INTERNATIONAL JOURNAL OF RESEARCH ISSN NO : 2236-6124

BIOSYNTHESIS AND ANTIBACTERIAL ACTIVITY OF CHEMICALLY MODIFIED STEVIA REBAUDIANA Merlin M1*, Chitra S1 1Department of Chemistry, Jeppiaar Engineering College, Chennai, India *Corres.author: [email protected]

Abstract Plants have been an important source of secondary metabolites known for their diverse biological activities; some have been shown to inhibit the development of certain pathogenic microorganisms. The need to explore novel antimicrobial drugs for the worldwide spread of drug resistant microbes is of supreme significance. Bioactive compounds extracted from plants were traditionally used as a source for medicines. The phytochemicals obtained from plants have exhibited antibacterial, antioxidant, anti-inflammatory and antimicrobial characters. Stevia Rebaudiana is an outstanding herb bearing leaves of very refreshing sweet taste and remarkable health promoting factors. S. Rebaudiana containing biologically active substances was studied widely due to its antibacterial and antimicrobial activities. S. Rebaudiana Bertoni produces diterpene glycosides that are low calorie sweeteners, and having therapeutic properties with antimicrobial activity. The aim of the present study was to characterize the biological activity of extracts prepared from dried S. Rebaudiana leaves with potential application to antibacterial activity. S. Rebaudiana was chosen, since it possesses potent antibacterial properties against gram positive bacteria and gram negative bacteria. Antibacterial activity of the sample was determined by well diffusion method on Muller Hinton agar (MHA) medium. The chemically modified S. Rebaudiana extract with glycine and SnCl2 shows antibacterial activity in close proximity to that of standard antibiotic. Keywords: Phytochemicals, S. Rebaudiana, antibacterial activity, well diffusion method, gram positive, gram negative bacteria.

1. INTRODUCTION

Medicinal plants are of great interest to the researchers in the field of biotechnology as most of the drug industries depend on plants for the production of pharmaceutical compounds[1]. The bioactive compounds obtained from plants have attracted researchers to discover drugs for medicinal applications[2]. It can be either directly extracted from plants or can be synthesized from plant sources. Hence the therapeutic values of plant extracts as well as phytochemicals had enlightened the importance in all times due to the presence of diverse range of bioactive molecules[3,4]. These molecules were traditionally used as the source for different types of medicines[5]. Most of the drugs used today in the traditional systems are obtained from natural sources or from synthetic derivatives of natural products[6]. The chemical substances present in plant extracts and its derivatives can act in human body physiologically. Particularly, alkaloids, tannins and flavonoids obtained from plants have been used as biologically active compounds[7.8]. Moreover the flavonoids and other biologically active molecules obtained from plants were reported as an antibacterial, antioxidant, anti- inflammatory and antimicrobial characters[9-12]. The reports of phytochemicals obtained from plants have even proved to act as anticancer agents[13.14]. Asteraceae is a widely distributed plant family reported to contain biologically active molecules[15-17]. Stevia Rebaudiana belongs to Asteraceae family commonly referred as honey leaf or sweet leaf was found to contain biologically active substances such as terpenes, flavonoids and phytochemicals such as austroinullin, β-carotene, dulcoside, nilacin, rebaudi oxides, riboflavin, steviol and stevioside[18]. It has been commercially used in energy drinks[19,20]. Leaves of this plant produce zero-calorie, a non-nutritive, high potency sweetener and substitute to sucrose[21]. A renewed interest of Stevia Rebaudiana being used as antibacterial activity emerged in the last

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decade[22]. S. Rebaudiana was chosen, since it possesses potent antibacterial properties against gram positive bacteria and gram negative bacteria. Here we have focused on the synthesis of modified derivative extract from the plant source. The antibacterial activity of the prepared S. Rebaudiana derivative was evaluated against bacteria such as Klebsiella pneumoniae and Staphylococcus aureus.

2. MATERIALS AND METHODS

Plant source The fresh leaves of S. Rebaudiana were received from nurseries in Moonar. The leaves were taxonomically identified and washed with sterile water, dried under shade. It was powdered well before extraction. Extract preparation The aqueous extract was obtained by the maceration technique, and 10g of the powder was added in 100 mL of distilled water. It was then boiled to 80oC in a magnetic stirrer at approximately 220 rpm for an hour and then cooled. The supernatant was used as extract. The crude extract was filtered twice, first with a Whatman No. 5 paper filter, and then with a Whatman No. 1 paper filter. The extract was stored in tagged, sterile amber bottle in the refrigerator for its later analysis. Preparation of modified S. Rebaudiana extract 10 ml of the aqueous extract of S. Rebaudiana was mixed with 500 mg of glycine solution in 100 ml beaker. The reaction mixture was heated to about 80°C in the magnetic stirrer for two hours. The green coloured extract was changed into dark brown coloured solution. Dark brown coloured crystals (SM1) were obtained by recrystallization. 50 ml of aqueous SnCl2 solution (0.02M) was taken in a 100ml beaker. It was stirred in a magnetic stirrer at 80°C for two hours by adding glycine stepwise till the solution reaches super saturation. The greenish coloured solution changed into light yellow coloured solution and the solution was filtered. Yellow coloured crystals (SM2) were obtained after recrystallization. To the 10 ml of aqueous extract of S. Rebaudiana, 45mL of SnCl2 solution (0.02M) was mixed in a magnetic stirrer and heated to about 80°C for two hours in 100 ml beaker. Glycine was added stepwise till the solution becomes supersaturated. Then the mixture was heated and glycine was completely soluble and then stirred for about 2 hours. The yellow solution obtained was kept for slow evaporation and pale yellow crystals (SM3) were obtained after recrystallization. Test Bacterial Species The bacterial species were obtained were maintained on nutrient agar media. And the antibacterial activity of S. Rebaudiana species such as SM1, SM2 and SM3 were synthesized chemically (vide infra). Preparation of Inoculum Stock cultures were maintained at 4°C on slant of nutrient agar. Active cultures for experiments were prepared by transferring a loop full of cells from the stock cultures to test tubes of nutrient broth for bacteria that were incubated at 24hrs at 37ºC. The Assay was performed by agar disc diffusion method. Agar disc diffusion method Antibacterial activity of the sample was determined by well diffusion method on Muller Hinton agar (MHA) medium. The Muller Hinton Agar medium was weighed as 3.8gms and dissolved in 100ml of distilled water and 1gm of agar was added. Then the medium is kept for sterilization. After sterilization the media was poured in to sterile petriplates and were allowed to solidify for 1hr. After the medium was solidified, the inoculums were spread on the solid plates with sterile swab moistened with the bacterial suspension. Wells were cut on these plates and loaded with 20 µl sample of respective concentrations (1000 µg, 500 µg, 250 µg, 125 µg) , negative control 20 µl of DMSO and positive control 10 µl (10 µg) streptomycin. These plates were incubated for 24 hrs at 37ºc .Then the microbial growth was determined by measuring the diameter of zone of inhibition. Four replicates of each treatment were carried out. The mean and standard deviation of the data obtained were

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determined by statistical tool.

Antibacterial Activity No. of Microorganism: 2 (Klebsiella pneumoniae, Staphylococcus aureus ) Sample concentration: 1000 µg, 500 µg, 250 µg, 125 µg Negative control: DMSO Positive control: Streptomycin (disc.10 µg)

Zone of Inhibition in mm Microorganisms S.No 1000 µg 500µg 250µg 125µg DMSO Streptomycin 10µg Klebsiella pneumoniae 1 Sm1 20 18 14 - - 22 2 Sm3 21 15 13 - - 22 3 16 14 12 12 - Sm2 22

Staphylococcus aureus

1 Sm1 21 21 16 - - 23 2 Sm3 23 18 15 - - 23 3 Sm2 20 15 13 10 - 23

Figure 2: Graphical representations of antibacterial activity of SM1, SM2 and SM3 against Klebsiella pneumoniae and Staphylococcus aureus

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3. RESULTS AND DISCUSSION From the literature, it is clear that the ethanolic extract of Stevia Rebaudiana show zone of

inhibition 11.2mm for Klebsiella pneumoniae and 5mm for Staphylococcus aureus but the aqueous extract of S. Rebaudiana show zone of inhibition 12mm and 9.3mm repectively[23,24]. Further Miranda-Arambula et al proves clearly the effect of the aqueous extract of S. Rebaudiana is maximum in comparison with other solvents like ethanol, CCl4, C6H6. Minimal inhibitory concentration (MIC) is maximum for aqueous extract of S. Rebaudiana Bertoni for different species of bacteria[25]. Hence we have chosen the aqueous extract for further modification. In the present work, aqueous extract of S. Rebaudiana was chemically modified by adding glycine and SnCl2. The chemically modified aqueous extract of S. Rebaudiana species synthesized was shown to have good inhibitory effects on microbial organisms such as Klebsiella pneumoniae and Staphylococcus aureus. The three chemically modified extracts shows different degree of antibacterial activity against Klebsiella pneumoniae and Staphylococcus aureus in comparison with the extract prepared from the plant[26]. The results shown in table prove that the chemically modified forms show antibacterial activity close proximity to that of the standard antibiotic streptomycin. It was found that the antibacterial activity of modified S. Rebaudiana is much higher than the plain plant extract and found to be closer to the standard antibiotic. Glycine stabilizes the plant extract and acts as a good complexing agent and thereby increases the antibacterial activity from 11 to 14 in 250 µg concentration. As the concentration increases the activity against Klebsiella pneumoniae increases to 18 and 20 in 500 µg and 1000 µg respectively, which is in very close proximity with the standard antibiotic. Similarly, the inhibition against Staphylococcus aureus was also found to increase with glycine addition and with the increase in concentration. Addition of SnCl2 with glycine and plant extract also has the same effect as the addition of glycine. Comparing the antibacterial activity results of SM2 and SM3, it is confirmed that S. Rebaudiana has a potent antibacterial activity. Further it is proven from the results that the glycine modified S. Rebaudiana extract (SM1) shows higher antibacterial activity than the SnCl2 modified extract (SM2). 4. CONCLUSION

The chemically modified form of the aqueous extract of S. Rebaudiana species was synthesized. The chemically modified S. Rebaudiana extract shows antibacterial activity in close proximity to that of standard antibiotic. Further the glycine modified S. Rebaudiana extract has higher antibacterial activity than the others. The increase of antibacterial activity with concentration is also confirmed. 5. ACKNOWLEDGEMENT

We thank the management of our college for providing the facilities and requisite support for this research work. We thank our department staff for their continuous support and encouragement. Special thanks to our family members.

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