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

be a potential source of antimicrobial compounds.

TABLE OF CONTENTS

Page

Acknowledgements .............................................................................................................

Abstract ................................................................................................................................

List of Figures ......................................................................................................................

List of Tables .......................................................................................................................

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 ………………………………………………....

Test for Alkaloids ………………………………………………………………….

Test for Flavonoids ………………………………………………………………...

Test for Glycosides …………………………………………………………………

Test for Saponins …………………………………………………………………...

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Test for Taninns …………………………………………………………………….

Test for Phenols …………………………………………………………………….

Test for Terpenoids …………………………………………………………………

Test for Phytosterols ………………………………………………………………..

Preparation of Bacterial Cultures ....................................................................................

Antimicrobial Activity Assay ..........................................................................................

Minimum Inhibitory Concentration Determination ........................................................

Data Analysis ..................................................................................................................

Results ……..........................................................................................................................

Discussion ...........................................................................................................................

Conclusion ………..............................................................................................................

Recommendations ...............................................................................................................

Literature Cited …...............................................................................................................

Appendix ………………………………………………………………………………….

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LIST OF FIGURES

Figur

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1 Antibacterial assay results showing the ZOIs of the methanol and n-hexane extracts of A. aciculatus ..........................................................................................

18

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.

aciculatus...................................................................................................................

3 T-test Analysis: Two-Sample Assuming Equal Variances ………………………...

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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; El-

Mahmood & 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 disease-

causing 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 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, β-

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, 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 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 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 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

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

(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.

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 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

Methanol StreptomycinMean 23.1 28.2Variance 5.9 0.9Observations 3 3Pooled Variance 3.4Hypothesized Mean Difference 0Df 4t Stat -3.4P(T<=t) one-tail 0.01t Critical one-tail 2.1P(T<=t) two-tail 0.03t Critical two-tail 2.8