IJBPAS, July, 2016, 5(7): 1655-1663
ISSN: 2277–4998
ANTIMICROBIAL AND PHYTOCHEMICAL ANALYSIS OF SOME MEDICINAL PLANTS FROM HIMACHAL PRADESH AGAINST ESCHERICHIA COLI KUMAR R1*, JANDAIK S2AND PATIAL P3 Department of Biotechnology Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India *Corresponding Author: E Mail:
[email protected] ABSTRACT An attempt was made to analyse the antimicrobial activity of 8 medicinal plants viz., garlic, neem, ginger, coriandrum, clove, rosemary, mint, turmeric and their antimicrobial activity was checked against E.coli. Antimicrobial study was carried out by well diffusion method. The overall antimicrobial activity recorded by zone of inhibition around the well which was ranging from 9-13mm in case of plant extracts and in case of antibiotics zone of inhibition recorded was from 19-23mm. Three concentrations of methanolic plant extracts i.e 50µg/ml, 100µg/ml and 200µg/ml were used for detection of antimicrobial activity and out of these three concentrations 200µg/ml concentration shows better antimicrobial activity against E.coli. The phytochemical analysis of these plant extracts were carried out to detect the presence of different phytocompounds present in the extract. Different phytocompounds detected in the extracts of all the plants were alkaloids, flavonoids, tannins, reducing sugars, phenols, saponins and terpenoids which contribute for the antimicrobial action of above medicinal plants. Keywords: Antimicrobial activity, phytochemical analysis, E.coli INTRODUCTION Medicinal plants are classified as greatest
bioactive
bio-resource of drugs. Plants are used from
medicinal plants led them to be used in
many years in traditional system of
industries such as pharmaceutical, food and
medicine,
cosmetic industries as botanical drugs,
in
nutraceuticals,
modern
medicines,
pharmaceutical
compounds
presents
in
the
and
dietry supplements and functional foods.
chemical entities in synthetic drugs.1 The
Plants are also used in ethno pharmacy for 1655
IJBPAS, July, 2016, 5(7)
Kumar R et al
Research Article
treatment of various diseases such as
Garlic has been used as a medicinal agent
hypertension, cholesterol and diarrhea and
from thousands of years. Garlic shows
scientific validation of these medicinal
antimicrobials, antithrombotic, antiarthritic
plants was provided by identifying and
and antitumor activity.8 Garlic is also used
isolating of the bioactive phytochemical
in hardening of the arteries and high blood
present in these plants.2
Phytochemical
pressure (Hypertension).8 Ginger is also
compounds presents in plants are known as
used in the treatment of migraine headache
secondary metabolites and have many
without causing any side effects.9 Turmeric
subgroups having various bioactivities such
helps in preventing coronary and heart
as
and
diseases. Turmeric is used in treatment of
anticancer activities.3 Recent research has
liver infections and it is also used in the
shown that natural products isolated from
treatment of jaundice and hepatitis.10,11
plants are used as an alternative to the
From many years medicinal properties of
existing drugs for the treatment of diseases
neem has been recognized in Indian
in developing countries.4 Medicines which
tradition. Every part of neem tree posseses
are extracted from plants has been used in
some medicinal properties.1
traditional health treatments for thousands
MATERIAL AND METHODS
of years in many parts of the world and
Collection of plant material:
they have agents to fight with microbial
Plants
diseases.5,6,7
selected for the study.
antioxidant,
antimicrobials
In
the
present
study
from
different
families
were
Plants were
methanolic extract of 8 medicinal plants
collected from physical garden of Shoolini
were studied for their antimicrobial activity
University, Solan and from the local
against
strains.
markets of Himachal Pradesh. A total of 8
Phytochemical screening of these extracts
plants were collected belonging to different
was
families and possess medicinal properties
Escherichia
also
done
for
coli
detection
of
phytochemicals present in them. The plants
and
shows
synergistic
discussed in the present study includes
different
garlic, ginger, turmeric, neem, mint, clove,
bacteria. Only the required plant parts are
coriandrum and rosemary. The therapeutic
collected either in dry or in fresh form and
use of these plants has been reported.
stored in sterile containers at 40C (Table 1).
antibiotics
activity
against
with
different
Table 1: List of plants selected for antimicrobial activity and phytochemical analysis. S.NO. 1 2 3 4 5
Plant Garlic Rosemary Turmeric Ginger Mint
Vernacular name
Adrak
Botanical name Allium sativum Rosamarinus officinalis Curcuma longa Zingiber officinale Mentha longifolia
Family Liliaceae Lamiaceae Zingiberaceae zingiberaceae Lamiaceae
Part used Bulb Leaf rhizome Rhizome Leaves
1656 IJBPAS, July, 2016, 5(7)
Kumar R et al 6 7 8
Neem Coriandrum Clove
Research Article
laung
Azadirachta indica Coriandrum satium Syzygium aromaticum
Meliaceae Umbelliferae Myrtaceae
Leaves Leaf Dried buds
Preparation of plant extract:
Schuster and Winfried V. Kern, Center for
The parts of plants used for extract
Infectious Diseases and Travel Medicine,
preparation were first washed with tap
University Hospital, and Department of
water and then washed with 0.1% hgcl2 to
Medicine,
remove the contamination and after that
Freiburg, Germany and from MTCC and
washed with distilled water. The plant parts
NCTC. The E.coli strains used were one
washed were then dried for 4 to 5 days in
knock out strain 1- DC14, wild type strain
shade. Then the dried plant parts were
K-12 AG100 and two standard strains with
grinded in to fine powder with the help of
NCTC number 12923 and MTCC number
mortar and pestle. Plants powder was
1302.
stored at 40c until use.
Antimicrobial activity:
Soxhlet extraction:
Antibacterial activity was measured using
Powdered plants were subjected to soxhlet
well
extraction with methanol as a solvent.
National
Methanol is widely used as a solvent,
Laboratory Standard. Tryptose soya broth
because many of the compounds dissolve in
was inoculated and incubated at 37ºC for
it easily, which is important for the plant
overnight. Presence of turbidity in broth
material,
was adjusted according to 0.5 McFarland
moreover
methanol
easily
Albert-Ludwigs-University,
diffusion
method
according
Committee
(McFarland
for
to
Clinical
evaporates. So, it can be separated from the
standards
extract and it is also easily available at low
prepared by dissolving 99.5 mL of 1%
cost. The initial concentration of 0.1g/ml
H2SO4 and 0.5 mL of 1.175% BaCl2 and
(200 ml methanol + 25 g powder) was
stored in dark at room temperature). Muller
made. Apparatus was run for 18-24 hours
Hinton agar plates were prepared. Sterilized
to get final concentrated slurry. Then
swabs
extract was poured in china dish. Methanol
bacterial suspension with inoculum size of
was evaporated from the extract by
1.5 x 108 cfu/mL prepared above and
incubation at 35-380c. Powders obtained
excess of culture was removed by turning
were weighed and stored in a sterile tube at
the swab against the side of the tube.
40c till use17.
Inoculum was spread evenly over the entire
Microbial strains:
surface of Muller Hinton Agar plates.
The strains of Escheritia coli used in the
These plates were allowed to dry for at
present study were procured from Sabine
least 15 min. Wells 6 mm in diameter were
were
dipped
solution
in
was
standardized
1657 IJBPAS, July, 2016, 5(7)
Kumar R et al
Research Article
punched in agar and were filled with 30µL
bath. 5 ml of 2N Hcl was added and stirred
of plant extract (3 concentrations 50µg/ml,
while heating on the water bath for 10 min.,
100 µg/mL and 200 µg/mL of plant extract
cooled, filtered and the filtrate was treated
were used) and antibiotic alone was used as
with few drops of mayer reagent. The
positive control and DMSO was used as
samples were then observed for the
negative control. The three replicates of
presence of turbidity or precipitation.
each plate was performed. The plates were (c) Test for Saponin: incubated at 37ºC for 24hrs.
About 2g of the powdered sample was
Phytochemical analysis:
boiled in 20 ml of distilled water in a water
The methanolic plant extracts which are
bath and filtered. 10 ml of the filtrate was
selected by screening were then analysed
mixed with 5 ml of distilled water and
for the presence of all the major groups of
shaken vigorously for a stable persistent
phytocompounds
standard
froth. The frothing was mixed with 3 drops
phytochemical assays. The phytochemical
of olive oil and shaken vigorously, then
screening of extracts from different plants
observed for the formation of emulsion.
by
using
was carried out to determine the presence (d) Test for Tannins: of active secondary plant metabolites. The
About 0.5 g of the dried powdered samples
plant extract were analysed for the presence
was boiled in 20 ml of water in a test tube
of reducing sugars, alkaloids, saponnin,
and then filtered. A few drops of 0.1%
flavonoids,
tannins,
anthraquinones,
ferric chloride was added and observed for
phlobatannin,
steroids,
terpenoids
brownish green or a blue black colour.
and
cardiac glycosides according to previously (e) Test for Flavonoids: described procedures.15,16
5 ml of dilute ammonia solution was added
(a) Fehling’s test for reducing sugars: The methanolic herbal extract (0.5 g in 5
to a portion of the aqueous filtrate of plant extract
followed
by
addition
of
ml of water) was added to boiling Fehling’s
concentrated H2SO4. A yellow colour
solution (A and B) in a test tube. The
observed in extract indicates the presence
solution was observed for a colour reaction.
of flavonoids.
Change in colour from
blue to red (f) Test for Anthraquinone:
indicates the presence of reducing sugars. (b) Test for Alkaloids:
10
ml
of N/2
potassium
hydroxide
containing 1 ml of 3% hydrogen peroxide
30 ml of sample extract was evaporated to
solution was added to 1 g of the powdered
dryness in an evaporating dish on water
plant material. The suspension was boiled
1658 IJBPAS, July, 2016, 5(7)
Kumar R et al
Research Article
for 3-5 min. then cooled, filtered and 5 ml (j) Test for Cardiac glycosides (Kellerof filtrate was acidified with 10 drops of
Killani test):
benzene. A 5 ml aliquot of the benzene
5 ml of extract was treated with 2 ml of
solution was shaken with 3 ml of 10%
glacial acetic acid containing one drop of
ammonium hydroxide solution and the two
ferric chloride solution. This was under
layers were allowed to separate. A pink to
layered with 1 ml of concentrated sulphuric
red colour of the alkaline layer indicates the
acid. A brown ring of the interface
presence of anthraquinone.
indicates a deoxysugar characteristic of cardenolides. A violet ring may appear
(g) Test for Phlobatannins: Deposition of a red precipitate when an
below the brown ring, while in the acetic
aqueous extract of plant sample was boiled
acid layer, a greenish ring may form just
with 1% aqueous hydrochloric acid was
gradually throughout thin layer.
taken as evidence for the presence of
RESULT
phlobatannins.
Antimicrobial activity of plants and
(h) Test for Steroids:
antibiotics:
2 ml of acetic anhydride was added to 0.5 g
Results obtained in the present study
methanolic extract of plant sample with 2
relieved that the tested eight medicinal
ml H2SO4. The colour changed from violet
plants
to blue or green indicates the presence of
antibacterial activity against Escherichia
steroids.
coli. When tested by the well diffusion
(i) Test for Terpenoids (Salkowski test):
extracts
exhibits
potential
method, the methanolic extracts of the
5 ml of extract was mixed with 2 ml
plants
viz.,
garlic,
chloroform, and concentrated H2SO4 (3
coriandrum,
ML) was carefully added to form a layer. A
turmeric showed significant activity against
reddish brown colour of the interface was
the tested micro organism (Table.2). The
formed to show the positive result for the
highest antibacterial activity recorded in
presence of terpenoids.
garlic (13mm) and lowest in mint and
clove,
neem, rosemary,
ginger, mint,
coriandrum (9mm) (table 2). S.n 1 2 3 4 5 6 7 8
Table 2: Effect of plant extract on Escherichia coli Plants Zone of inhibition in mm Garlic 13 ±1 Neem 11 ± 1 Ginger 10 ±2 Rosemary 11 ±1 Mint 9 ±1 Clove 10 ±0 Coriandrum 9±0 Turmeric 11±1
1659 IJBPAS, July, 2016, 5(7)
Kumar R et al
Research Article Table 3: Effect of antibiotics on Escherichia coli Antibiotics Zone of inhibition in mm Tetracycline 23±2 Ciprofloxacin 20±1 Erythromycin 19±1
S.NO 1 2 3
-ve
E
-ve
E E
-ve
A
A
A
-ve
E A
Fig 1: A- Antibiotic alone, E= Extract only and –ve = negative control (DMSO). Antimicrobial activity expressed in terms of clear zone (mm) produced around the well (6 mm) by different plant extract and antibiotics after incubation at 370C for 24 hours of incubation.
Phytochemical analysis: The methanolic extracts of medicinal plants revealed the following phytochemicals (Table.4). S.No 1 2 3 4 5 6 7 8
Table 4: Different group of phytochemicals present in methanolic extract of plants Phytochemical Phenols Tannins Reducing sugars Saponins Flavonoids tests (fehling’s test Garlic + + + Neem + + + + Ginger + Coriandrum + Mint + + + + Clove + + + Rosemary + + + Turmeric + + + + + + = positive, - = negative
Terpenoids + + + + + -
DISCUSSION
attempted to check whether they possess
Traditional medicines involve a large
antimicrobial activity or not. Antibacterial
number of plant species. In the present
activity and phytochemical analysis of 8
study we selected a small subset of 8 plants
medicinal plants were studied against
belonging to different families and just
Escherichia coli. Extract of all 8 medicinal
1660 IJBPAS, July, 2016, 5(7)
Kumar R et al
Research Article
plants possesses good inhibitory activity
Escherichia coli. They can be used in
against Escherichia coli. Garlic shows
combination with different antibiotics for
higher antibacterial activity against E.coli
the treatment of infections. The present
as shown in earlier studies as well.13,14
study leads to the establishments of some
Screening of these 8 medicinal plants
valuable compounds which can be used in
suggest that these plants are potential
the formation of new and more potent
source of antimicrobial
agents. This in
antimicrobial drugs of natural origins.
vitro study corroborated the antimicrobial
Further experimental studies are needed on
activity of these 8 medicinal plants i.e.
plants to identify new compounds which
Garlic, ginger, neem, mint, coriandrum,
show activity against pathogenic bacteria
turmeric, rosemary and clove in Ayurveda.
responsible for diseases in humans.
Results from our study suggest that leaves,
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