Phytochemical Screening and In Vitro Antibacterial Activity ofCrude Extracts from Andropogon aciculatus retz. (Poaceae) by ELYROSE KIM C. RUIZO A research paper submitted to the Division of Natural Sciences and Mathematics University of the Philippines Visayas Tacloban College, Tacloban City As partial fulfillment of the requirements for the Degree of B.S. BIOLOGY April 2013 Permission is given for the following people to have access to this research: Available to the general public Available only after consultation with author/adviser Available only for those bound by confidentiality agreement Student’s signature: Signature of Research Adviser: Yes No No This is to certify that this research paper, entitled: “Phytochemical screening and in vitro antibacterial activity of crude extracts from Andropogon aciculatus Retz. (Poaceae)” and submitted by ELYROSE KIM C. RUIZO to fulfill part of the requirements for the Degree of Bachelor of Science in Biology is hereby endorsed. IRENE L. TAN Research Adviser The Division of Natural Science and Mathematics (DNSM) accepts this research paper in partial fulfillment of the requirements for the Degree Bachelor of Science in Biology. ROBERTO E. CAPON DNSM Chair ACKNOWLEDGEMENTS First of all, I would like to thank our Heavenly Father, for giving me this challenge that has become a lesson in my life and also for keeping me strong and determined which led me to do all things possible. I would also like to extend my heartfelt gratitude to all the people who were there to help me make this research study a success: To my Mama Rodelle and the rest of my family, who have always believed that I can still make it through despite all the challenges that I may have encountered. Thank you for your support, guidance, motivation and also for being an inspiration; To my adviser, Prof. Irene L. Tan, for having the patience to keep on motivating and guiding me all through out the conduct of my study; To Prof. Marjhun Ricarte and Kenneth, for assissting me on the use of the rotary evaporator, thereby allowing me to move on with this research; To Ate Gen and Kuya Rey, for not only accommodating my requests regarding the use of equipments, reagents, and glasswares during the conduct of my study, but also for motivating me to finish my study; To my BioHaniti family, for the support, encouragement, and advices you have given me; To all the scientists, for having the time and effort to respond to my request for reprints; and, Lastly, I offer all of this in memory of my Papa Ely, who continues to be an inspiration of my family. aciculatus showed antimicrobial activity against the gram-positive bacterium. subtilis was at 23. the recorded mean MIC of the methanol extract against B. aureus. Furthermore. Results revealed that A.ABSTRACT The study was conducted to screen for phytochemicals present and the antimicrobial activity in methanol and n-hexane extracts of Andropogon aciculatus. subtilis was at 25 g/L. . and phytosterols while the n-hexane extract only contains tannins. phenols. aciculatus against the gram-positive bacteria.4 mm. B.1±2. Streptomycin (200 mg/L) served as the positive control and sterile distilled water as the negative control. P. terpenoids. The mean diameter of the ZOI±SD of the methanolic extract against B. The mean diameter of the zone of inhibition (ZOI) was then recorded. B. aeruginosa and S. and the gram-negative bacteria. marcescens. Preliminary phytochemical screening of the plant revealed that the methanol extract contains phytochemicals such as saponins. aciculatus can be a potential source of antimicrobial compounds.Bauer method of disc diffusion susceptibility test was used to evaluate the antimicrobial activity of the crude extracts of A. subtilis and S. terpenes. Kirby. subtilis. tannins. and phytosterols. This method was done only to the extract that showed antimicrobial activity. Broth macrodilution method was conducted for the minimum inhibitory concentration (MIC) determination. Only the methanol extract of A. ................................................................................................................... 6 Methodology …………............... vi List of Tables .................................................. iv List of Figures ............................................ 10 Test for Alkaloids …………………………………………………………………............................................................................................................................................................................................ 9 Preparation of Plant Material ……………………….......................................................................................... 10 Test for Flavonoids ………………………………………………………………...................... ...................................... vii Introduction ....................................................................................... 11 ... 1 Review of Literature .................................................... 9 Preparation of Extracts ………................................... iii Abstract ............. 3 Biology and Importance of Andropogon aciculatus Retz......... 10 Test for Glycosides ………………………………………………………………… 10 Test for Saponins …………………………………………………………………..... 3 Antimicrobial Activity Testing Protocol ................................................................................................. 9 Preliminary Phytochemical Screening ………………………………………………....................................................................... 3 Antimicrobial Activity of Plants ………………….................................................................................TABLE OF CONTENTS Page Acknowledgements .......................................................................................................................... ...................................................................................... 13 Minimum Inhibitory Concentration Determination ..Test for Taninns ……………………………………………………………………................................................................................................................................................................................................ 20 Conclusion ………................................................. 15 Results ……................................................ 23 Recommendations ...................... 16 Discussion ................................................................................... 24 Literature Cited …........................................................................................................................................................................................ 25 Appendix …………………………………………………………………………………................ 30 .................. 12 Preparation of Bacterial Cultures .. 11 Test for Terpenoids ………………………………………………………………… 12 Test for Phytosterols ………………………………………………………………....................................................... 11 Test for Phenols ……………………………………………………………………......... 14 Data Analysis ....................................................................................... 12 Antimicrobial Activity Assay .......................................................................... .............................. aciculatus ............LIST OF FIGURES Figure 1 2 Page Antibacterial assay results showing the ZOIs of the methanol and n-hexane extracts of A......................... aciculatus against B.................... MIC assay results showing the MIC of the methanolic extract of A......... subtilis ……................................................................... 18 19 ........................ ...... 30 ............................................................LIST OF TABLES Table 1 Page 2 Preliminary phytochemical analysis of the methanol and n-hexane extracts of A.............................................. 3 T-test Analysis: Two-Sample Assuming Equal Variances ………………………..................................... 17 aciculatus...... 16 Antimicrobial activity of the methanol and n-hexane extracts of A.................................. aciculatus .............................................. Researchers continue to screen for antibacterial properties of plants (Islam et al. 2008. 2008). Some of these compounds include the secondary metabolites such as phenols. Nakamura. & Vasuki. Padhi. alkaloids.d). 2007. Sri Vijaya Kirubha. & Dias Filho. terpenoids and essential oils. Pathak. 2006) using different solvents for extraction (Hidayathulla. n. 2008. lectins and polypeptides. flowers. . A phytochemical study of the A. Medicinal uses of these plants have been a form of treatment known to humans (Islam. 2010). usually in open places (“Chrysopogon aciculatus”. Senthamarai. commonly known as „amor-seco‟ or love grass belongs to the family Poaceae. It is a common weed that can be found throughout the Philippines. Panda. 2008). aciculatus‟ flowers conducted by Chua (1978) suggested substantial amounts of sterols and terpenes present in the sample.INTRODUCTION Plants have been widely used as a healing purpose since the ancient times (Cowan. 2000) have been reported to have antimicrobial activity. roots. and polyacetylenes (Cowan. Islam et al. Marino. et al. & Chandrashekar. Tanaka. stems. & Dutta. Barua. & Ahmed. aciculatus has been used as a traditional medicine in the Philippines and in other countries (“Amor-seco”.. Germano. Those compounds responsible for antibacterial activity can be evaluated by conducting phytochemical analysis of the plants used.. Selvadurai. Andropogon aciculatus Retz. D'Angelo. ElMahmood & Doughari. Das.. This plant is famous for its seeds adhering to trousers and dresses. 2011. Some are also extracted with the use of the entire plant (Masoodi. 2007). 2008. and fruits (Parekh & Chanda. de Oliveira. Satapathy. Sterols (Ragasa & Lim. da Silva. Saraswathy. Extraction of plants can be done from different parts such as leaves. & Vora. 1999). 2011). & Cannatelli. A. 1999). Chandra. 2005) and terpenes (Nostro. 2011. Khan. 3. . aciculatus Retz. extracts against selected diseasecausing bacteria such as Bacillus subtilis. to determine the antibacterial activity of A. extracts using broth macrodilution method. Staphylococcus aureus. Pseudomonas aeruginosa. aciculatus Retz. and. to screen for the phytochemicals present in A. to determine the minimum inhibitory concentration of A. methanol and n-hexane extracts.These are the main objectives of the study: 1. and Serratia marcescens using Kirby-Bauer method. 2. aciculatus Retz. aciculatus has been used in traditional medicine. In India. decoction of the roots of A. ashes of the roots are used for rheumatism (“Amor-seco”. In the Philippines. It is a common weed that can be found throughout the Philippines.). Andropogon aciculatus Retz.d. leafy perennial grass. with short horizontal stems. A. Results showed a significant . which belongs to the family Poaceae. it is used as a poison antidote.LITERATURE REVIEW Biology and Importance of Andropogon aciculatus Retz. was done against Escheria coli. n.d. aciculatus has been used as an antidiarrheal alternative and the decoction of the entire plant served as a diuretic (“Amor-seco”. The inflorescence measures 7-10 cm long with numerous slender branches. Cynodon dactylon (Bermuda grass). The leaf blades measure 3-12 cm long and 5 mm wide. creeping and branching below. usually in open places (“Chrysopogon aciculatus”. Staphylococcus aureus & Streptococcus pyogenes. is a dense.). in vitro evaluation of antibacterial activity of the crude extract from the whole plant. And in Malaysia. 2005). n. (2011). commonly known as amor-seco or love grass. In Indonesia. 2007). In the study of Kumar et al. This plant is also used as an alternative medicine in other Asian countries. the rhizome is also pounded as a cure for stomachache and other gastric disorder (Mitra and Mukherjee. Antimicrobial Activity of Plants Antimicrobial activity of some plants from the family Poaceae has already been evaluated. . 2011. Other studies (Hindumathy. cosmetic.antibacterial activity against the test bacteria. & Ebibeni. 2006). The antimicrobial activity of plants has been a great focus because of substances. 2011). The study conducted by Jananie and Vijayalakshmi (2011) on the determination of bioactive components of C. also known as lemongrass. Singh. Singh. Results revealed an effective antimicrobial activity on the selected microorganisms that suggested the plant‟s essential oil as a safe natural food preservative. Pseudomonas aeruginosa.. citratus. Proteus vulgaris) and two gram-positive bacteria Bacillus subtilis and Staphylococcus aureus at four different concentrations (1:1. pharmaceutical and insecticide industries (Negrelle & Gomes. Singh. Vazirian. which have versatile applications. biological experiments of plant extracts can be done to ensure its efficacy and safety. This activity was accounted for the presence of alkaloid and phenols based from the phytochemical analysis done on the plant (Hindumathy. 2011. et al. 1:5. in the study of Vazirian et al. 1:10 and 1:20) using disc diffusion method. There must be compounds present in a plant which make it a potential antimicrobial agent. citratus essential oil was tested against food-borne pathogens. Thus. C. citratus extracts showed a significant antibacterial activity against four gram-negative bacteria Escherichia coli. (2012). has been widely used as a traditional medicine and its essential oil is used in food. the antimicrobial activity of C. These are just some important factors so that these plant extracts will be accepted as valid medicinal agents.. 2007). 2012) that involve the screening for antimicrobial activity of Cymbopogon citratus (Poaceae) were conducted. C. derived from it (Baris et al. dactylon has revealed components that can justify the antibacterial activity of this plant. Cowan (1999) described some of the major compounds known as the secondary metabolites that contributes to the antimicrobial property of a plant. Furthermore. Klebsiella pneumoniae. (2000). which is to tan leather or precipitate gelatin from solution (Cowan. “Tannin” is the term given for a group of substances capable for astringency. In a study conducted by Ignacimuthu. & Dhanabalan. 1999). tannins.butanone and other terpenoid contents were . the components of essential oil from spikes and stems of Artemisia annua. These are phenols containing one carbonyl group (Cowan. octanal and 1. were identified using chromatography–mass spectrometry (GC/MS). which are examples of the flavonoid variants. These are secondary metabolites that are highly enriched in compounds based on an isoprene structure. 6-(4. & Liu (2011). which belong to the phenol group. Compounds of pharmacological interest specifically. 7 dihydroxy-heptyl) quinone. 1999). were isolated from the plant as active constituents against Staphylococcus aureus. 2009). & Muthu (2009) the active extract present in Pergularia daemia leaves was separated by column chromatography and one fraction yielded a new compound. Raja. Cinnamic and caffeic acids are common representatives of this wide group of compounds. As described by Cowan (1999). an aerial plant in China. which showed antibacterial activity against some pathogenic bacteria. 4-Diphenyl-2. It was found out that compounds from the spike oil such as piperitone. In the study of Li. which have aromatic rings with two ketone substitutions and is naturally highly reactive (Cowan. are tannins. Duraipandiyan. Other compounds are the flavonones. Pavunraj. apigenin and luteolin. Hu.One of the major compounds consists of a single substituted phenolic ring known as the phenols. Zheng. were isolated from Solanum trilobatum Linn and assayed against some bacteria have exhibited antibacterial activity (Doss. Mohammed Mubarack. One compound that belongs to this class includes the quinones. another major group of compounds are essential oils and terpenoids. 1999). Other compounds. Zhu. In the study conducted by Sato et al. & Palmery. & Okoh. For the broth dilution method. The assay is done on a medium. the broth and agar dilution. Another major group of compounds discussed by Cowan (1999) is the heterocyclic nitrogen compounds called alkaloids. (2001) for in vitro AST. which is broth for broth dilution and agar for agar dilution with dissolved specified antimicrobials (Manual of antimicrobial susceptibility testing. and bacteriostatic activity in liquid medium by MIC values. βhydrastine. are dilution and diffusion methods. in ethnopharmacology research. (Ranunculaceae) was evaluated by measuring the “killing time” on a low density bacterial inoculum. Two major methods discussed by White et al. the tubes are observed for visible bacterial growth by comparing the turbidity to the positive and . Results showed that the plant can be a possible traditional medicine (Scazzocchio. Bactericidal activity of the major alkaloids (berberine. canadine and canadaline) isolated from Hydrastis canadensis L. two-fold serial dilutions of the substance to be assayed are prepared and transferred into the base medium. Antimicrobial Activity Testing Protocol The antimicrobial susceptibility test (AST) is an essential technique used in pathology to determine resistance of microbial strains to antimicrobials. Cometa. Tomassini. 2005). 2001). this technique is used to determine the efficacy of antimicrobials against microorganisms (Ncube. For the dilution method there are two different techniques.higher than that from the stem oil which has revealed a more active antimicrobial property of the spike oil than that of the stem oil. Also. Tubes containing the medium and dilutions are inoculated with a standardized bacterial suspension of 1-5 x 105 CFU/mL After an overnight incubation at 35 °C. Afolayan. 2008). Mueller-Hinton agar (MHA) medium is the only susceptibility test medium that has been validated by the committee (EUCAST. MIC of an antimicrobial agent is the lowest concentration of the antimicrobial agent that inhibits the growth of bacterial isolate in the test system (Manual of antimicrobial susceptibility testing. the famous Kirby-Bauer method has been recommended by the National Committee for Clinical Laboratory Standards (NCCLS). & Smânia Jr. Clear white zones on the TLC plate indicate antimicrobial activity of the extracts. Sharma. however. 2008). Desired volumes of the plant extract are placed into the wells (Valgas. This method is same as the disc diffusion. & Batra.. Rani. 2008). This method is utilized as a preliminary phytochemical screening for the . de Souza. 2010).. 2010). wells between 6 and 8 mm are aseptically punched on the agar using a sterile cork borer. Another method known as bioautography is a variation of agar diffusion method (Ncube et al. Paper discs are saturated with the desired amount of the extract and are placed onto the plates preinoculated with 1 x 108 CFU/mL test bacteria (Baris et al... & Shrivastava. 2005). The results of the Kirby-Bauer method are based on the measurement of the diameter of the zone of inhibition (ZOI) and presented qualitatively and quantitatively (White et al. The plant extract is absorbed onto a Thin Layer Chromatography plate and developed with a solvent system. Agar dilution is similar with broth dilution. 2001). 2007). different concentrations of the substance to be assayed are incorporated into the agar (Ncube et al. 2009). Results of the broth dilution are presented as the minimal inhibitory concentration (MIC). Another diffusion method is the agar well diffusion. Saxena. For the diffusion methods. 2006. however.negative controls (Das. Smânia. Tiwari. Rajore.. Tetrazolium salts serve as the microbial indicator which is sprayed onto the plates and is reincubated at 25 °C for 24 hrs. Bacterial suspension of the test bacteria are sprayed onto the TLC plate and incubated at 25 °C for 48 hrs. 2008).. .. 2010. This method was concluded to be practical and easy to perform (Das et al.extracts since it can isolate and detect active components. Ncube et al. It was then filtered through Whatman No. The solvent was removed under vacuum using a rotary evaporator (Vital & Rivera. These samples were air dried and kept in a vacuum sealed container until further use. It was filtered through Whatman No. . The residue was again mixed with 50 ml of each solvent (methanol and n-hexane) and was allowed to stand for another 24 hours. Preparation of Extracts Ten grams of the powdered plant material were mixed with 50 ml of each solvent (methanol and n-hexane) and were allowed to stand for 24 hours. Air dried samples were cut and pulverized using the Osterizer to achieve a powder-like material. 1 filter paper. Samples were authenticated by Prof.METHODOLOGY Preparation of Plant Materials Plant samples of Andropogon aciculatus Retz. The filtrate was added to the first filtrate and stored. Samples were then washed with running tap water thoroughly and with distilled water once. 1 filter paper and the filtrate was stored at 4°C in airtight bottles. 2009). Teresa Mahinay from the Division of Natural Sciences and Mathematics of the University of the Philippines Visayas Tacloban College. were harvested from low land areas of Tacloban City. The solution was mixed and added with 1 ml concentrated H2SO4.Preliminary Phytochemical Screening Qualitative preliminary phytochemical screening of the crude extracts was determined using different tests with modifications adapted from Pathak. The mixture was then filtered and 1 ml of the filtrate was added with 2 drops of 1% picric acid solution. violet.2 g of the sample and was boiled in a steam bath. Formation of yellow precipitates indicate the presence of alkaloids.5 ml of 10% HCl were mixed with the sample. The mixture was then added with Mg metal. Formation of a reddish color indicates the presence of flavonoids. & Vora (2010). Saraswathy. Test for Glycosides Five milliliters of the extract was added with 2 ml of glacial acetic acid with one drop FeCl3. . Test for Flavonoids One milliliter of 10% ethanol and 0. green) indicates the presence of glycosides. Formation of three different ring layers (brown. Test for Alkaloids Five milliliters of 2% HCl was added to 0. 2 g of extract. The solution was shaken vigorously. One milliliter of the filtrate was added with 1 ml distilled water and 2 drops FeCl3. It was then cooled and filtered.2 g of extract. The mixture was boiled for five minutes.1 g of the extract. One milliliter of the filtrate was added with 4 ml of distilled water. It was boiled for five minutes and then filtered. Bluish black color formation indicates the presence of phenols. Test for Tannins Five milliliters of 45% ethanol were added to 0. The persistence of a stable froth indicates the presence of saponins. Formation of greenish to black precipitates indicates the presence of tannins. Test for Phenols Three to four drops of FeCl3 solution was added to 0.Test for Saponins Five milliliters of distilled water was added to 0. . These cultures were grown in nutrient agar plates and were placed inverted into 37°C incubator for 24 hours prior to performing the assay.Test for Terpenoids Five milliliters of the extract was added with 2 ml of chloroform. Staphylococcus aureus. Test for Phytosterols This test was done according to the Salkovski‟s test. Preparation of Bacterial Cultures Bacterial cultures of some common disease-causing bacteria such as Bacillus subtilis. Pseudomonas aeruginosa and Serratia marcescens were prepared from stock cultures available in the laboratory. . Five milliliters of the extract was added with chloroform. Formation of a golden yellow color indicates the presence of sterols. The solution was then shaken and let stand. Reddish brown precipitates indicate the presence of terpenoids. The mixture was then filtered and the filtrate was added with few drops of concentrated H2SO4. It was then mixed and added with 3 ml of concentrated H2SO4. 2008). inoculum was obtained using a sterile cotton swab by dipping it into the suspension and pressing firmly against the inside wall of the tube just above the fluid level. MHA was prepared according to the manufacturer‟s instructions. 1 filter paper. 3-10 well-isolated colonies were selected and were transferred into tubes of Mueller Hinton Broth (MHB) using a sterile inoculating loop. the inoculum was swabbed all around the edge of the agar surface. If the bacterial suspensions do not appear to have the same density as the McFarland 0.Antimicrobial Activity Assay The antibacterial activity of the different plant extracts were evaluated by Kirby-Bauer method using Mueller Hinton Agar (MHA).5 McFarland standard. This cotton swab was streaked over the entire surface of the MHA plate three times. Within 15 minutes after adjusting the turbidity of the bacterial suspensions. The discs were dried for 15 minutes inside the incubator at 35°C. These paper discs were impregnated with 20 µl of the extracts using the mechanical pipette. rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculum. which were sterilized in the autoclave together with the Petri plates (CDC. the discs were placed individually onto the MHA plates containing the inoculum using sterile forceps. After incubation at 35°C overnight. Finally. the diameter of the zone of inhibition (ZOI) was . Prior to performing the assay. The bacterial suspensions were compared to the 0. paper puncher was used to prepare paper discs of approximately 6 mm in diameter from Whatman No. After incubation. Each bacterial culture tested was streaked onto an MHA plate to obtain isolated colonies. while rotating the swab to remove excess liquid.5. These plates were placed inverted in the incubator at 35°C for 16 to 18 hours. the opacity was reduced by adding sterile broth or increased by adding more bacterial growth. After incubation. A two-fold serial dilution range of 100 mg/L–0. One milliliter of the bacterial suspension was added to the MHB+Extract dilution tubes and was incubated at 35–37°C for 18–20 hours.5 McFarland standard.4. 1999). One milliliter of extract dilution and 20 ml of MHB were mixed. sterile distilled water was used as the negative control and streptomycin (200 mg/L) as the positive control. Inoculated MHB served as the negative control. it was discarded and a new batch was prepared. Bacterial suspension was prepared using the growth method. It was then diluted in MHB (1:100) giving a bacterial concentration of 105 CFU/ml. Minimum Inhibitory Concentration (MIC) Determination The Minimum Inhibitory Concentration (MIC) of the extract that has the least inhibition was determined by broth macrodilution method. The lowest concentration of the extract at which no visible growth was observed was recorded as the MIC. After reaching the desired pH. the broth was autoclaved then allowed to cool to 50°C. Mueller Hinton broth (MHB) was prepared according to the manufacturer‟s instructions.measured using Vermier caliper and was recorded in millimetres (CDC. This method is adapted from Andrews (2001). MHB+Extract dilution tubes were prepared by transferring 1 ml of the mixture into sterile screw capped tubes. while uninoculated MHB served as the positive control. Turbidity was adjusted to be the same as the 0. The pH of the broth was checked to see if it lied between 7.20 mg/L of extracts was used for this assay. . If the broth‟s pH lied outside the given range. For each bacterial strain.2-7. . And t-test was used to analyze the mean ZOIs of the bacteria which were susceptible to the extracts. Same test was performed for the mean MICs of the extracts. The mean and standard error values were calculated and were recorded.Data Analysis The assays were done in triplicates. A one-way analysis of variance was used (One-way ANOVA) to analyze the mean ZOIs for the Kirby-Bauer test. violet.RESULTS In this study. Results revealed that the methanol extract of the plant contains phytochemicals such as saponins. aciculatus was done in both of the methanol and n-hexane extracts. . diameters of the ZOIs were measured. aciculatus. tannins. After an overnight incubation. terpenes. phenols. terpenoids. the antimicrobial activity of A. were applied onto the plate. using methanol and nhexane as the solvent. 6 mm Table 1. Mueller-Hinton plates were prepared and swabbed with the test bacteria. and phytosterols while the n-hexane extract only contains tannins. Preliminary phytochemical analysis of the methanol and n-hexane extracts of A. Paper discs impregnated with the methanol and n-hexane extracts. and phytosterols (Table 1). preliminary phytochemical screening of A. were also assessed through the Kirby-Bauer method of disc diffusion susceptibility testing. aciculatus Retz. as well as the positive (streptomycin) and negative (distilled water) controls. green) - - Saponins persistence of froth + - Tannins dark green solution + + Phenols bluish black solution + - Terpenoids reddish brown precipitate + + Phytosterols golden yellow solution + + In this study. NAME OF THE TEST OBSERVATION METHANOL N-HEXANE Alkaloids yellow precipitate - - Flavonoids reddish color formation - - Glycosides different layers of color (brown. The mean diameter of the ZOI±SD of the methanolic extract against B. marcescens 0 0 21.Results of the test revealed that only the methanolic extract of the entire plant showed antimicrobial activity.8 0 S. aureus 0 0 24. Furthermore.7±1. subtilis but not against the gram-negative bacteria. the methanolic extract was effective only against the gram-positive bacteria. aeruginosa 0 0 9.7 0 P. T-test 6 mm on the mean ZOI of the bacteria which was susceptible to the extract showed that there is a significant (p<0. subtilis was at 23.05) difference between the methanolic extract to the positive control (Appendix). Antimicrobial activity of the methanol and n-hexane extracts of A.1±2. ZONE OF INHIBITION (mm) Methanol extract (100g/L) N-hexane extract (100g/L) Positive control (streptomycin: 200g/L) Negative control (distilled water) B.4 0 28. aureus (Figure 1).2±0.9 0 S.4 mm (Table 2). aeruginosa and the other gram-positive bacteria. marcescens and P.9±1. B. Table 2. aciculatus (disc diameter is 6 mm). Also.1 0 BACTERIA Gram-positive Gram-negative . subtilis 23. S. the test bacteria that showed antibacterial activity were considered to be susceptible to the extract.0±1.1±2. S. and (D). aciculatus (disc diameter is 6 mm). d . aureus (B).negative control (distilled water).methanol extract. and gram-negative bacteria: P. b . subtilis (A).positive control (streptomycin) . aeruginosa (C). a .A 6 mm B 6 mm C 6 mm D 6 mm Figure 1. S.n-hexane extract. Gram-positive bacteria: B. Antibacterial assay results showing the ZOIs of the methanol and n-hexane extracts of A. c . MHB+Extract dilution tubes were prepared and inoculated with the test bacteria. MIC assay results showing the MIC of the methanolic extract of A. Uninoculated MHB tube was used as the positive control and MHB tube inoculated with the test bacteria served as the negative control. In this study. inoculated MHB: negative control. subtilis was at 25 g/L (Figure 2).20g/L was used. positive control negative control Figure 2. aciculatus against B.The extract that showed antibacterial activity and the test bacteria on which they were active were subjected to MIC assay using the broth macrodilution method.20 g/L. subtilis. The lowest concentration of the extract at which no visible bacterial growth was observed was recorded as the MIC. Mean MIC of the extract was at 25 g/L. The method was done in triplicates. the mean MICs of the methanolic extract against B. Two-fold serial dilution range: 100 g/L – 0. uninoculated MHB: positive control. . A two-fold serial dilution of the extract from 100g/L – 0. aciculatus revealed that the plant has several active compounds that may have contributed to the activity. 2007. tannins. . Salmonella typhi.DISCUSSION Antimicrobial activity was observed only in the methanol extract of A. All of the other compounds have been reported to have antimicrobial activities (Cowan. Souza. 1999).. A. D'Angelo. Salawu. 2011. 2011. Vanitha. were isolated from Solanum trilobatum Linn and were assayed against Staphylococcus aureus. aciculatus. 2005) and terpenes (Nostro. Pereira. & Akindahunsi. Sterols (Ragasa & Lim. Ogundare. and phytosterols. et al. In the phytochemical study conducted by Chua (1978). 1999). Streptococcus pyrogenes. Results revealed that tannins have exhibited antibacterial activity against all of the test organisms. B. Phenols (Nitiema. Ola-Salawu. terpenes. & Traore. & Dhanabalan (2009). Savadogo. Mohammed Mubarack. compounds of pharmacological interest specifically. 2011) are known to show antimicrobial activity against wide range of bacteria. N-hexane extract contains tannins. terpenoids. and phytosterols.. only the methanol extract showed antimicrobial activity against the gram-positive bacterium. tannins. In the study of Doss. However. phenols. Preliminary phytochemical screening of A. Germano. The methanol extract of the plant showed that it contains phytochemicals such as saponins. Kalra. Saravanakumar. This activity may be due to the presence of compounds in the plant sample (Cowan. & Cannatelli. & Saxena. Dianou. Proteus vulgaris and Escherichia coli using agar diffusion method. Marino. & Ganesh. 2012. Venkateshwaran. et al. aciculatus‟ flowers contained substantial amounts of sterols and terpenes. subtilis. Pseudomonas aeruginosa. 2009) and terpenoids (Gupta. 2000) have been reported to have antimicrobial activity. Simpore. 1991). 2005.. gram-negative bacteria have an outer membrane which regulates the entry of molecules into it (Manual of antimicrobial susceptibility testing. Piot. These factors include inoculum density. This periplasm contains degradative enzymes that can hydrolyze antibiotics and other large molecules (Manual of antimicrobial susceptibility testing. Gram-positive bacteria have a different cell wall structure that makes it susceptible to antibiotics as compared to gram-negative bacteria. Rollins & Joseph. Furthermore. Thus. Gram-positive bacteria have a thicker peptidoglycan layer than gram-negative bacteria. Engbaek. such as methanol (Parekh et al. incubation time. temperature of incubation. aureus to the methanolic extract as compared to B. These may have been a great influence to the inactivity of the methanol extract against the gram-negative bacteria. Resistance of S. and the inability to cause the extract to dissociate. N-hexane is a non-polar solvent. ease of evaporation at low heat. 2008) stated some of the properties a good solvent must have during plant extraction which include low toxicity. Hughes (cited in Ncube et al. certain molecules responsible for antimicrobial activity may not have been able to pass through the membrane and act against the bacteria.N-hexane extract of A. 2005). amount of extracts and the extract‟s diffusibility on the agar. The amount of the extract being tested against the specified bacteria may not have been enough to obtain a considerable antimicrobial activity. Lastly. In addition. P. timing of disc application. the gram-negative bacteria have a space between the outer and the inner membranes of the cell wall called the periplasm. 2006). & Heuck. aciculatus did not reveal antimicrobial activity against any of the test bacteria. subtilis may be accounted to several factors. marcescens. However. depth of agar medium (Vandepitte. 2000). the extract may not have been able to diffuse through .. aeruginosa and S. most of the antimicrobial active compounds are often obtained when using polar solvents. MHA has been the base medium used for Kirby-Bauer test because of its low sulphonamide. This was done using the broth macrodilution method. MHA. Other studies (Baris et al. & Kurt. Ozturk. used in this study. aureus may have acquired or developed intrinsically to resist the extract (Sibanda & Okoh. tetracycline. aciculatus using methanol as the extraction solvent. The mean MIC of the extract was at 25 g/L. and enzymatic degradations to the components of the extract may have been several reasons that the methanolic extract did not have any activity against S. aureus as compared to its activity to B. Parekh and Chanda. 2007. and trimethoprim inhibitors which result in the satisfactory growth of most bacteria and it has been recommended by the Clinical and Laboratory Standards Institute (CLSI). Taskin. subtilis. which include isolation of the antimicrobial compounds from this plant. MIC was used to determine the lowest concentration of the extract that showed antimicrobial activity. However. 2007. are necessary to confirm this. Tenover. 2006). However. 2006.. . 2007) have shown similar results wherein the other bacteria with the same kind did not reveal any response to the antimicrobial compound being tested. subtilis were subjected to the MIC assay. In this study. This study reports a potential source of antimicrobial compounds from A. There are three mechanisms that S. alteration of the target sites in the bacterium. Further studies. Mechanisms such as the active efflux of the active component in the extract. only the methanol extract and B.the medium. these results are insufficient to support the use as herbal medicine to treat bacterial infections. phenols. . Antimicrobial activity of A. aciculatus in methanol and n-hexane extracts was determined using Kirby-Bauer method of disc diffusion susceptibility testing and B. aciculatus can be a potential source of antimicrobial compounds. and phytosterols while the n-hexane extract only contains tannins. terpenes. subtilis was found to be susceptible to the methanolic extract of the plant. Results indicate that A. tannins. and phytosterols. terpenoids.CONCLUSION Preliminary phytochemical screening of A. aciculatus methanol and n-hexane extracts revealed that the methanol extract contains phytochemicals such as saponins. MIC determination was done using broth macrodilution method and revealed that 25 g/L of the extract can inhibit the growth of the test bacterium. further antimicrobial activity assay is also suggested on the isolated active component/s for verification purposes. .RECOMMENDATIONS It is recommended that an extensive evaluation of the phytochemical constituent of the plant should be conducted for the identification of the active component. Also. Techniques for evaluation of medicinal plant products as antimicrobial agent: current methods and future trends. Turkish Journal of Biology. M. Journal of Antimicrobial Chemotherapy.. Indian Journal of Science and Technology. Coyle.. 5-16. 2(2).. Ozer.. Doss. K. (2006). (2010). Mohammed Mubarack. Kilic.LITERATURE CITED Andrews.gov/ncidod/dbmd/diseaseinfo/cholera/ch9.. Plant products as antimicrobial agents. 104-111.. M. Marie B. Manual of antimicrobial susceptibility testing. (2001). R. . African Journal of Pharmacy and Pharmacology. D. R. 41-43. 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