Antifungal Activity of Cleome rutidosperma Aerial Parts

A. Khuntia¹* and S.K. Mohanty²

¹Jeypore College of Pharmacy, Rondapalli, Jeypore, Koraput-764002, Odisha.

²Jayamukhi Institute of Pharmaceutical Sciences, Warangel, A.P.

*Corresponding Author E-mail: anuphchemist@gmail.com

ABSTRACT:

Cleome rutidosperma, Linn [Family-Cleomaceae] is claimed to relieve earache, pain. However thorough literature survey indicated that different plants of the same genus available in tropical parts of India have various reported biological activities like antifungal activity, antibacterial activity. So it is reasonable to expect that this plant might have similar kind of biological activity. The present study was undertaken to explore the antifungal potential of different solvent extracts of aerial parts against selected fungal species.

The aerial parts of the plant Cleome rutidosperma was successively extracted with ethanol (90%), petroleum ether, diethyl ether, ethyl acetate and N-butanol. The extracts were vaccum dried and studied for in vitro antifungal activity against Aspergillus niger, Penicillium notatum, Candida albicans, Helminthosporium solani. The ethanolic extract exhibited prominent antifungal activity against all selected strains .Minimum inhibitory concentration of the extracts was performed by broth dilution method and the zone of inhibition was studied by agar disc diffusion method at concentrations 2, 5, 10 mg/ml in DMSO. Cotrimazole (25 µg/ml) was used as reference control for antifungal studies. The present study indicates the potential usefulness of Cleome rutidosperma aerial parts as antifungal agent.

KEYWORDS: Antifungal activity, Cleome rutidosperma, aerial parts, DMSO

INTRODUCTION:

Fungal diseases are major cause of morbidity and mortality worldwide. The no of multidrug resistant microbial strains and the appearance of strains with reduced susceptibility to antibiotics are continuously increasing. This increase has been attributed to indiscriminate use of broad spectrum antibiotics and immunosuppressive agent¹. This situation provided the impectus to the search for new antifungal substances from various sources like medicinal plants². Synthetic drugs are not only expensive and inadequate for the treatment of diseases, but are also often with serious side effects. Traditional medicine has made use of many different plant extracts for treatment of fungal infections and some of these have been tested for in vitro antifungal activity³.

Cleome rutidosperma grows principally at low altitudes in humid conditions. It occurs up to 400m altitude, in areas with an annual rainfall of 1700-3000nmm occasionally. It is found as a weed up to 1200m altitude. Flowering and fruiting plants can be found throughout the year, although most abundantly in the rainy season.

It is an erect annual herb up to 70cm tall, branched from the base; stem is finely pubescent or glandular pubescent, green purplish. Leaves are alternate with 3-foliolate containing petiole up to 7cm long; leaflets are elliptical, glabrous to sparsely setulose-pubescent. Inflorescence is racemose, bracts, similar to leaves. Flowers are small, violet-blue coloured, which turn pink as they age, found singly. They are bisexual, regular, 4-merous with pedicel up to 3.5 cm long; sepals are linear to lanceolate, 2-4.5 mm long, glandular; petals are oblanceolate, 6-11 mm long, usually white, sometimes pinkish; stamens are 6 in number; ovary is superior, cylindrical, 1-celled.Fruit is cylindrical capsule; with stalk 5-13 mm long, sub glabrous, dehiscing with 2 valves; when ripe splits into two scattering many seeds. Seeds are aglobular-reniform, 2 mm in diameters, orange-brown-black in colour with fine longitudinal striations and low irregular transverse ridges.

Cleome rutidosperma Linn [Family-Cleomaceae] is claimed to relieve earahe, pain, skin diseases. In Ghana Gabon and DR Congo leaf sap is applied to cure earache and deafness. The plant is used as antimalarial by traditional healers in Cameroon. The plant is used in the treatment of paralysis, epilepsy, convulsion and spasm^4,5.

However thorough literature survey indicated that different plants of the same genus available in tropical parts of India have various reported biological activities like analgesics, antipyretic, anthelmintic activity, antifungal activity, antibacterial activity, antimycobacterial activity and antiyeast activity, cytotoxic activity etc. Thus it was reasonable to expect that this plant might have some similar or other kinds of biological activity, which remained unexplored because of lack of work. The present study was undertaken to explore the antifungal potential of different solvent extracts of the aerial parts against selected fungus species, since no such work has been reported earlier.

MATERIAL AND METHODS:-

Plant Material;- The plant was identified by the taxonomists of Botanical survey of India, Shibpur, Howrah. After authentication, fresh aerial parts were collected in bulk from young matured plants, washed, shade dried (11.2% w/w of fresh plant) and then milled into coarse powder by a mechanical grinder. The powder was passed through sieve number 40 and used for further studies.

Preparation of extract:- The powdered plant material was extracted with 90% ethanol using soxhlet apparatus. The solvent was removed under reduced pressure, which gave a greenish-black coloured sticky residue (yield- 12% w/w on dried material basis). A portion of dried ethanolic extract was suspended in water and fractionated successively with petroleum ether (40-600c), diethyl ether, ethyl acetate and N-butanol. The yields of the fractions were found to be 26.64%, 8.95%, 6.39% and 16.33% w/w respectively of the ethanolic extract. All the fractions were dried by distillation under reduced pressure^6,7.

Determination of phytoconstituent:-

Preliminary phytochemical screening was performed as per standardized procedure and the various phytoconstituents identified are amino acid, terpenes, lipids, steroids, flavonoids. Alkaloids and glycosides were absent in all the extracts.

Drugs used:- Cotrimazole was used as reference standard for antifungal studies.

Microorganisms used:- For the present study, the microorganisms used include⁸ Aspergillus niger, Penicillium notatum, Candida albicans, Helminthosporium solani respectively.

Antifungal activity:- The antifungal activities of the extracts were tested using various pathogenic fungi. The antifungal activities of the extracts were performed by agar disc diffusion method⁹ with the disc diameter 6mm.The extracts were separately dissolved in DMSO at concentration of 2,5 and 10 mg/ml. Cotrimazole (25 µg/ml) in Dimehyl sulphoxide (DMSO) was also served as reference control for antifungal study. Solvent control (only DMSO) was also maintained throughout the experiment.PDA media was used for the antifungal study. The molten media was then inoculated with 200µl of the inoculum (1x108Cfu) and poured into the sterile Petri plates. The disc was saturated with 20 µl of the extract separately, allowed to dry and was introduced on the upper layer of the seeded agar plate. The plates were incubated for 48hrs at 280c and the zone of inhibition was measured.

Minimum inhibitory concentration (MIC) of the extract was performed by broth dilution method¹⁰ at concentrations of the extracts ranging from 25µg/ml to 500 µg/ml in DMSO against all test microorganisms. The results of the zone of inhibition values were depicted in Table no.1 and MIC values in Table no.2.

RESULT:-

The result of the preliminary phytochemical analysis revealed presence of tannins, steroids, alkaloids and flavonoids.

Table No.1 depicts the antifungal activity of the extracts of Cleome rutidosperma aerial parts. The results of MIC study revealed the antifungal activity of the extracts against the tested strains of microorganisms between concentration ranges of 150 and 350 µg/ml. The results of zone of inhibition study revealed that the extract possesses antifungal activity in concentration dependant manner against test microorganisms and were comparable with the standard drug.

Table No.1:- Zone of inhibition (mm) of various extracts of aerial parts of Cleome rutidosperma

*Sl. No.*	*Test substance*	Concentration (mg/ml)	*Aspergillus niger*	*Penicillium notatum*	*Candida albicans*	*Helminthosporium solani*
1.	Petroleuem ether extract	2	5.2	-	-	-
		5	8.3	-	-	-
		10	11.4	-	-	-
2.	Diethyl ether extract	2	-	4.6	3.9	-
		5	-	7.9	6.8	-
		10	-	10.8	9.7	-
3.	Ethyl acetate extract	2	-	-	-	-
		5	-	-	-	-
		10	-	-	-	-
4.	N-butanol extract	2	-	-	-	-
		5	-	-	-	-
		10	-	-	-	-
5.	Ethanol extract	2	7.8	7.9	7.6	7.3
		5	11.3	11.7	10.9	11.1
		10	12.5	13.7	14.1	14.3
6.	Cotrimazole	25(µg/ml)	27.0	24.0	24.0	26.0

*Values are man of three readings, Standard used for antifungal studies; Cotrimazole -25µg/ml; (-) indicates no activity.

Percentage yield of various extracts confirm that petroleum ether extract has maximum yield (26.64% w/w) whereas ethyl acetate extract has minimum yield (6.39%w/w) .Ethyl acetate and N-butanol extract showed no antifungal activity.

Table No. 2:-MIC values of different microorganisms

S.L. No.	Microorganisms	MIC values(µg/ml)
1.	Aspergillus niger	225
2.	Penicillium notatum	175
3.	Candida albicans	175
4.	Helminthosporium solani	150

DISCUSSION:-

Aspergillus niger was inhibited by petroleum ether extract and ethanol extract Penicillium notatum was inhibited by di ethyl ether and ethanol extract. Candida albicans was inhibited by diethyl ether and ethanol extract. Helminthosporium solani was inhibited by ethanol extract.

Petroleum ether extract was most active against Aspergillus niger. Diethyl ether extract was most active against Penicillium notatum and Candida albicans. Ethanol extract was most active against all fungal species.

There were reports in the literature that ethanol is a better solvent for consistent extraction of antifungal and antimicrobial agent from medicinal plants .The antifungal activities of medicinal plants are attributed due to presence of flavonoids and tannins in the ethanolic extract of aerial parts of Cleome rutidosperma, confirm its potential against all selected pathogens. This suggests that of aerial parts of Cleome rutidosperma have a broad spectrum antifungal activity, although the degree of susceptibility could differ between different organisms. The broad spectrum of antifungal activity found in this present study may be attributed to the presence of secondary metabolites of various chemical types present in the plant material either individually or in combination. Our result indicates the potential usefulness of Cleome rutidosperma as antifungal agent.

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Received on 26.03.2011 Modified on 05.04.2011

Research J. Pharm. and Tech. 4(7): July 2011; Page 1103-1105