EVALUATION OF IN VITRO ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF MORINGA OLEIFERA L. LEAVES

Abstract

Natural plants have been receiving wide attention as source of biological active substances. In the present study, Moringa oleifera L. leaves are tested for their in vitro antioxidant and antimicrobial activities. Chloroform, ethanol and aqueous extracts of Moringa leaves were tested for scavenging of DPPH, Superoxide and FRAP radicals. Antimicrobial activity of the l ea f extracts was evaluated against five bacterial strains by using cylinder plate assay. Concentration dependent antioxidant activity was observed by all the tested extracts, The extracts also induced significant zone of inhibition a t 500µg/ml against tested bacterial strains. The highest zone of inhibition was shown by ethanolic extract against Klebsiella pneumonia at 500µg/ml dose.

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Int. Res J Pharm. App Sci., 2014; 4(3):4-8 ISSN: 2277-4149
Neelima Routhu et al., 2014 4
International Research Journal of Pharmaceutical and Applied
Sciences (IRJPAS)
Available online at www.irjpas.com
Int. Res J Pharm. App Sci., 2014; 4(3):4-8
EVALUATION OF IN VITRO ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF
MORINGA OLEIFERA L. LEAVES
Neelima Routhu, Bandaru V Rao, Bhavani Y and Monika Singh
Department of Botany, Andhra University, Visakhapatnam 530 003, India
*Corresponding Author: Bandaru V Rao; Email: bandaruvrao@yahoo.co.in
Abstract: Natural plants have been receiving wide attention as source of biological active substances. In the present study,
Moringa oleifera L. leaves are tested for their in vitro antioxidant and antimicrobial activities. Chloroform, ethanol and aqueous
extracts of Moringa leaves were tested for scavenging of DPPH, Superoxide and FRAP radicals. Antimicrobial activity of the
leaf extracts was evaluated against five bacterial strains by using cylinder plate assay. Concentration dependent antioxidant
activity was observed by all the tested extracts, The extracts also induced significant zone of inhibition at 500µg/ml against
tested bacterial strains. The highest zone of inhibition was shown by ethanolic extract against Klebsiella pneumonia at
500µg/ml dose.
Key words: Moringa oleifera L., leaves, in vitro antioxidant, antimicrobial activity
INTRODUCTION
Plants have the major advantage of being the
most effective and cheaper alternative source of drugs
1
.
Historically, pharmacological screening of compounds of
natural origin has been the source of innumerable therapeutic
agents
2 - 4
. The problem of bacterial resistance to antibiotics
has become a growing concern worldwide
5 - 9
. This led to
the resurgence in search of the antimicrobial role of herbs
against resistant strains
10-12
. In search of new, safe and
effective antimicrobial agents from natural products, the
present investigation was carried out to evaluate in vitro
antioxidant and antimicrobial activity of Moringa oleifera
leaves extracts
13-14
. Moringa oleifera L. is commonly
known as the drum stick plant, belongs to family
Moringaceae. Earlier studies on Moringa oleifera
have been reported to contain different phytochemical
compounds like lupeol, moriginine, and a bacteriocide,
spirochin, β-sitosterol, daucosterol, stigmasterol
15
.
MATERIALS AND METHODS
Preparation of extracts from leaves of Moringa leifera
The leaves of Moringa oleifera. were collected,
shade dried, powdered and separately extracted in a
Soxhlet apparatus for 6 hrs successively with three
different solvents viz., hexane, chloroform and methanol.
Each extract dried at 45°C under vacuum by using rotary
evaporator (Buchi, Switzerland. And the samples were
weighed in order to make concentrations of 100µg, 250µg,
500µg and 1000 µg each of water, chloroform and ethanol
samples. These samples were further stored in air tight
bottles for their use in antioxidant studies.
IN VITRO ANTIOXIDANT ACTIVITY
The alcoholic extracts of Moringa leaves were
screened for free radical scavenging activity against
DPPH, Superoxide and FRAP radicals at different
concentrations. The Percentage Inhibition was calculated.
All experiments were performed thrice and the mean values
are summarized. DPPH radical scavenging activity was
measured according to the method of Braca et al
16
. An
aliquot of 3ml of 0.004% DPPH solution in ethanol and
0.1ml of plant extract at different concentrations were mixed
and incubated at 37˚c for 30 min. and absorbance of the
test mixture was read at 517nm. Positive controls are treated
in the same way as test sample except sample replaced by
positive standard. Superoxide radical scavenging activity of
the extracts was measured according to McCord and
Fridovich method
17
. All the solutions were prepared in
phosphate buffer (pH 7.8). FRAP working reagent (3 ml)
was taken in a test tube and added 100µl of plant extract.
The solution is vortex mixed and the absorbance was
read at 593nm against a reagent blank at a
predetermined time after sample–reagent mixture. The
results are expressed as ascorbic acid equivalents (µ
moles/ml) or FRAP units
18
.
Calculation of percentage of inhibition
The percentage inhibition of superoxide production
by the extract was calculated using the formula:
Inhibitory ratio = (Ao-A
1
)×100
A
0
Where, A
0
is the absorbance of control;
A
1
is the absorbance with addition of plant
extract/ascorbic acid.
Test organisms
The microorganisms used in the experiment were
procured from MTCC, IMTECH-Chandigarh. The Gram-
positive bacterial strains employed in the present study are
Staphylococcus aureus and Bacillus subtilis and the Gram-
negative strains are Escherichia coli, Klebsiella pneumonia
and Proteus vulgaris.
Research Article

Int. Res J Pharm. App Sci., 2014; 4(3):4-8 ISSN: 2277-4149
Neelima Routhu et al., 2014 5
Evaluation of in vitro antimicrobial activity
The cylinder plate assay of drug potency is
based on measurement of the diameter of zone of
inhibition of microbial growth surrounding cylinders
(cups), containing various dilutions of test extracts. A
sterile borer was used to prepare the cups of 6 mm
diameter in the agar medium spread with the micro-
organisms and 0.1 ml of inoculums. These cups were
spread on the agar plate by spread plate technique.
Accurately measured (0.05 ml) solution of each
concentration and reference standards were added to the
cups with a micropipette. All the plates were kept in a
refrigerator at 2 to 8°C for a period of 2 hours for
effective diffusion of test compounds and the standards.
Later, they were incubated at 37°C for 24 hours. The
presence of definite zone of inhibition of any size around
the cup was measured to assess the antimicrobial activity
19
.
RESULTS
Antioxidant activity
The results of in-vitro antioxidant activity of
methanolic leaf extract clearly indicate the presence of free
radical scavenging activity and it produced dose dependent
inhibition of free radical generation of DPPH, Superoxide
and FRAP radicals. Graphs were plotted from the
observed values to find the percentage inhibition of the
leaf extracts (Tables 1-3 &Figs 1- 3).
Evaluation of antimicrobial activity
Among all the tested extracts, ethanol extracts
have shown significant antimicrobial activity when
compared to that of chloroform and aqueous extracts. All
the extracts had produced a minimum zone of inhibition
against tested bacterial strains at 500µg/ml dose. However,
aqueous extract did not show significant effect on both
S.aureus and B.subtilis (Diameter of Zone of Inhibition
1mm) and E.coli (Dia 2mm) and completely inactive on P.
vugaris. The highest zone of inhibition was shown by
ethanolic extract against Klebsiella pneumonia(Dia 22mm)
at 500µg/ml. Chloform extracts generated a minimum zone
of inhibition against B.subtilis(Dia 10mm) and P.vulgaris
(Dia 14mm). The results are summarized in Table 4.
Table-1. Percentage inhibition of
DPPH radical by leaf extracts of M. oliefera.
Conc. of plant
extract
% inhibition of DPPH radical
Aqueous extract
Chloroform extract
Ethanol extract
100µg
22.002±0.02
27.046±0.12
29.140±0.15
250 µg
25.225±0.11
27.605±0.18
31.324±0.11
500 µg
26.232±0.13
28.571±0.14
34.598±0.13
1000 µg
26.800±0.11
32.011±0.15
37.102±0.12
Values are means ± SD (n=3)
Table 2: Percentage inhibition of Superoxide radical by leaf extracts of M.oliefera.
Conc. of plant
extract
% inhibition of Superoxide radical
Aqueous extract
Chloroform extract
Ethanol extract
100µg
11.195±0.08
19.191±0.02
39.487±0.06
250 µg
18.705±0.02
38.461±0.08
64.487±0.02
500 µg
20.261±0.05
69.465±0.04
83.462±0.06
1000 µg
24.421±0.05
71.830±0.06
90.642±0.06
Values are means ± SD (n=3)
Table 3: Total Antioxidant Assay-FRAP Method
Plant Name
FRAP units in µM
Moringa oleifera
780±0.06
Values are means ± SD (n=3).
Table 4: Antimicrobial activity of M. oleifera L. leaf extracts
Organism
Diameters of zones of inhibition in mm
(E)
(C)
(A)
Rifampicin
(50 µg/ml)
E.coli
19
9
2
24
B.subtilis
9
10
1
22
S.aureus
21
14
1
24
K. pneumonia
22
9
21
25
P.vulgaris
20
14
0
23
A- Aqueous extract; C- Chloroform extract; E- Ethanol extract of M. oliefera.

Int. Res J Pharm. App Sci., 2014; 4(3):4-8 ISSN: 2277-4149
Neelima Routhu et al., 2014 6
Fig-1 : Percentage inhibition of DPPH radical by leaf extracts of M. oliefera.
Positive Control:
Compound
% of Inhibition
Ascorbic acid
68.0±0.13
Fig 2: Percentage inhibition of Superoxide radical by leaf extracts of M. oliefera.

Int. Res J Pharm. App Sci., 2014; 4(3):4-8 ISSN: 2277-4149
Neelima Routhu et al., 2014 7
Fig 3: In-vitro total antioxidant assay (FRAP) by leaf extracts of M. oliefera.
DISCUSSION
By and large, the three tested extracts showed
good percentage of inhibition of DPPH, Superoxide and
FRAP radicals. Of the three tested extracts ethanolic extract
exerted highest activity followed by chloroform and aqueous
extracts. The activity of all the three extracts was dose
dependent. Ethanol and aqueous extracts generated
considerable size of zone of inhibition on K. pneumoniae .
On the other hand aqueous extract did not show any effect
on E.coli, S.aureus and P.vulgaris compared to the zone of
inhibition generated by Rifampicin at 50 µg/ml (Table 4)
at the current dose levels employed in the present study,
the non-activity of aqueous extract on the test bacterial
strains could be attributable to presence of active
compound(s) in insufficient quantities
20
. Lack of activity
can thus only be further confirmed with non- activity even
with higher doses
21
. Alternatively, even if the active
principle is present in high enough quantities, there could
be other constituents exerting antagonistic effects or
negating the positive effects of the bioactive agents
22
.
CONCLUSION
All the extracts exhibited antioxidant activities in a
dose dependent manner. The antimicrobial activity by the
three different extracts was relatively low a when compared
to that of control.
ACKNOWLEDGEMENT
The third and fourth authors are thankful to U.G.C
for financial assistance under special assistance programme
to the department of Botany, Andhra University,
Visakhapatnam.
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