In Vitro Antimicrobial Activity

of Cynodon dactylon (L.) Pers. (bermuda)

Against Selected Pathogens
S. Abdullah, J. Gobilik and K. P. Chong

Abstract Cynodon dactylon (L.) Pers. is a type of perennial grass that possesses
great medicinal values. In this study, the antimicrobial activity of the plant crude
extract from seven different solvents (acetone, chloroform, diethyl ether, ethanol,
ethyl acetate, methanol, and n-pentane) was investigated against some pathogens
(Bacillus cereus, Bacillus subtilis, Escherichia coli, Klebsiella spp., Pseudomonas
aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus
pneumonia) using disc diffusion method and thin-layer chromatographic (TLC)
bioassay for plant-SPE extracts against Aspergillus niger. Crude extraction showed
that ethanolic extraction produced highest yield (7.065 %) followed by methanolic
(5.420 %) and chloroform (3.550 %) extraction. The lowest yield was obtained
from n-pentane extraction (0.500 %). The antimicrobial study revealed broad
spectrum of antimicrobial activity from ethanol (7.010.0 0.01.0 mm) and
ethyl acetate (7.012.0 0.01.0 mm) extracts against all of the bacterial
pathogens. Both methanol and acetone extracts showed activity to B. cereus
(8.0 0.0 mm) and B. subtilis (7.0 0.0 mm), while chloroform extract showed
activity to B. subtilis (7.0 0.0 mm) and S.pyogenes (8.3 0.6 mm), respectively. Diethyl ether extraction showed activity only to S. pyogenes
(7.3 0.6 mm), while no activity was observed from n-pentane extraction. Great
antimicrobial activity were observed for both ethyl acetate and ethanol SPE-based
extracts (SBE) with size of inhibition ranging from 8.0 0.0 mm to
15.7 0.6 mm for ethyl acetate SBE and 8.0 0.0 mm to 13.0 0.0 mm for
ethanol SBE. No significant antimicrobial activity was observed from thin-layer
chromatographic bioassay against A. niger.
S. Abdullah  K. P. Chong (&)
Sustainable Palm Oil Research Unit (SPOR), School of Science and Technology,
Universiti Malaysia Sabah, 88400 Kota Kinabalu, Sabah, Malaysia
e-mail: chongkp@ums.edu
J. Gobilik
School of Sustainable Agriculture, Universiti Malaysia Sabah,
Sandakan Campus, Mile 10, Sg. Batang 90000 Sandakan, Sabah, Malaysia
e-mail: jgobilik@ums.edu.my

R. Pogaku et al. (eds.), Developments in Sustainable Chemical

and Bioprocess Technology, DOI: 10.1007/978-1-4614-6208-8_29,
 Springer Science+Business Media New York 2013

227

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S. Abdullah et al.

Keywords Cynodon dactylon Crude extract SPE-based extract TLC bioassay

Antimicrobial

Introduction
Medicinal plants play a very important role in pharmaceuticals industry in
developing alternative drugs to overcome the pitfalls possessed by the synthetic
drugs (Yadav et al. 2010). The development of drug-resistant pathogens that are
mostly involved in nosocomial infection has raised concern among medicinal
practitioners (Adalet et al. 2011). It was believed that the intense used of a number
of synthetic antimicrobial drugs which contributed to the development. Besides the
tougher jobs to search for more effective drugs against the pathogens, it also
created the problem in controlling the growth of infectious diseases caused by the
pathogens. Meanwhile, most of the synthetic drugs possess side effect to the
consumers (Chaudhari et al. 2011). Given the alarming incidence of antibiotic
resistance in pathogens raises concern among the medical practitioners, there is a
constant need for new and effective therapeutic agents. Hence, there is a need to
develop alternative antimicrobial drugs for the treatment of infectious diseases
from medicinal plants. Plants produce large number of organic compounds such as
alkaloids, flavonoids, glycosides, tannins, terpenoids, and phenolics as secondary
metabolites which are used as defensive mechanism against respective pathogens
(Francisco and Pinotti 2000; Zwenger and Basu 2008; Chong et al. 2011). These
compounds possess medicinal values and become an attractive subject for
researchers to develop new antibiotics (Joy et al. 2001). Previous studies show that
many plant secondary metabolites act as bioactive compounds, chemotherapeutic,
bactericidal, and bacteriostatic agents (Singh and Gupta 2008; Pongsak and
Parichat 2010; Syahriel et al. 2012). As a result, considerable amount of attention
of antimicrobial substances derived from higher plants is immerging in recent
years. However, the assessments on plant-derived antibiotics are still under
investigation. Cynodon dactylon is a perennial, pan-tropical species of grass which
belongs to the family Poaceae. It is found almost everywhere in tropical, subtropical, and even in semi-arid climates (Watson and Dallwitz 1992). It is not only
widespread, but also widely used by human. Scientifically, it is tested to have
antidiabetic effect, diuretic activity, antioxidant, anticancer potentials, antiulcer
activity, right heart failure protective activity, and allergic effect (Singh et al.
2007; Albert-Baskar and Ignacimuthu 2010). Traditionally, C. dactylon is used as
a rejuvenator and for wound healing (Mangathayaru et al. 2009). Besides used in
remedy, the plant is also used as forage for animals and as turfgrass (Wu et al.
2007). This study will report the antimicrobial activity of C. dactylon against some
common pathogens such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, which are highly associated with nosocomial infection.

In Vitro Antimicrobial Activity

229

Methodology
Plant Collection (Cynodon Dactylon)
Wild ecotype of C. dactylon was collected near the Universiti Malaysia Sabahs
(UMS) area in Kota Kinabalu, Malaysia, sowed, and maintained in a mini-nursery
in campus. Voucher (jgobilik 1090/2011) was kept at School of Sustainable
Agriculture, UMS, and a duplicate was submitted to BORH Herbarium, Institute of
Tropical Biology and Conservation, UMS. Samples were collected in the evening
during the daylight time. Prior to extraction, the plant samples were cleaned
thoroughly with distilled water to remove soil and dirt.

Plant Extraction
The whole part of C. dactylon which was cleaned by distilled water was shadedried for 24 h in a drying chamber at 4050 C and powdered using a mechanical
blender (Waring Commercial Blender). Approximately, 100 g of plant powder
was later soaked into 200 mL of different solvents (acetone, chloroform, diethyl
ether, ethanol, ethyl acetate, n-pentane, and methanolMerck) and shaken on a
platform shaker (LabCompanionTM) at 150 rpm with temperature of 25 C to
obtain various plant extracts. The soaking process was repeated three times for
each extraction to obtain a complete extraction. The extracts obtained were then
evaporated and concentrated under reduced pressure (7687 mmhg) using RotaVaporTM (BUCHI) to obtain 1 ml of extract per 10 g of plant sample. Aliquots
were then kept in -20 C temperature for further use.

Solid-Phase Extraction
StrataTM-X 33um Polymeric Sorbent reverse-phase (200 mg/6 mL) (Phenomenex)
cartridges with 12-cartridge manifold system was used. Methanol absolute (1 ml)
was used to activate the sorbent and further equilibrated with sterile deionised
distilled water (1 ml). Samples were then loaded into the cartridges and washed
with 1 % methanol (1 ml) to remove any impurities from the samples and finally
eluted with 2 ml of methanol:acetonitrile (1:1; v/v). The aliquots were taken to
dryness using purified nitrogen gas. Dried aliquots were stored in -20 C for
further bioassays. In this study, only ethyl acetate and ethanol extracts were used
for SPE-based extract bioassay.

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S. Abdullah et al.

Test Microorganisms
Pure cultures of eight different pathogens (Bacillus cereus, Bacillus subtilis,
E. coli, Klebsiella spp., P. aeruginosa, S. aureus, Streptococcus pyogenes, and
Streptococcus pneumonia) were obtained from Queen Elizabeth Hospital, Kota
Kinabalu, Malaysia. The microbial cultures were preserved in 30 % glycerol stock
solution at -85 C temperature. Prior to antimicrobial activity study, the test
microorganisms were subcultured on nutrient agar (NA), incubated at 37 C for
24 h before grown in nutrient broth (NB) and adjusted according to McFarland
standard to achieve approximately 1 9 108 CFU/ml before introducing to the test
media. For thin-layer chromatographic (TLC) bioassay, Aspergillus niger was
obtained from stock culture of UMS. The A. niger was cultured on potato dextrose
agar (PDA) for approximately 710 days, before it is suspended in potato dextrose
broth (PDB) prior to TLC bioassay.

Antimicrobial Activity Study

Thin-layer chromatographic bioassay: Thin-layer chromatography (TLC) (Silica
Gel 60 F254Merck) bioassay coupled with A. niger culture was used as preliminary screening for antimicrobial activity. Approximately, 2 mg of C. Dactylon
ethanol and ethyl acetate extracts were applied on TLC plate which developed in
ethyl acetate:hexane (1:9) solvent system and then examined under UV light (265
and 325 nm) to spot the presence of compounds of interest. The plates later were
sprayed with 710 day-old A.niger which was suspended in PDB. TLC plates were
incubated in a moist chamber for 48 h at 2527 C to spot any antimicrobial
property of the separated compounds from the plant.
Disc diffusion bioassay: Antimicrobial activity of the plant extracts was evaluated using disc diffusion method according to Kirby-Bauer method as described
by Lalitha (2011). Sterile discs of Whatman No.3 paper (6 mm diameter) were
prepared for the disc diffusion bioassay. All extract concentrations were standardised to 100 mg/mL, and 60 ll (6 mg) of the extracts were loaded to each disc.
For the negative control discs, 60 lL of the respective solvents was loaded to
separate discs. Chloramphenicol antibiotic discs (1 mg/ml, 30 lg/disc) were used
as positive control to compare the antimicrobial activity. Mueller-Hinton Agar
(MHA) medium was prepared and sterilised in an autoclave at 121 C for 15 min
at 15 psi before transferring it to sterilised petri dish. Approximately, 0.1 ml
culture of bacterial pathogens adjusted according to McFarland standard was
placed on the MHA media and spread throughout the plate using spread plate
technique. The discs loaded with test extracts, their corresponding solvents, and
the antibiotic discs were placed on the media with the help of a sterile forceps
carefully with adequate spacing between each other. The plates were kept at room
temperature for 30 min, which helps to diffuse the extract into the medium.

In Vitro Antimicrobial Activity

231

The plates were then incubated at 37 C for 24 h in an incubator to determine the

antibacterial activity of the respective solvent extraction of C. dactylon. After
incubation, zone of inhibition in diameter was measured and recorded.

Results
Extraction Yield
Different solvents have different resolving strength towards the plant constituents
which resulted in different yield as shown in Table 1. From all of the extractions,
ethanolic extraction produced the highest yield (7.065 %) followed by methanolic
extraction (5.420 %) and chloroform extraction (3.550 %). Most of the extractions
using non-polar solvents produced lower yield. The lowest yield was obtained
from n-pentane extraction with only 0.500 % of yield. The other extraction yield
results are as shown in the Table 1.

Antimicrobial Activity
Thin-layer chromatographic bioassay: Separation of bioactive compounds from
C. dacylon for ethanol and ethyl acetate extracts was the best in ethyl acetate:hexane (1:9) solvent system which produces higher resolution and greater
separation, as shown in Fig. 1. However, the antimicrobial properties from both
ethanol and ethyl acetate extracts were not detected (Fig. 2). The extracts were
unable to inhibit the growth of A. niger after incubated for 48 h.
Disc diffusion bioassay (crude extract): Ethanol and ethyl acetate extracts of the
plant exhibited broad spectrum of antimicrobial activity. From Table 2, both
ethanol and ethyl acetate extracts showed significant effect to almost all the tested
pathogens with the size of inhibition between 7.0 0.0 mm and 10.0 1.0 mm
for ethanol extract and 7.0 0.0 and 12.0 1.0 mm for ethyl acetate extract. The
greatest activity observed was against S. pyogenes (10.0 1.0 mm) for ethanol
extract while B. cereus (12.0 1.0 mm) for ethyl acetate extract. The least
activity observed was against B.subtilis (7.0 0.0 mm) for ethanol extract while
S. pneumonia (7.0 0.0 mm) for ethyl acetate extract. Methanol extract showed
very weak activity against Streptococcus pneumoniae (7.0 0.0 mm), while both
methanol and acetone showed weak activity against B. cereus (8.0 0.0 mm) and

Table 1 Extraction yield of C. dactylon with different solvents

Solvent
Acetone Chloroform Diethyl ether Ethanol Ethyl acetate Methanol n-pentane
Yield (%) 2.9605

3.550

0.923

7.065

1.218

5.420

0.500

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S. Abdullah et al.

Fig. 1 Separation of
bioactive compounds from
C.dactylon by TLC,
E.A:Hexane (2:18) solvent
system, observed under UV
light (i = 254 nm;
ii = 365 nm). a Ethyl acetate
extract; b: Ethanol extract

Fig. 2 Thin-layer
chromatographic bioassay of
ethyl acetate (E.A) and
ethanol (EtOH) extracts of C.
dactylon. A. niger was able to
grow on the plate after
incubated for 48 h. a E.A
extract; b EtOH extract

B. subtilis (7.0 0.0 mm). Chloroform extract showed inhibition to two pathogens tested, B. subtilis (7.0 0.0 mm) and S. pyogenes (8.3 0.6 mm). Diethyl
ether extract exhibited the least activity only to S. pyogenes (7.3 0.6 mm).
Meanwhile, n-pentane extract did not inhibit any of the tested pathogens. In the
present study, B. subtilis (except for the diethyl ether and n-pentane extracts) and
S. pyogenes (except for acetone and n-pentane extracts) were more sensitive
towards most of the extracts. Meanwhile, E. coli, P. aeruginosa, S. aureus,

8.0 0.0
7.0 0.0
n.d
n.d
n.d
n.d
n.d
n.d

n.d
7.0 0.0
n.d
n.d
n.d
n.d
8.3 0.6
n.d

Chloroform
n.d
n.d
n.d
n.d
n.d
n.d
7.3 0.6
n.d

Diethyl ether
9.0 0.0
7.0 0.0
8.3 0.6
8.3 0.6
8.0 0.0
9.0 1.0
10.0 1.0
7.3 0.6

Ethanol
12.0 1.0
8.0 0.0
8.0 0.0
8.0 0.0
9.3 0.6
10.0 0.0
8.0 0.0
7.0 0.0

Ethyl acetate
8.0 0.0
7.0 0.0
n.d
n.d
n.d
n.d
7.0 0.0
n.d

Methanol

n.d
n.d
n.d
n.d
n.d
n.d
n.d
n.d

n-pentane

*Values presented are means of three replicates, stand. dev.; each disc loaded with approx. 60 ul or 12 mg/disc of plant extract
n.d = not detected
C CHL = Chloramphenicol (1 mg/ml, 30 lg/disc)

Bacillus cereus
Bacillus subtilis
Escherichia coli
Klebsiella spp.
Pseudomonas aeruginosa
Staphylococcus aureus
Streptococcus pyogenes
Streptococcus pneumoniae

Acetone

Table 2 Antimicrobial activity of C.dactyloncrude extracts with the respective solvents against pathogens
Tested microbial pathogens
Size of inhibition zone (mm) of the plant extract with the respective solvents*
24.0
22.3
23.3
20.0
24.0
22.3
24.0
21.0

CHL

1.0
0.6
0.6
1.0
1.0
0.6
0.0
1.0

In Vitro Antimicrobial Activity

233

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S. Abdullah et al.

Table 3 Antimicrobial activity of C. dactylon EtOH- and EA SPE-based extraction against

pathogens after 17-h incubation
Tested microbial pathogens Size of inhibition zone (mm)*
A
Bacillus cereus
Bacillus subtilis
Escherichia coli
Klebsiella spp.
Pseudomonas aeruginosa
Streptococcus pyogenes
Streptococcus pneumonia
Staphylococcus Aureus

8.0
7.0
n.d
7.0
7.0
7.0
n.d
n.d

B
0.0
0.0
0.0
0.0
0.0

8.0
7.0
n.d
8.0
7.0
n.d
8.0
7.0

0.0
0.0
0.0
0.0
0.0
0.0

CHL

15.7 0.6
11.0 0.0
9.0 0.0
8.0 0.0
10.0 0.0
8.0 0.0
10.0 0.0
10.0 0.0

13.0 0.0
8.0 0.0
8.0 0.0
7.0 0.0
7.0 0.0
11.0 1.0
8.0 0.0
9.0 0.0

23.7
20.0
24.3
25.7
19.0
19.0
18.0
18.0

0.6
0.0
0.6
0.6
1.0
0.0
0.0
1.0

*Values presented are means of three replicates, stand. dev.; each disc loaded with approx.
60 ul or 12 mg/disc of plant extract; A EA SPE-based extract (elute portion), B EtOH SPE-based
extract (elute portion), C EA SPE-based extract (flush portion), D EtOH SPE-based extract
(flush portion); n.d = not detected; EA = Ethyl acetate, EtOH = Ethanol, SPE = Solid-Phase
Extraction
CHL = Chloramphenicol (1 mg/ml, 30 lg/disc)

Klebsiella spp, and S. pneumoniae were only sensitive to ethanol and ethyl acetate
extracts. No growth or activity was observed on negative discs. Strong inhibition
to all the pathogens growth observed on standard chloramphenicol disc proved
none of the microorganisms tested were chloramphenicol resistant.
Disc diffusion bioassay (for solid-phase extraction-based extract, SBE): Flush
portion from both ethyl acetate and ethanol SPE-based extracts exhibited greater
antimicrobial properties in contrast to the elute portion of the extracts. As shown in
Table 3, both ethyl acetate and ethanol SBEs showed significant activity to almost
all the tested pathogens with size of inhibition ranging from 8.0 0.0
to 15.7 0.6 mm for ethyl acetate SBE and from 13.0 0.0 mm to
8.0 0.0 mm for ethanol SBE. In accordance to the bioassay of the ethyl acetate
crude extract, the greatest activity for ethyl acetate SBE was observed against
B. cereus with 15.7 0.0 mm of inhibition diameter, higher than the crude
extract (12.0 1.0 mm) followed by B. subtilis (11.0 0.0 mm), P. aeruginosa,
S. pneumonia, and S. aureus with 10.0 0.0 mm of inhibition diameter, respectively. The weakest activity was observed against Klebsiella spp. and S. pyogenes
with 8.0 0.0 mm of inhibition diameter, respectively. The greatest activity for
ethanol SBE was against B. cereus with 13.0 0.0 mm of inhibition diameter, in
contrast to ethanol crude extract in which the greatest activity was observed
against S. pyogenes (10.0 1.0 mm). Ethanol SBE also showed activity against
S. pyogenes, with 11.0 1.0 mm of inhibition diameter, and some activity against
S. aureus (9.0 0.0 mm). The weakest activity for ethanol SBE was observed
against Klebsiella spp. and P. aeruginosa with 7.0 0.0 mm of inhibition
diameter, respectively.

In Vitro Antimicrobial Activity

235

Discussion
Higher plants consist of wide range of bioactive compounds, such as alkaloids,
terpenoids, flavonoids, saponins, tannins, etc. which were utilised by the plants
themselves as a defensive mechanism and to maintain the plant biological activities (Zwenger and Basu 2008). From the extraction yield in Table 1, polar solvents were able to produce higher yield. This implies that most of the plant
constituents are polar compounds such as saponins and the phenolics. Previously,
the plant has been studied to contain many bioactive constituents which contributed to its antimicrobial activity (Syahriel et al. 2012). Biological actions are
primarily due to these components in a very complicated concert of synergistic or
antagonistic activities. Mixtures of such chemicals show a broad spectrum of
biological effects and pharmacological properties. To a large extent, the age of the
plant, percentage humidity of the harvested material, situation and time of harvest,
and the method of extraction are possible sources of variation for the chemical
composition, toxicity, and bioactivity of the extracts (Mandal et al. 2007). From
the previous study, the abundance of glycoside and its derivatives were identified
from the plant (Syahriel et al. 2012). Cardiac glycosides from C. dactylon were
previously studied to possess antiarrhythmic activity against ischaemia or reperfusion-induced arrhythmias and cardioprotective properties in tested rat (AlbertBaskar and Ignacimuthu 2010; Najafi et al. 2008). A large number of constitutive
plant compounds have been reported to have antimicrobial activity. Due to the
great extent of pharmacological effects exert by the plant glycoside, the antimicrobial activity exhibited by the plant in the present study could be induced by the
derivatives from this carbohydrate constituent. Carbohydrate and fatty acid
derivatives from natural source have been proven to possess broad-spectrum
antimicrobial activity (Nobmann et al. 2009). The antibacterial activity of ethanol
extract was believed to be due to the presence of active principle in the extracts
such as saponins, phenolics, and terpenoids which might be responsible for the
broad spectrum of antibacterial activity compared to the other extracts (Kafaru
1994; Singh and Gupta 2008). Higher resolving strength of ethanol in regards to its
yield percentage consequently enables it to resolve comparatively more bioactive
compounds which might explain the considerable antimicrobial activity compared
to the other solvents. Meanwhile, other solvents such as ethyl acetate were able to
resolve other trace bioactive constituents which are not being able to be resolved
by ethanol in greater amount, explaining the significant antimicrobial activity
which levelled to the ethanolic extraction. Generally, gram-negative bacteria were
more resistant to antibiotics than gram-positive bacteria. The resistance is due to
the differences in their cell wall composition. In gram-negative bacteria, the outer
membrane acts as a great barrier to many environmental substances including
antibiotics. Presence of thick murine layer in the cell wall prevents the entry of the
inhibitors (Madigan et al. 2009). The present study revealed that there is no
significance difference between gram-negative and gram-positive bacteria in terms
of susceptibility to the crude extracts although for the extracts other than ethanol

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S. Abdullah et al.

and ethyl acetate extracts, the activity was mostly observed against gram-positive
bacteria. However, no significant difference in the antimicrobial activity between
gram-positive and gram-negative bacteria was observed for both ethanol and ethyl
acetate extracts. It may be due to the presence of broad-spectrum antibiotic
compounds in C. dactylon which are able to penetrate or deteriorate the defensive
or growth mechanism of the microorganisms. The potential of antimicrobial
compound sources from the plant extract is worth to be further explored by
identifying the bioactive constituents responsible for the action.
Acknowledgments The authors acknowledge their profound gratitude to Ministry of Higher
Education (MOHE), Malaysia for funding the research (Grant code FRG0288) and the School of
Science and Technology, Universiti Malaysia Sabah for providing the facilities for research work.
We are indebted to Dr. Katrina and the team, Department of Pathology, Queen Elizabeth Hospital
for their support in supplying the biological specimens.

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