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Antimicrobial activity of Some Ethno-medicinal Plants used by
Baiga Tribes from Amarkantak, India
Jitendra Malviya1* Vaibhavi Joshi2 Kiran Singh3
1.
Department of Biotechnology , Indira Gandhi National Tribal University ,Amarkantak M.P.
2.
Department of Microbiology , Barkatullah University , Bhopal
M.P.
* E-mail of the corresponding author: jitmalviya123@gmail.com
Abstract
Antimicrobial activity of 05 ethnomedicinal plants extracts were evaluated against six bacterial strains
Staphylococcus aureus , Niesseria gonorrohae , Pseudomonas aeruginosa, Escherichia coli Streptococcus
pyogenes Bacillus subtilis. The collected ethnomedicinal plant were used in folk medicine in the treatment
of diuretic stomachache , urinary tract, piles ,high fever , leprosy , ulcers, sexual diseases like gonorrhea
and spermatorrhoea, dogbite ,snakebite, skin diseases, respiratory problems asthma , bronchitis, nervous
diseases and blood purification. Plants were collected from mekal hills, sonmura ,mai ki bagi,
sammbhudhara ,laxmandhara , panchdhara ,dense vegetative forest ,dense rain isolated eco-system,
remote valleys and islands and the ethnomedicinal data were gathered from traditional healers who inhabit
the study area. The traditional, chemosystematics and methanol extract were obtained by maceration method
with antimicrobial activity was found using disc diffusion method phytochemical anaylysis, Minimal
inhibitory concentration (MIC) and minimal bactericidal concentration (MBC). All microorganism were
obtained from (ATCC), American type culture collection.The result indicate that out of 05 plants
exhibited antimicrobial activity against one or more of the tested microorganism we observed that extract
concentrations spanning from 25 µg/ml to 2 mg/ml or even 40 mg/ml. Concentration of alkaloids,
carbohydrates, flavonoids, saponins, terpens steroids associated to antioxidant activities and thus have
curative properties. MIC and MBC for all 5 crude extracts (72%) showed positive results against bacterial
strains.This study evaluate the antimicrobial ,phytochemical and Minimal inhibitory concentration (MIC)
and minimal bactericidal concentration (MBC) activity of the some ethnomedicinal plants used in folkoric
medicine compared to all test showed significant activity against tested microorganism . This study also
showed that Selaginella bryopteris (Amarbatoooti Sanjivini), Lygodium flexuousm (Kalijar) Adiantum
philippense (Kalijhant), Drypteris eochleata (Jatashankari), Tectacria coadunate (Jatamasi) could be
potential sources of new antimicrobial agents.
Keywords: key words, ethnomedicinal plant, Antimicrobial, Phytochemical,diseases
1. Introduction
More than 80% of the world’s population uses natural medicines and depends on medicinal plants
for health care. In recent years, the growing demand for herbal products has led to a quantum jump in
volume of plant materials traded within and across the countries. At present, 90% collection of herbal raw
drugs used in the manufacture of Ayurveda, Siddha, Unani, and Homeopathy systems of medicine is largely
from the wild out of which 70% collection involves destructive harvesting. Due to this spurt, medicinal
plants are being overexploited and many of them are pushed to the brink of extinction. Many medicinal
plants are highly sensitive to the level of harvest and fragility of the ecosystem (Pandey A K et al. 2010).
Amarkantak is one of the world’s focuses for conservation located at 22.67°N 81.75°E. It has an
average elevation of 1048 metres (3438 ft) from sea level this biomass should be studied in terms of
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Advances in Life Science and Technology
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pharmacological or biological activity. Approximately 40% of the plants found in the world have been
submitted to pharmacological or biological test, and a substantial number of new antibiotics introduced on
the market are obtained from natural or semi synthetic resources. The systematic screening of antibacterial
plant extracts represents a continuous effort to find new compounds with the potential to act against
multi-resistant bacteria. The temperate climate and the equitable distribution of rain make Amarkantak an
ideal plateau for dense vegetation cover 21 pteridophytes 7 of these species were new for Central India and
14 for Madhya Pradesh (Khare P B 1989 and Saxena O P 1970).
In isolated eco-system, such as remote valleys and islands, there is an extremely wide variety of floristic
genera, but with the proportion of species to genera being very small. This is because each genus is unique to
itself and does not proliferate and subdivide into many species. The eco-system of Amarkantak is truly
unique, closely resembling that of isolated valleys or islands, because whereas the proportion of species
genera is 13:1 in the world, in India it is 7:1 and in Amarkantak it is 15:1. This makes every genus in
Amarkantak of great medicinal importance because if a particular plant becomes extinct the genus itself will
die because it does not live through any of its variant species are now gravely endangered, especially because
it is highly localised around Amarkantak, and it requires total protection. The region is tribal dominant and
tribes are Gonds, Bharia, Bhils and Baiga’s they are dependent on forest products for their survival they used
their traditional knowledge and indigenous systems of medicine for the treatment of various diseases (Sweta
Singh et al. 2005, Dixit R D 1947). Plants used for traditional medicine contain a wide range of substances
that can be used to treat chronic as well as infectious diseases. A vast knowledge of how to use the plants
against different illnesses may be expected to have accumulated in areas where the use of plants is still of
great importance. The medicinal value of plants lies in some chemical substances that produce a definite
physiological action on the human body (Kloucek P et al. 2005). The most important of these bioactive
compounds of plants are alkaloids, flavanoids, tannins and phenolic compounds (Sandhya B et al. 2006).
Rural communities in particular baiga tribes of Amarkantak depends on plant resources,claim that their
medicine is cheaper and more effective than modern medicine.
The reports on the use of plants in traditional healing through tribes interview, discussion, personal contacts
and keen observations i.e. Selaginella bryopteris, (Amarbatoooti Sanjivini;used for gonorrhea and venereal
diseases) Lygodium flexuousm,(Kalijar; leaves applied for piles, rheumatism, sprains, ulcers, cut wounds
and scabies also cure gonorrheao ,spermetaorrhoea and menorrhagia ,spores cure high fever. ) Adiantum
philippense (Kalijhant; uses for leprosy, dysentery, ulcers, asthma, bronchitis as well as dogbite and
snakebite), Drypteris eochleata, ( Jatashankari ; rhizomes shows antifungal activity and used as antidote
and also applied for cuts, wounds ,ulcers, blood purification) Tectacria coadunate (Jatamasi; used in
-respiratory disorders).
The principal aim of the present study was to screen extracts obtained from the Amarkantak forest
for antibacterial activity against the phytochemical research based on ethnopharmacological informations is
generally considered an effective approach in the discovery of new anti-infective agents from higher
plants (Kloucek P et al. 2005). The development of drug resistance in human pathogens against commonly
used antibiotics has necessitated a search for new antimicrobial substances from other sources including
plants (Erdogrul OT 2002) screening of medicinal plants for antimicrobial activities and phytochemicals is
important for finding potential new compounds for therapeutic use. This paper reports the results of a
survey that was done based on folk uses by traditional practitioners in Amarkantak Mekal Plataue along
with bioassay test for antimicrobial activity.
2.
Material and Methods
2.1 Plant collection and extract preparation
All the plant were collected with the Amarkantak mekal plataue and Achanakmar biosphere region of
Chhattisgarh five plants were used for organic and aqueous extracts were obtained from plants native to
the tribal region. Plants were collected using a traditional5 and chemosystematic6 approach. Plant parts were
collected according to the biomass availability. Plant material was dried and ground before being submitted
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to 24-h maceration with methanol: dichloromethane (1:1) followed by 24-h maceration with water,
resulting in four extracts from each plant material viz, Selaginella bryopteris (Amarbatoooti Sanjivini),
Lygodium flexuousm (Kalijar) Adiantum philippense (Kalijhant), Drypteris eochleata (Jatashankari),
Tectacria coadunate (Jatamasi).
2.2 Phytochemical test
Mayer test for Alkaloids take 0.135g of mercury bichloride, and 0.05g potassium iodide, are dissolved in
100 mL of water; this is used as a test for alkaloid, with which it gives a white precipitate. FeCl3 test for
Tannin take FeCl3 solution and add 2 ml of extract. It gives deep blue color. This indicates the presence of
tannin. Frothing test for Saponin take 300mg of extract and boil with 5ml water for 2 minutes. Mixture was
cooled and mixed vigorously and left for 3 minutes. The formation of froth indicates the presence of
saponin. Salkowski test for Steroids take 2ml of plant extract and add 2ml of chloroform and 2ml of conc.
sulfuric acid. Shake it well. Chloroform layer appears red and acid layer shows yellow fluorescence.
Sodium hydroxide test for flavonoids addition of increasing amount of sodium hydroxide to the residue
shows yellow coloration which decolorizes after the addition of acid ( Turnidge JD et al. 2003). Benedicts's
test for Carbohydrates add 1 ml of the solution to be tested to 5 ml of Benedict's solution, and shake each
tube. Place the tube in a boiling water bath and heat for 3 minutes. Remove the tubes from the heat and
allow them to cool. Formation of a green, red, or yellow precipitate is a positive test for reducing sugars
(Skene et al. 2006, Andrews, J M 2001 and Policegoudr RS et al. 2007).
2.3 Antimicrobial assay
Broth microdilution method was used to screen the 5 plant extracts. The inoculums was prepared at the
concentration of 10-2 CFU/ml, starting from a 0.5 McFarland (or 108 CFU/ml) prepared from fresh colonies
of bacteria as described below7. Staphylococcus aureus, Niesseria gonorrohae, Pseudomonas aeruginosa,
Escherichia coli Streptococcus pyogenes Bacillus subtilis were the bacterial strains tested (HiMedia
Laboratories Pvt. Limited Mumbai). The bacterial inoculum of each was obtained from fresh colonies
grown on Mueller Hinton agar plates. Each strain was inoculated into 5 ml of Muellen Hinton broth in
order to obtain a concentration of 1.5 x 108 CFU/ml. The inoculums were then diluted to 1.5 x 102
CFU/ml. One hundred and ninety microliters of this suspension was transferred to each microplate well.
Ten microliters of each extract solution was added to the microplate wells and incubated at 35ºC for 18 to
20 h. Extracts were prepared to 20 times the desired test concentration (2 mg/ml) in water or 50% DMSO
solution. The extracts were screened at a concentration of 100 µg/ml. Extracts that showed inhibitory
activity at this concentration were submitted to a subculture of the broth media in Mueller Hinton agar
(HiMedia Laboratories Pvt. Limited Mumbai ) in order to evaluate bacterial growth (Macfoy C A et al.
1990, Maikere-Faniyo R et al. 1989, Biavatti MW et al. 2001, Alves TM et al . 2000, Gnan SO et al. 1999
and Nakamura CV et al . 1999).
2.4 Determination of minimal inhibitory concentration and minimal bactericidal concentration
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined
for the extracts that showed total growth inhibition using the protocol described above. Extract
concentrations of 10 to 100 µg/ml in steps of 10 µg were evaluated. The concentration at which there was
no visually detectable bacterial growth was taken as the MIC, and the concentration at which there was no
bacterial growth after inoculation in Mueller-Hinton agar was taken as the MBC (Eloff JN 1998).
3. Results
The result of phytochemical screening is presented in (Table 1.) this reveals moderate concentration
of alkaloids, carbohydrates, flavonoids, saponins, terpens and steroids some of which chemical compounds
have been associated to antioxidant activities and thus have curative properties. Bacterial growth inhibition
was confirmed after inoculation in Mueller-Hinton agar. Many extracts showed some degree of inhibition
of bacterial growth at concentrations of 100 µg/ml, represented by “++”. The MIC and MBC of these
extracts are currently being obtained. (Table 2) lists the five plant extracts that showed antibacterial activity
against Niesseria gonorrohae ATCC 43069 , Staphylococcus aureus ATCC BAA 1026 , Pseudomonas
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aeruginosa ATCC 9027 (HiMedia Laboratories Pvt. Limited Mumbai )and their respective MIC and MBC.
(Eloff JN 1998 and Suffredini IB et al. 2002)
4. Discussion
Plant extracts have been studied against bacteria for years, but in a more intensified way in the last three
decades. During this period, a lot of antimicrobial screening evaluations have been published based on the
traditional use of Chinese, African, and Asian plant drugs (9-15). During the late 1990’s, a large number of
manuscripts describing methodologies of screening took part and resulted in more than 30 articles
representing antibacterial extracts obtained from Asian and African native plants, but only a few studies
relating antibacterial activity of Amarkantak plant extracts were found. Amarkantak is home to more than
20% of the world’s biodiversity and only a few species have been submitted to any sort of large-scale
biological screening. We collected a substantial amount of information about the antimicrobial activity of 5
Amarkantak plant extracts which have been collected. In order to establish the extract concentrations to be
used during the screening, we observed that extract concentrations spanning from 25 µg/ml ( Eloff JN 1998)
to 2 mg/ml(Loganga Otshudi A et al. 2000) or even 40 mg/m (Gnan SO et al. 1999) have been used. We
screened the crude extracts using a concentration of 100 µg/ml (Suffredini IB et al. 2002). Such
concentration is nowadays considered the proper concentration an antimicrobial extract should present
(Table 3). The dilution test is a very precise technique that permits us to work with such a low
concentration.
Only 3 of 5 extracts showed bactericidal activity: extract Selaginella bryopteris, Lygodium flexuousm
against N. gonorrhea Lygodium flexuousm against S. aureuas
and Adiantum philippense ,Drypteris
eochleata, Tectacria coadunate against P.aeugoginosa, Several crude extracts apparently inhibited bacterial
growth. Among them, we are currently determining the MIC and MBC for all 5 crude extracts (72%)
showed positive results against S. aureus, N. gonorrhea, P. aeruginosa. Extract (MIC and MBC), obtained
from the stem of a Selaginella bryopteris, Lygodium flexuousm plant, showed antibacterial activity against
N. gonorrhea (MIC = 160 and 140 µg/ml respectively; MBC = 180 and 170 µg/ml). Extract Adiantum
philippense ,Drypteris eochleata, Tectacria coadunate (MIC = 90 ,80,60 µg/ml; MBC = 140 , 80 ,70 µg/ml
respectively ), obtained from the stem of a plant, showed activity against P. auroginosa. The three active
extracts are going to be bioguide fractionated in order to identify their active substances as well as the
remaining extracts, whose MICs were ≤500 µg/ml (Kim HK et al.1970, De Tommasi N et al.1993 and
Hamilton-Miller JM et al. 1995). All the species have been studied phytochemically and biologically, and
beta-sitosterol, betulinic acid and sericic acid have been isolated from their stem bark extracts. (Hess SC et
al. 1999). These substances have shown antibacterial activity against P.auroginosa and S. aureus. It is a
matter of major national interest to study the potential of Amarkantak forests in offering new lead
antibacterial compounds that can be further synthesized and have their activity improved. Thus, we strongly
hope to contribute to the conservation and protection of the biodiversity of our forests and to the
development of the Amarkantak community as a whole. Further phytochemical studies are required to
determine the types of compounds responsible for the antibacterial effects of these species. The results also
indicate that scientific studies carried out on medicinal plants having traditional claims of effectiveness
might warrant fruitful results. Several plants used by Baiga tribe exhibit some degree of antibacterial
activity
towards
gram-positive
bacteria
such
as,
Bacillus
subtilis, Staphylococcus
aureus and Staphylococcus epidermidis. These plants could serve as useful sources for new antimicrobial
agents.
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anti-oxidant and anti-bacterial compound from mango-ginger (Curcuma amada Roxb) rhizome.
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Advances in Life Science and Technology
ISSN 2224-7181 (Paper) ISSN 2225-062X (Online)
Vol 4, 2012
Table No.1.
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Amarkantak flora are Ethnomedicinaly used by local tribes.
S.No.
Botanical
Name/Family
Natural habitat
collection site
1.
Selaginella
bryopteris
(L.)Baker
Heavy
rocks
boulders , deep
forest
Forest
Amarkantak
Panchdhara
(Selaginellacea)
Local
Name:
Sanjivini
Amarkantak
Lygodium
flexuousm (L) Sw.
(Lygodiaceae)
Grow on Bushes
and trees, Sandy
Soils
2.
Plants parts
used
of
Adiantum
philippense
(L)
Adiantaceae Local
Under
moist
condition ,
Low
Name:Kalijhant
mountainous
region
Paste of leaves used in gonorrhea, spermatorrhoea,
leucorrhoea, diuretic, stomachache, urinary tract
inflammation in children.
Tobacco
Forest
of
Amarkantak Mai ki
Bagia
Local
Name :Kalijar
3.
Leaves
dried plant
with
Ethnomedicinal uses
Forest
of
Amarkantak near
Sambhudhara
Stem,
Rhizome
leaves,
Antibacterial property. Boiled fronds used for
Carbuncles, rheumatism ,sprains, scabies ulcers and
cuts wounds.
spores
Extract of stem and rhizome is taken for curing
sexual disease like gonorrhoea and spermatorrhoea.
Fresh leaves extract for piles. Spores cure high fever.
Rhizomes,
leaves
Fronds
extract
for
fever
,
asthma
,
bronchitis ,dysentery ,epileptic fits ,leprosy ulcers and
erysipelas. Powder of rhizome as an antitode against
dogbite and snakebite.
Leaves extract applied for stomach for clear and
early release of urine. Died rhizome for women
during menstrual period sterility.
4.
Drypteris eochleata
D.C.
Stream
sides,Amanala,
Amanala
Laxmandhara
Rhizome,
Leaves
Rhizome as Antifungal property ,used as antitode.
Extarct of dried rhizome is given in epilepsy and
(Ham.ex.D .Don )
C.
chr.
Dryopteridaceae
laterite soils
Panchdhara
stems stipe
leprosy . Paste of fresh rhizome ,stem stipe is applied
for cuts, wounds, ulcers, swelling and pains.
Fresh Paste and rhizome and fronds used in
Local
Name:Jatashankar
i
5.
snakebite and dogbite.
The decoction of dried rhizome tem stipe used for
blood purification.
Tectacria
coadunate
(Wall.ex.Hook.et
Rocks Crevices
boulders in moist
places along the
grev.)
C.chr
(Tectariaceae)
Local
Name:
stream
Sonmura
Bagia
Mai
ki
Rhizome
Leaves
Anthelmintic, fresh rhizome and fronds paste is used
in insect bites or getting relief in centipede bite. Fresh
rhizome and stipe in stomach pain, Gastrointestinal
disorders of worm in children .Extraxction of dried
rhizome ,stem and stipe is used in respiratory
disorders like cold,cough ,asthma and bronchitis.
Jatamasi
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ISSN 2224-7181 (Paper) ISSN 2225-062X (Online)
Vol 4, 2012
Table No. 2. Phytochemical screening of the given plant
S.No. Contituents
Leaves
Extracts
1. Alkaloids Meyer’s, test
++
2. Carbohydrates, Benedict’s test
++
3. Flavonids ,Sodium hydroxide test
++
4. Saponins , Frothing test
++
5. Steroids ,Salkowski test
++
6. Tannins ,FeCl3 test
+
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Rhizome
stem
Roots
+
++
+
++
++
++
++
+
+
+
++
++
++
++
++
+
+
+
Key (- )= Negative (absent),(+) = Positive (Slightly present) (++)
= Positive (moderately
present)
Table No. 3. Antimicrobial activity of plants extract from the Amarkantak forest that showed strong
activity, and their corresponding minimal inhibitory and minimal bactericidal concentrations.
Lygodium Adiantum
Drypteris
Tectacria
Plants Samples
Selaginella
flexuousm philippense eochleata
coadunate
bryopteris
(Kalijhant) (Jatashankari) (Jatamasi)
(Amarbatoooti (Kalijar)
Sanjivini)
Number of extracts evaluated
4
4
4
4
4
+ve
-ve
-ve
-ve
Extract classified as ++++ +ve
against Niesseria gonorrohae
ATCC 43069
Extract classified as ++++ -ve
+ve
-ve
-ve
-ve
against Staphylococcus aureus
ATCC BAA 1026
Extract classified as ++++ -ve
-ve
+ve
+ve
+ve
against
Pseudomonas
aeruginosa ATCC 9027
Minimal
inhibitory 160
140
90
80
60
concentration (µg/ml)
Minimal
Bactericidal 180
170
140
80
70
concentration (µg/ml)
Activity was measured by the micro dilution broth assay ++++ indicate that the extracts caused total growth
inhibition
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Computer Engineering and Intelligent Systems
CEIS@iiste.org
Innovative Systems Design and Engineering
ISDE@iiste.org
Journal of Energy Technologies and Policy
JETP@iiste.org
Information and Knowledge Management
IKM@iiste.org
Control Theory and Informatics
CTI@iiste.org
Journal of Information Engineering and Applications
JIEA@iiste.org
Industrial Engineering Letters
IEL@iiste.org
Network and Complex Systems
NCS@iiste.org
Environment, Civil, Materials Sciences
PAPER SUBMISSION EMAIL
Journal of Environment and Earth Science
JEES@iiste.org
Civil and Environmental Research
CER@iiste.org
Journal of Natural Sciences Research
JNSR@iiste.org
Civil and Environmental Research
CER@iiste.org
Life Science, Food and Medical Sciences
PAPER SUBMISSION EMAIL
Journal of Natural Sciences Research
JNSR@iiste.org
Journal of Biology, Agriculture and Healthcare
JBAH@iiste.org
Food Science and Quality Management
FSQM@iiste.org
Chemistry and Materials Research
CMR@iiste.org
Education, and other Social Sciences
PAPER SUBMISSION EMAIL
Journal of Education and Practice
JEP@iiste.org
Journal of Law, Policy and Globalization
JLPG@iiste.org
New Media and Mass Communication
NMMC@iiste.org
Journal of Energy Technologies and Policy
JETP@iiste.org
Historical Research Letter
HRL@iiste.org
Public Policy and Administration Research
PPAR@iiste.org
International Affairs and Global Strategy
IAGS@iiste.org
Research on Humanities and Social Sciences
RHSS@iiste.org
Developing Country Studies
DCS@iiste.org
Arts and Design Studies
ADS@iiste.org
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