ANTI-BACTERIAL ACTIVITIES OF VOLVARIELLA VOLVACEA

: Extracts of twenty- one basidiomycctc fungi were tested against the bacteria, Staphylococcus aureus, Streptococcus. Pseuciornor~c~s mruginosa, Escherichia coli, and Klebsiella. Among them Volvariella oolvacea showed promising activity against E. coli. Ethyl acetatc extract of Voluariella uobacea wax fractiotiatcd to hexane and purified on normal phase silica gel column to obtain ergostcro; 1)croxide which showed comparable activity against E. coli as that of the crucic csxacc.


INTRODUCTION
Fungi belonging to the subdivision Basidiomycotina possess a great variety of fruit bodies having different shapes and co1ours.l Some of these basidiomycete fungi have been shown to have anti-bacterial, anti-fungal, anti-viral, anti-tumour, hypercholesterolaemic and hyperglycemic a c t i v i t i e~.~"~.~.~.~~o r k has also shown that gram-positive bacteria are more sensitive to the basidiomycete fungi than gramnegative specie^.^ Another important observation is that Micrococcus pyogenes, a penicillin resistant bacterium, is sensitive to the inhibitory action of a large number of basidiomycete~.~ A number of anti-microbial compounds have been isolated from basidiomycete fungi.gn They include illudin, collybial, melleolide B, 5-methoxy-p-toluquinone, linoleic acid, pleurotin, crinipellin A, drosophilin A and frustulosin all of which are antibacterial compounds. Furthermore about sixteen antifungal compounds have been isolated from basidiomy~etes.~ Thus basidiomycetes appear to be a good source for new anti-microbial compounds.
Several basidiomycete fungi isolated -from different localities in Sri Lanka were screened for anti-bacterial activity and among them Volvariella volvacea showed promising activity against E. coli. I? volvacea is an edible mushroom, belonging to the order Agaricales. The vitamin and mineral content of V: volvacea is comparable to other edible mushrooms.1u Bioassay guided fractionation was done to isolate and characterize the biologically active compounds from V: volvacea.

METHODS AND MATERIALS
Organisms: Fruiting bodies (21) of basidiomycetes were collected from different localities in Sri Lanka. The fungi were identified using the characters of fruiting bodies.ll Some were identified upto generic level while a few were identified only upto the order level.
Screening of basidiomycete specimens for antibacterial activity: Five grams of each fruit body (fresh weight) was crushed and soaked in 50% methanol (MeOH) in methylene chloride (CH2C12) for 3 days at room temperature. Thereafter the solvent was filtered and rotary-evaporated under vacuum. Stock solutions (10000 ppm in acetone) were prepared using these extracts.
The bacteria Staphylococcus aureus (NCTC 8532), Streptococcus, Pseudomonas aeruginosa (NCTC 10662), E.coli (NCTC 10148) and Klebsiella were inoculated to separate nutrient broths and incubated a t 37OC for 24 hours. Thereafter, broth of the test bacterium (0.1 ml).was evenly spread on a nutrient agar plate under sterile conditions.
The required stock solution (20~1) was absorbed onto sterile filter paper discs (7 mm) and allowed to dry for a few minutes in a sterile Petri dish. Each disc was placed a t the center of a nutrient agar plate, which was earlier inoculated with the appropriate bacterium. Filter paper discs having 2 0~1 of acetone were used as the control. The Petri dishes were incubated a t 37OC. After 24 hours the diameter of any clear inhibition zone around the discs was measured. All experiments were triplicated.
Antibacterial activity of Volvariella volvacea: Fruiting bodies of V: volvacea were collected from the Export Development Board Research Center, Ratmalana, Sri Lanka. V: volvacea (900 g) was crushed and soaked in ethyl acetate (EtOAc) for five days at room temperature. Thereafter, the solvent was filtered and the filtrate evaporated to dryness in a rotavapor to obtain 30 g of EtOAc extract. The EtOAc extract was re-extracted to hexane (50 mg) and purified on a normal phase silica gel column packed in hexane and eluted with a gradient of hexane and EtOAc to obtain lOml fractions which were numbered from 1-50.
Crystalline 46th fraction, which is active against E.coli (15 mg), eluted with 20% EtOAc in hexane was purified on a normal phase preparative silica gel TLC using 33% hexane in EtOAc as the solvent. Band a t R, = 0.3 was extracted to CH,Cl,,dichloro methane. Evaporation of CH2C1, gave a crystalline compound.
The residue was soaked in methyl alcohol (MeOH) for five days, filtered and rotary-evaporated to obtain 80g of MeOH extract. The MeOH extract was reextracted into hexane to remove the non-polar compounds. The remaining residue (79.6 g) was fractionated by passing through a column of Sephadex, and eluted with MeOH.
One fraction active against E. coli was chromatographed over a column of reverse phase (C,,) silica gel packed in acetonitrile and eluted with acetonitrile, acetone, MeOH, H,O gradient.
To facilitate the characterization, fractions were acetylated with lml pyridine and 1 ml acetic anhydride for 24 hours. Acetylated reaction mixture was dried by nitrogen flushing. The dried reaction mixture was dissolved in CH2Cl, and concentrated. The concentrated CH,Cl, extract (135mg) was chromatographed over a normal phase silica gel column packed in CH2C12 and eluted with CH,C12, EtOAc solvent gradient. Compounds 2,3,4,5,6 were isolated and characterized using spectroscopic data.

Antibqcterial activity of the basidiomycete extracts:
The first four specimens in figure 1 belongs to the order, Tullasnellales, the next 10 specimens belongs to the order Agaricales and the last 7 specimens belongs to the order Aphyllophorales. The extract of V: volvacea was the most active extract against Escherichia coli. Extract of Agaricus and Schizophyllum possessed significant antibacterial activity against Klebsiella. Pseudomonas aeruginosa was not significantly inhibited by any of the fungal extracts. Staphylococcus aureus was moderately inhibited by the extracts of Cantharellus, Clytocybe and two bracket fbngi. Streptococcus was sensitive t o the extracts ofAmanita.

Antibacterial activity of I? volvacea
Bio-assay guided fractionation of the ethyl acetate extract of V volvacea resulted in the isolation of compound 1, which sliowed promising activity against E.coli. Compound 1 was identified as ergosterol peroxide and'the assignments were made using lH NMR, 13C NMR (BB, DEPT), COSY, Hetcorr and HMQC.
Compound 2 was identified as a mixture of a and P -1, 2, 3, 4, 6pentaacetyl glucose. 13C NMR d a t a of t h e compound 2 a n d t h e known a a n d Pglucosepentaacetates were compared13 and the data found to have good correlation.

DISCUSSION
Extract of V volvacea had significant antibacterial activity, but the activity was confined to one bacterium, E. coli. The non-polar fraction of the extract of V volvacea was subjected to bio-assay guided fractionation and the most active fraction contained ergosterol peroxide. This is the first report on the isolation of ergosterol peroxide from V: volvacea and the activity against E. coli of ergosterol peroxide. Linoleic acid and some triglycerides were also isolated from the biologically active fractions.12 The polar fraction of the extract of V volvacea was also fractionated and towards the final stages in identification of the structures of active compounds, compounds were acetylated to facilitate the purification and structure elucidation.
It was noted that the most active fraction contains a mixture of sugars and nucleosides; a and 0-D-glucose, trehalose, uridine and adenosine. Moreover this fraction contains 3-deoxyascorbic acid. This compound is not available in the un-acetylated form to test against E. coli.
According to the available literature this is the first report on isolation of 3deoxyascorbic acid from a basidiomycete fungus.