Datta Samaresh et al. IRJP 2013, 4 (1)
INTERNATIONAL RESEARCH JOURNAL OF PHARMACY
www.irjponline.com ISSN 2230 – 8407
EXPLORATION OF ANTIMICROBIAL POTENTIAL OF METHANOL EXTRACT OF STEMS OF
Datta Samaresh1*, Nayak Siva S2, Dinda Subas C3
1A.N.D. College of Pharmacy, Gonda, Uttar Pradesh, India-271 313
2College of Pharmaceutical Sciences, Mohuda, Odisha, India -760 002
3School of Pharmaceutical Education & Research, Berhampur, Orissa, India-760 007
Article Received on: 19/10/12 Revised on: 21/11/12 Approved for publication: 11/12/12
The antimicrobial efficacy of methanol extract of stem of the plant Euphorbia neriifolia (Family: Euphorbiaceae) was evaluated against selected pathogenic
bacterial strains (Staphylococcus aureus ATC-2245, Streptococcus aerugenosa U59, Escherichia Coli K88, Pseudomonas aeruginosa, Salmonella typhi 12,
Proeus vulgaris CC-52, Aspergillus niger 36 and Candida albicans. The antimicrobial activity was evaluated by disc diffusion and micro dilution assay
methods. Streptomycin and ampicilin were used as standard antibacterial drugs whilst amphotericin B was used as standard antifungal drug. Results of both
assays ensured that the stem possess significant antimicrobial activity in terms of antibacterial and antifungal activity. Results are comparable to that of
standard drugs selected. It is also evident from results that methanol extract showed better activity against pathogenic bacteria than fungi.
Keywords: Euphorbia neriifolia, Antimicrobial activity, Disc diffusion assay, Minimum inhibitory concentration (MIC)
Herbal medicines have been used since the dawn of
civilization to maintain health and to treat disease. There is a
tremendous historical legacy in folklore uses of plant
preparations in medicines. Scientific studies on plants used in
ethnomedicine led to the discovery of many valuable drugs1.
Antimicrobial properties of medicinal plants are being
increasingly reported from different parts of the world2,3. The
World Health Organization estimated that about 80 % of the
world’s population still believes in herbal drugs for their
primary health care4. There are indiscriminate uses of
synthetic antimicrobial drugs for the treatment of infectious
diseases and as a result drug resistance developed in human
beings as well as in plant also5,6. Sometimes antibiotics cause
adverse reaction like hypersensitivity, immunosuppression
and allergic reactions. Therefore, there is a need to develop
alternative antimicrobial drugs for the treatment of infectious
diseases from various sources, including medicinal plants7,8.
Euphorbia neriifolia Linn. (Euphorbiaceae) commonly
known as “Sehund or thohar” in Hindi, is found throughout
the Deccan Peninsula of India and grows luxuriously around
the dry, hilly, rocky areas of North, Central and South India.
Ayurveda describes the plant as bitter, pungent, laxative,
carminative, improves appetite useful in abdominal troubles,
bronchitis, tumors, loss of consciousness, delirium,
leucoderma, piles, inflammation, enlargement of spleen,
anaemia, ulcers and fever9,10 . As far as our literature survey
could ascertain, no information was available on the
antimicrobial activities of the stem of E. neriifolia. Therefore,
the aim of this current investigation was to explore the
antimicrobial potential of methanol extract of stem of E.
neriifolia against some pathogenic organism.
MATERIALS AND METHODS
The stem of Euphorbia neriifolia was collected from the rural
region of Midnapore, West Bengal, India. The plant was
authenticated by the Botanical Survey of India (BSI), Shibpur
(W.B), and India. Air dried whole stem (500 g) were
powdered in a mechanical grinder and the powdered
materials was extracted by methanol using Soxhlet extraction
apparatus. The solvent was completely removed under
reduced pressure in a rotary vacuum evaporator. The
concentrated extract (yield 35.42%) was stored in vacuum
desiccators for further use.
Preliminary phytochemical studies
The extracts were subjected to various phytochemical tests to
determine the active constituents present in the different
crude extract following modified method of Kar et al.,
Bacterial and fungal stain
The antimicrobial activity of the methanol extract was
screened against two gram positive bacteria such as:
Staphylococcus aureus (ATC-2245) and Streptococcus
aerugenosa (U59) and four gram negative bacteria such as:
Escherichia Coli (K88),
Salmonella typhi (12), Proeus vulgaris (CC-52) and two
fungi such as: Aspergillus niger (36), Candida albicans. All
strains were preserved in freeze dried state and at 4 °C in stab
slant agar 12. The microorganism’s cultures were maintained
on nutrient agar (NA) medium for 18 h at 37±1 °C.
Media Preparation and antibacterial Activity
The antimicrobial assay of stem was performed by agar disc
diffusion method of methanol extract. A loop full of the strain
was inoculated in 30 ml of nutrient broth in a conical flask
and incubated on a rotary shaker for 24 h to activate the
Muller Hinton Agar (MHA) (3.8 g/100ml) was weighed and
dissolved in 100 ml of distilled water in a sterile conical
flask. The medium was sterilized by autoclaving and was
allowed to cool at room temperature. The molten Muller
Hinton Agar was inoculated with 200 μl of the inoculum and
poured into the Petri plate.
For Agar disc diffusion method, the disc (0.6 cm) was
saturated with 50 μl of the compound, allowed to dry and was
introduced on to the upper layer of the medium with bacteria.
Antibiotic paper (streptomycin, ampicillin) discs were used
as positive control. These test discs was placed on MHA plate
swabbed with the culture of microorganisms. The plates were
incubated at 37 °C or overnight. For each bacterial strain,
Datta Samaresh et al. IRJP 2013, 4 (1)
controls were maintained where pure solvents were used
instead of the extract. The result was obtained by measuring
the zone diameter. The experiments were carried out in
triplicate. The results (mean value n = 3) were recorded by
measuring the zone of growth inhibition around the discs13.
The antifungal assays against yeasts were performed as the
antibacterial assays described above with the replacement of
SDA as an assay medium and the minimum fungicidal
concentrations (MFCs) were recorded. Amphotericin B
(Bristol-Myers, Germany) was used as a positive control.
Plates were incubated at 35ºC for 24 h (C. albicans) and 48 h.
Minimum Inhibitory Concentration (MIC)
MIC is defined as the lowest concentration where no visible
turbidity is observed in the test tube
(Bacteriostatic concentration). According to Haniffa, 2012
method with slight modification; Briefly the broth dilution
technique was utilized where the plant extract was prepared
to the highest concentration in sterile distilled water and
serially diluted (two-fold) to a working concentration using
nutrient broth and later inoculated with 0.2 ml suspension of
bacterial strains. After 18 hours of incubation at 37º C, the
test tubes were observed for turbidity. The least where no
turbidity was observed was determined and noted as the
minimum inhibitory concentration (MIC) value.
Table 1: Phytochemical screening of Euphorbia neriifolia extract
Terpenoids Tannin Saponin Steroid Flavonoid
+ ++ +++ - +
Table 2: Antibacterial activity of the methanol extract of Euphorbia neriifolia by disc diffusion assay
Zone of Inhibition
50 mg/ml 100 mg/ml 200 mg/ml
Values are mean of triplicates experiment
Table 3: Antifungal activity of the methanol extract of Euphorbia neriifolia by disc diffusion assay
50 mg/ml 100 mg/ml
Table 4: MIC and MBC value of Methanol extract of Euphorbia neriifolia (MEEN)
Organism Extract MEEN
Values are mean of triplicates experiment
Streptomycin (50 µl)
Ampicillin (50 µl)
Zone of Inhibition
RESULTS AND DISCUSSION
Preliminary phytochemical screening of the methanol extract
of Euphorbia neriifolia stem revealed the presence of various
bioactive components of which alkaloid, saponin, tannin and
cardiac glycosides were the most prominent and the result of
phytochemical test has been summarized in table-1. All these
phytochemicals possess good antioxidant activities and has
been reported to exhibit multiple biological effects including
anti-inflammatory, antitumor activities. The results of disc
diffusion assay of both methanol and water extracts of the
stem of Euphorbia neriifolia have been tabulated in table-2
and table-3. It is evident from table-2 that the both methanol
extract was found to be active against the bacteria like
Escherichia coli K88, Staphylococcus aureus ATC 2245,
Pseudomonas aeruginosa and Salmonella typhi 12.
The results of disc diffusion assay of the crude extracts were
compared with that of standard antibiotic Streptomycin
(10μg/disc) and ampicilin (10μg/disc) also recorded. Table-3
indicates that the extract is also potent for its antifungal
efficacy. The extracts have shown profound antifungal
activity with respect to fungal stains namely Aspergillus
niger 36, Candida albicans and results are comparable to that
of standard antifungal agent Amphotericin B (10μg/disc).
Among bacteria Escherichia coli K88 and among fungi
Pseudomonas aeruginosa are most susceptible to the extracts.
The antibacterial activity and inhibition activity of E.
neriifolia extracts could be attributed to the chemical
compounds. The phytochemical investigations demonstrated
the presence of alkaloid, saponin and tannin in methanol
extract of E. neriifolia 14.
Table-4 represents MIC and MBC/MMC values of the
methanol extract against bacterial and fungal strains and
results were compared with that of standard antibiotics
streptomycin and amphotericin B for bacteria and fungi
From above results it can be concluded that the methanol
extract of plant Euphorbia neriifolia possess significant
antimicrobial activity in term of antibacterial and antifungal
Datta Samaresh et al. IRJP 2013, 4 (1) Download full-text
effects. This antimicrobbial property against bacteria and
fungi surely is due to presence of some antimicrobial
substances in stems. Now our study will be directed to
explore the lead compound responsible for aforementioned
activity from this plant.
The authors are thankful to the authority of A.N.D. College
of pharmacy, Babhnan, Gonda for providing necessary
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Source of support: Nil, Conflict of interest: None Declared
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