International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 2(3), 23-27, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 23 Phytochemical Screening and Antimicrobial Activity of Leaf Extract of Wrightia tomentosaPenchala Srinivas, Talari Samatha, Guguloth Valya, Azmeera Ragan and Nanna Rama Swamy2*Department of Botany, Kakatiya University, Warangal-506009, AP, INDIA Department of Biotechnology, Kakatiya University, Warangal-506009, AP, INDIAAvailable online at: www.isca.in Received 20th December 2012, revised 2nd January 2013, accepted 2nd February 2013Abstract Phytochemical screening and antimicrobial activities of solvent extracts of leaf of Wrighitia tomentosa (Roxb.) Roem and Schult. have been studied for the presence of secondary metabolites and to find out their activity against microbes. The results revealed that the aqueous, chloroform, ethyl acetate, petroleum ether and methanolic leaf extracts of W. tomentosa contain alkaloids, ellagic acids, iridoids, methylene dioxy compounds, steroids, tannins and triterpenoids. The antimicrobial activity of these leaf extracts were tested against gram positive and gram negative bacteria through well diffusion method. Among all the extracts of leaf the highest sensitivity was recorded for methanolic, ethyl acetate and chloroform extracts. None of the aqueous extract exhibited any antimicrobial activity. The results provided evidence that the species W. tomentosa can be used as a potential source of antimicrobial agent. Keywords: Phyotochemical screening, antimicrobial activity, leaf extract, zone of inhibition. Introduction Plants have been the basis of traditional medicines throughout the world for thousands of years and continue to provide new remedies to mankind. Aromatic and medicinal plants are known to produce certain bioactive molecules which react with other organisms in the environment inhibiting bacterial or fungal growth2,3. About 30% of drugs used worldwide are based on natural products. Emergence of multiple drug resistant strains of microorganisms due to indiscriminate use of antibiotics to treat infectious diseases has generated a renewed interest in herbal medicine. The increasing prevalence of multi-drug resistant strains of bacteria and the recent appearance of strains with reduced susceptibility to antibiotics raises the spectre of untreatable bacterial infections and adds urgency to the search for new infection combating strategies and new effective therapeutic agents. The medicinally important secondary metabolites produced by plants offer a new source of novel drugs that can act against many diseases and microbes. The phytochemical research based on ethno-pharmacological information is generally considered an effective approach in the discovery of new anti-infective agents from higher plants. The results of different studies provide evidence that some medicinal plants might indeed be potential sources of new antibacterial agents even against some antibiotic resistant strains. One among such plants possessing a number of biologically active compounds is Wrightia tomentosa (Roxb.) Roem and Schult.(Apocynaceae) an endangered medicinal tree with many medicinal uses. W. tomentosa is widely used to treat stomach ache, tooth ache, fever, hemorrhage, arthritis and snake bite. The purpose of the present study was to investigate the presence of phytochemicals and antimicrobial properties of W. tomentosa. In this research article we report the results of antimicrobial activity of leaf extracts on bacterial pathogens in order to orient future investigations towards the finding of new, potent and safe antimicrobial compounds. The antimicrobial activities of plant extract can be determined by various methods such as disc diffusion, agar well diffusion and twofold serial dilution techniques. The agar well diffusion technique for screening of the antimicrobial activity of medicinal plant is normally considered10. Our present investigation envisages the collection, identification, extraction and antimicrobial evaluation of W. tomentosa. Material and MethodsCollection and Preparation of plant material: Fresh leaves of W. tomentosa were collected from Kakatiya Arboretum, Department of Forest Research and Development, Warangal (AP), India. The collected plant material was washed thoroughly under running tap water followed by distilled water to remove surface contaminants and was shade dried at room temperature for two weeks. The dried plant material was ground into fine powder using an electric blender and stored in air tight containers at ambient pressure until further use.Extraction of the plant material: The powdered plant material (250g) was placed in Soxhlet apparatus. Then materials were extracted with different solvents such as petroleum ether, International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 23-27, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 24 chloroform, ethyl acetate methanol (MeOH) and water at 72 cycles (8 h per day for 9 days) and when the solvent was drained colourless, the extraction process was stopped. The extracts were filtered using Whatman No.1 filter paper and then concentrated in vaccum at 40ºC using a rotatory evaporator. A semisolid mass is obtained for analytical study.Phytochemical screening: The phytochemical tests were performed to find out the presence of biologically active chemical constituents such as alkaloids, ellagic acid, iridoids, lignans, methylene dioxy compounds, steroids, tannins and triterpenoids by following the procedures given below.Alkaloids: For testing the presence of alkaloids, a small quantity of the methanolic extract of the plant was taken into a test tube followed by the addition of 1% HCl and tested with Dragendorff’s and also with Mayer’s reagents. When a precipitate forms or when the solution turns turbid, the reaction was considered as positive for the presence of alkaloids. Ellagic acid: For the detection of ellagic acid, the methanolic extract was treated with a few drops of each of 5% acetic acid and 5% sodium nitrate solution. The extract turns yellow olive brown, niger brown or deep chocolate, depending upon the amount of ellagic acid present. Iridoids: 1ml of Trion-Hill reagent was added to the concentrated methanolic leaf extract. It was then heated for a few minutes. The presence of iridoids was inferred when the solution turned to blue-green or red. Lignans: Using Badouni test, the presence or absence of lignans was determined. In this process, 2% furfuraldehyde was added to 2ml of methanolic extract when this was acidified with HCl, the development of red colour was conceived as positive reaction for the presence of lignans. Methylene-dioxy compounds: Gallic acid was added to the methanolic extract of leaf acidified with few drops of concentrated HSO. The development of blue green colour was the indication for the presence of methylene dioxy compounds. Steroids: Salkowski test was used to detect the presence or absence of steroids. Conc.HSO and chloroform were added to the methanolic leaf extract in a test tube. When the wine red colour developed, the reaction was considered as positive and the presence of steroids was inferred. Tannins: The presence of tannins was detected by adding 1% potassium dichromate solution to equal volume of concentrated methanolic extract of leaf, the development of generous precipitate is considered to be positive for the presence of tannins. Triterpenoids: Libermann-Burchard11,12 test was adopted to know the presence of triterpenoids and steroids. Conc.HSOwas added to the mixture of methanolic leaf extract and acetic anhydride. Development of bluegreen colour was the indicative for the presence of triterpenoids. Antimicrobial activity: The antimicrobial activity was carried out by agar well diffusion method. For this, the different solvent extracts of leaf of W. tomentosa have been tested against gram positive and gram negative bacteria to find out the antibacterial activity. A nutrient broth was prepared (beef extract-3gm/L; Peptone-5gm/L; NaCl-5gm/L; Agar-16gm/L) and then sterilized in an autoclave at 15lb pressure for 15 minutes. The sterilized medium was poured into petridishes. The solidified plates were bored with 6mm diameter sterilized cork borer. The plates with wells were used for the antibacterial studies. Various solvent extracts of leaf of W. tomentosa were subjected to individual microbiological tests to ascertain their antimicrobial activity against six species of microorganisms, Bacillus cereus, Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium for their antimicrobial activity. The evaluation of antimicrobial activity of the extracts was done by observing the clear zone around disks and measuring the diameter of the zone of inhibition (ZI) exhibited by the extracts around disks in mm by sliding digital vernier caliper. Well diffusion method: Antibacterial activity of various solvent extracts of leaf of W. tomentosa was tested using agar well diffusion method13,14. The bacterial strains (Streptococcus sp., B.cereus, A. hydrophila V.cholerae and MRSA33) were inoculated in the nutrient broth under aspectic condition and incubated at 37C for 18 hours forantimicrobial analysis of Root extracts of Pseudarthria viscida Wight and Arn and Desmodium gangeticum (Linn) DC15. Wells were made on the agar surface with 6 mm cork borer. The extracts were poured into the well using sterile syringe. The plates were incubated at 37ºC±2ºC for 24 hours for bacterial activity. The plates were observed for the zone clearance around the wells. Gentamycin was also used as standard antibiotics. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well (in mm) including the well diameter. Results and DiscussionThe phytochemical constituents and the percentage yield of extracts prepared from leaf extracts of W. tometosa are shown in tables 1-2. The methanolic leaf extract screened for the presence of various biologically active constituents were responded positive for the alkaloids, ellagic acid, iridioids, lignans, methylene dioxy compounds, steroids, tannins and triterpenoids. Zaki et al16 have also reported to possess unsaturated sterols, terpenes, flavonoids, phenolic acids and alkaloids. Similarly phytochemical constituents were also observed from different solvent extracts of leaf and bark extracts of W. tometosa17. During the present investigations the highest yield was found in petroleum ether, methanol and aqueous extracts of leaf followed International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 23-27, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 25 by ethylacetate and chloroform (table-2). The difference in the yield of extraction products may be dependent on the polarity difference of solvents used for extraction, solubility of various ingredients, and type of extraction method used18. The antimicrobial potential of leaf extracts was tested against three gram positive and three gram negative bacterial pathogens viz., B. cereus, S. aureus, B. subtilitis and E. coli, K. pneumonia, S. typhimurium respectively (table-3). The ZI was highest in chloroform extracts against E. coli, followed by S. typhimurium, B. subtilitis, S. aureus, K. pneumoniae and B. cereus. Ethyl acetate extract showed highest ZI against S. aureus, followed by B. subtilitis, E. coli, S. typhimurium, S. aureus and B. subtilis, aqueous extract has recorded lowest ZI against S. aureusfollowed by K. pneumonia, and S. typhimurium and showed zero against B. cereus, B. subtilis, E. coli. The gram positive bacteria were more sensitive to methanol (S. aureus) and chloroform (B. subtilitis) extracts. The higher sensitivity of gram-positive bacteria could be attributed to their outer peptidoglycan layer which is not an effective permeability barrier19 Cinnamomum zeylanicum essential oil is mainly composed in cinnamaldehyde,cinnamyl acetate and cinnamyl benzoate. This composition canjustify the significant antimicrobial activities observed20. The secondary metabolites detected in different solvent extracts could be responsible for the observed antimicrobial activity. These metabolites have been shown to be responsible for various therapeutic activities of medicinal plants21. ConclusionIn conclusion, the present investigation of phytochemical screening and antimicrobial activity of leaf extracts of W. tomentosa reports that the various solvent extracts of leaf contains biologically active secondary metabolites which showed the antimicrobial activity against bacterial pathogens of both gram positive and gram negative. Thus it can be employed as an antibacterial agent against many bacterial diseases. Further investigations on other medicinal activities are in progress. Table-1 Showing various secondary metabolites present in the methanolic leaf extract of W.tomentosaLeaf extract Alkaloids Ellagic acid Iridoids Lignans Methylene dioxy compounds Steroids Tannins Triterpenoids W.tomentoa +ve +ve +ve +ve +ve +ve +ve +ve Table-2 Showing the percentage yield of different solvent extracts of leaf of W.tomentosaPlant extract Weight of raw material (gm) Weight of the extract (gm) Percentage of yield Petroleum ether 250 20 8.0 Methanol 250 20 8.0 Ethyl acetate 250 18 7.2 Chloroform 250 15 6.0 Aqueous 250 20 8.0 Table-3 Data showing antibacterial activity of leaf extracts of W.tomentosa by agar well diffusion methodName of the solvent Test microorganism and zone of inhibition (in mm) +ve Bacteria Test microorganism and zone of inhibition (in mm) –ve Bactria Leaf Extract Bacillus cereus Staphylococcus aureus Bacillus subtilis Escherichia coli Klebsiella pneumoniae Salmonella typhimurium Chloroform 8 9 11 15 9 13 Pet-ether 4 2 3 4 9 7 Ethyl Acetate 3 8 12 10 3 10 Methanol 5 14 9 9 13 10 aqueous - 3 - - 2 2 Gentamycin (Control) 18 18 19 20 20 20 International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 2(3), 23-27, March (2013) Int. Res. J. Biological Sci. International Science Congress Association 26 Figure-1 Showing the zone of inhibition of Bacillus cereus against various solvent extracts of leaf of W. tomentosaFigure-2 Showing the zone of inhibition of Staphylococcus aureusagainst various solvent extracts of leaf of W. tomentosaFigure-3 Showing the zone of inhibition of Bacillus subtilis against various solvent extracts of leaf of W. tomentosaFigure-4 Showing the zone of inhibition of Escherichia coli against various solvent extracts ofleaf of W. tomentosaFigure-5 Showing the zone of inhibition of Klebsiella pneumoniaeagainst various solvent extracts of leaf of W. tomentosaFigure-6 Showing the zone of inhibition of Salmonella typhimuriumagainst various solvent extracts of leaf of W. tomentosa AcknowledgementWe thank the University Grants Commission, New Delhi for providing the funds under UGC-SAP-DRS-I program to the Department of Biotechnology, Kakatiya University, Warangal, India. 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