Pakistan J. Agric. Res. Vol 24 No.1-4, 2011.

ANTIMICROBIAL PROPERTY OF GLIRICIDIA SEPIUM PLANT

EXTRACT

Rahila Nazli, Tehmina Sohail, Bushra Nawab and Zahra Yaqeen*

ABSTRACT:- Quart for the new antimicrobial agent is still there and the present
work is an attempt in this regard, Ethanolic extract of Gliricidia sepium in differ-
ent concentration was used to investigate its antimicrobial activity against gram
+ve and gram-ve bacteria. The study was also extended to some species of fungi.
Results showed that the activity was more pronounced against gram +ve organ-
isms and fungi. Maximum inhibition activity was calculated against all the groups
of organisms, which was between 0.5 and 1mg ml-1 against bacteria and 2.5 mg
ml-1 against fungi. It is therefore concluded that G. sepium provide a lead towards
the exploration of new antimicrobial agent.

Key Words: Gliricidia sepium; Antibacterial Actvity; Antifungal Activity, Pakistan.

INTRODUCTION creases in weight and milk production in

Gliricidia sepium is a leguminous tree both large and small ruminants when
and belongs to the family Fabeacae Gliricidia forage is used as a supplement.
(Chadhokar, 1982). It is originated in Cen- (Nochebueno and O’ Donovan, 1986).
tral America and is used in many tropical Gliricidia means mouse or rat killer, which
and sub-tropical countries. This plant was is derived from its bark and leaves which
introduced in Philippines and Sri Lanka in when cooked with grain can be used as
1600s and 1800s, respectively to provide poisonous bait for rodents. Though poison-
shade to tea plants. Seeds of G. sepium were ous to rodent and insect, the leaves con-
introduced in Pakistan in 1996 from Sri tain 3-4% dry weight of nitrogen and small
Lanka to provide green manure for coco- amount of phosphorus, potassium, calcium
nut plants and shadow for beetle leaf plant and magnesium, so they can be used as
in the Plant Research Center of Coastal Re- excellent green manure and fodder.
search Station, Karachi. Research showed In another study the antimicrobial
that the plant can be cultivated in the properties of extracts from the leaves of
plains of Sindh and Punjab in addition to Gliricidia sepium was tested. It was effec-
the coastal areas of Sindh where irrigation tive against bacteria and fungi causing der-
facilities are available. matitis. Plant oils and extracts have been
For the first time in Pakistan research used for various purposes for many thou-
on Gliricidia sepium is being carried out at sands of years (Jones, 1996). In particular,
PCSIR Labs Complex, Karachi especially in the antimicrobial activity of plant oil and
the area of mosquito repellent and nem- extracts has formed the basis of many ap-
aticidal characteristics of this plant. plications, including raw and processed food
The plant is used for fuel wood, ani- preservation, pharmaceutical, alternative
mal feed, green manure, shade, living medicine and natural therapies (Lis-
fences and as support plants (Csurhes and Balchin and Deans, 1997).Traditionally
Edward, 1998). The leaves of G. sepium have used medicinal plants produce a compound
a high feeding value with crude protein of known therapeutic properties (Iyengar
comprising 20-30% of the dry matter, a 1981; Chopra et al., 1992, Harborne and
crude fiber content of about 15% and in Baxter, 1995). The substance that can ei-
vitro dry matter digestibility of 60-65% ther inhibit the growth of pathogens or kill
(Adejumo and Ademosun, 1985; Gohl, them and have no or least toxicity to host
1981). There are numerous reports of in- cell are considered candidate for develop-
*PCSIR Lab Complex, Karachi, Pakistan.

51
 RAHILA NAZLI ET AL.
ing new antimicrobial drugs. In the present Tryptic soya agar (Merck) was used to
study Gliricidia sepium was selected for test bacteria and Sabouraud Dextrose agar
screening against some pathogenic bacte- was used for fungi. Bacterial cultures
ria and fungi. The selection of this medici- freshly grown at 37°C and fungal culture at
nal plant is based on their traditional uses. 25°C. Bacterial cultures were appropriately
Present work is an attempt to screen diluted in sterile normal saline solution to
antimicrobial agents from plant origin obtain the cell suspension at 106CFU ml-1.
(Clark, 1996). These agents may have
many therapeutic effects for the treatment
Antibacterial Activity
of disease and infections. The side effect
associated with these diseases by the us- This activity was carried out by agar
age of synthetic drugs may also be reduced. well diffusion method (Ahmad et al., 1998).
Antimicrobial agents from plants may also According to this method, 0.1 ml of diluted
help to reduce the multiple drug resistance inoculums (106 CFU ml-1) of test organism
burdens. was thoroughly mixed with 20 ml of mol-
ten sterile tryptic soya agar and poured into
pre-sterilize Petri dishes under sterile con-
MATERIALS AND METHODS dition. All plates were left to set at 4°C for
Gliricidia sepium plant leaves were col- 30-40 minutes. Holes of 6 mm diameter
lected from PARC-SARC, Karachi. All the were made in the center of each seeded
leaves samples were preserved in wax plates. Holes were then filled aseptically
quoted paper bags and brought to the labo- with 0.1 ml of test solution (various extract
ratory for biological assays. in various conc.) reference standard and
The fresh leaves of G. sepium (5kg) was negative control (i.e., solvent only) respec-
ground and soaked in ethanol (commercial, tively and marked accordingly. All plates
doubly distilled 50 l). The filtrate was con- were then incubated at 37 ±1°C for 24h and
centrated under reduced pressure at 40ºC zone of inhibition exhibited by the differ-
to a gum. ent extracts in various concentration mea-
Alcoholic extract was dissolved in 6% sured and recorded accordingly. All plates
dimethylformamide(DMF) to make stock were run in triplicates.
solution of 20mgml-1 by which further dilu-
tions were made and used for testing. Ampi- Antifungal Activity
cillin and Nystatin (1mgml-1) were used as This activity was determined by agar
a reference standard. While 6% tube dilution method (Paxton, 1991). Test
dimethylformamide was used as negative
tubes having sterile sabouraud dextrose
control. The said activity was assessed
agar were inoculated with test solution of
against gram +ve and -ve microorganisms.
different concentration and kept in slant-
All microorganisms used in the present
ing position at room temperature for solidi-
study were taken from Department of Mi-
fication. Test fungal cultures were inocu-
crobiology, University of Karachi, Karachi.
lated on slant incubated at 25°C for 7 days
The clinical isolates were biochemically
and growth inhibition were observed after
characterized by standard methods. The
7 days incubation period (Washington and
organisms used in this study were:
Sutter, 1980). Nystatin was used as stan-
Bacillus subtilus, B. pumilus, B. cereus,
dard antifungal drug.
Staphylococcus aureus, Streptococcus in-
termedius. Escherichia coli, Proteus
mirabilis, Salmonella typhi, Klebsiella MIC Determination
pneumoniae, Shigella flexneri, Fusarium The MIC values of ethanolic extract of
solani, Trichophyton rubrum, Aspergillus Gliricidia sepium were determined against
effuses, Rhizomucor pusillus, Trichophy- the gram +ve and -ve bacteria and fungi
ton sclerosis, Macrophomnia phaseolina (106 CFU ml-1) by the serial dilution tech-
and Rhizoctonia solani. nique (Reiner, 1982). Nutrient agar and
52
 ANTIMICROBIAL PROPERTY OF GLIRICIDIA SEPIUM
Nutrient broth were used as bacteriological RESULTS AND DISCUSSION
media. A set of tubes with different concen- In the present study, Ethanol extract
trations (0.25, 0.5, 1, 2, 2.5, 5, 10 and 20 mg of leaves of Gliricida sepium were tested
ml-1) were prepared. The tubes were inocu- against some pathogenic bacteria and
lated with test organisms incubated at 37oC fungi. The antibacterial activity of extract
for 24 h. After incubation time, plates were was quantitatively assessed by the pres-
analyzed visually for the presence of growth. ence or absence of inhibition zone and
Growth is seen to diminish as the concen- diameter, respectively (Figure 1).
tration of extract increased and eventually G. sepium extract showed activity
that concentration was observed at which against all gram-ve organisms at 20mg
growth fails to occur. ml-1 concentration while at 10 mgml-1 and
5 mgml-1 concentration showed good and
Statistics low activity respectively all these con-
The data are analyzed as mean + S.E. and centration showed significant difference
compared by applying t-test using Sigma Plot (p> 0.05). At 5mg ml-1 concentration Kleb-
software version 11.2. The value less than siella pneumoniae was found resistant as
0.5% is considered as significant. concentration increases extract showed

A B C D E F G H I J

Figure 1. Antibacterial activity exhibited by G. sepium gram +ve and -ve organisms (A=
Staphylococcus aureus, B= Streptococcus intermedius, C= Bacillus pumilus, D=
Bacillus subtilus, E= Bacillus sereus, F= Escherichia coli, G= Salmonella typhi,
H= Klebsiella pneumoniae, I= Proteus mirabilis, J= Shigella flexneri)

Figure 2. Antifungal activity exhibited by G. sepium (A= Fusarium solani, B=

Trichophyton rubrum, C= Aspergillus effuses, D= Rhizomucor pusillus, E=
Trichophyton sclerosis, F= Macrophomnia phaseolina , and G=
Rhizoctonia solani)

53
 RAHILA NAZLI ET AL.
significant inhibitory activity. In gram +ve activity at highest concentration 20mg
organisms, extract showed significant ac- ml-1. At this concentration, Fusarium solni,
tivity at 20mg ml-1 concentration compa- Rhizomucor pusillus, Trichophyton sclerosis,
rable to standard antibiotic. These findings Macrophomnia phaseolina and Rhizoctonia
were in conformity with Jhon et al. (2006) solani showed good activity while Aspergil-
and Abulude and Adebote (2009) who also lus effuses showed low activity at 20mgml -
reported that ethanol extract of G.sepium 1
and was resistant at 10 and 5mg ml-1. In
exhibited good antimicrobial activity some studies G. sepium showed significant
against Staphylococcus aureus, Bacillus activty against Candida albican.
subtilus and Escherichia coli (Figure 2). An attempt has therefore been made
Similarly Kakuko et al. (2005) also reported to determine the minimum inhibitory con-
that 22 Mexican medicinal plants showed centration of ethanol extract against gram
antibacterial activity against Staphylococ- + ve and -ve bacteria as well as fungi (Table
cus aureus, and Escherichia coli. However 1 and 2). In gram + ve bacteria (Staphylo-
some research studies established on dif- coccus aureus, Streptococcus intermedius, Ba-
ferent solvents like chloroform and metha- cillus pumilus, B. subtilus and B. cereus) MIC
nol extract of the bark of G.sepium and on value was found 1mg ml-1. Against the gram
essential oils of leaf and flower part of -ve bacteria (Salmonella typhi, Escherichia
G.sepium also showed significant activity coli, Klebsiella pneumoniae, Proteus
against different bacterial strains (Salud mirabilus and Shigella flexneri), MIC value
et al., 2007; Beena and Reddy, 2010). was found to be 0.5mgml-1. Against fungi
Among fungi none showed significant MIC value was 2mgml-1 except Aspergillus

Table 1. Minimum inhibitory concentrations of ethanolic extract against gram

-ve and+ve bacteria
Test organism Concentration (mg ml-1)
20 10 5 2.5 2 1 0.5 0.25
Staphylococcus aureus - - - - - - + +
Streptococcus intermedius - - - - - - - +
Bacillus pumilus - - - - - - + +
Bacillus subtilus - - - - - - + +
Bacillus cereus - - - - - - + +
Escherichia coli - - - - - - - +
Salmonella typhi - - - - - - - +
Klebsiella pneumoniae - - - - - - - +
Proteus mirabilis - - - - - - - +
Shigella flexneri - - - - - - - +

Table 2. Minimum inhibitory concentrations of ethanolic extract against gram –ve

and +ve yeast and fungi
Test organism Concentration (mg ml-1)
20 10 5 2.5 2 1 0.5 0.25
Fusarium solani - - - - + + + +
Trichophyton rubrum - - - + + + + +
Aspergillus effuses - + + + + + + +
Rhizomucor pusillus - - - - + + + +
Trichophyton sclerosis - - - - + + + +
Macrophomnia phaseolina - - - - - + + +
Rhizoctonia solani - - - + + + + +

54
 ANTIMICROBIAL PROPERTY OF GLIRICIDIA SEPIUM
effuses which is somewhat resistant. edn). Taylor and Francis, London.
It may be concluded safely that etha- Iyenger, M.A. 1981. Study of Crude Drugs,
nol extract of G.sepium have the most (2nd edn). Manipal Power Press, Manipal,
active antibacterial components than an- India. p. 13-78.
tifungal and can be a good source of chemi- Jhon, J.R. Veronica, J.O. Soul, A.O.and
cal compound. John, F.M. 2006. Screening for antibac-
terial activity of ten medicinal plants
LITERATURE CITED used in Colombia folkloric medicine: a
Abulude, F.O. and Adebote, V.T. 2009. Anti- possible alternative in the treatment of
bacterial investigation of crude extracts non somial infection. BMC Complemen-
of the root, bark of Gliricidia sepium. Con- tary and Alternative Medicine 10/1186/
tinental J. Microbiol. 3:23-26. 1472.
Ahmed I. Mehmood, Z. and Mohammad, Jones, F.A.1996. Herbs useful plants role
F.1998. Screening of some Indian me- in history and today. European J. Gas-
dicinal plants for their antimicrobial troenterology and Hepatology, 8: 1227.
properties. J. Ethanopharmacology, Kakuko, Y. Fumiko, A. Ogayama, A. N.
62:183-193. Hikaru, O. Lucio, L. P. Edith, L. Eliza-
Adejumo, J. O. and Ademosun, A. A.1985. beth, M. Abigail, A. and Ricardo,
Effect of plants age at harvest and cut- R.C.2005. Antibacterial activity of crude
ting time frequency and height on the extract from Mexican medicinal plants
dry matter yield and nutritive value of and purified coumarins and xanthones
Gliricidia sepium and Cajanus cajan. J. J. Ethnopharmacology, 97(2): 293-299
Anim. Prod. Res. 5:1-12. Lis- Balchin, M. and Deans, S.G.1997. Bio-
Beena, J. and Reddy, L.J. 2010. Evaluation activity of selected plants essential oil
of antibacterial activity of the leaf and against Listeria monocytogenes. J. Appl.
flower essential oil of Gliricidia sepium Bacteriol. 82: 759-762.
from South India. Intern. J. Appl. Phar- Nochebuena,G. and O’ Donovan P.B. 1986.
maceutics, 2(2):20-22. The nutritional value of high- protein
Chopra, R.N. Nayer, S.L. and Chopra, forages from Gliricidia sepium. World
Anim. Rev. 57:48-49.
I.C.1992. Glossary of Indian Medicinal
Paxton, J.D. 1991. Assay for anti
Plants, (3rd edn.) Council of Scientific
fungal activities.In:Dey,T.M. and
and Industrial Research, New Delhi.p.7-
Harborne, J.B. (eds.) Methods in
246.
Plant Biochemistry, Academic Press,
Clark, A. M. 1996. Natural product as a London,UK.
source for new drugs. Pharm. Res. Reiner, R. 1982. Detection of antibiotics
13:1133-1141. activity: In: Antibiotic an Introduction.
Chadokar, P A. 1982.Gliricidia maculate, a Roche Scientific Service, Switzerland.
promising legume forage plant. World p. 21-25.
Anim. Rev. 44:36-43. Salud, P.G. Miguel, A. Zavala, S. Lucina,
Csurhes, S. and Edwards, R. 1998. Poten- A.G. Cuachtemoe P. G. Rosa, M. and
tial environmental weeds in Australia; Perez, G. 2007. Antibacterial study of
candidate species for preventative con- bark from five tree species.
trol. Queensland Department of Natural Phytotherapy Res.15(4): 356-359.
Resources p.164. Washington, J.A. and Sutter, V.L. 1980.Di-
Gohl, B. 1981. Tropical feeds; feed informa- lution susceptibility test: Agar and
tion summaries and nutritive values. microbroth dilution procedures In:
FAQ Animal Production and Health Lennette E.H. Balws, A. Hauster, W.J Jr.
Series, No. 12. FAQ, Rome, Italy, 529p. and Traunt, J.P. (eds.) Manual of Clini-
Harborne, S.B. and Baxter, H. 1995. Photo- cal Microbiology, American Society for
chemical dictionary. A handbook of Microbiology,Washington DC, USA.p.
bioactive compounds from plants, (2nd 453-458.
55