Indian Journal of Experimental Biology Vol. 40, March 2002, pp. 3 14-3 1 8

Fungistatic activity of Semecarpus anacardium Linn. f nut extract

Kanika Sharma, Sunil Dutt ShukJa*, Pooja Mehta & Maheep Bhatnagar*

Department of Botany and Zoology * , M.L.Sukhadia University, Udaipur, 3 1 3001 , India Received 22 September 2000; revised 7 November 2001

Alcoholic extract of dry nuts of S.anacardium showed dose dependent antifungal activity in vitro against Aspergillus Jumigatus and Candida albicans. At 400 mg/ml concentration, growth of both the fungi was inhibited and considerable re­duction in size of cells and hyphae was observed. Sporulation also decreased.

Semecarpus anacardium L.f. , a deciduous tree of family Anacardiaceae is found in Sub-Himalayan re­gions and hotter parts of Indial .The fruit, commonly called marking nut or Bhallataka, is also known as Dhobi nut as its liquid is used to mark laundry. The nut extract is used frequently as home remedy for in­flammation, arthritis, warts and rheumatism2• Its anti­cancer, immuno-modulatory and antibacterial activity has also been reported 3.5.

Not much work has been done on antifungal activ­ity of the S.anacardium nut extract. In this view, a study on the effect of nut extract on two common fungal pathogens viz. Aspergillus and Candida was planned. Aspergillus, commonly found in soil, air and water is pathogenic to humans. It causes Aspergillo­ses-a disease of lungs which is quite common in patients with malignancies specially leukemia and lymphoma and in patients who receive immuno­supressants.Out of eight species of Aspergillus, Afumigatus accounts for most infections6.Candida albicans causes Candidiasis, skin and nail infection. Oral candidiasis is very common in new borns, debili­tated and aged patients. Other diseases, physiological disorders, use of broad-spectrum antibiotics for a long duration creates conditions under which C.albicans is converted into a pathogen6. It is also common in housewives, fruit canners, diabetics and people who are required to immerse their hands in water fre­quently for long durations. Pulmonary candidiasis leads to low grade fever, coughing, mucoid and blood streaked sputum, pleurisy and in severe cases pneu­moma.

Correspondent author : Telephone : 0294-441 250 E-mail: mbhalnagar@yahoo.com

An upsurge in number of patients succumbing to fungal infections has resulted in demand for new herbal antifungal compounds. Present study reports the fungistatic activity of S.anacardium nut extract against Afumigatus and C.albicans.

Material and Methods The antifongal activity of crude alcoholic extract in

vitro was determined using tissue method of biologi­cal assay technique'. Extract preparation was done following modified method of Shadomy and Ingrofr. Dried nuts of S. anacardium were crushed with silica and ground into fine powder. 20g powder was sus­pended in 100 ml absolute alcohol over night and sub­sequently centrifuged at 3000 rpm for 30 min. Super­natant was evaporated at 45°C and residue was re­dissolved in alcohol to obtain seven different concen­trations of extract viz. 20, 40, 60, 80, 100, 200 and 400 mg/ml. Similarly aqueous extract was also pre­pared. One ml extract of each of the above mention concentrations was added to culture tubes containing 20 ml potato dextrose agar (PDA) medium. The tubes were autocIaved at 1 2 1 °C and 1 5 psi to eliminate mi­crobial growth. AutocIaving also results in evapora­tion of alcohol leaving only the extract in the medium. AutocIaved medium was then inoculated with pure cultures of Aspergillus and Candida and incubated at 36°C. Growth of fungus was observed on 3,d, 6th, 10th

and I th days. Tube dilution technique9 was used for determining the antifungal activity. Tubes containing extract free autocIaved PDA medium were used as control. Five replicates of each concentration were maintained and the experiment was repeated thrice.

Cultures of A fumigatus and C. albicans were pro­cured from RNT medical college, Udaipur and sub­cultured by serial dilution to get single spore culture.

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SHARMA et al.: FUNGISTATIC ACTIVITY OF SEMECARPUS ANA CARDIUM NUT EXTRACT 3 15

Pure cultures thus produced were used for inocula­tion. Fungal mycelium and fungal cells were stained with cotton blue and mounted in lactophenol for mi­croscopic observation.

Microscopic measurements of the spores were done using standard methods of micrometry at 100X. Stan­dard deviation was calculated using computerized programme.

Results No inhibitory activity was observed with aqueous

extract. Abundant growth was visible in control as well as low concentrations of the alcoholic extract. Decrease in the growth was observed as concentration of the extract increased. 400 mg concentration proved inhibitory for both fungi. Inhibitory effect was noticed even after 1 2 days. Similar results were observed in all replicates and in successive repetitions, suggesting 100% inhibition.

Profuse growth of C. albicans was seen in control as it contains extract free medium (Fig. 1 ; a). No sig­nificant decrease in growth was observed at 20, 40, and 60 mg concentrations of extract · (Fig� 1 ; b, c, d). Gradual decrease in growth was visible from 80 mg concentration onwards (Fig. 1 ; e, f, g, h).The order of decrease of growth was concomitant with the concen­tration of extract in these tubes. Maximum inhibition was observed at 400 mg concentration (Fig. 1 ; h) sig­nifying that the inhibitory action is directly propor­tional to increasing concentration of extract.

A.fumigatus showed abundant growth at lower concentration of extract. Slight decrease in growth

could be observed at 40 mg concentration although it was not very significant (Fig. 2; a, b). This suggests that the extract is ineffective at this and lower concen­trations. Subsequently, progressive decrease in growth was observed at 80 mg concentration onwards, sig­nificant inhibition was noticed at 400mg (Fig 2; d, e, f, g). Concurrent with the inhibitory effect the colony colour also changed. Colony at 400 mg/ml extract was whitish (Fig. 2; g). whereas in all the other tubes the colony was dark green in colour (Fig. 2; a-f).

On microscopic examination, at 400 mg concentra­tion, the hyphae and the cytoplasmic material of A.fumigatus appeared shrunken (Fig. 4) as compared to the control (Fig. 3). The average mycelium width at this concentration was 3 . 1 1 Jl whereas in the control was 9.9 1 Jl (Fig. 1 1 ; a) . Spore number, size and spo­rangium size was also reduced as compared to control (Figs. 3, 4, 5 and 6). Sporangium diameter in the con­trol was 49.45 Jl but at 400mg concentration, it was reduced to 20.54 Jl (Fig. 1 1 ; a). Similarly the average spore diameter was reduced from to 2.97 Jl in the con­trol to 1 .59 Jl at 400 mg concentration (Fig. 1 1 ; a).

In C.albicans, pseudo mycelium, blastospores and chlamydospores were observed in control (Fig 7,8). At 200 and 400 mg concentrations only chlamy­dospores could be seen (Fig 9 , 10) . As compared to control, chlamydospores size decreased with increas­ing concentration of extract. The average chlamy­dospore diameter in control was 5 .79 Jl, at 200mg it was 4.54 Jl and at 400 mg it was further reduced to 2.54Jl (Fig. 1 1 ; b) .

Fig. 1 - Inhibitory effect of various concentrations (mglml extract) of S. anacardium nut extract on C. albicans after 1 2 days (a: control, b: 20, c: 40, d: 60, e: 80, f: 1 00, g: 200, h: 400). Fig. 2-Inhibitory effect of various concentrations (mg/ml extract) of S. anacardium nut extract on A. fumigatus after 1 2 days (a: control, b: 40, c: 60, d: 80, e: 100, f: 200, g: 400).

3 16 INDIAN J EXP B IOL, MARCH 2002

Figs 3 and 4-Mycelium and spores of A. fumigatus after 1 2 days of experimentation (3: control ; 4: following 400 mg/ml treatment) x 1 0

Figs 5 and 6-Sporangiophore of A. fumigatus after 1 2 days of experimentation (5 : control; 6: following 400 mg/ml treatment) x 20

Figs 7- IO-Psuedomycelium, blastospores and chlamydospores of C. albicans after 1 2 days of experimentation (7 and 8: control; 9 : following 200 mg/ml treatment; 10 : following 400 mg/ml treatment) x 40 [M=mycelium; C=chlamydospores; sp= sporangium; S= spore]

SHARMA et al. : FUNGISTATIC ACTIVITY OF SEMECARPUS ANA CARDIUM NUT EXTRACT 3 17

70 (0) 60 D Conlrol

• Experimental 50

E 40 ::I.

:!l 30 Vi 20

1 0

0 Sporangiophore Mycelium width spore size

size

7 ( b ) E l :200mg/ml extr.ct cone. 6

E2:400mglml extr.ct conc.

6

� 4

II N 3 iii

2

o Control E1 E2

Figs I I -(a) Effect of 400 mg/ml extract concentration on spo­rangium diameter, mycelium width and spore size of A. fumigatus after 1 2 days of experimentation. (b) Effect of 200 and 400 mg/ml extract concentration on cell size of C. albicans after 1 2 days of experimentation.

Discussion

There was a remarkable decrease in growth and sporulation of A.fumigatus following treatment with S.anacardium extract.This deuteromycetous fungus reproduces mostly by producing spores. A reduction in their number as well as size, shrunken mycelium suggests that the extract may be fungistatic in nature. The spores are dark green and are responsible for the colour of the colony.'Whitish colony in the tube con­taining 400mg extract correlates with the microscopic observation of reduced sporulation.53.75%, 58.56% and 46.46% reduction in mycelial width, sporangium and spDre size respectively indicates inhibitory action of extract.

C.albicans normally occurs in two states. When the fungus grows as a commensal, it exists as unicellular yeast phase and when it becomes pathogenic, the thal-

Ius becomes psedomyceJial lD. As observed the thallus is pseudomyceJial in the control (Figs 7,8) but at 200 and 400 mg concentrations of the extract, only unicel­lular chlamydospores were seen (Figs 9, 10). Thus it can be said the fungus may have converted to the commensal nonpathogenic form.2 1 .59% and 57 .68% reduction in the chlamydospore size at 200mg and 400 mg extract concentration suggests that the extract is inhibitory.

The antimicrobial activity of the alcoholic extract can be attributed to the presence of naturally occur­ring antimicrobial phenolic compounds like catechols, oleoresins and secondary metabolites 1 1 . 14. Phenolic compounds alter membrane permeability and act as protein denaturing agents 1 5 . Phenolic compounds are soluble in alcohol only, which explains the inactivity of aqueous extract. Reports of antitumerous activity of S.anacardium suggest that the extract and the oil of the nut are growth inhibitory'6. In vitro cytotoxic ac­tivity of nut oil has been suggested3, 16. Antimicrobial activity of S.anacardium has also been reported.5

The results of present study thus suggest that alco­holic extract of S.anacardium is fungistatic in nature as it inhibits growth and reproduction.

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