Prabhat Kumar Jena et al. / Journal of Pharmacy Research 2012,5(9),4723-4725 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Physical Evaluation and Antibacterial Activity of Various Leafy Extracts of zeylanica Linn

Prabhat Kumar Jena1, Subas Chandra Dinda 1 and P. Ellaiah 2 1Department of Pharmaceutical Technology, School of Pharmaceutical Education and Research, Berhampur University, Bhanja Bihar, Berhampur – 760007, Odisha, 2Department of Pharmaceutical Technology, Jeypore College of Pharmacy, Rondapalli, Jeypore – 764002, Koraput, Odisha, India Received on:12-06-2012; Revised on: 17-07-2012; Accepted on:26-08-2012

ABSTRACT The antibacterial activity of different leafy extracts of S. zeylanica was tested by disc diffusion method, against 4 human microbial pathogens. All leafy extracts at 80 mg concentration showed zone of inhibition ranging from 03.8-12.33 mm. S. aureus was found to be highly susceptible forming highest zone of inhibition, suggesting that S. zeylanica was strongly inhibitory towards this organism. These pathogens were highly sensitive to the ethanol extract forming 10.0 to 13.0 mm zone of inhibition suggesting that the ethanol extract of S. zeylanica was more effective than other extracts against most of the microbes tested.

Key words: Antibacterial activity, Human pathogens, Tetracycline, physical evaluation, S. zeylanica. INTRODUCTION The increasing failures of chemotherapeutics and antibiotic resistance Preparation of Extract exhibited by pathogenic microbial infectious agents have lead to the screening In the extraction procedure, a total amount of 1.5 Kg dried leaves were made of several medicinal for their potential antimicrobial activity[1, 2, 3, 4]. coarse powder and were extracted with each solvent (Petroleum ether, ethyl Smilax zeylanica Linn () commonly known as Jangliashbha acetate, n-butanol and ethanol) by using soxhlet apparatus. For each sol- (Hindi) is widely distributed in Indian forests. It is a brambled, woody vine vent, 50 cycles were run. Each extract was filtered and concentrated by that grows up to 50 m long. It produces small flowers and black, blue, or red distilling the solvent to obtain the crude extract. Then each crude extract berry-like fruits which are eaten greedily by birds. Plants flower in May was dried by rotary evaporator. The successive solvent extraction of leaves and June with white/green clustered flowers. If pollination occurs, the of S. zeylanica with different solvents resulted in separation of constituents will produce a bright red to blue-black spherical berry fruit about 5-10 mm of different polarities. All solvent extracts were stored in refrigerator for in diameter that matures in the fall[5, 6]. The literature survey reveals that further studies. various parts of S. zeylanica have been used as a folklore medicine for curing various ailments like veneral diseases (root and plant); impotency, Test organisms analgesic and anthelmintic activity (leaf); as a carminative and in dropsy Various Gram-positive and Gram-negative bacteria including both standard (plant), for relief in burning sensation in the feet accompanied by vesicular and clinical isolates were used as test strains. The Gram-positive bacteria watery eruptions (plant) [7]. But reports on antibacterial activity of S. like Staphylococcus aureus (NCTC 3761) and Bacillus subtilis (NCTC 5677) zeylanica were scanty, particularly on these strains of microorganisms and were isolated from patients in Institute of Microbial Technology (IMTECH), their biochemical processes. Therefore an attempt has been made to study Chandigarh, India. The Gram- negative bacteria like Pseudomonas aeruginosa the antibacterial activity of various leafy extracts of S. zeylanica on certain (NCTC 8201) and Escherichia coli (NCTC 9001) were laboratory strains, pathogenic microorganisms. obtained from National Institute of Immunology (NII), New Delhi, India. The organisms were maintained on soybean casein digest agar (HIMEDIA) MATERIALS AND METHODS and transferred onto fresh slants on a regular basis.

Drugs and Chemicals Physical Evaluation All the chemicals were procured from different suppliers. Tetracycline (Micro Lab. Ltd., Goa), Petroleum Ether AR (60-80°C, Thomas Baker Extractive Value Chemical Pvt. Ltd, ethyl acetate and n-butanol (Loba Chemicals, Mumbai) The extractive value determination will help in evaluation of the plant prod- and ethanol AR (Merck Pvt. Ltd., Mumbai) and dimethyl sulfoxide (DMSO) ucts, specifically with reference to the solubility in different solvents. Evalu- which was used as a control. ation of drug basically needs its identification and can be done by morpho- logical or microscopically characters. Many a times, the drug identified by Plant material its diagnostic characters is of substandard in quality due to either faulty The leaves of S. zeylanica Linn were collected from local area of Baipariguda collection or incorrect storage. So to prove its acceptability as a drug extrac- (Dt. Koraput) in the month of July - August 2008. The plant was identified tive value determination can be applied, wherever possible. and authenticated by the Biju Pattnayak Medicinal Plants Garden and Research Centre, Dr. M.S. Swami Nathan Research Foundation, Jeypore, About 5gm of coarsely powdered drug was macerated with 100 ml of each Koraput (District), Odisha (Letter no. MJO8/DBT/575, date, 03.12.2008). specified solvent in a closed flask for 24 hr, shaking frequently for first six The leaves were shade dried under normal environmental condition. The hours and allowed to stand for eighteen hours. The extract was then filtered dried leaves were powered and stored in a closed container for further use. and the filtrate was evaporated to dryness in tared flat bottomed shallow dish on boiling water bath. Weight of the extractive was determined and *Corresponding author. from that the %w/w of extractive value was calculated [9, 10, 13]. Prabhat Kumar Jena Jeypore College of Pharmacy, Alcohol Soluble Extractives Rondapalli, Jeypore – 764002, About 5 gm of shade dried powdered drug was macerated with 100 ml of Koraput, Odisha, India Journal of Pharmacy Research Vol.5 Issue 9.September 2012 4723-4725 Prabhat Kumar Jena et al. / Journal of Pharmacy Research 2012,5(9),4723-4725 95% alcohol in a closed flask for twenty four hours, shaking frequently for six Antibacterial Activity hours and allowed to stand for eighteen hours. It was then filtered rapidly taking precautions against loss of alcohol. About 25ml of the filtrate was evaporated Inoculum preparation to dryness in tared flat bottomed shallow dish, dried at 1050C and weighed. The Inoculum of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus percentage of the alcohol soluble extractive was calculated with reference to the and Bacillus subtilis were prepared in nutrient broth medium, same as for broth air dried drug. dilution method and kept for incubation at 35°C for 8 hrs.

Determination of Ash Value Inoculation of Test Plates The ash value is meant for detecting low grade products, exhausted drugs and A sterile cotton swab was dipped into the turbid culture suspension. The swab sandy or earthy matter. It can also be utilized as a mean for detecting the was rotated several times and pressed firmly on the inside wall of the tube chemical constituents by making use of water soluble ash and acid insoluble above the fluid level. This will remove excess inoculums from the swab. ash. The dried surface of a Nutrient agar plate was inoculated by swabbing the swab Determination of Total Ash over the entire sterile agar surface. This procedure was repeated by swabbing Accurately about 3 gm of air dried powder was weighed in a tared silica crucible two more times, rotating the plate approximately 60°C each time to ensure an and incinerated at a temperature not exceeding 4500C until free from carbon, even distribution of Inoculum. As a final step, the rim of the agar was swabbed. cooled and weighed. Then the percentage of total ash with reference to the air dried powdered drug was calculated [9, 10, 13]. The lid may be left aside for 3 to 5 minutes, but not more than 15 minutes, to allow for any excess surface moisture to be absorbed before applying the plant Determination of Acid Insoluble Ash extract in well. The above obtained total ash was boiled for 5 minutes with 25ml of dilute hydrochloric acid. The residue was collected on an ash less filter paper and Three Wells were made on the agar surface with 8mm cork borer in each plate. washed with hot water, ignited and weighed. The percentage of acid insoluble The extracts (80 micro gram), Tetracycline (30 microgram) as standard and ash was calculated with reference to the air dried drug. DMSO (80 microliter) as a control were loaded to their respective well using sterile syringe. Determination of Water soluble Ash The total ash was boiled for 25 minutes with 25 ml of water. The insoluble The plates were incubated at 37ºC+2ºC for 24 hours for antibacterial activity. matter was collected in a Gouch crucible or on an ash less filter paper, washed The plates were observed for the zone clearance around the wells. with hot water and ignited to a constant weight at low temperature. The weight of insoluble matter was subtracted from the weight of the ash. The difference in RESULTS AND DISCUSSION weights represents the water soluble ash. The percentage of water soluble ash The inhibitory effects of Petroleum ether, ethyl acetate, n-butanol and ethanol with reference to the air dried drug was calculated. extracts of Smilax zeylanica against different test organisms are shown in Table 2 and Figure 1 . The extracts exhibited significant antibacterial activity (growth Determination of Sulphated Ash inhibition zone diameters ranging from 3 to 13 mm) against both Gram-positive About 5 gm of the drug accurately weighed and moistened with sulphuric acid, and Gram-negative bacteria. The results indicate that the ethanolic extract was ignited gently, again moistened with sulphuric acid, reignited, cooled and weighed. more potent than the other three extracts against all the micro-organism except The percentage of sulphated ash with reference to the air dried drug was Pseudomonas aeruginosa. The order of antibacterial activity was ethanol ex- calculated. tract > ethyl acetate extract > n-butanol extract > pet-ether extract. Among different organisms, Staphylococcus aureus is found to be more sensitive to Determination of Moisture Content (loss on drying) ethanolic and ethyl acetate extracts. The Petrolium ether extract was less effec- About 10 gm of drug (without preliminary drying) was accurately weighed and tive against Pseudomonas aeruginosa as it formed 3.8 mm zone of inhibition. placed in tared evaporating dish and dried at 1050C for 5 hours, and again That indicating the active principle against this organism was present in the weighed. Drying and weighing were continued at one hour intervals until leafy extracts. The physical evaluation of Smilax zeylanica shown in Table 1. difference between two successive weighing was corresponded to not more Table-1 Physical Evaluation of S. zeylanica. than 0.25 per cent. Constant weight is reached when two consecutive weightings Specification Min Max* after drying for 30 minutes and cooling for 30 minutes in a desiccator, showing (%w/w) (%w/w) not more than 0.01g difference[9, 10, 13]. Foreign matter 0.02 0.13 Alcohol Soluble Extractives 0.11 15.24 Determination of foreign matter Water Soluble Extractives 0.91 8.90 The sample (50 g) was spread in a thin layer and the pieces of foreign matter Ether Soluble Extractives 0.30 12.32 were sorted out by visual inspection. The powder of the foreign matter was Total Ash 1.22 8.23 Acid Insoluble Ash 0.3 0.7 sifted through a 250 micron sieve. All portions of the foreign matter were Water soluble Ash 3.7 4.6 [9, 10, 13] pooled and weighed . Sulphated Ash 0.6 0.9 Determination of water content Loss on drying 7.60 11.32 Water content 7.12 10.40 The powdered sample (50 g) in water-saturated toluene (200 ml) was subjected Volatile oil content - to azeotropic distillation. As soon as the water was completely distilled, the inside of the condenser tube was rinsed with toluene, and the distillation was Table-2 Antibacterial activity of various leafy extracts of Smilax continued for 5 more minutes. The heat was then removed, and the receiving zeylanica Linn. tube was allowed to cool to room temperature. The water and toluene layers Plant extracts Diameter of Inhibition zone in millimeter (mm) were allowed to separate, and then the volume of water was read off [9, 10, 13]. Staphylococcus Bacillus Pseudomonas Escherichia aureus subtilis aeruginosa coli Determination of Volatile oil content Volatile oil distillation was performed on the ground sample (100 g) in water +ve control 12.25 11.25 12.33 11.48 -ve control No zone 4.0 No zone (600 ml) using a Clevenger apparatus. When the distillation was complete, the P1 Ethanolic 12.25 12.8 No zone 12.24 heat was removed, and the receiving tube was allowed to cool to room P2 n-butanol 6.75 4.0 No zone 4.18 temperature. The volatile oil and water layers were allowed to separate, and P3 Ethyl acetate 11.0 8.5 8.0 8.98 then the volume of volatile oil was read off [9, 10, 13]. P4 Pet. Ether 4.5 5.8 3.8 5.86 Journal of Pharmacy Research Vol.5 Issue 9.September 2012 4723-4725 Prabhat Kumar Jena et al. / Journal of Pharmacy Research 2012,5(9),4723-4725

Fig– P1 Zone of inhibition of P. aeruginosa Fig– P1 Zone of inhibition of E. coli

Fig– P4 Zone of inhibition of P. aeruginosa Fig– P4 Zone of inhibition of E. coli Figure-1. Zone of Inhibition of different Bacterial species

CONCLUSION It is concluded that the ethanolic extract showed most potent antibacterial activity. The results of this study support the use of these plants for human and animal disease therapy and reinforce the importance of the ethno botanical approach as a potential source of bioactive substances.

Fig– P2 Zone of inhibition of S. aureus Fig– P2 Zone of inhibition of B. subtilis Further studies are required to identify the actual chemical constituents that are present in the crude extracts of this plant which are responsible for antibacterial activity and to establish the effectiveness and pharmacological rationale for the use of Smilax zeylanica as an antibacterial drug.

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Source of support: Nil, Conflict of interest: None Declared Fig– P4 Zone of inhibition of S. aureus Fig– P4 Zone of inhibition of B. subtilis Journal of Pharmacy Research Vol.5 Issue 9.September 2012 4723-4725