Phytochemical Phytochemical Screening and In Vitro Antibacterial Activity of Crude Extracts from An dropogo dropogon n acicul acicul atus retz. 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: Sign atur e of Res Research Adviser Adviser :
Yes No No
This is to certify that this research paper, entitled: “Phytochemical screening and in vitro antibacterial activity of crude extracts from Andr opogo opogon n acicul acicul atus 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
This is to certify that this research paper, entitled: “Phytochemical screening and in vitro antibacterial activity of crude extracts from Andr opogo opogon n acicul acicul atus 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 bein g 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.
ABSTRACT
The study was conducted to screen for phytochemicals present and the antimicrobial activity
in
methanol
and
n-hexane
extracts
of Andropogon
aciculatus. Preliminary
phytochemical screening of the plant revealed that the methanol extract contains phytochemicals such as saponins, tannins, phenols, terpenoids, and phytosterols while the n-hexane extract only contains tannins, terpenes, and phytosterols. Kirby- Bauer method of disc diffusion susceptibility test was used to evaluate the antimicrobial activity of the crude extracts of A. aciculatus against the gram-positive bacteria, B. subtilis and S. aureus, and the gram-negative bacteria, P. aeruginosa and S. marcescens. Streptomycin (200 mg/L) served as the positive control and sterile distilled water as the negative control. The mean diameter of the zone of inhibition (ZOI) was then recorded. Broth macrodilution method was conducted for the minimum inhibitory concentration (MIC) determination. This method was done only to the extract that showed antimicrobial activity. Only the methanol extract of A. aciculatus showed antimicrobial activity against the gram-positive bacterium, B. subtilis. The mean diameter of the ZOI±SD of the methanolic extract against B. subtilis was at 23.1±2.4 mm. Furthermore, the recorded mean MIC of the methanol extract against B. subtilis was at 25 g/L. Results revealed that A. aciculatus can be a potential source of antimicrobial compounds.
TABLE OF CONTENTS
Page Acknowledgements .............................................................................................................
iii
Abstract ................................................................................................................................
iv
List of Figures ......................................................................................................................
vi
List of Tables .......................................................................................................................
vii
Introduction ..........................................................................................................................
1
Review of Literature ............................................................................................................
3
Biology and Importance of Andropogon aciculatus Retz. .............................................
3
Antimicrobial Activity of Plants ………………….........................................................
3
Antimicrobial Activity Testing Protocol .........................................................................
6
Methodology …………........................................................................................................
9
Preparation of Plant Material ………………………......................................................
9
Preparation of Extracts ……….......................................................................................
9
Preliminary Phytochemical Screening ………………………………………………....
10
Test for Alkaloids ………………………………………………………………….
10
Test for Flavonoids ………………………………………………………………...
10
Test for Glycosides …………………………………………………………………
10
Test for Saponins …………………………………………………………………...
11
Test for Taninns …………………………………………………………………….
11
Test for Phenols …………………………………………………………………….
11
Test for Terpenoids …………………………………………………………………
12
Test for Phytosterols ………………………………………………………………..
12
Preparation of Bacterial Cultures ....................................................................................
12
Antimicrobial Activity Assay ..........................................................................................
13
Minimum Inhibitory Concentration Determination ........................................................
14
Data Analysis ..................................................................................................................
15
Results ……..........................................................................................................................
16
Discussion ...........................................................................................................................
20
Conclusion ………..............................................................................................................
23
Recommendations ...............................................................................................................
24
Literature Cited …...............................................................................................................
25
Ap pendix ………………………………………………………………………………….
30
LIST OF FIGURES
Figure 1
2
Page Antibacterial assay results showing the ZOIs of the methanol and n-hexane extracts of A. aciculatus .......................................................................................... MIC assay results showing the MIC of the methanolic extract of A. aciculatus against B. subtilis …….............................................................................................
18
19
LIST OF TABLES
Table 1 2
3
Page Preliminary phytochemical analysis of the methanol and n-hexane extracts of A. aciculatus .................................................................................................................. 16 Antimicrobial activity of the methanol and n-hexane extracts of A. 17 aciculatus................................................................................................................... T-test Analysis: Two-Sample Assuming Equal Variances ………………………...
30
INTRODUCTION
Plants have been widely used as a healing purpose since the ancient times (Cowan, 1999). Medicinal uses of these plants have been a form of treatment known to humans (Islam, Barua, Das, Khan, & Ahmed, 2008). Researchers continue to screen for antibacterial properties of plants (Islam et al., 2008; Tanaka, da Silva, de Oliveira, Nakamura, & Dias Filho, 2006) using different solvents for extraction (Hidayathulla, Chandra, & Chandrashekar, 2011; Padhi, Panda, Satapathy, & Dutta, 2011; Pathak, Saraswathy, & Vora, 2010). Extraction of plants can be done from different parts such as leaves, roots, stems, flowers, and fruits (Parekh & Chanda, 2007; ElMahmood & Doughari, 2008; Islam et al., 2008). Some are also extracted with the use of the entire plant (Masoodi, et al., 2008; Selvadurai, Senthamarai, Sri Vijaya Kirubha, & Vasuki, 2011). Those compounds responsible for antibacterial activity can be evaluated by conducting phytochemical analysis of the plants used. Some of these compounds include the secondary metabolites such as phenols, alkaloids, terpenoids and essential oils, lectins and polypeptides, and polyacetylenes (Cowan, 1999). Andropogon aciculatus Retz. 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, usually in open places (“Chrysopogon aciculatus”, 2007). This plant is famous for its seeds adhering to trousers and dresses. A. aciculatus has been used as a traditional medicine in the Philippines and in other countries (“Amor-seco”, n.d). A phytochemical study of the A. aciculatus‟ flowers conducted by Chua (1978) suggested substantial amounts of sterols and terpenes present in the sample. Sterols (Ragasa & Lim, 2005) and terpenes (Nostro, Germano, D'Angelo, Marino, & Cannatelli, 2000) have been reported to have antimicrobial activity.
These are the main objectives of the study: 1. to screen for the phytochemicals present in A. aciculatus Retz. methanol and n-hexane extracts; 2. to determine the antibacterial activity of A. aciculatus Retz. extracts against selected diseasecausing bacteria such as Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens using Kirby-Bauer method; and, 3. to determine the minimum inhibitory concentration of A. aciculatus Retz. extracts using broth macrodilution method.
LITERATURE REVIEW
Biology and Importance of An dropogon aciculatus Retz.
Andropogon aciculatus Retz. commonly known as amor-seco or love grass, which belongs to the family Poaceae, is a dense, leafy perennial grass, creeping and branching below, with short horizontal stems. The leaf blades measure 3-12 cm long and 5 mm wide. The inflorescence measures 7-10 cm long with numerous slender branches. It is a common weed that can be found throughout the Philippines, usually in open places (“Chrysopogon aciculatus”, 2007). A. aciculatus has been used in traditional medicine. In the Philippines, decoction of the roots of A. aciculatus has been used as an antidiarrheal alternative and the decoction of the entire plant served as a diuretic (“Amor-seco”, n.d.). This plant is also used as an alternative medicine in other Asian countries. In India, the rhizome is also pounded as a cure for stomachache and other gastric disorder (Mitra and Mukherjee, 2005). In Indonesia, it is used as a poison antidote. And in Malaysia, ashes of the roots are used for rheumatism (“Amor-seco”, n.d.).
Antimicrobial Activity of Plants
Antimicrobial activity of some plants from the family Poaceae has already been evaluated. In the study of Kumar et al. (2011), in vitro evaluation of antibacterial activity of the crude extract from the whole plant, Cynodon dactylon (Bermuda grass), was done against Escheria coli, Staphylococcus aureus & Streptococcus pyogenes. Results showed a significant
antibacterial activity against the test bacteria. The study conducted by Jananie and Vijayalakshmi (2011) on the determination of bioactive components of C. dactylon has revealed components that can justify the antibacterial activity of this plant. Other studies (Hindumathy, 2011; Singh, Singh, Singh, & Ebibeni, 2011; Vazirian, et al., 2012) that involve the screening for antimicrobial activity of Cymbopogon citratus (Poaceae) were conducted. C. citratus, also known as lemongrass, has been widely used as a traditional medicine and its essential oil is used in food, cosmetic, pharmaceutical and insecticide industries (Negrelle & Gomes, 2007). C. citratus extracts showed a significant antibacterial activity against four gram-negative bacteria Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris) and two gram-positive bacteria Bacillus subtilis and Staphylococcus aureus at four different concentrations (1:1, 1:5, 1:10 and 1:20) using disc diffusion method. This activity was accounted for the presence of alkaloid and phenols based from the phytochemical analysis done on the plant (Hindumathy, 2011). Furthermore, in the study of Vazirian et al. (2012), the antimicrobial activity of C. citratus essential oil was tested against food-borne pathogens. Results revealed an effective antimicrobial activity on the selected microorganisms that suggested the plant‟s essential oil as a safe natural food preservative. The antimicrobial activity of plants has been a great focus because of substances, which have versatile applications, derived from it (Baris et al., 2006). Thus, biological experiments of plant extracts can be done to ensure its efficacy and safety. These are just some important factors so that these plant extracts will be accepted as valid medicinal agents. There must be compounds present in a plant which make it a potential antimicrobial agent. Cowan (1999) described some of the major compounds known as the secondary metabolites that contributes to the antimicrobial property of a plant.
One of the major compounds consists of a single substituted phenolic ring known as the phenols. Cinnamic and caffeic acids are common representatives of this wide group of compounds. One compound that belongs to this class includes the quinones, which have aromatic rings with two ketone substitutions and is naturally highly reactive (Cowan, 1999). In a study conducted by Ignacimuthu, Pavunraj, Duraipandiyan, Raja, & Muthu (2009) the active extract present in Pergularia daemia leaves was separated by column chromatography and one fraction yielded a new compound, 6-(4, 7 dihydroxy-heptyl) quinone, which showed antibacterial activity against some pathogenic bacteria. Other compounds are the flavonones. These are phenols containing one carbonyl group (Cowan, 1999). In the study conducted by Sato et al. (2000), apigenin and luteolin, which are examples of the flavonoid variants, were isolated from the plant as active constituents against Staphylococcus aureus. Other compounds, which belong to the phenol group, are tannins. “Tannin” is the term given for a group of substances capable for astringency, which is to tan leather or precipitate gelatin from solution (Cowan, 1999). Compounds of pharmacological interest specifically, tannins, were isolated from Solanum trilobatum Linn and assayed against some bacteria have exhibited antibacterial activity (Doss, Mohammed Mubarack, & Dhanabalan, 2009). As described by Cowan (1999), another major group of compounds are essential oils and terpenoids. These are secondary metabolites that are highly enriched in compounds based on an isoprene structure. In the study of Li, Hu, Zheng, Zhu, & Liu (2011), the components of essential oil from spikes and stems of Artemisia annua, an aerial plant in China, were identified using chromatography – mass spectrometry (GC/MS). It was found out that compounds from the spike oil such as piperitone, octanal and 1, 4-Diphenyl-2- butanone and other terpenoid contents were
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. Another major group of compounds discussed by Cowan (1999) is the heterocyclic nitrogen compounds called alkaloids. Bactericidal activity of the major alkaloids (berberine, βhydrastine, canadine and canadaline) isolated from Hydrastis canadensis L. (Ranunculaceae) was evaluated by measuring the “killing time” on a low density bacterial inoculum, and bacteriostatic activity in liquid medium by MIC values. Results showed that the plant can be a possible traditional medicine (Scazzocchio, Cometa, Tomassini, & Palmery, 2001).
Antimicrobial Activity Testing Protocol
The antimicrobial susceptibility test (AST) is an essential technique used in pathology to determine resistance of microbial strains to antimicrobials. Also, in ethnopharmacology research, this technique is used to determine the efficacy of antimicrobials against microorganisms (Ncube, Afolayan, & Okoh, 2008). Two major methods discussed by White et al. (2001) for in vitro AST, are dilution and diffusion methods. For the dilution method there are two different techniques, the broth and agar dilution. The assay is done on a medium, which is broth for broth dilution and agar for agar dilution with dissolved specified antimicrobials (Manual of antimicrobial susceptibility testing, 2005). For the broth dilution method, two-fold serial dilutions of the substance to be assayed are prepared and transferred into the base medium. Tubes containing the medium and dilutions are inoculated with 5
a standardized bacterial suspension of 1-5 x 10 CFU/mL After an overnight incubation at 35 C, °
the tubes are observed for visible bacterial growth by comparing the turbidity to the positive and
negative controls (Das, Tiwari, & Shrivastava, 2010). Agar dilution is similar with broth dilution; however, different concentrations of the substance to be assayed are incorporated into the agar (Ncube et al., 2008). Results of the broth dilution are presented as the minimal inhibitory concentration (MIC). 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, 2005). For the diffusion methods, the famous Kirby-Bauer method has been recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Mueller-Hinton agar (MHA) medium is the only susceptibility test medium that has been validated by the committee (EUCAST, 2009). Paper discs are saturated with the desired amount of the extract and are placed 8
onto the plates preinoculated with 1 x 10 CFU/mL test bacteria (Baris et al., 2006; Sharma, Saxena, Rani, Rajore, & Batra, 2010). 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., 2001). Another diffusion method is the agar well diffusion. This method is same as the disc diffusion; however, wells between 6 and 8 mm are aseptically punched on the agar using a sterile cork borer. Desired volumes of the plant extract are placed into the wells (Valgas, de Souza, Smânia, & Smânia Jr., 2007). Another method known as bioautography is a variation of agar diffusion method (Ncube et al., 2008). The plant extract is absorbed onto a Thin Layer Chromatography plate and developed with a solvent system. Bacterial suspension of the test bacteria are sprayed onto the TLC plate and incubated at 25 C °
for 48 hrs. Tetrazolium salts serve as the microbial indicator which is sprayed onto the plates and is reincubated at 25 C for 24 hrs. Clear white zones on the TLC plate indicate antimicrobial °
activity of the extracts. This method is utilized as a preliminary phytochemical screening for the
extracts since it can isolate and detect active components. This method was concluded to be practical and easy to perform (Das et al., 2010; Ncube et al., 2008).
METHODOLOGY
Preparation of Plant Materials
Plant samples of Andropogon aciculatus Retz. were harvested from low land areas of Tacloban City. Samples were authenticated by Prof. Teresa Mahinay from the Division of Natural Sciences and Mathematics of the University of the Philippines Visayas Tacloban College. Samples were then washed with running tap water thoroughly and with distilled water once. These samples were air dried and kept in a vacuum sealed container until further use. Air dried samples were cut and pulverized using the Osterizer to achieve a powder-like material.
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. It was filtered through Whatman No. 1 filter paper and the filtrate was stored at 4°C in airtight bottles. The residue was again mixed with 50 ml of each solvent (methanol and n-hexane) and was allowed to stand for another 24 hours. It was then filtered through Whatman No. 1 filter paper. The filtrate was added to the first filtrate and stored. The solvent was removed under vacuum using a rotary evaporator (Vital & Rivera, 2009).
Preliminary Phytochemical Screening
Qualitative preliminary phytochemical screening of the crude extracts was determined using different tests with modifications adapted from Pathak, Saraswathy, & Vora (2010).
Test for Alkaloids
Five milliliters of 2% HCl was added to 0.2 g of the sample and was boiled in a steam bath. The mixture was then filtered and 1 ml of the filtrate was added with 2 drops of 1% picric acid solution. Formation of yellow precipitates indicate the presence of alkaloids.
Test for Flavonoids
One milliliter of 10% ethanol and 0.5 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.
Test for Glycosides
Five milliliters of the extract was added with 2 ml of glacial acetic acid with one drop FeCl3. The solution was mixed and added with 1 ml concentrated H2SO4. Formation of three different ring layers (brown, violet, green) indicates the presence o f glycosides.
Test for Saponins
Five milliliters of distilled water was added to 0.1 g of the extract. It was boiled for five minutes and then filtered. One milliliter of the filtrate was added with 4 ml of distilled water. The solution was shaken vigorously. The persistence of a stable froth indicates the presence of saponins.
Test for Tannins
Five milliliters of 45% ethanol were added to 0.2 g of extract. The mixture was boiled for five minutes. It was then cooled and filtered. One milliliter of the filtrate was added with 1 ml distilled water and 2 drops FeCl3. Formation of greenish to black precipitates indicates the presence of tannins.
Test for Phenols
Three to four drops of FeCl3 solution was added to 0.2 g of extract. Bluish black color formation indicates the presence of phenols.
Test for Terpenoids
Five milliliters of the extract was added with 2 ml of chloroform. It was then mixed and added with 3 ml of concentrated H2SO4. Reddish brown precipitates indicate the presence of terpenoids.
Test for Phytosterols
This test was done according to the Salkovski‟s test. Five milliliters of the extract was added with chloroform. The mixture was then filtered and the filtrate was added with few drops of concentrated H2SO4. The solution was then shaken and let stand. Formation of a golden yellow color indicates the presence of sterols.
Preparation of Bacterial Cultures
Bacterial cultures of some common disease-causing bacteria such as Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens were prepared from stock cultures available in the laboratory. These cultures were grown in nutrient agar plates and were placed inverted into 37°C incubator for 24 hours prior to performing the assay.
Antimicrobial Activity Assay
The antibacterial activity of the different plant extracts were evaluated by Kirby-Bauer method using Mueller Hinton Agar (MHA). MHA was prepared according to the manufacturer‟s instructions. Each bacterial culture tested was streaked onto an MHA plate to obtain isolated colonies. After incubation at 35°C overnight, 3-10 well-isolated colonies were selected and were transferred into tubes of Mueller Hinton Broth (MHB) using a sterile inoculating loop. The bacterial suspensions were compared to the 0.5 McFarland standard. If the bacterial suspensions do not appear to have the same density as the McFarland 0.5, the opacity was reduced by adding sterile broth or increased by adding more bacterial growth. Within 15 minutes after adjusting the turbidity of the bacterial susp ensions, 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, while rotating the swab to remove excess liquid. This cotton swab was streaked over the entire surface of the MHA plate three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of the inoculum. Finally, the inoculum was swabbed all around the edge of the agar surface. Prior to performing the assay, paper puncher was used to prepare paper discs of approximately 6 mm in diameter from Whatman No. 1 filter paper, which were sterilized in the autoclave together with the Petri plates (CDC, 2008). These paper discs were impregnated with 20 µl of the extracts using the mechanical pipette. The discs were dried for 15 minutes inside the incubator at 35 C. After incubation, the discs were placed individually onto the MHA plates °
containing the inoculum using sterile forceps. These plates were placed inverted in the incubator at 35°C for 16 to 18 hours. After incubation, the diameter of the zone of inhibition (ZOI) was
measured using Vermier caliper and was recorded in millimetres (CDC, 1999). For each bacterial strain, sterile distilled water was used as the negative control and streptomycin (200 mg/L) as the positive control.
Minimum Inhibitory Concentration (MIC) Determination
The Minimum Inhibitory Concentration (MIC) of the extract that has the least inhibition was determined by broth macrodilution method. This method is adapted from Andrews (2001). A two-fold serial dilution range of 100 mg/L – 0.20 mg/L of extracts was used for this assay. Mueller Hinton broth (MHB) was prepared according to the manufacturer‟s instructions. The pH of the broth was checked to see if it lied between 7.2-7.4. If the broth‟s pH lied outside the given range, it was discarded and a new batch was prepared. After reaching the desired pH, the broth was autoclaved then allowed to cool to 50 C. One milliliter of extract dilution and 20 ml of MHB °
were mixed. MHB+Extract dilution tubes were prepared by transferring 1 ml of the mixture into sterile screw capped tubes. Bacterial suspension was prepared using the growth method. Turbidity was adjusted to be the same as the 0.5 McFarland standard. It was then diluted in MHB 5
(1:100) giving a bacterial concentration of 10 CFU/ml. 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. °
Inoculated MHB served as the negative control, while uninoculated MHB served as the positive control. The lowest concentration of the extract at which no visible growth was observed was recorded as the MIC.
Data Analysis
The assays were done in triplicates. The mean and standard error values were calculated and were recorded. A one-way analysis of variance was used (One-way ANOVA) to analyze the mean ZOIs for the Kirby-Bauer test. 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.
RESULTS
In this study, preliminary phytochemical screening of A. aciculatus was done in both of the methanol and n-hexane extracts. Results revealed that the methanol extract of the plant contains phytochemicals such as saponins, tannins, phenols, terpenoids, and phytosterols while the n-hexane extract only contains tannins, terpenes, and phytosterols (Table 1).
6 mm
Table 1. Preliminary phytochemical analysis of the methanol and n-hexane extracts of A. aciculatus. NAME OF THE TEST
OBSERVATION
METHANOL
N-HEXANE
Alkaloids
yellow precipitate
-
-
Flavonoids
reddish color formation
-
-
Glycosides
different layers of color (brown, violet, green)
-
-
Saponins
persistence of froth
+
-
Tannins
dark green solution
+
+
Phenols
bluish black solution
+
-
Terpenoids
reddish brown precipitate
+
+
Phytosterols
golden yellow solution
+
+
In this study, the antimicrobial activity of A. aciculatus Retz, using methanol and nhexane as the solvent, were also assessed through the Kirby-Bauer method of disc diffusion susceptibility testing. Mueller-Hinton plates were prepared and swabbed with the test bacteria. Paper discs impregnated with the methanol and n-hexane extracts, as well as the positive (streptomycin) and negative (distilled water) controls, were applied onto the plate. After an overnight incubation, diameters of the ZOIs were measured.
Results of the test revealed that only the methanolic extract of the entire plant showed antimicrobial activity. Also, the test bacteria that showed antibacterial activity were considered to be susceptible to the extract. Furthermore, the methanolic extract was effective only against the gram-positive bacteria, B. subtilis but not against the gram-negative bacteria, S. marcescens and P. aeruginosa and the other gram-positive bacteria, S. aureus (Figure 1). The mean diameter of the ZOI±SD of the methanolic extract against B. subtilis was at 23.1±2.4 mm (Table 2). T-test 6 mm
on the mean ZOI of the bacteria which was susceptible to the extract showed that there is a significant (p<0.05) difference between the methanolic extract to the positive control (Appendix).
Table 2. Antimicrobial activity of the methanol and n-hexane extracts of A. aciculatus (disc diameter is 6 mm). ZONE OF INHIBITION (mm)
Methanol extract (100g/L)
N-hexane extract (100g/L)
Positive control (streptomycin: 200g/L)
Negative control (distilled water)
B. subtil is
23.1±2.4
0
28.2±0.9
0
S. aur eus
0
0
24.0±1.7
0
P. aer ugi nosa
0
0
9.9±1.8
0
S. mar cescens
0
0
21.7±1.1
0
BACTERIA
Gram-positive
Gram-negative
A
C
6 mm
B
6 mm
6 mm
D
6 mm
Figure 1. Antibacterial assay results showing the ZOIs of the methanol and n-hexane extracts of A. aciculatus (disc diameter is 6 mm). Gram-positive bacteria: B. subtilis (A), S. aureus (B); and gram-negative bacteria: P. aeruginosa (C), and (D). a - methanol extract; b - n-hexane extract; c - negative control (distilled water); d - positive control (streptomycin)
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. A two-fold serial dilution of the extract from 100g/L – 0.20g/L was used. MHB+Extract dilution tubes were prepared and inoculated with the test bacteria. The lowest concentration of the extract at which no visible bacterial growth was observed was recorded as the MIC. Uninoculated MHB tube was used as the positive control and MHB tube inoculated with the test bacteria served as the negative control. The method was done in triplicates. In this study, the mean MICs of the methanolic extract against B. subtilis was at 25 g/L (Figure 2).
positive control
negative control
Figure 2. MIC assay results showing the MIC of the methanolic extract of A. aciculatus against B. subtilis. Two-fold serial dilution range: 100 g/L – 0.20 g/L; uninoculated MHB: positive control; inoculated MHB: negative control. Mean MIC of the extract was at 25 g/L.
DISCUSSION
Antimicrobial activity was observed only in the methanol extract of A. aciculatus. This activity may be due to the presence of compounds in the plant sample (Cowan, 1999). Preliminary phytochemical screening of A. aciculatus revealed that the plant has several active compounds that may have contributed to the activity. The methanol extract of the plant showed that it contains phytochemicals such as saponins, tannins, phenols, terpenoids, and phytosterols. Phenols (Nitiema, Savadogo, Simpore, Dianou, & Traore, 2012; Pereira, et al., 2007; Salawu, Ogundare, Ola-Salawu, & Akindahunsi, 2011; Saravanakumar, Venkateshwaran, Vanitha, & Ganesh, 2009) and terpenoids (Gupta, Kalra, & Saxena, 2011; Souza, et al., 2011) are known to show antimicrobial activity against wide range of bacteria. In the study of Doss, Mohammed Mubarack, & Dhanabalan (2009), compounds of pharmacological interest specifically, tannins, were isolated from Solanum trilobatum Linn and were assayed against Staphylococcus aureus, Streptococcus pyrogenes, Salmonella typhi, Pseudomonas aeruginosa, Proteus vulgaris and Escherichia coli using agar diffusion method. Results revealed that tannins have exhibited antibacterial activity against all of the test organisms. All of the other compounds have been reported to have antimicrobial activities (Cowan, 1999). N-hexane extract contains tannins, terpenes, and phytosterols. In the phytochemical study conducted by Chua (1978), A. aciculatus‟ flowers contained substantial amounts of sterols and terpenes. Sterols (Ragasa & Lim, 2005) and terpenes (Nostro, Germano, D'Angelo, Marino, & Cannatelli, 2000) have been reported to have antimicrobial activity. However, only the methanol extract showed antimicrobial activity against the gram-positive bacterium, B. subtilis.
N-hexane extract of A. aciculatus did not reveal antimicrobial activity against any of the test bacteria. N-hexane is a non-polar solvent. Furthermore, most of the antimicrobial active compounds are often obtained when using polar solvents, such as methanol (Parekh et al., 2006). Hughes (cited in Ncube et al., 2008) stated some of the properties a good solvent must have during plant extraction which include low toxicity, ease of evaporation at low heat, and the inability to cause the extract to dissociate. Gram-positive bacteria have a different cell wall structure that makes it susceptible to antibiotics as compared to gram-negative bacteria. Gram-positive bacteria have a thicker peptidoglycan layer than gram-negative bacteria. However, gram-negative bacteria have an outer membrane which regulates the entry of molecules into it (Manual of antimicrobial susceptibility testing, 2005; Rollins & Joseph, 2000). Thus, certain molecules responsible for antimicrobial activity may not have been able to pass through the membrane and act against the bacteria. In addition, the gram-negative bacteria have a space between the outer and the inner membranes of the cell wall called the periplasm. This periplasm contains degradative enzymes that can hydrolyze antibiotics and other large molecules (Manual of antimicrobial susceptibility testing, 2005). These may have been a great influence to the inactivity of the methanol extract against the gram-negative bacteria, P. aeruginosa and S. marcescens. Resistance of S. aureus to the methanolic extract as compared to B. subtilis may be accounted to several factors. These factors include inoculum density, timing of disc application, temperature of incubation, incubation time, depth of agar medium (Vandepitte, Engbaek, Piot, & Heuck, 1991), 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, the extract may not have been able to diffuse through
the medium, MHA, used in this study. However, MHA has been the base medium used for Kirby-Bauer test because of its low sulphonamide, tetracycline, 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). There are three mechanisms that S. aureus may have acquired or developed intrinsically to resist the extract (Sibanda & Okoh, 2007; Tenover, 2006). Mechanisms such as the active efflux of the active component in the extract, alteration of the target sites in the bacterium, 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. subtilis. Other studies (Baris et al., 2006; Parekh and Chanda, 2007; Taskin, Ozturk, & Kurt, 2007) have shown similar results wherein the other bacteria with the same kind did not reveal any response to the antimicrobial compound being tested. MIC was used to determine the lowest concentration of the extract that showed antimicrobial activity. This was done using the broth macrodilution method. In this study, only the methanol extract and B. subtilis were subjected to the MIC assay. The mean MIC of the extract was at 25 g/L. This study reports a potential source of antimicrobial compounds from A. aciculatus using methanol as the extraction solvent. However, these results are insufficient to support the use as herbal medicine to treat bacterial infections. Further studies, which include isolation of the antimicrobial compounds from this plant, are ne cessary to confirm this.
CONCLUSION
Preliminary phytochemical screening of A. aciculatus methanol and n-hexane extracts revealed that the methanol extract contains phytochemicals such as saponins, tannins, phenols, terpenoids, and phytosterols while the n-hexane extract only contains tannins, terpenes, and phytosterols. Antimicrobial activity of A. aciculatus in methanol and n-hexane extracts was determined using Kirby-Bauer method of disc diffusion susceptibility testing and B. subtilis was found to be susceptible to the methanolic extract of the plant. 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. Results indicate that A. aciculatus can be a potential source of antimicrobial compounds.
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, further antimicrobial activity assay is also suggested on the isolated active component/s for verification purposes.
LITERATURE CITED
Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy, 48, 5-16.
Baris, O., Gulluce, M., Sahin, F., Ozer, H., Kilic, H., Ozkan, H., . . . Ozbek, T. (2006). Biological activities of the essential oil and methanol extract of Achillea biebersteinii Afan. (Asteraceae). Turkish Journal of Biology, 30, 65-73.
Center for Disease Control and Prevention. 2008. Antimicrobial susceptibility testing (agar disk diffusion method). [20 Aug 2011]. Available from: http://www.cdc.gov/ncidod/dbmd/diseaseinfo/cholera/ch9.pdf.
Cowan, M. M. (1999). Plant products as antimicrobial agents. Clinical Microbiology Reviews, 12(4), 564-582.
Coyle, Marie B. (2005). Manual of antimicrobial susceptibility testing. American Society for Microbiology.
Das, K., Tiwari, R. K., & Shrivastava, D. K. (2010). Techniques for evaluation of medicinal plant products as antimicrobial agent: current methods and future trends. Journal of Medicinal Plants Research, 4(2), 104-111.
Doss, A., Mohammed Mubarack, H., & Dhanabalan, R. (2009). Antibacterial activity of tannins from the leaves of Solanum trilobatum Linn. Indian Journal of Science and Technology, 2(2), 41-43.
El-Mahmood, A. M., & Doughari, J. H. (2008). Phytochemical screening and antibacterial evaluation of the leaf and root extracts of Cassia alata Linn. African Journal of Pharmacy and Pharmacology, 2(7), 124-129.
EUCAST. (2009, December). Disk Diffusion Method for Antimicrobial Susceptibility Testing. Retrieved from European Society of Clinical Microbiology and Infectious Diseases: http://www.eucast.org
Gupta, N., Kalra, S., & Saxena, G. (2011). Antimicrobial activity pattern of certain terpenoids. International Journal of Pharmaceutical and Biological Sciences, 2(1), 87-91.
Hidayathulla, S., Chandra, K. K., & Chandrashekar, K. R. (2011). Phytochemical evaluation and antibacterial activity of Pterospermum diversifolium blume. International Journal of Pharmacy and Pharmaceutical Sciences, 3(2), 165-167.
Hindumathy, C. K. (2011). In vitro study of antibacterial activity of Cymbopogon Citratus. World Academy of Science, Engineering and Technology.
Ignacimuthu, S., Pavunraj, M., Duraipandiyan, V., Raja, N., & Muthu, C. (2009). Antibacterial activity of a novel quinone from the leaves of Pergularia daemia (Forsk.), a traditional medicinal plant. Asian Journal of Traditional Medicines, 4(1), 36-40.
Islam, M. J., Barua, S., Das, S., Khan, M. S., & Ahmed, A. (2008). Antimicrobial activity of some indigenous medicinal plants. Journal of Soil and Nature, 2(3), 26-28.
Kumar, A., Kashyap, P., Sawarkar, H., Muley, B., & Pandey, A. (2011). Evaluation of antibacterial activity of Cynodon dactylon (L.) Pers. International Journal of Herbal Drug Research, 1(2), 31-35.
Li, Y., Hu, H.-b., Zheng, X.-d., Zhu, J.-h., & Liu, L.-p. (2011). Composition and antimicrobial activity of essential oil from the aerial part of Artemisia annua. Journal of Medicinal Plants Research, 5(16), 3629-3633.
Masoodi, M. H., Ahmed, B., Zargar, I. M., Khan, S. A., Khan, S., & P., S. (2008). Antibacterial activity of whole plant extract of Marrubium vulgare. African Journal of Biotechnology, 7 (2), 86-87.
Ncube, N. S., Afolayan, A., & Okoh, A. I. (2008). Assessment techniques of antimicrobial properties of natural compounds of plant origin: current methods and future trends. African Journal of Biotechnology, 7 (12), 1797-1806.
Negrelle, R., & Gomes, E. (2007). Cymbopogon citratus (DC.) Stapf : chemical composition and biological activities. Brazilian Journal of Medicinal Plants, 9(1), 80-92.
Nitiema, L. W., Savadogo, A., Simpore, J., Dianou, D., & Traore, A. S. (2012). In vitro antimicrobial activity of some phenolic compounds (coumarin and quercetin) against gastroenteritis bacterial strains. International Journal of Microbiological Research, 3(3), 183-187. doi:10.5829/idosi.ijmr.2012.3.3.6414
Nostro, A., Germano, M., D'Angelo, V., Marino, A., & Cannatelli, M. (2000). Extraction methods and bioautography for evaluation of medicinal plant antimicrobial activity. Letters in Applied Microbiology, 30, 379-384.
OSWALDASIA (2007). Chrysopogon aciculatus (Retz.) Trin. - POACEAE - Monocotyledon. [ONLINE] Available at: http://www.oswaldasia.org/species/c/chrac/chrac_en.html. [Last Accessed January 12, 2013].
Padhi, L., Panda, S., Satapathy, S., & Dutta, S. (2011). In vitro evaluation of antibacterial potential of Annona squamosa L. and Annona reticulata L. from Similipal Biosphere Reserve, Orissa, India. Journal of Agricultural Technology, 7 (1), 133-142.
Parekh, J., & Chanda, S. V. (2007). In vitro antimicrobial activity and phytochemical analysis of some indian medicinal plants. Turkish Journal of Biology, 31, 53-58.
Pathak, P., Saraswathy, D., & Vora, A. S. (2010). In vitro antimicrobial activity and phytochemical analysis of the leaves of Annona muricata. International Journal of Pharmaceutical Research and Development, 2(5), 1-6.
Pereira, A. P., Ferreira, I. C., Marcelino, F., Valentão, P., Andrade, P. B., Seabra, R., . . . Pereira, J. A. (2007). Phenolic compounds and antimicrobial activity of olive (Olea europaea L. Cv. Cobrançosa) leaves. Molecules, 12, 1153-1162.
Ragasa, C. Y., & Lim, K. (2005). Sterols from Cucurbita maxima. Philippine Journal of Science, 134(2), 83-87.
Rollins, D., & Joseph, S. (2000, August). BSCI 424 — PATHOGENIC MICROBIOLOGY . Retrieved from University of Maryland: http://life.umd.edu/classroom/bsci424
Salawu, S. O., Ogundare, A. O., Ola-Salawu, B. B., & Akindahunsi, A. A. (2011). Antimicrobial activities of phenolic containing extracts of some tropical vegetables. African Journal of Pharmacy and Pharmacology, 5(4), 486-492. doi:10.5897/AJPP10.317
Saravanakumar, A., Venkateshwaran, K., Vanitha, J., & Ganesh, M. (2009). Evaluation of antibacterial activity, phenol and flavonoid contents of Thespesia populnea flower extracts. Pakistan Journal of Pharmaceutical Sciences, 22(3), 282-286.
Sato, Y., S., S., T., N., M., K., H., S., & T, H. (2000). Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against methicillin-resistant Staphylococcus aureus. Journal of Ethnopharmacology, 72(3), 483-488.
Scazzocchio, F., Cometa, M., Tomassini, L., & Palmery, M. (2001). Antibacterial activity of Hydrastis canadensis extract and its major isolated alkaloids. Planta Medica, 67 (6), 561564. doi:10.1055/s-2001-16493
Selvadurai, S., Senthamarai, R., Sri Vijaya Kirubha, T., & Vasuki, K. (2011). Anitimicrobial activity of ethanolic extract of the whole plant of Sida Spinosa Linn. (Malvaceae). Journal of Natural Products and Plant Resources, 1(2), 36-40.
Sharma, A., Saxena, S., Rani, U., Rajore, S., & Batra, A. (2010). broad-spectrum antimicrobial properties of medicinally important plant Jatropha curcas L. International Journal of Pharmaceutical Sciences Review and Research, 4(3), 11-14.
Sibanda, T., & Okoh, A. I. (2007). The challenges overcoming antibiotic resistance: plant extracts as potential sources of antimicrobial and resistance modifying agents. African Journal of Biotechnology, 6 (25), 2886-2896.
Singh, B. R., Singh, V., Singh, R. K., & Ebibeni, N. (2011). Antimicrobial activity of lemongrass (Cymbopogon citratus) oil against microbes of environmental, clinical and food origin. International Research of Pharmacy and Pharmacology, 1(9), 228-236.
Souza, A. B., Martins, C. H., Souza, M. G., Furtado, N. A., Heleno, V. C., de Sousa, J. P., . . . Ambrósio, S. R. (2011). Antimicrobial activity of terpenoids from Copaifera langsdorffii Desf. against cariogenic bacteria. Phytotherapy Research, 25(2), 215-220. doi:10.1002/ptr.3244
Tanaka, J. C., da Silva, C. C., de Oliveira, A. J., Nakamura, C. V., & Dias Filho, B. P. (2006). Antibacterial activity of indole alkaloids from Aspidosperma ramiflorum. Brazilian Journal of Medical and Biological Research, 39, 387-391.
Taiwo, S. S., Oyekanmi, Y. O., Adesiji, O., Opaleye, O., & Adeyeba, O. A. (2007). In vitro antimicrobial activity of crude extracts of Citrus aurantifolia Linn and Tithonia diversifolia Poaceae on clinical bacterial isolates. International Journal of Tropical Medicine, 2, 113-117.
Taskin, E., Ozturk, M., & Kurt, O. (2007, December 17). Antibacterial activities of some marine algae from the Aegean Sea (Turkey). African Journal of Biotechnology, 6 (24), 27462751.
Tenover, F. C. (2006). Mechanisms ofantimicrobial resistance in bacteria. The American Journal of Medicine, 119(6A), S3-S10.
Valgas, C., de Souza, S. M., Smânia, E. F., & Smânia Jr., A. (2007). Screening methods to determine antibacterial activity of natural products. Brazilian Journal of Microbiology, 38, 369-380.
Vandepitte, J., Engbaek, K., Piot, P., & Heuck, C. C. (1991). Technical factors influencing the size of the zone in the disc diffusion method. Basic laboratory procedures in clinical bacteriology. World Health Organization.
Vazirian, M., Kashani, S. T., Ardekani, M. R., Khanavi, M., Jamalifar, H., Fazeli, M. R., & Toosi, A. N. (2012). Antimicrobial activity of lemongrass (Cymbopogon citratus (DC) Stapf.) essential oil against food-borne pathogens added to cream-filled cakes and pastries. Journal of Essential Oil Research, 24(6), 579-582. doi:10.1080/10412905.2012.729920
Vital, P. G., & Rivera, W. L. (2009). Antimicrobial activity and cytotoxicity of Chromolaena odorata (L. f.) King and Robinson and Uncaria perrottetii (A. Rich) Merr. extracts. Journal of Medicinal Plants Research, 3(7), 511-518.
White, D. G., Acar, J., Anthony, F., Franklin, A., Gupta, R., Nicholls, T., . . . Costarrica, M. L. (2001). Antimicrobial resistance: standardisation and harmonisation of laboratory methodologies for the detection and quantification of antimicrobial resistance. Revue Scientifique et Technique, 20(3), 849-858.
