), saponosides (Bouchet et al., 1986; Favel et al., 1994) and essential oils (Gupta, 1976; Youssef and Tawil, 1980; Thompson, 1986). The n-butanol extract was particularly effective on growth of Candida species and afforded after silica gel column chromatography eight fractions. Fractions 3 and 4 were very active on C. albicans (Table 3). We suppose that the activity was due to heterosides. The MIC values obtained were consistently higher for isolates growing on Casitone medium than on YNBG, thus the culture medium is an important factor to consider for MIC determination. Several environmental factors, of which the most frequently cited are inoculum size, duration of incubation and medium composition (Block et al., 1973; Brass et al., 1979; Cook et al., 1990), are known to influence the outcome of susceptibility tests in vitro. The analgesic experimental results show that S. nodiflorum aerial parts extracts administered at an oral dose of 100 mg/kg in mice treated i.p. with 0.6% acetic acid were less effective when compared with 100 mg/kg acetyl salicylic acid (74%). The plant extracts were well tolerated up to a dose of 5 g/kg when given orally to mice.

5. Conclusion The present study tends to confirm the folk medicine indications of S. nodiflorum aerial parts as being safe and useful in the treatment of mouth ulcer and stomatitis (buccal candidiasis). Identification of the individual chemicals from these extracts and the confirmation of their activity are now under investigation.

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Acknowledgements Authors wish to thank Prof. A. Moulay, head of Military Hospital, Marrakesh, and members of Microbiology Laboratory, Military Hospital of Marrakesh, for partial technical assistance and Dr. A. Ouhammou, Laboratory of Plant Ecology, Faculty of Sciences Semlalia, Marrakesh, for authenticating the plant material.

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