Phytochemical Screening and In Vitro Antibacterial Activity of Crude 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:
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Phytochemical Screening and In Vitro Antibacterial Activity of Crude Extracts from Andropogon aciculatus retz. (Poaceae)
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Phytochemical Screening and In Vitro Antibacterial Activity of Crude Extracts from Andropogon aciculatus retz. (Poaceae)
by
ELYROSE KIM C. RUIZO
A research paper submitted to theDivision of Natural Sciences and Mathematics
University of the Philippines VisayasTacloban College, Tacloban City
As partial fulfillment of the requirementsfor the Degree ofB.S. BIOLOGY
April 2013
Permission is given for the following people to have access to this research:
Available to the general public YesAvailable only after consultation with author/adviser No
Available only for those bound by confidentiality agreement No
Student’s signature:
Signature of Research Adviser:
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. TANResearch 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. CAPONDNSM 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.
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
Literature Cited …...............................................................................................................
Appendix ………………………………………………………………………………….
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LIST OF FIGURES
Figur
e
Page
1 Antibacterial assay results showing the ZOIs of the methanol and n-hexane extracts of A. aciculatus ..........................................................................................
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2 MIC assay results showing the MIC of the methanolic extract of A. aciculatus against B. subtilis …….............................................................................................
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LIST OF TABLES
Table Page
1 Preliminary phytochemical analysis of the methanol and n-hexane extracts of A. aciculatus .................................................................................................................. 16
2 Antimicrobial activity of the methanol and n-hexane extracts of A.
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
(1:100) giving a bacterial concentration of 105 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.
6 mm
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).
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 - -
Glycosidesdifferent layers of color (brown, violet, green)
- -
Saponins persistence of froth + -
Tannins dark green solution + +
Phenols bluish black solution + -
Terpenoidsreddish brown
precipitate+ +
Phytosterols golden yellow solution + +
In this study, the antimicrobial activity of A. aciculatus Retz, using methanol and n-
hexane 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
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).
BACTERIA
ZONE OF INHIBITION (mm)
Methanol extract (100g/L)
N-hexane extract (100g/L)
Positive control (streptomycin:
200g/L)
Negative control (distilled water)
Gram-positive
B. subtilis 23.1±2.4 0 28.2±0.9 0
S. aureus 0 0 24.0±1.7 0
Gram-negative
P. aeruginosa 0 0 9.9±1.8 0
S. marcescens 0 0 21.7±1.1 0
6 mm
A
D
B
C
6 mm
6 mm
6 mm
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)
positive control negative control
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).
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.
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 necessary 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.
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