Download Statistical Analysis of the Antibacterial Activity of Glyricidia Sepium

Asian Journal of Biochemical and Pharmaceutical Research Issue 3(Vol. 3) 2013
ISSN: 2231-2560
CODEN (USA): AJBPAD
Research Article
Asian Journal of Biochemical and Pharmaceutical Research
Statistical Analysis of the Antibacterial Activity of Glyricidia Sepium Bark, Leaf and
Flower Extracts
Beena Jose1*, Alice Thomas1 and L. Joji Reddy2
1*
Department of Chemistry, Vimala College, Thrissur, Kerala, 680009, India.
1
Department of Statistics, Mercy College, Palakkad, Kerala, India.
2
Department of Biotechnology, Loyola Academy Degree & P.G. College, Alwal,Secunderabad, Andhra Pradesh, 500010, India.
Received: 19 May 2013; Revised: 04 August 2013; Accepted: 19 September. 2013
Abstract: The antibacterial activities of methanol, ethyl acetate, chloroform and aqueous extracts of the bark,
flower and leaf of Gliricidia sepium against ten pathogenic bacteria such as Bacillus cereus, Enterobacter
faecalis, Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Streptococcus faecalis, Proteus
vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa and Serratia marcescens were statistically
evaluated. G. sepium flower methanol extract (Gsfa) was found to be the most potent extract and its activity is
quite comparable with the standard antibiotics such as tobramycin and ofloxacin screened under similar
conditions. So, methanol extract of the Gliricidia sepium flower can be used as a potential external antiseptic
and can be incorporated into drug formulations.
Key words: Gliricidia sepium, antibacterial activity, statistical analysis, standard antibiotics, drug formulation.
INTRODUCTION:
Gliricidia sepium (Leguminosae family) is a medium sized tree introduced into India from the
American continent. This tree is used in Mexico as shade for cocoa and coffee plantations and for this
reason it is called ‘Madrecacao’ (mother of cocoa). It is also used as a poison for rodents and in fact
the Latin name Gliricidia means rodent poison. It is used as a hedge plant and the flowers are utilized
as food in some places in Mexico[1]. In Panama, the decoction of G. sepium leaves used in utricaria,
rash and also in burns and erysepalas. In Guatemala and Costa Rica, bark decoction is used against
bacterial and protozoal infections2. Branches of Gliricidia sepium is used to reduce fever in children
and adults. It has also been used to treat infections produced by Microsporum canis, Trychophyton
mentagrophytes and Neisseria gonorrohae[2]. Sharma and Qadry[4] investigated the larvicidal activity
of the crude ethanol extract of Gliricidia sepium bark and leaves. Various phytochemicals like
flavanoids[5], triterpenoid saponins [6], stigmastanol glucoside [7], rhamnogalactoside of
kaempferol[8], coumarin, coumaric acid and melilotic acid[9] have been isolated and characterised
from various parts of this plant. Allelochemicals from Gliricidia sepium leaves were extracted,
identified and quantified using HPLC [10]. Rastrelli [11] isolated a new 12a-hydroxy rotenoids from
the methanolic extract of Gliricidia sepium bark.
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Asian Journal of Biochemical and Pharmaceutical Research
Issue 3 (Vol. 3) 2013
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In the present study antibacterial activities of the crude extracts of Gliricidia sepium bark,
flower and leaf were analyzed statistically so as to evaluate the medicinal potential of these plant
extracts.
MATERIALS AND METHODS:
Plant Material: Bark, flower and leaf of Gliricidia sepium were collected from Kerala, South India
and authenticated by Dr. A.K. Pradeep, Dept. of Botany, Calicut University. Voucher specimen is
deposited in the specially maintained herbarium, Department of Chemistry, Calicut University.
Preparation of plant Extracts: Fifty grams of each of powered plant material were extracted
successively with 150ml of chloroform, ethyl acetate, methanol and water as solvents for 24 hours by
Soxhlet equipment.
Antibacterial Activity Assay: The agar diffusion method is used for the antimicrobial evaluations.
Wells of 8mm (0.8cm) diameter were dug on the inoculated nutrient agar medium with sterile cork
borer and 50µl of the chloroform, ethyl acetate, methanol and aqueous extracts of the bark, flower and
leaf of Gliricidia sepium were added in each well. The diameter of the inhibition zone was measured
in mm[12].
RESULTS AND DISCUSSION:
Statistical Analysis: Let us assume that the mean activity of G. sepium flower methanol extract (Gsfa)
and G. sepium leaf methanol extract (Gsla) are same. Also the alternate hypothesis is that the mean
activity of Gsfa is greater than Gsla. By applying student’s t-test, it can be seen that the calculated tvalue is greater than tabled value at eighteen degrees of freedom with significance level 0.05. Hence
the assumption that two extracts having same activity is rejected and the mean activity of Gsfa against
ten pathogenic bacteria is found to be higher than Gsla.
Similarly by applying t-test, the mean activity of G. sepium bark, leaf and flower extracts in
various solvents are compared. The mean activity of Gsfa>Gsba (activity of Gsfa greater than Gsba),
Gsbb>Gslb, Gsfb>Gsbb, Gsfb>Gslb, Gsfc>Gslc, Gslc>Gsbc, Gsfc>Gsbc, Gsfd>Gsbd, Gsfd>Gsld
while that of Gsba-Gsla and Gsbd-Gsld are almost same. The interval estimation of the mean activity
of G. sepium bark, leaf and flower extracts in various solvents are given in table 1.
The correlation studies suggests that Gsfd-Gsbd and Gsfd-Gsld are highly correlated (0.95)
and hence the mean activity of these extracts against ten pathogenic bacteria increases simultaneously
whereas Gsba-Gsla, Gsfa-Gsla and Gsfa-Gsba are negatively correlated and the activity of these
extracts against the bacteria changes in an inverse manner. Ethyl acetate (b) and chloroform (c)
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extracts of G. sepium bark, leaf and flower also showed appreciable positive correlation though not
high.
From table (2), it can be seen that the mean activity of Gsba>Gsbc, Gsbb>Gsbc, Gsbd>Gsbc,
Gsla>Gslb, Gsla>Gslc, Gsfa>Gsfb, Gsfa>Gsfc, Gsfa>Gsfd, Gsfb>Gsfd and Gsfc>Gsfd while the
mean activity of Gsbb-Gsba, Gsba-Gsbd, Gsbb-Gsbd, Gslc-Gslb, Gslb-Gsld, Gslc-Gsld, Gsfb-Gsfc
and Gsla-Gsld are almost same as the t-value is less than tabled value.
The correlation studies showed that Gsfa-Gsfb is highly correlated whereas Gsfa-Gsfc, GsfaGsfd, Gsfb-Gsfc, Gsfb-Gsfd, Gsfc-Gsfd, Gslb-Gsld, Gsbb-Gsbc, Gsba-Gsbd and Gsbb-Gsba showed
low positive correlation. Gsba-Gsbc, Gsbb-Gsbd, Gsbd-Gsbc, Gsla-Galb, Gsla-Galc and Gsla-Gsld
showed negative correlation and the mean activity of these extracts against ten bacteria varies in an
inverse manner. The activity of Gsfa and activity of standard antibiotics such as tobramycin, ofloxacin
are statistically analysed and found that they have the same activity against ten pathogenic bacteria and
hence G. sepium flower methanol extract (Gsfa) can be incorporated into the drug formulations.
CONCLUSION:
Statistical analysis proved that the activities of G. sepium bark ethyl acetate (Gsbb), methanol
(Gsba) and distilled water (Gsbd) extracts are almost same and greater than that of chloroform (Gsbc)
extract. The activity of Gsla is higher than Gslb, Gslc and Gsld. The activity of Gsfa is higher than
Gsfb, Gsfc and Gsfd. Hence the activity of Gsla, Gsfa, Gsbb, Gsba and Gsbd are compared and found
that the activity of Gsfa is greater than the activity of all other extracts. The activity of Gsfa is quite
comparable with the activity of standard antibiotics such as tobramycin and ofloxacin and hence G.
sepium flower methanol extract (Gsfa) can be incorporated into the drug formulations.
The antimicrobial potency of the G. sepium flower methanol extract can be attributed to the
presence of phenolic compounds13 and flavonoids5. It is interesting to note that even crude extract of
this plant showed prominent activity against various pathogenic bacteria where modern therapy has
failed. The variation of the susceptibility of the tested microorganisms could be attributed to their
intrinsic properties that are related to the permeability of their cell surface to the extracts.
ACKNOWLEDGEMENT:
One of the authors (Beena Jose), sincerely acknowledge University Grants Commission, New
Delhi, for providing financial assistance to carry out the study.
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Table1: Interval estimation of the mean antibacterial activity of G. sepium bark, leaf and flower
extracts
G. sepium bark, leaf Mean activity
Standard Deviation
and flower extracts
Interval estimation at 5%
level of significance
Gsba
11.8
6.512
11.8 + 4.04
Gsla
12.3
4.498
12.3 + 2.79
Gsfa
27.4
3.134
27.4 + 1.94
Gsbb
13.0
9.055
13.0 + 5.61
Gslb
4.00
6.599
4.00 + 2.48
Gsfb
22.1
3.281
22.1 + 2.03
Gsbc
3.80
6.143
3.80 + 2.35
Gslc
7.30
6.308
7.30 + 3.90
Gsfc
20.0
3.399
20.0 + 3.39
Gsbd
9.50
6.770
9.50 + 4.19
Gsld
8.10
7.200
8.10 + 4.46
Gsfd
14.5
2.320
14.5 + 1.44
Gsb: G. sepium bark; Gsl: G. sepium leaf; Gsf: G. sepium flower
a: methanol extract; b: ethyl acetate extract ; c: chloroform extract
d: aqueous extract
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Table2: Student’s t-test: Mean activities of G. sepium bark, leaf and flower extracts
G. sepium bark, leaf Calculated value of ‘t’
and flower extracts
Correlation coefficient
Gsba-Gsla
0.341
-0.2215
Gsfa-Gsla
8.263
-0.0999
Gsfa-Gsba
6.476
-0.2208
Gsbb-Gslb
2.727
0.0320
Gsfb-Gsbb
2.834
0.0023
Gsfb-Gslb
7.367
0.2717
Gsfc-Gslc
5.317
0.4910
Gslc-Gsbc
1.193
0.1879
Gsfc-Gsbc
12.41
0.1745
Gsbd-Gsld
0.425
0.3530
Gsfd-Gsbd
4.750
0.9595
Gsfd-Gsld
2.537
0.9564
Gsbb-Gsba
0.322
0.2676
Gsba-Gsbc
2.681
-0.1788
Gsba-Gsbd
0.734
0.3806
Gsbb-Gsbc
2.522
0.3795
Gsbb-Gsbd
0.928
-0.3860
Gsbd-Gsbc
1.870
-0.0988
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Asian Journal of Biochemical and Pharmaceutical Research
G. sepium bark, leaf and Calculated value of ‘t’
Issue 3 (Vol. 3) 2013
CODEN(USA) : AJBPAD
Correlation coefficient
flower extracts
Gsla-Gslb
3.117
-0.6213
Gsla-Gslc
1.936
-0.3559
Gsla-Gsld
1.484
-0.4091
Gslb-Gslc
-1.084
0.0534
Gslb-Gsld
-1.259
0.1215
Gslc-Gsld
-0.251
0.0799
Gsfa-Gsfb
3.504
0.8060
Gsfa-Gsfc
4.801
0.6258
Gsfa-Gsfd
9.923
0.2291
Gsfb-Gsfc
1.333
0.3985
Gsfb-Gsfd
5.672
0.1969
Gsfc-Gsfd
4.008
0.2675
Gsb: G. sepium bark; Gsl: G. sepium leaf; Gsf: G. sepium flower; a: methanol extract; b: ethyl
acetate extract ; c: chloroform extract., d: aqueous extract Tob: tobramycin; Gen: gentamicin
sulphate; Oflo: ofloxacin; Cip: ciprofloxacin
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*Correspondence Author: Beena Jose, Department of Chemistry, Vimala College, Thrissur, Kerala, ,
INDIA.
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