ABSTRACT: The objective of present paper was to understand the antimicrobial and antifungal potential of Stevia rebaudiana which is popularly known as Stevia and synonymously known as 'sugar substitute' belonging to family Asteraceae. Antibacterial and antifungal compounds of Stevia leaves were extracted in four solvents acetone, chloroform, ethyl acetate and distilled water. These chemical extracts were subjected to microbial assay using four pathogenic bacteria namely Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Staphylococcus aureus and two food spoiling pathogenic fungi which are Aspergillus niger, Aspergillus terreus isolates. Ethyl acetate and chloroform extracts had greater antimicrobial potential for strains of Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Staphylococcus aureus and acetone extract also proved to have antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa. However acetone along with ethyl acetate extract had antifungal potential for Aspergillus niger, Aspergillus terreus, thus indicating that Stevia leaf extract can serve ideal plant for further research in to its role in food preservations and pharmaceutical due to their antifungal and antimicrobial activities.

Key words: Stevia rebaudiana, antibacterial, antifungal, ethyl acetate, chloroform, acetone.

IN VITRO ANTIMICROBIAL AND ANTIFUNGAL ACTIVITES OF STEVIA REBAUDIANA BERT.

LAKSHMI GIRISH, DEVYANI TOPTE AND SHARDA VAIDYADepartment of Botany, Smt. C. H. M. College, Ulhasnagar, Thane 421003

Email : sharda.vaidya@yahoo.inReceived on :20.02.2013 and Accepted Revised on : 29.10.2013

INTRODUCTION:

The plants are nature's gift and even nature biggest chemical laboratory, where a wide variety of compounds are synthesized. The natural products will provide a clue to synthesis of new antimicrobial chemicals. These chemicals are relatively safe to man and his environment. Different medicinal plants and their medicinal values are widely used for various ailments throughout the world. Now days natural products are an integral part of human health care system, because there is now popular concern over toxicity and resistance of modern drugs. Humans have relied on bacterial and fungal sources for these activities. There is an alarming increase in the incidence of new and reemerging infectious diseases and development of resistance to the antibiotics in current clinical use. Plant extracts are rich source of new drugs which is effective in treatment of several diseases. The various phytochemicals are well known for its antimicrobial properties can be of great significance in therapeutic treatments. The medicinal value of plants lies in some chemical substances that produce a definite physiological action on the human body. The most important of these bioactive compounds of plants are alkaloids, flavanoids, tannins and phenolic compounds.

Leaves of many plants have antimicrobial principles such as tannins, essential oils and other aromatic compounds. Various chemical constituents isolated and characterized from Asteraceae family is well known. One of the potent members of the Asteraceae family is Stevia rebaudiana (commonly referred to as Honey leaf, Candy leaf and Sweet leaf). It is rich in terpenes and flavanoids. The phytochemicals present in Stevia rebaudiana are austroinullin, b- carotene, dulcoside, niacin, rebaudi oxides, riboflavin, steviol, stevioside and tiamin. Stevia has important industrial uses in beverages, energizers as well as medicinal uses such as low uric acid treatment, vasodilator cardiotonic, anesthetic and antiinflammatory. Stevia rebaudiana has also been reported to present antimicrobial activity against a broad range of

microorganisms (Jayaraman et al, 2008). Over 50% of all modern clinical drugs are of natural origin and natural products play an important role in drug development in the pharmaceutical industry (Baker et al, 1995). The effects of plant extract on bacteria have been studied by a very large number of researchers in different parts of the world (Digrak et al., 1999). Much work has been done on ethano-medicinal plants in India (Erdogrul, 2002, Ates et al., 2003).

There are number of reports available on the antibacterial activities of different terrestrial plants which proved to be very effective against variety of pathogenic bacteria (Shahidi, 2008, Ezeifeka, 2004). However limited literature is available on the antimicrobial activity of aquatic angiosperms. Nature has been a source of medical agents for thousand of year and an impressive number of modern drugs have been isolated from natural sources; many of these isolation were based on the uses of the agents in traditional medicine. The present study was carried out to evaluate the antimicrobial activity of Stevia rebaudiana leaves extracted using various solvents.

MATERIALS AND METHODS :

Plant material: Stevia rebaudiana leaves were obtained from Pathare nursery, Kalyan. The leaves were washed with sterile distilled water, dried in shade, finely powdered & stored in air tight bottles.Test organisms: Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella pneumoniae, Staphylococcus aureus, were used to test antibacterial activity while Aspergillus niger, Aspergillus terreus were used to assess antifungal activities. Preparation of Extracts: The Stevia rebaudiana leaves were dried in shade and grined into powder was used for extraction. 25 g of air-dried powder of leaves was immersed in 100 mL of organic solvents such as ethyl acetate, acetone, chloroform and distilled water in conical flask. It was incubated at room temperature for 48 hours at 150 rpm on an orbital shaker. The suspension was filtered and concentrated to dryness at 40 C in hot air oven and the residue was dissolved

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in 0.1 ml of ethanolic solvent was used for antimicrobial activities. Screening of Antibacterial Activity:Media used: Nutrient agar (Hi Media)Preparation of inoculum :Stock cultures were maintained at

o4 C on nutrient agar (Hi Media) slants. Active cultures for experiments were prepared by transferring a loopful of culture

0to nutrient Agar slant (Hi Media), incubated at 37 C for 24 hours for bacterial proliferation. Paper disc method (Gould and Bowie, 1952) was employed for testing antibacterial activity of Stevia rebaudiana leaves. The final concentration of all the extracts was made to 50 mg/ mL. Twenty four hour old cultures of test organisms (0.05 mL) were spread on Mueller Hinton agar (Hi Media) plate. Paper disc (5 mm in diameter) soaked into plant extract were placed onto the

oplates and the plates were incubated at 37 C for 24 hours to allow the bacterial growth. The antibacterial activity of the plant extract was determined by comparing the diameter of the zone of inhibition with that of respective controls (acetone, ethyl acetate, chloroform and distill water). For each bacterial strain, controls were maintained where pure solvents were used instead of the extract. The experiment was conducted in triplicate and the mean values are represented.Screening of antifungal activityMedia used - Potato dextrose agar (Hi Media) Preparation of inoculum :Potato dextrose agar (HiMedia) was plated. After solidification about 0.5 ml of culture suspension was spread on the agar medium. All the plates were incubated at

o25 C for 4 days. The experiments were performed in triplicates. The growth of the fungal cultures was measured and compared with the respective control plates.

RESULTS AND DISCUSSIONS :

Table.1. Antimicrobial activity (Zone of Inhibition in mm)

Table.2. Antifungal Activity (Zone of inhibition in mm)

Among the four extracts tested, ethyl acetate extract had greater antibacterial potential, followed by chloroform extract and acetone extracts (Table. 1.). Similar results were already reported by Balasubramanian (2012) in Nyctanthes arbor-tritis. There was no antimicrobial activity was observed in distilled water extract. The higher antibacterial activity of the ethyl acetate extracts may be due to the greater solubility of more of the active antimicrobial phytoconstituents in this extract, consequently displaying the highest relative antibacterial activity as has been reported by De Boer et al (2005). The largest zones of inhibition were observed for ethyl acetate extract against Proteus vulgaris (18 mm) and Staphylococcus aureus (17 mm). Jayaraman et al (2008) reported maximum zone of inhibition in acetone extract against Staphylococcus aureus (19 mm). Chloroform extract was slightly effective and distilled water ineffective against the test organism. This finding is similar to that of Tadhani and Subhash (2006), Jayaraman et al (2008) who also recorded very low antibacterial activity for water extracts of Stevia rebaudiana leaves. Several workers have reported that water extracts do not have much activity against bacteria.

In the present study, all the four extracts showed inhibitory effect against fungal test organisms (Table.2.). Among the four extracts studied, ethyl acetate extract showed maximum zone of inhibition against Aspergillus niger followed by acetone and chloroform extracts and least by distilled water extract. As the incubation period was prolonged the inhibitory effect of ethyl acetate was slightly more in A. terreus. The increase in the inhibitory effect of extracts with increase in the incubation period was already reported by Jayaraman et al. (2008). Many biological activities and antibacterial effects have been reported for tannins and flavanoids (Scalbert, 1991., Chung et al., 1998). The antimicrobial activity of Stevia rebaurdiana leaves may be due to the presence of phytochemicals such as flavanoids, terpenes etc.

CONCLUSION :

The broad-spectrum antibacterial and antifungal activities of the plant extract may be possibly due to the various identified phytoconstituents. Overall, the study revealed that Stevia rebaudiana, a medicinal herb, with its ethyl acetate extracts having very high potency against bacteria and fungi. Bio active substance from this plant can therefore be employed in the formulation of antimicrobial and antifungal agents for the treatment of various infections.

ACKNOWLEDGEMENT :

The authors are grateful to the Principal, Smt. C. H. M. College, Ulhasnagar, for support and encouragement to carry out this research activity.

REFERENCES :

Ates, D.A and Erdogrul, O.T. (2003): Antimicrobial activities of various medicine and commercial plant extracts. Turk J Biol., 27:157-162

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Chung, K. T, Wong, T. Y, Wei, Y, Huang, Y. W, Lin, Y. (1998). Tannins and human health. A review. Crit Rev Food Sci Nutr, 8: 421-464.

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Jayaraman, S, MuthuSaravanan, M. Seethalakshmi, I. (2008). In-vitro Antimicrobial and Antitumor Activities of Stevia rebaudiana (Asteraceae) Leaf Extracts. Tropical Journal of Pharmaceutical Research, 7 (4): 1143-1149.

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