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:
Available to the general public
Available only after consultation with author/adviser
Available only for those bound by confidentiality agreement
Students 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.

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 ZOISD of the
methanolic extract against B. subtilis was at 23.12.4 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 ..........................................................................................................................

Review of Literature ............................................................................................................

Biology and Importance of Andropogon aciculatus Retz. .............................................

Antimicrobial Activity of Plants .........................................................

Antimicrobial Activity Testing Protocol .........................................................................

Methodology ........................................................................................................

Preparation of Plant Material ......................................................

Preparation of Extracts .......................................................................................

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

Appendix .

30

LIST OF FIGURES

Figure
1

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

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 Andropogon 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 plants
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
chromatographymass 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, hydrastine, 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
a standardized bacterial suspension of 1-5 x 105 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
onto the plates preinoculated with 1 x 108 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, Smnia, & Smnia 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 4C 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 of 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 Salkovskis 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 37C 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 manufacturers
instructions. Each bacterial culture tested was streaked onto an MHA plate to obtain isolated
colonies. After incubation at 35C 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 suspensions, 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
l of the extracts using the mechanical pipette. The discs were dried for 15 minutes inside the
incubator at 35C. 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
35C 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/L0.20 mg/L of extracts was used for this assay.
Mueller Hinton broth (MHB) was prepared according to the manufacturers instructions. The pH
of the broth was checked to see if it lied between 7.2-7.4. If the broths 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 50C. 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 3537C for 1820 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 ZOISD of the methanolic extract against B. subtilis was at 23.12.4 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. subtilis

23.12.4

28.20.9

S. aureus

24.01.7

P. aeruginosa

9.91.8

S. marcescens

21.71.1

BACTERIA

Gram-positive

Gram-negative

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)

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 extracts 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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APPENDIX

Table 3. T-Test Analysis : Two-Sample Assuming Equal Variances

Mean
Variance
Observations
Pooled Variance
Hypothesized Mean Difference
Df
t Stat
P(T<=t) one-tail
t Critical one-tail
P(T<=t) two-tail
t Critical two-tail

Methanol
23.1
5.9
3
3.4
0
4
-3.4
0.01
2.1
0.03
2.8

Streptomycin
28.2
0.9
3