ANTIFUNGAL ACTIVITY OF SOME MEDICINAL PLANTS OF SRI LANKA

Thirty six medicinal plants (63 extracts) have been screened for antifungal activity against Cladosporium cladosporioides. Extracts of the following plants have displayed significant antifungal activity: Butea monosperma, Costus speclosus, Curcuma zedoaria, Eupatorium riparium, Pleiospennium alatum. Theobroma cacao i and Z~ngrber zerumbet. A preformed antifungal constituent of C. speciosus has been identified as methyl 3-(4-hydroxypheny1)-2 (E)-propenoate (1). The andfungal activity of this compound has been evaluated against Aspergillus niger, G. cladosporioides, Collerotrichum gloeosporioides, Curvularia sp. and Penicillium sp. 1.. Introduction Development of specific fungicides for use in the treatment of plant diseases is important. Plants are known t o contain antifungal compounds9, and' are a potentially useful source of these compounds. .The present study describes the investigation of 63 plant extracts derived from 36 medicinal plants of Sri Lanka. Isolation of an active constituent from the rhizome of Costus s p e c i o s u s 6 , a potential source for the diosgenin--derived steroidal drugs7 is also described. 2.1 Plant Material Plant materials used in this study, were mature (reproductive maturity) specimens collected (1-5 kg) from different localities of Sri Lanka specialIy from Central Province (Table 1). All plant varieties collected were true species. Plant specimens were identified by comparison at the National Herbarium, Royal Botanic Gardens, Peradeniya. The specimens were immediately "washed in running water tb remove contaminated soil etc. and cut into small pieces about 3-6 cm in length. The specimens were immediately airdxied and powdered in a laboratory mill. 2 B. M. R. Bandara et al. 2.2 Preparation of Plant Extracts. The air--dried plant materials (100 g) were extracted successively with (500 ml) hot hexanellight petroleum (40-60°C) dichloromethane, ethyl acetate and methanol, in a Soxhlet apparatus, or extracted directly with cold ethyl acetate and cold methanol in a bottle shaker, for a period of 48 hrs. . The solubles, were concentrated t o dryness separately using a rotavapor (below 45OC). The extracts were subjected to Cladosporium TLC-bioassay for antifungal screening as described below. 2.3 Cladosporium TLC-Bioassay Extracts (2 mg) were spotted on tlc plates (silica gel 60 PF254-366' 0.50 mm x 20 cm x 20 cm) and the plates were developed in dichloromethane. After air-drying overnight the plates were sprayed with a suspension of conidia of Cladosporium cladosporioides in Czapek-DOX nutrient solution. Plates were then incubated in a moist chamber at 25 + 2 ' ~ for 48 h.3 Inhibition areas appeared white against a background of green mycelia. The diameter of zones in which the growth was inhibited, which were approximately circular, was measured (in mm). The extracts which showed inhibition are given in the Table 1, with the Rf value and the diameter of the zone of inhibition. Benlate (0.2 mg, in methanol, 50% active ingredient, methyl-l-(butylcarbonyl)-2-benzimidazolecarbamate. Du Pont, USA) was spotted on each tlc plate as the standard fungicide.


1.. Introduction
Development of specific fungicides for use in the treatment of plant diseases is important.
Plants are known t o contain antifungal compounds9, and' are a potentially useful source of these compounds..The present study describes the investigation of 63 plant extracts derived from 36 medicinal plants of Sri Lanka.Isolation of an active constituent from the rhizome of Costus s p e c i o s u s 6 , a potential source for the diosgenin--derived steroidal drugs7 is also described.

Plant Material
Plant materials used in this study, were mature (reproductive maturity) specimens collected (1-5 kg) from different localities of Sri Lanka specialIy from Central Province (Table 1).All plant varieties collected were true species.Plant specimens were identified by comparison at the National Herbarium, Royal Botanic Gardens, Peradeniya.The specimens were immediately "washed in running water tb remove contaminated soil etc. and cut into small pieces about 3-6 cm in length.The specimens were immediately airdxied and powdered in a laboratory mill.

Preparation of Plant Extracts.
The air--dried plant materials (100 g) were extracted successively with (500 ml) hot hexanellight petroleum (40-60°C) dichloromethane, ethyl acetate and methanol, in a Soxhlet apparatus, or extracted directly with cold ethyl acetate and cold methanol in a bottle shaker, for a period of 48 hrs.
. The solubles, were concentrated t o dryness separately using a rotavapor (below 45OC).The extracts were subjected t o Cladosporium TLC-bioassay for antifungal screening as described below.

Cladosporium TLC-Bioassay
Extracts (2 mg) were spotted on tlc plates (silica gel 60 PF254-366' 0.50 mm x 20 cm x 20 cm) and the plates were developed in dichloromethane.After air-drying overnight the plates were sprayed with a suspension of conidia of Cladosporium cladosporioides in Czapek-DOX nutrient solution.Plates were then incubated in a moist chamber at 25 + 2 ' ~ for 48 h.3 Inhibition areas appeared white against a background of green mycelia.The diameter of zones in which the growth was inhibited, which were approximately circular, was measured (in mm).The extracts which showed inhibition are given in the   with the Rf value and the diameter of the zone of inhibition.Benlate (0.2 mg, in methanol, 50% active ingredient, methyl-l-(butylcarbonyl)-2-benzimidazolecarbamate.Du Pont, USA) was spotted on each tlc plate as the standard fungicide.
. in a suspension of conidia (50 x 10' conidia per ml) of Gloeosporium mangiferae, a non-pathogenic fungus on C. speciosus, and another 10 g were placed in sterile distilled water.Two days after incubation at 26OC the tissue discs were taken out and extracted with hot methanol under the same conditions.Each extract (4 mg).wasrtested for antifungal activity using the TLC-bioassay technique.Both the extracts produced inhibition areas of similar diameter at Rf 0.28 -0.29.1).x Penicillium sp.

Biological screening of the Extracts'
L Most of the plant~~listed in Table 1 are used in ethnomedical preparations.Some plants have been selected for the present study on the basis that they are widely distributed and apparently free of pest attack.Eupatorium and w Strobilanthes species, for example, are found abundantly in the hill country.Sometimes selection has been made on the basis that useful compounds and/or biological properties have been reported from th.e p l y t previously and information on the antifungal properties would enhance the industrial or medicinal utility of the species.Costus speciosus has been, for example, reported t o possess antifertility1 3., estrogenic' *' and anti-inflammatory5 properties and has been identified as a source of diosgenin.4 3 7 . 8 3 1 1 the 36 plants (  The low polar'.antifungalcompound in the extracts of .B. monosperma has been identified as (-)Tmedicarpin ( z ) .~ m he active constituent in the hexane extract of P. alatum root bark has been obtained by chromatographic fractionation and characterized as seselin (3.

.Active constituent of Costus speciosus
Considering the economic potential of C. speciosus as a source of diosgenin and its medicinal value, bioassay-directed fractionation of the .rhizomeextract was carried out in order t o isolate'the antifungal constituent.
A part of the methanol extract was chromatographed over silica gel.The column fraction eluted with 60% chloroform in petroleum inhibited the growth of C. cladosporioides.Further purification of this fraction by column chromatography on silica gel yielded a white crystakline solid, m.p. 133-1 3 4 ' ~ which was found to be responsible for the antifungal" activity.The IR absorption bands of this compound at 3425, 1685 and 1610.cm-'corres- ponded to -OH, C=O and C=C groups, respectively.The UV 1 ; ., at 305 h m J suggested the presence of an cw,P punsaturated carboxyl system with extended conjugation.The D,O exchangeable proton at 6 8.33 in the H NMR , spectrum confirmed the presence of a hydroxy group in the molecule.The doublets at 6 7.43 and 6.87 (J=8 Hz) corresponded to two ortho coupled protons.Two protons of a trans olefinic double bond was indicated by the doublets at 6 7.63 and 6.27 (J=16 Hz).The singlet at 6 3.77 was assignable to a methoxy group.The molecular ion at g / g 178 in the mass spectrum was consistent with a formula of CIoH1 ,,03.The mass peaks at m_/g 147 and 119 corresponded M--OCH3 and M-C0,CH3 fragments.The compound was thus identified as methyl 3-(4-hydroxypheny1)-2-(E)-propenoate

(1).
The possibility that this compound (1) was an artifact formed by transesterrification of a labile natural product during the methanol extraction was eliminated on the following grounds.The rhizome of C. speciosus was separately extracted with methanol, ethanol and dicbloromethane under reflux conditions.All three extracts produced an inhibition zone at the same Rf value (0.30 e1uant:dichloromethane) in the Cladosporium TLC-plate.The three extracts were subjected to repeated TLC separation, and the bioactive fraction compared with an authentic sample of compound (1).
The results showed that all three extracts contained the active compound(L).
The fresh rhizome was inoculated with a fungus Gloeosporium mangiferae and incubated at 2 8 ' ~ over a period of 2 days.Equal amounts of the inoculated rhizome and the untreated rhizome were separately extracted with hot methanol under identical conditions.Equal amounts of each extract produced inhibition zones of similar area in the Cladosporium TLC-plate suggesting that the inoculation had not increased the amount of the active compound.This indicated that the ester (I) was a preformed antifungal compound and not a phytoalexin.

Figure 1 .
Figure 1.Inhibition of fungal growth by the ester1 and Benlate

Figure - 1 .
Figure -1.The ester 1 inhibited the growth of all the fungi tested.Both the ilatural compound l a n d Benlate displayed comparable activities against the saprophytic soil fungus Curvularia sp. and the plant pathogenic fungus C. gloeosporioides.

niger, Cladosporium cladosporioides, Colletotrichum gloeosportoides, Curvularia sp. and Penicillium sp.
25 and 10 p1 in each plate using micropipettes.The undeveloped plates were a i d r i e d overnight.Each plate was sprayed with one of the above fungi in Czapek-Dox nutrient solution and kept in a moist chamber at 2 8 -+ 2 ' ~ for 48 hrs.The diameters of the inhibition zones were measured (Figure Compound(1)Against Aspergillus