ANTIFUNGAL ACTMTY OF FICUS RACEMOSA LEAF EXTRACT AND ISOLATION OF THE ACTIVE COMPOUND

: The 50% methylene chloride in hexane flash column fraction of the extract ofthe leaves ofFicus racenzosa was found to have antifungal activity. The extract inhibited the growth of several plant pathogens (Curvularia sp, Colletotriclzun~ gloeosporioides, Alternaria sp, Corynespora cassiicola and Fusariun sp). ~soralen was identified as the active compound and was shown to be biodegradable, having the potential to be developed as a fungicide against pathogens causing diseases on crops of economic importance. of tlze active coinpo7zent: Fifteen ml of potato dextrose liquid medium was introduced into 100 ml Erlenmeyer flasks and each flask was inoculated with a 0.5 cm2 agar square of Colletotriclzu~ngloeosporioides obtained from a 7-day old culture on PDA at room temperature. To each liquid culture medium, 20 mg of pure psoralen in 2 ml acetone was added immediately after inoculation. In the control experiments, the pure psoralen was incorporated into liquid medium without inoculating with the fungus. The absorbance of the culture filtrate was measured at 330nm. Biodegradatioil was estimated Gy calculating the percentage decreasein absorbanceL0 a t 33Onm assuming that the product has no significant absorbance. The percen.tage decrease in absorbance a t 330nm was


INTRODUCTION
Tropical forest plant species have served as a source of medicine for the people of the tropics for millennia. Sri Lanka with its great diversity of flora and fauna, has many plants of medicinal value.'-"Discovery of metabolites showiilg biological activity has led to research programmes aimed a t the isolation and characterization of biologically. active metabolites from Sri Lankan plants. Many extracts of local plants have been screened for antifungal activity."--11 Ficz~s racernosa which belongs to the family Moraceae, is a n evergreen tree 18-20 m high, with glabrous, pubescent or scaberulous shoots. I t flowers in November and the flowers are unisexual. The tree is distributed in India, Burma and Sri Lanka. I n Sri Lanka it is common in the banks and streams i n the moist low country upto a n altitude of 610 m. The fruit is reddish and about 3.5 cm long4 Many uses of the fruit, root and bark in traditional medicine are r e p~r t e d :~ The powdered leaves are used for bilious ailments.
There are no previous studies on the antifungal activity of the leaf extracts of F. racemosa. We report here the effect of the 1:l methylene chloride : hexane flash column fraction of the leaf extract of F. racemosa on several fungal pathogens causing diseases in crops of economic importance. The active compound was isolated and its biodegradation was also investigated.

METHODS AND MATERIALS
Preparation of the leaf extract: Ficus racemosa leaves (1.3 kg; collected from the University of Colombo) were washed under running water and air dried, cut into small pieces, ground in a laboratory mill and kept immersed in methanol (51) for 2 weeks. The methanol extract was filtered through cotton wool and solvent removed under reduced pressure a t 40-45OC to yield a crude extract ( 165 g). The , crude extract was subject to flash chromatography using G-6 silica gel and solvent systems of increasing polarity(hexane, methylene chloride in hexane, methanol in methylene chloride). These fractions were concentrated under reduced pressure at 40-45OC using a rotary evaporator and tested for antifungal activity by Cladosporium thin layer chromatography (tlc) bioassay using Cladosporium cladosporioides as the test f~n g u s .~W l l solvents were distilled before use.
Cladosporium tlc-bioassay: Test solutions were spotted on tlc plates (Aldrich, -1 tlc grade silica pre-coated to a thickness of 0.5mm on 20x20 cm glass plates). Six sets of plates were separately developed in a solvent system of 1:l methylene chloride : hexane in solvent tanks equilibrated with eluant. After air drying at ambient temperature for 24 hours, the plates were sprayed with a conidia suspension of Cladosporiuln cladosporioides and incubated as described by Smith. l2 The 50% methylene chloride in hexane flash column fraction (containing about 5.3g of compound) showed antifungal activity against Cladosporiz~m cladosporioides and was used to study antifungal activity against other plant pathogens.
Agarplate assay: Fifteen ml portions of sterile molten PDA were cooled to 45°C and were mixed with volumes oftest solution (20 mg/ml oftest sample in acetone) so that the final concentration of the test compounds were either 0.01%, 0.02% or 0.05% by weight, and then poured into sterile petri dishes. A 0.5 cm2 agar square obtained from a periphery of a 7d old fungal culture growing on PDA at room temperature was placed a t the centre of the medium. Three plates were used per test fungus. In the control experiments the medium was preparedusing only the corresponding volume of acetone. All plates were incubated at room temperature and the diameter of the colony was measured as described in Senaratna et a1. 13 at 24 h intervals for 7d; growth on day 4 was used for the calculations. The experiments were carried out in triplicate.

Ficus racemosa Leaf Exract
The percentage inhibition was calculated as follows: Growth area in reference -Growth area in sample % Inhibition = x 100 Growth area in reference Isolation of the active comnpoz~nd: The 1:l methylene chloride : hexane flash column fraction was concentrated under vacuum (40-45°C) and column chromatographed on silica gel (Aldrich 70-230 mesh) column, eluting with mixtures of methylene chloride in hexane of increasing polarity. Fractions were collected, pooled together after tlc monitoring and these combined fractions were subjected to Cladosporiz~m bioassay. The fractions that showed significant activity and were almost pure with respect to tlc analysis were combined together and the solvent evaporated under vacuum (40-45OC). The crystalline compound obtained was further purified by recrystallisation with metllano1 in methylene chloride. The structure was elucidated by spectroscopic methods.
Densitolnetric stz~dies o f tlze crz~de extract used for agar plate assays: 6.0 mg active compound and 47.0 mg of crude sample were used in this study. Th.e active compound (6.0 mg) was dissolved in 10.0 ml of CHCI,. Crude sample (47.0 mg) was dissolved in 5.0 ml of CHC1,. These samples were applied on to a tlc plate (0.5 mm x 20 cm x 10 cm Merck precoated with silica gel GF254) and the plate was developed with ethyl acetate : hexane 2:l to give a R, = 0.57 for the pure compound. lop1 of the crude sample ( i n triplicate ) and 6~~1,8pl,lOpl, 12yl of pure compound (each in triplicate) was spotted on a tlc plate and. scanned by a densitometer (hex = 365 nm and h ,,,,,,, = 480 nm). A calibration curve of volume (111) vs peak area was obtained. This was used to calculate the conceiltration of the active compoun.d in the crude extracts.
Degradation of tlze active coinpo7zent: Fifteen ml of potato dextrose liquid medium was introduced into 100 ml Erlenmeyer flasks and each flask was inoculated with a 0.5 cm2 agar square of Colletotriclzu~ngloeosporioides obtained from a 7-day old culture on PDA a t room temperature. To each liquid culture medium, 20 mg of pure psoralen in 2 ml acetone was added immediately after inoculation. I n the control experiments, the pure psoralen was incorporated into liquid medium without inoculating with the fungus. The absorbance of the culture filtrate was measured a t 330nm. Biodegradatioil was estimated Gy calculating the percentage decreasein absorbanceL0 a t 33Onm assuming that the degradation product has no significant absorbance. The percen.tage decrease in absorbance a t 330nm was calculated as follows: Initial absorbance -Absorbailce after harvest Percentage decrease = x 100 in absorbance Initial absorbailce

S.A. Deraniyagala et nl.
Spectroscopic analysis: N M R was recorded in CDC1, solution using a Brucker AC 200 MHz spectrometer. Mass spectra was recorded using a Hewlett Packard 5989A GC-MS spectrometer operatinginthe EI mode. IR spectrum was recorded in a JASCO 5300 FT-IR spectrometer. UV absorbance was measured in 1 cm cells using a JASCO V560 UVNisible spectrometer. The Densitometer was a Shimadzu CS 9000 Dual-wavelength scanner.

Antifungal activity of tlze leaf extract:
The preliminary screening of the flash col.umn fractions for antifungal activity was done by tlc plate bioassay method using Cladosporiz~m cladosporioides. Two of the flash columi~ fi-actions (1:l methylene chloride : hexane and 4:l hexane : methylene chloride) showed inhibitory zones i n the bioassay. The I: 1 methylene chloride : hexane fraction was chosen for the study ofthe inhibitory action against plant pathogens because of its higher inhibition and greater weight of residue (5.3 g). The fraction inhibited the growth of all fungi examined. The % illhibition of' each fungus by 1: 1 methylene chloride: hexane fraction of F. raceinosa leaf' extract obtained using agar plate bioassay is given in Table 1. The highest inhibition was against Curvz~laria sp. The inhibitory activity of the test samples increased with. increase in concentration except in C. cassiicolu. Isolation of tlze active compound: The active compound that was separated by chromatography on a silica gel columll and purified by recrystallisation with methanol in methylene chloride was identified as psoralen (1)  Densitometry study: Densitometric study on the crude extract used for the bioassay showed that the 0.01%, 0.02% and 0.05% concentrations contained 93, 186.4 and 466.0 pg respectively of active compound.

Degradation of the active compound:
The results in Table 2 show a decrease in absorbance a t 330 nm when incubated with the fungus Colletotriclzum gloeosporioides. The decrease in absorbance a t 330 nmindicated the degradation of the active compound psoralen. In the control experiment, without the fungus a decrease in absorbance was not observed.

DISCUSSION
Isolation and characterization of secondary metabolites of plants whi.ch have inhibitory activity against fungal plant pathogens, would enable the development of inexpensive fungicides based on locally available natural products. The 1:l methylene chloride: hexane flash colun~n fraction of the leaf extract of F. racemosa had inhibitory activity against several plant pathogens examined.
The active compound was identified as psoralen (1). Coumarins such as pso1areli.s and xanthotoxins are of mediciilal interest in the control of 1eucodermia.l" Psoralens are powerful phototoxic agents in animals and humans. Psoralen containing pulp of fruits and vegetables can be applied to the slrin directly or in the form of cosmetics. It can also be taken orally by ingestion, of certain. common fruits and vegetables or by the use of certain psoralen containing d.rug formulations. Psoralen and its derivatives such as 8-methoxy psoralen are increasingly used in photochemotherapy for management of disorders such as vitiligo, psoriasis and mycons fungaids.'" Psoralen has been isolated from the leaves, stem bark7 and U~I-ipe fruit shell%f Limonia acidissima and has been reported to inhibit the growth of Aspergillus niger, Colletotrichumgloeosporioides, Cvrvularia sp., and Penicilliz~m sp.I Here we have isolated psoralen from the leaves of F. racemosa and shown it to be active against several fungal pathogens (Cz~rvularia sp, Colletotrichr~ln gloeosporioides, Alternaria sp, Coryizespora cassiicola and Fz~sariz~nz sp.) of plants which cause important diseases in major crops of Sri Lanka. Psoralcn underwent biodegradation when incubated with C.gloeosporioides in liquid culture. The decrease in absorbance at 330 nm 8000 ) shows that the degradation occurs in the psoralen nucleus. In conclusion, we show that psoralen which has antifungal activity against several plant patl~ogens eausing diseases in crops of economic importance to Sri Lanka, could be isolated from the leaves ofF. racemosa. The pathogenic fungus C. gloeosporioides was shown to degrade psoralen. Therefore it has the potential to be developed as an effective fungicide.