Antimicrobial Studies on Selected Medicinal Plants in Khandesh Region, Maharashtra, India
M.A. Chaudhari1*, S.M. Sarode2, Y.A. Chaudhari3, G.Vidyasagar2 and M.K. Kale1
1Department of Pharmacology, K.Y.D.S.C.T’s College of Pharmacy, Sakegaon-
425201
2Veerayatan
Institute of Pharmacy, Bhuj, Gujarat.
3TVES’S Hon’ble L. M. C. College of Pharmacy, Faizpur
ABSTRACT:
Medicinal plants contribute
in human health care system. Most of the plants utilized by
village peoples as a folk medicine. Now we are turned in to medicinal
plant analysis of active compounds and conservation aspect. In the present
study we had select the four important medicinal plants in the Khandesh region. Such plants are widely used in this region
for ayurvedic purpose by villagers and local herbal health masters. We have colleted four medicinally important medicinal plants such
as Acalypha indica,
Cassia auriculata, Eclipta
alba and Phyllanthus
niruri for antimicrobial studies. The experiment
carried out in the selected medicinal plants leaves and roots. The results are
discussed with the available literature.
INTRODUCTION:
In India, the use of different parts of several medicinal
plants to cure specific ailments has been in vague from ancient times. The
indigenous system of medicine namely Ayurvedic, Siddha
and Unani have been in existence for several
centuries. These systems of medicine cater to the needs of nearly seventy percent
of our population residing in the villages. In Homeopathy system, 70% of the
medicines are prepared from plants. As Homeopathy originated in Europe
naturally, majority of the drugs prepared from plants are of exotic origin1.
The plants supply us with large number of excellent “chemicals” which form
sources for different types of drugs. The present trend in modern medicine is
towards a change from the use of cellulose coated medicinal pills to extracts
of plant supplied either in pure forms or in synthetic versions for curing many
human ailments. Thus plants have provided the blue prints for the modern
medicine.
From over 3, 00,000 species of higher plants to occur in
nature, only about 2 percent have been screened so for. Extract of plants from
157 families have been reported to be active against microorganisms2.
Plants are rich in alkaloids and other phytochemical
contents and many of them are effectively used to cure a wide range of
ailments. The alkaloids and phytochemical contents
may be present in all organs of the plant including roots, stems, buds, leaves,
flowers and fruits.
All plant parts syntheses some chemicals with themselves
which metabolite their physiological activities. These phytochemicals
were used to cure the disease in herbal and homeopathic medicines. Now a day
most of the peoples like to use the traditional methods to cure general
diseases. Plant based to
control major diseases and the need to discover
new molecular structures as lead compounds from the plant kingdom3.
This worldwide interest in medicinal plants reflects recognition of the
validity of many traditional claims regarding the value of natural products in
health care and the development of microbial resistance to the available
antibiotics has led the authors to investigate the antimicrobial activity of
medicinal plants.
MATERIALS AND METHODS:
Plant materials are collected from the Revenue Village of
Pal, Jalgaon District, Khandesh region. Plants are identified and confirmed
with the authentic. Plant selected for the present study was Acalypha indica, Cassia auriculata,
Eclipta alba and Phyllanthus niruri. Fresh
leaves and roots are collected and shade dried under room temperature. The
dried leaves and roots are grained into a coarse powder and used for further
investigations.
The dried powdered leaves and roots were macerated by using
mortar and pestle. The collected Sam herbarium specimen available in the Botany
Department of D.N. College, Faizpur was analyzed for
photochemical analysis by quantitative analysis.
Extraction: A soxhlet
apparatus were used for the extracting antimicrobial active compounds from the
plant leaves and roots. The collected plant leaves and roots were shade dried
and powdered separately. 20 gm of dried powder was packed with thimble and then
subjected to extraction with the water and ethanol separately. The collected
extracts were concentrated by evaporation under room temperature. The collected
extracts were then chosen for antibacterial activity.
Bacterial Inoculum
Preparation: Bacterial cultures
used in this study were obtained from MTCC, Chandigar.
Bacterial cultures included in this study were Escherichia coli, Proteus vulgaris, Staphylococcus aureus
and Bacillus subtilis. All the cultures were grown in
Muller - Hilton agar medium. The inoculum was used
for antibacterial assay.
Antibacterial Assay: The media and the test bacterial cultures
were poured into dishes. [Muller - Hilton agar media].
The test strain (0.2 ml) was inoculated into the media to inoculum
size (108cells/ml) when the temperature reached 40-42°C. Care was taken to
ensure proper homogenization. The plant extracts were tested for antibacterial
activity in the agar well diffusion assay, against Escherichia coli, Proteus vulgaris, Staphylococcus aureus
and Bacillus subtilis
Agar Well Diffusion Method: The antibacterial activity was tested
against (Ethanol, Aqueous) leaves and roots of Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri. The inoculation of microorganism
was prepared from bacterial culture4. About 15-20 ml of
Muller-Hilton agar medium was poured in the sterilized petridish
and allows solidifying. One drop of bacterial strains was spread over the
medium by a rod. Wells of 6nm in diameter and about 2 cm apart punctured in the
culture medium using sterile Cork borers. About 100 ml of plant extracts was
added to the wells. Plates were incubated in air at 37°C for 24 hours. Antibacterial
activities were evaluated by measuring inhibition zone diameters.
RESULTS:
Medicinally important plant species viz., Acalypha indica, Cassia auriculata,
Eclipta alba and Phyllanthus niruri were
selected for screening of secondary metabolites. During this investigation, an
attempt has been made to decipher the effect of these secondary metabolites
towards its antibacterial activities. Ethanol and aqueous extracts from the
leaf and root of Acalypha indica,
Cassia auriculata,
Eclipta alba and Phyllanthus niruri
exhibit antibacterial activity against Escherichia coli, Proteus vulgaris, Staphylococcus aureus
and Bacillus subtilis.
Antibacterial Activity: The effect of different extracts of four
test plants on Escherichia coli was shown in Tables 1 and 2. The results
clearly showed that plant extracts were specific in action against the growth
of bacteria. Ethanol extract was most effective followed by aqueous extract.
Escherichia coli were more sensitive for ethanol extract of roots and leaves of
all the tested plants. Aqueous extracts did not show much inhibition against
the tested organism compared to ethanolic extract.
The extracts of leaf of Phyllanthus niruri
exhibit relatively higher zone of inhibition followed by Cassia auriculata, Eclipta alba and Acalypha
indica.
Table 1: Effect of ethanolic extract of root and leaf samples of certain
medicinal plants on Escherichia coli (RMI-cm2)
|
Name of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
2.4 |
4.8 |
0.5 |
1.9 |
3.8 |
|
Cassia auriculata |
0.5 |
1.9 |
3.8 |
0.5 |
2.2 |
4.4 |
|
Eclipta alba |
0.5 |
2.6 |
5.2 |
0.5 |
1.8 |
3.6 |
|
Phyllanthus niruri |
0.5 |
2.5 |
5.0 |
0.5 |
2.2 |
4.4 |
A1 = Area of well in cm2;
A2 = Area of Zone of inhibition in cm2 (including area of well); RMI
= A2/A1
Table 2: Effect of aqueous extract of
root and leaf samples Of certain medicinal plants on Escherichia
coli (RMI-cm2)
|
Name
of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
1.3 |
2.6 |
0.5 |
1.4 |
2.8 |
|
Cassia auriculata |
0.5 |
1.6 |
3.2 |
0.5 |
2.1 |
4.2 |
|
Eclipta alba |
0.5 |
1.6 |
3.2 |
0.5 |
1.6 |
3.2 |
|
Phyllanthus niruri |
0.5 |
1.9 |
3.8 |
0.5 |
2.2 |
4.4 |
A1 = Area of well in cm2;
A2 = Area of Zone of inhibition in cm2 (including area of well); RMI
= A2/A1
Tables 3 and 4 showed the antibacterial activity of ethanolic and aqueous extracts (root and leaf) of the
tested plants on the selected bacteria Proteus vulgaris.
The results showed inhibition diameters ranging from 1.2 cm to 2.1 cm. Aqueous
leaf extract from all the four plants were found to have a higher antibacterial
activity compared to ethanolic leaf extracts. Except Eclipta alba aqueous
extract of all the three plants exhibit relatively higher zone of inhibition
compared to ethanolic extract. The susceptibility of
the test organism Staphylococcus aureus to the test
plants was given in Tables 5 and 6. Ethanolic
extracts (leaf and root) were most effective followed by aqueous extracts.
Maximum inhibition was recorded in ethanolic root
extract of Cassia auriculata.
This was followed by ethanolic leaf extracts of Phyllanthus niruri, Eclipta alba and Cassia auriculata.
The test organism was resistant to the aqueous root extracts of Acalypha indica and Phyllanthus niruri.
Table 3: Effect of ethanolic
extract of root and leaf samples of certain medicinal plants on Proteus vulgaris (RMI-cm2)
|
Name
of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
1.1 |
2.2 |
0.5 |
1.2 |
2.4 |
|
Cassia auriculata |
0.5 |
1.3 |
2.6 |
0.5 |
1.9 |
3.8 |
|
Eclipta alba |
0.5 |
1.4 |
2.8 |
0.5 |
1.5 |
3.0 |
|
Phyllanthus niruri |
0.5 |
1.3 |
2.6 |
0.5 |
1.7 |
3.4 |
A1 = Area of well in cm2; A2
= Area of Zone of inhibition in cm2 (including area of well); RMI =
A2/A1
Table 4: Effect of aqueous extract of
root and leaf samples of certain medicinal plants on Proteus vulgaris (RMI-cm2)
|
Name
of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
1.7 |
3.4 |
0.5 |
1.4 |
2.8 |
|
Cassia auriculata |
0.5 |
1.6 |
3.2 |
0.5 |
1.6 |
3.2 |
|
Eclipta alba |
0.5 |
2.1 |
4.2 |
0.5 |
1.2 |
2.4 |
|
Phyllanthus niruri |
0.5 |
2.1 |
4.2 |
0.5 |
1.4 |
2.8 |
A1 = Area of well in cm2;
A2 = Area of Zone of inhibition in cm2 (including area of well); RMI
= A2/A1
Table 5: Effect of ethanolic
extract of root and leaf samples of certain medicinal plants on Staphylococcus
aureus (RMI-cm2)
|
Name
of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
1.2 |
2.4 |
0.5 |
1.9 |
3.8 |
|
Cassia auriculata |
0.5 |
1.9 |
3.8 |
0.5 |
2.4 |
4.8 |
|
Eclipta alba |
0.5 |
1.9 |
3.8 |
0.5 |
1.8 |
3.6 |
|
Phyllanthus niruri |
0.5 |
2.1 |
4.2 |
0.5 |
1.7 |
3.4 |
A1 = Area of well in cm2;
A2 = Area of Zone of inhibition in cm2 (including area of well); RMI
= A2/A1
Table 6: Effect of aqueous extract of
root and leaf samples of certain medicinal plants on Staphylococcus aureus (RMI-cm2)
|
Name
of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
1.7 |
3.4 |
0.5 |
- |
- |
|
Cassia auriculata |
0.5 |
1.9 |
3.8 |
0.5 |
1.7 |
3.4 |
|
Eclipta alba |
0.5 |
1.8 |
3.6 |
0.5 |
1.7 |
3.4 |
|
Phyllanthus niruri |
0.5 |
1.9 |
3.8 |
0.5 |
- |
- |
A1 = Area of well in cm2;
A2 = Area of Zone of inhibition in cm2 (including area of well); RMI
= A2/A1
The effect of aqueous and ethanolic
(root and leaf) extracts against Bacillus subtilis
were depicted in Tables 7 and 8. The highest antibacterial potentials were
observed from the ethanolic root extracts of Acalypha indica, Cassia auriculata
and Eclipta alba which inhibited
2.6, 2.5 and 2.3 cm of the tested microorganism.
Table 7: Effect of ethanolic
extract of root and leaf samples of certain medicinal plants on Bacillus subtilis (RMI-cm2)
|
Name
of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
1.9 |
3.8 |
0.5 |
2.6 |
5.2 |
|
Cassia auriculata |
0.5 |
2.3 |
4.6 |
0.5 |
2.5 |
5.0 |
|
Eclipta alba |
0.5 |
2.1 |
4.2 |
0.5 |
2.3 |
4.6 |
|
Phyllanthus niruri |
0.5 |
1.7 |
3.4 |
0.5 |
1.8 |
3.6 |
A1 = Area of well in cm2;
A2 = Area of Zone of inhibition in cm2 (including area of well); RMI
= A2/A1
Table 8: Effect of aqueous extract of root and leaf
samples of certain medicinal plants on Bacillus subtilis
(RMI-cm2)
|
Name of Medicinal Plant |
Leaf |
Root |
||||
|
A1 |
A2 |
RMI |
A1 |
A2 |
RMI |
|
|
Acalypha indica |
0.5 |
- |
- |
0.5 |
1.6 |
3.2 |
|
Cassia auriculata |
0.5 |
1.7 |
3.4 |
0.5 |
1.8 |
3.6 |
|
Eclipta alba |
0.5 |
1.2 |
2.4 |
0.5 |
2.7 |
5.4 |
|
Phyllanthus niruri |
0.5 |
1.8 |
3.6 |
0.5 |
2.1 |
4.2 |
A1 = Area of well in cm2;
A2 = Area of Zone of inhibition in cm2 (including area of well); RMI
= A2/A1
Cassia auriculata and Eclipta alba ethanolic
leaf extracts exhibit more inhibition than Acalypha indica and Phyllanthus niruri. Aqueous leaf extract of Acalypha indica did
not respond for its antibacterial activity against Bacillus subtilis.
Aqueous root extract of Eclipta alba exhibit
more relative magnitude of inhibition [RMI] value compared to the other three
plants studied.
The growth of bacteria was suppressed by phytochemical
compounds of ethanol and aqueous extracts of all the plants with different
magnitudes. Cassia auriculata
possess higher antibacterial activity among the tested plants. This was
followed by Eclipta alba, Phyllanthus niruri and Acalypha indica.
The growth of Escherichia coli was inhibited more with ethanolic extracts of Eclipta alba, followed by Phyllanthus niruri and Acalypha indica. Proteus vulgaris
showed maximum inhibition with Eclipta alba and Phyllanthus niruri aqueous leaf extracts. Staphylococcus aureus was more sensitive to ethanolic
root extract of Cassia auriculata. Bacillus subtilis
was resistant to aqueous leaf extract of Acalypha indica compared to all the other three
plants studied.
DISCUSSION:
In the present study, four medicinal plant species viz., Acalypha indica, Cassia auriculata,
Eclipta alba and Phyllanthus niruri were
screened to detect the presence or absence of several bioactive compounds which
are reported to cure different diseases and ailments. It was confirmed that the
presence of antimicrobial substances in all the tested plants in one form (or)
other. The antimicrobial compounds may be found as anthroquinone,
alkaloids, catachols, flavonoids,
phenolic compounds, saponins,
steroids, tannins and triterpenoids, whose presence
may be attributed to the medicinal properties of plants5-8.
In the present study, Escherichia coli was sensitive to all
the four plant extracts and exhibited more or less same Relative magnitude of
inhibition (RMI). This is in consonance with the earlier report9 in
the seed extract of Citrullus vulgaris
and Eclipta alba, in Allium sativum, Citrullus vulgaris and Eclipta alba and in Melia
azadirachta against Escherichia coli10-13.
On contrary, Escherichia coli was more resistant to
acetone extract of Acorus calamus,
Zingiber officinal, Cinnamomum
zeylanicum, Moringa olifera and Ocimum sanctum14.
Proteus vulgaris was sensitive with ethanolic extracts of Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri. The results obtained in this
study indicated a considerable difference in antibacterial activity between
extracts obtained with ethanol and water. Aqueous leaf extract was more active
than the ethanol extract. The widest zone of inhibition was observed with
aqueous leaf extracts of Eclipta alba and Phyllanthus niruri. It is
in corroboration with the earlier study in the aqueous extract of neem against Salmonella weltevreden15. Reported
water extracts of oriental herbal medicines were more antibacterial against
Staphylococcus aureus and Bacillus subtillis. On contrary, Bacteria and yeast were more
resistant to aqueous extract of Micromeria nerosa16.
In the present investigation the ethanolic
extract of all the four plants exhibited higher Relative magnitude of
inhibition (RMI) against staphylococcus aureus. This
credit to ethanol extraction was supposed to be because ethanol is an organic
solvent and will dissolve organic compounds better than aqueous extract and
also liberate the active component required for antibacterial activity. The
volatile components of acetone extracts of Ocimum
sanctum were more effective against all the tested microorganisms viz.,
Staphylococcus aureus, Escherichia coli and Klebsiella speciesVarious organic
extracts of Eclipta prostrata
against Escherichia coli, Staphylococcus aureus,
Salmonella typhi, Shigella dysenteriae and Bacillus subtilis
and Cassia elata leaf extract against Staphylococcus aureus and Escherichia coli17-19.
Ethanol and aqueous extracts of Acalypha indica, Cassia auriculata, Eclipta alba and Phyllanthus niruri
against Bacillus subtilis revealed that ethanolic root extract seem to be more active compared to
aqueous extract. Seed extracts of Citrullus vulgaris, Eclipta alba, Sida acuta
and Achyranthus aspera on
Bacillus subtilis, Escherichia coli and Salmonella
typhimurium20.
It was clear from this study that the solvent of extraction
and method of extraction affected the degree of antimicrobial activity. Other
factors such as the environmental and climatic conditions of the plants also
affected the degree of antimicrobial activity. Successful prediction of
botanical compounds from plant material is largely dependent on the type of
solvent used in the extraction procedure. Traditional healers use primarily
water as the solvent but in our studies we found that plant extract in organic
solvent provided more consistent antimicrobial activity compared to those
extracted in water.
Many substances may be antimicrobial, but only a few of them
will be potential therapeutic agents for the simple reason that mammalian cells
are more sensitive to chemical inhibition than microbial cells21.
Moreover, emphasized the need for toxicity testing of drugs derived from
medicinal plants because the crude products obtained from such cheaper sources
are often associated with a large number of compounds that have discomforting
abilities22. Hence the herbal drugs have to be subjected to
extensive pharmacological, toxicological and clinical tests to conform the
prescribed status. Thus the ethnobotanical approach
will be like a search for molecular diversity subjecting a wide variety of new
molecules from plant sources and testing them with as many different tests as possible22.
The present study has shown a spectrum of antibacterial
activities, which provides a support to some traditional uses of these few
medicinal plants. But the effective biomolecules
which act as antibacterial have to be identified isolated and subjected to
extensive scientific and pharmacological screening that can be used as sources
for new drugs.
ACKNOWLEDGEMENT:
The authors are gratefully acknowledged The Secretary,
Principal and The Head, Department of Botany, D. N. College, Faizpur. and College of Pharmacy, Sakegaon. Maharashtra, India and who are encourage and
given their valuable suggestion during the study period.
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Received
on 24.06.2010
Accepted on 07.07.2010
© A&V Publication all right reserved
Research Journal of Pharmacognosy and Phytochemistry.
2(5): Sept.-Oct. 2010, 386-390