Phytochemical Screening and Antibacterial Activity of Leaf Extracts of Gymnema sylvestre against Pathogenic Bacteria

The aim of our present study to investigate the phytochemical screening and antibacterial activity of Etholnolic and Chloroform leaf extracts of Gymnema sylvestre against some important pathogenic bacteria. The Ethonoloic and Choloroform leaf extracts of G.sylvestre showed significant antibacterial against some clinically important gram negative and gram positive bacteria such as Staphylococcus aureus (ATCC6538), Enterococcuc faecalis (ATCC 29212) and Escheria coli (ATCC8739), Salmonella enterica (ATCC 10798) respectively. The in-vitro antibacterial activity was performed by agar well diffusion method (25, 50, 75 and 100 μg/ml ). The maximum inhibition zone of ethonolic leaf extract was found in 100 μg /ml against gram negative bacteria such as Salmonella enterica (26mm), Escheria coli (24mm) and gram positive bacteria such as Enterocococcus faecalis (26mm) and Staphylococcus aureus (24mm). The maximum inhibition zone of chloroform leaf extracts found in 100 μg/ml (25,50,75, and 100 μg/ ml) against gram negative bacteria such as Salmonella enterica (24mm), Escheria coli (24mm) and gram positive bacteria Enterocococcus faecalis (23mm) and Staphylococcus aureus (23mm). The phytochemical tests were carried out and showed that the antibacterial activity of plant Gymnema sylvestre leaves were due to presence of phytochemical compounds such as Alkaloids, Flvanoids, Glycosides, Reducing sugar, Tannins, Saponins, Polysaccharides and Phenols. The ethonolic and choloroform leaf extracts of Gymnema sylvestre showed maximum inhibition against Staphylococcus aureus, Enterocococcus faecalis, Salmonella enterica and Escheriacoli. The Ciprofloxacin was used as a positive control. KeywordsGymnema sylvestre, pathogenic microorganisms, antibacterial activity, bioactive components


I. INTRODUCTION
In the recent years, an increasing number of infectious agents are becoming more resistant to commercial antimicrobial components [1,2].India is a paradise of medicinal plants and traditionally used many medicinal plant components are known as therapeutic properties from India [3,4].The development of new antibacterial agent especially from the natural source is based on the pharmacological properties [5].The Gymnema sylvestre is one of the most important medicinal plants that grow in tropical forests in Western India, Africa, Australia, South East Asia and India [6,7].The plant has been reported to possess antimicrobial [8] and ethnoveterinary medicinal properties.The active ingredients are present in leaves and roots used in traditional medicine and available pharmaceutical products to treat various ailments like diabetes, asthuma and etc. [9].In the Ayurvedic system of medicine, G. sylvestre is referred to as "mesarngi" and both the dried leaf and dried root are used therapeutically.The plant leaves are used in ayurvedic as antiviral, diuretic, antiallergic, hypoglycemic, hypolidemic, for the treatment of obesity and dental caries [10].The antioxidant and antimicrobial activities of plant extracts have formed the basis of applications in pharmaceuticals, alternative medicines and natural therapy and growing demand for G. sylvestre leaves in pharmaceutical trade [11].Gymnemic acid is the active ingredient of this plant, the extracted from leaves and used widely as an anti-diabetic [12] anti-sweetner [13] and antihypercholesterolemia [14].It has also stomachic, diuretic and cough suppressant properties [15].
The several studies are reported that leaf extract of G. sylvestre has protective role against gram-positive bacteria such as S. mitis and S. mutans [16] and used to treating infectious diseases caused by Escherichia coli and Staphylococcus aureus [17] Bhuvaneshwari et.al., [18] demonstrated that the methonlic extracts of G. sylvestre were assessed for antimicrobial activity of leaves and root separately.The results revealed that the methanol extracts in acidic range have good activity towards all the pathogens.In a similar study carried out by Satdive et al., [8] the antimicrobial activity of ethanolic extracts of G. sylvestre against B. subtilis, B. pumilus, S. aureus and P. aeruginosa showed good antimicrobial activity.Hence, the present study was planned to find out the phytochemical properties and antimicrobial activities of ethanol and chloroform extracts of Gymnema sylvestre.The prepared extracts were screened for their antibacterial activity against clinically important pathogenic bacteria such as Staphylococcus aureus, Enterococcus enteric, Escheria coli and Salmonella enterica.The presence of phytochemical components and antibacterial properties can lead to detect new sources of antibacterial agents.

Collection of plant
The leaves of Gymnema sylvestre were collected from Cheyyar, Thiruvannamalai District, Tamil Nadu and India.The species was identified and authenticated by the Department of Botany, Arignar Anna Government Arts College, Cheyyar, Thiruvannamalai District,Tamil Nadu.

Preparation of plant extracts
The leaves of Gymnema sylvestre were cleaned and dried in shade under dark room, then cut into small pieces and coarsely powdered by mixer.The course powder was used for extraction with various solvents.In 1 kg of dried leaf powder added 2.5 liters of solvents (water, ethanol, and chloroform) was taken in individual aspirator bottle, were used and the mixtures were shaken occasionally in Orbital shaker for 72 hours.Then the extract was filtered.This procedure was repeated three times and all extracts were decanted and pooled.The extracts were filtered before drying using Whatmann filter paper no.1 on a Buchner funnel and the solvent was removed by vacuum distillation in a rotary evaporator at 40˚C, the extracts were placed in pre-weighed flasks before drying.Finally the aqueous, ethanol and chloroform extracts of Gymnema sylvestre leaves were used for the preliminary phytochemical screening and antibacterial study.

Phytochemical Screening
Phytochemical screening in the Gymnema sylvestre leaf extract using different solvent systems (aqueous, ethanol and chloroform) were carried out as per the standard method of Dey and Raman [19].

Antibacterial Activity
Antibacterial activity of Aqueous, Ethanol and Chloroform extracts of Gymnema sylvestre were done by the method of Gillespe et al., [20,21] by using disc diffusion method with both Gram negative and positive bacteria.

Procedure
Aqueous, Ethanol and Chloroform extracts (10% and 50%) solution were prepared by dissolving 1 and 5 g in 10 ml each of distilled water.10 ml each of the prepared concentrations were pipette into sterile test tubes.Bacterial aliquots of the test organisms were made by scooping 2 colonies each of a 24 hours growth of the bacteria into 4 ml of sterile distilled water.0.2 ml of each of the aliquots containing approximately 5 × 104 bacterial cells or colony forming units was transferred into both of the extract concentrations and allowed to stand for an hour for reaction to take place between the extracts and the bacterial organisms.The mixtures were then inoculated on separate nutrient agar plates and incubated at 37°C for 24 hours.Ethanolic and aqueous extracts of the leaves at 10 and 50% concentration of both chloroform and aqueous extracts of leaves had inhibitory effect, hence the same concentrations were chosen as the working concentration.Results are expressed in area of inhibition zone in mm.

III. RESULTS AND DISCUSSION
The phytochemical analysis of Gymnema sylvestre leaf extract using Aqueous, Ethanolic and Chloroform were showed in Table 1.From the observation of phytochemical analysis, reducing sugar was found in Gymnema sylvestre in all three solvents.The Ethanol and Chloroform leaf extracts of Gymnema sylvestre showed the presence of alkaloids, flavanoids, glycosides, tannins, saponins, polysaccharide, phenols and absence of terpenoids, phytosterols but which were present in aqueous leaf extract.Aqueous extracts of Gymnema sylvestre have flavanoids, glycosides, reducing sugar, polysaccharides, phtoseterois and phenols.The presences of above bio active components are responsible for broad pharmacological profile, including antibacterial activity [22].
The Aqueous, Ethanol and Chloroform leaf extracts were tested using agar plate disc diffusion method against gram positive bacteria such as Staphylococcus aureus (ATCC6538), Enterococcus enteric (ATCC 2912) and gram negative bacteria such as Escheria coli (ATCC8739), Salmonella enterica (ATCC 10798.Table 2 and 3 showed antibacterial activity of ethanol leaf extract of Gymnema sylvestre found maximum inhibitory zone (26mm in 75 and 100µg/ml) against gram positive bacteria Enterococcus faecalis followed by 24mm, 25mm in 25 and 50µg/ml respectively.The Staphylococcus aureus, maximum zone of inhibition (24mm in 75 and 100 µg/ml) and followed by 21mm, 23mm in 25 and 50 µg/ml respectively (Figure 1).The antibacterial activity of Gymnema sylvestre found maximum inhibition zone (26mm) against gram negative Salmonella enterica bacteria followed by 24mm ,25mm in 25 and 50µg/ml and Escherichia coli maximum zone of inhibition (24mm in 100 µg/ml) and followed by 19mm ,21mm and 23mm in 25,50 and 75 µg /ml respectively (Table 4,5 and Figure 2).
The Choloroform leaf extract of G .sylvestre were observed the maximum inhibition zone (23mm in 75 and 100 µg/ml) against gram positive bacteria Staphylococcus aureus and Enterococcus faecalis followed by 23mm, 23mm in 75 and 100 µg/ml respectively (Figure 3).The maximum inhibition zone of Salmonella enterica and Escheria coli were 24mm in 75 and 100 µg/ml respectively (Table 4, 5 and Figure 4).
This experimental studies explained about the presence of bioactive compounds in the Gymnema sylvestre leaf extract from different solvent extracts such as flavanoids, glycosides, reducing sugar, polysaccharides, phytosterols and phenols source of which compound have been associated to antibacterial activity against pathogens [6].The G. sylvestre leaf extract were used in this study of different extracts and their antibacterial efficacy depend on the extractive solvent [23].The ethanol leaf extracts of G. sylvestre more potential activity than the chloroform leaf extrat.The phytochemical analysis showed the presence of various bioactive compounds like alkaloids, flavanoids, glycosides, tannins, saponins, polysaccharides and phenols.

IV. CONCLUSION
The present studies were revealed that the ethanol and chloroform leaf extract of Gymnema sylvestre showed significant activity against gram positive bacteria such as Staphylococcus aureus and Enterococcus faecalis and gram negative bacteria Escheria coli and Enterococcus faecalis.The ethanol leaf extracts of G .sylvestre more potential activity than the chloroform leaf extract.The phytochemical analysis showed the presence of various bioactive compounds like alkaloids, flavanoids, glycosides, tannins, saponins, polysaccharides and phenols.This studies may further leads to the formulation of a new drug having the active compound.

Figure 1 .Figure 2 .
Figure 1.Antibacterial activity of ethanolic extract of Gymnema sylvestre in Gram positive bacteria Escherichia coli Salmonella enterica

Figure 3 .Figure 4 .
Figure 3. Antibacterial activity of chloroform extract of Gymnema sylvestre in Gram positive bacteria