In Vitro Antimicrobial Activity of Stevia Rebaudiana Bertoni Leaves

Purpose: The aim of the present study was to investigate antimicrobial activity of the various extracts of Stevia rebaudiana leaves. Method: Stevia rebaudiana Bertoni leaves were extracted in water, methanol, ethyl acetate and hexane and their antimicrobial activities were examined against few selected microorganisms including B. subtilis, S. aureus, M. luteus, S. marcenscens, P. aeruginosa, B. megaterium, E. coli, P. vulgaris, Yeast, A. niger and R. oligoporus using cup plate method. Results: Water extract of Stevia leaf showed activity against B. subtilis and S. aureus only. Methanol extract gave the highest zone of inhibition against P. aeruginosa whereas minimum zone of inhibition was found against S. aureus and yeast. B. megaterium and yeast were found to be highly susceptible towards ethyl acetate and hexane extracts, respectively whereas A. niger and B. subtilis were found to be least susceptible against ethyl acetate and hexane extracts, respectively. Hexane extract showed the highest activity against yeast among the tested microorganisms. Conclusion: The study confirms the possible antimicrobial potentiality of the leaf extract of Stevia


INTRODUCTION
The use of natural products with therapeutic properties has a long history, plant, animal, and mineral products were the main source of medicines 1 .Many efforts have been made to discover new antimicrobial compounds from various kinds of sources such as microorganisms, animals, and plants.Systematic screening of them may result in the discovery of novel effective antimicrobial compounds 2 .Plants can possess antimicrobial natural products to protect themselves from microbial infection and deterioration 3 .In the developing countries, synthetic drugs are not only expensive and inadequate for the treatment of diseases but are also often with adulterations and side effects 4 .In recent years, concern over pathogenic and spoilage microorganisms in foods has increased due to the increase in outbreaks of food borne disease 5 .There are growing interests in using natural antimicrobial compounds, especially extracted from plants, for the preservation of foods.In addition, there are more consumers who tend to question the safety of synthetic additives and would prefer natural foodstuffs [6][7] .There is therefore the need to search for plants of medicinal value.The plant used in the present study was Stevia rebaudiana (Asteraceae), which is used traditionally for the source of natural sweetener.The dry extract from the leaves of stevia (Stevia rebaudiana Bertoni) contains sweet diterpene glycosides, flavonoids, alkaloids, water-soluble chlorophylls and xanthophylls, hydroxycynnamic acids (caffeic, chlorogenic, etc.), neutral water-soluble oligosaccharides, free sugars, amino acids, lipids, essential oils, and trace elements 8 .To the best of our knowledge, nobody has investigated the potential antimicrobial effect of Stevia rebaudiana leaves in India until now.Therefore, the presents study was planned to study the antimicrobial activity of water, methanolic, ethyl acetate and hexane extracts of Stevia rebaudiana leaves against the selected microorganisms.

MATERIALS AND METHODS
Plant Material and Microorganisms: Dried stevia leaves were supplied by Growmore Biotech Ltd., Hosur, Tamilnadu, India.Leaves were packed in Polyethylene bags and stored at -18º C until used.The bacterial strains used were obtained from the stock culture of the Department of Microbiology, V.P. Science College, Vallabh Vidyanagar, India.The organisms included in the present study were B. subtilis, S. aureus, M. luteus, S. marcenscens, P. aeruginosa, B. megaterium, E. coli, P. vulgaris, Yeast, A. niger and R. oligoporus.All the bacterial strains used for the experimental purpose were grown and maintained on nutrient agar medium.Yeast was isolated from curd sample on Sabouraud agar medium in the laboratory and maintained on the same medium.R. oligosporus and A. niger were grown and maintained on potato dextrose agar medium.

Preparation of extracts:
Dry powdered stevia leaves were extracted separately with water, methanol, ethyl acetate and hexane (all solvents were of HPLC grade) using environmental shaker (New Brunswick, USA) at 250 rpm for 24 hours.Then, the extracts were filtered using Whatman No. 1 filter paper and the filtrates were then evaporated to dryness under reduced pressure.Residues were stored in labeled sterile screwcapped bottles at -20°C.

Antimicrobial Assay:
The yield of water, methanol, ethyl acetate and hexane extracts of the leaf were found to be 22.44, 30.20, 10.31 and 1.74g %, respectively, on dry weight basis.Respective solvents were used to prepare a final concentration of 50 mg ml -1 and sterilized by filtration through a 0.45 µm nylon membrane filter.Various extracts of Stevia leaves were subjected to antimicrobial assay using the cup-plate method 9 .Nutrient agar plates were prepared by pouring 20 ml of nutrient agar in sterile Petri dishes for antibacterial assay.Similarly, potato dextrose agar plates were prepared for antifungal assay.These were allowed to solidify.The bacterial cultures used for assay were 24 hours old whereas fungus cultures were 4 to 5 days old.Concentration of these organisms was prepared Trop J Pharm Res, June 2006; 5 (1)  to contain approximately 1× 10 6 cfu/ml.Sugar tubes containing molten agar (10 ml) were sterilized and cooled to about 40-42 o C. The tubes were then inoculated with 0.1 ml of the appropriate culture suspension of bacterium or fungus, mixed gently and poured onto previously solidified nutrient agar or potato dextrose agar plates, respectively.After setting, a cup borer (6 mm diameter) was properly sterilized by flaming and used to make four uniform cups in each Petri dish.The cups were then filled with different Stevia leaf extracts and allowed to diffuse for 45 minutes.The solvents used for extraction were analyzed similarly as control.Ciprofloxacin (10 µg/disc) and amphotericin-B (100 units/disc) were used as standards for bacteria and fungi, respectively.The plates were incubated at 37 o C for 24 hours.At the end of the period, inhibition zones formed on the medium were evaluated in mm using a scale.The experiment was carried out in triplicates.
Statistical analysis: Analysis of variance (ANOVA) and Duncan's test were carried out using SPSS package for statistical analysis.

DISCUSSION
Antimicrobial activities of various herbs and spices in plant leaves, flowers, stems, roots, or fruits have been reported by many workers 3-6-10 .It is worthwhile to note that there are no data in the literature to indicate previous investigation of the antimicrobial activity of Stevia rebaudiana leaves for comparison.Water extract showed activity against only two microorganisms among the selected microorganisms.Methanol and ethyl acetate extracts have more or less similar activities but hexane extract showed significantly higher activity compared to other extracts against most microorganisms tested.The results of present study indicate that the stevia leaf extracts have inhibitory activities against microorganisms, although their antibacterial activities are lower than that of the standard (ciprofloxacin).However, in the case of fungi, hexane extract had significantly higher activity than the standard (amphotericin-B).The results of the present work indicate that stevia leaf extracts may be an ideal candidate for further research into their uses for food preservation as well as pharmaceutical and natural plant-based products.

Table 1 :
Antimicrobial activity of water, methanol, ethyl acetate and hexane extracts of Stevia rebaudiana leaves (zone of inhibition in mm) Values are the mean of three tests ± SEM a-d Mean values with the same superscript within a row do not differ significantly (p>0.05).* Indicate significant difference ( p<0.05).