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International Journal of Health and Pharmaceutical SciencesISSN 2278 - 0564
Research Paper
PRELIMINARY PHYTOCHEMICAL ANALYSIS AND ANTI BACTERIAL
ACTIVITY OF TINOSPORA CORDIFOLIA LEAF EXTRACTS
Venkanna Lunavath1, Raju Porika
1, Sri Priya
2and Estari Mamidala*
1Metabolic Disorders and Infectious Diseases Research Lab, Department of Zoology,Kakatiya University, Warangal-506009, (A.P), India2Department of Biotechnology, Dravidian University, Kuppam. (A.P), India
*estari08@gmail.com
ABSTRACT:
In India, different parts of several medicinal plants or their extracts are used for the treatment ofvarious diseases. Several antibiotics used for the treatment of human infections, which have
limited antimicrobial spectrum. The present study was undertaken to explore the phytochemical
constituents and antibacterial activity of leaves of Tinospora cordifolia extracts on somecommon bacterial species. Preliminary phytochemical screening revealed the presence of
Alkaloids, Carohydrates, Glycosides, Saponins and Flavonoids. T. cordifolia showed high
activity acrossEscherichia coli,Psuedomonas aeuruginosa andStaphylococcus aureusbacteria.The results suggest aqueous and chloroform extracts fractions of this plant with moderent
antibacterial activity and secondary metabolites of this plant used for wound healing property
and other forms of bacterial infections. The results confirm thatTinospora cordifolia can be used
as source of drugs to fight infections caused by susceptible bacteria.
KEY WORDS:
Antibacterial activity, Methanolic extract, phytochemical,Tinospora cordifolia.
INTRODUCTION:
The plant kingdomis a treasure house of potential drugs and in recent years there has been
anincreasing awareness about the importance
of medicinal plants. Drugs from the plants are easily available, less expensive, safe, and ef
ficient and rarely have side effects.
The plants which have been selected for medical use over thousands of yearsconstitute the most obvious choice
for examining the current search for
therapeutically effective new drugs suchas anticancer drugs, (Dewick, 1996), antimicr
obial drugs (Phillipson and Wright, 1996)and
anti hepatotoxic compounds (Evans, 1996).
About 80% of the worlds population depends on herbal medicines and the governments of
third world countries unable to sustaina com
plete coverage with western type drugs haveencouraged the rational development of tradit
ional Treatments.
Man has earlier discovered within hisenvironment the wealth of importance ofplants as therapeutic agents. This knowledge
together with their toxic potentials had
passed down from generation to generation.Of the 300,000 plant species acclaimed
worldwide only about 5% have been
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investigated scientifically for their medicinalpurposes (Sanusi and Rabo, 2004).
Researchers have reported that
developing countries rely mainly on plantsfor the treatment of their prevailing ailments
especially in areas where hospitals are not
accessible (Lambo, 1979).
Plant based drugs are beingincreasingly preferred in medical science.
The curative parts of a medicinal plant are
not simply its woody stem or its leaves, butthe number of chemical compounds
(phytochemicals) produced and uses for its
own growth and development. Thetherapeutic value and pharmacological action
of a drug is due to the presence of certain
chemical constituents such as carbohydrates,derivatives of carbohydrates, gums,
mucilages, pectins, various forms ofglycosides, tannins, phenolic compounds,
lipids, fixed and volatile oils, resins, variouskinds of alkaloids etc. These phytochemicals
are of immense importance to mankind.
Phytochemical investigation of plants is aninteresting area of research, leading to the
isolation of several new compounds. In the
present study, the hexane, chloroform, carbontetrachloride and aqueous extract fractions of
Tinospora cordifolia leaves were screened
for phytochemical constituents andantimicrobial activity againstStaphylococcusaureus and Enterococcus faecalis
Psuedomonas aeuruginosa and Escherichia
coli.
MATERIALS AND METHODS:Leaves of Tinospora cordifolia are
collected from Parvatagiri Village of Torrur
Mandal, Warangal district, Andhra Pradesh.Voucher specimens were prepared and
identified at the Department of Botany,Kakatiya University, Warangal. The
Tinospora cordifolia leaves were collectedand left at room temperature for two weeks to
dry, then ground into powder and extraction
with Soxhlet techniques with methanol.Obtaining methanolic crude extracts of
Tinospora cordifolia were then fractionated
successively using solvents of increasing
polarity, such as,n-hexane (HX), carbontetrachloride (CT), and chloroform (CF) and
aqueous fractions (AQ). All the four
fractions (HXF, CTF, CFF and AQF) wereevaporated to dryness by using rotary
evaporator at low temperature (390C).
The major secondary metabolites
classes such as alkaloids, carbohydrates,glycosides, saponins, proteins and Amino
acids, Phytosteroids, Fixed oils and Fats,
Phenolic compounds and Flavanoids werescreened according to the common
phytochemical methods described by Kokate
(1994) and Kokateet al(1995).Bacterial species selected for the
study were the four pathogens, namely, two
Gram-positive Staphylococcus aureus and
Enterococcus faecalis and two Gram-
negative Psuedomonas aeuruginosa andEscherichia coli. All the cultures were
maintained on Mueller-Hilton agar at 400C.
The cells were inoculated and incubated at
370C in broth for 12 hours prior to the
screening procedure.The serial microplate dilution method
developed by Eloff (1998) was used to
determine the minimum inhibitoryconcentration (MIC) for plant extracts using
tetrazolium violet reduction as an indicator of
growth. Residues of the different extractswere re-dissolved in methanol to aconcentration of 1 mg/ml. for each of the four
bacteria used, 100l of each plant extract
tested were two-fold serially diluted with 100l sterile distilled water in a sterile 96-well
microtitre plates. A similar two- fold serial
dilution of gentamicine (0.1mg/ml) was usedas a positive control against each bacterium.
One hundred microlitres of each bacterial
culture were added to each well. The plates
were covered and incubated overnight at 370
C. To indicate bacterial growth 40 l of 0.2
mg/ml p-iodonitrotetrazolium violet (INT)
were added to each well and the platesincubated at 37
0C for 30 minutes. Bacterial
growth in the wells was indicated by a red
colour, where as clear wells indicatedinhibition of the bacterial growth by the plant
extracts.
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RESULTS:
The yield of the methanol crudeextract of Tinospora cordifolia was 45g
(9%). The percentage yield of methanolic
crude extract fractions ofTinospora
cordifolia were showed in the table-1. TheCFF fractions obtained highest yield (2.6%)
when compared to other fractions. 0.7% yield
obtained in CTF fraction which is lowest.
S.No Fractions Yield (%)
1 HXF 1.42 CTF 0.7
3 CFF 2.6
4 AQF 1.0
Table-1 Percentage of yield
In the present study, preliminary phy
tochemical testing shows (Table2) the presence of high amountofglycosides, alkaloids,
phenolics and other all the principal secondary metaboliteswere detected in different
fractions of Tinospora cordifolia. AqueousExtract of the drug shows the presence ofalkaloids, saponins, phenolic compounds
and flavonoids in high concentration and
where as the glycosides, carbohydrates and
oils are present in lower concentration.
Alkaloids are present in all fractions ofT.
Cordifolia. Carbohydrates are moderatelypresent in CFF. Protein, amino acids and oil
are moderately present in CTF fraction ofT.
cordiofolia. Phenolic compounds and
flavonoids are present in low concentrationin HXF fraction of T. cordifolia. The
presences of organic constituents areconsidered to be responsible for therapeutic
action.
Table-2 Phytochemical screening for presence of different Phytoconstituents in Tinospora
cordifolia leaves extract fractions
Sl. No. Phytochemicals test HXF CTF CFF AQF
I Test for Alkaloidsa. Mayers Test + + + +
b. Wagners Test + + + +
c. Hagers Test ++ ++ ++ ++
D Dragendorffs Test + + + +
II Test for Carbohydrates
A Molishs Test - + ++ -
B Fehlings Test - - ++ -
C Barfoeds Test - - ++ -
D Benedicts Test - - ++ +
III Test for Glycosides
a. Borntragers Test - + + +B Legals Test - + + +
IV Test for Saponin
A Foam Test + + +++ ++
V Test for Proteins and Amino acids
A Millons Test - ++ + -
B Biurets Test + ++ - -
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C Ninhydrin Test + + - -
VI Test for Phytosteroids
A Libermann Burchards Test + + + +
VII Test for fixed oils and fats
A Spot Test + ++ - +
B Saponification Test - + - +
VIII Tests for Phenolic Compounds and Flavanoides
A Ferric chloride Test + - + +
B Gelatin Test + - ++ +
C Lead acetate Test + - - ++
D Alkaline Rgt. Test + - + +
E Magnesium Test + - + +
+++ Prominently Present, ++ Moderately Present, + Slightly Present, - Absent
The MIC values and total activity of
the four fractions of methanol crude extract
of Tinospora cordifolia plant against all thetested bacteria are presented in Table 3.T.
cordifolia had better activity against all the
tested organisms compared to other extractfractions.T. cordifolia AQF had MIC value
of 0.08 mg/ml againstS. aureus only.
In this studyT .cordifolia had better
activity againstS. aureus. These substantial
differences might be due to seasonal andgeographical variations. Other conditions
that might influence variation in biological
activity includes genetic variability, plantage and growth climatic conditions.
Plant species Ec Ef Pa Sa
T. cordifolia
HXF
CTFCFF
AQF
0.08
0.150.15
0.25
0.15
0.080.15
0.15
0.08
0.080.15
0.15
0.08
0.150.30
0.08
Gentamicin (g/ml) 8.0 1.6 0.2 0.3
Table:3 MIC values (mg/ml) of the Tinospora cordifolia plant extracts fractions of the
study: T. cordifolia against four bacteria: Escherichia coli (Ec),Enterococcus faecalis (Ef),
Pseudomonas aeuruginosa (Pa) and Staphylococus aureus (Sa). Gentamicin was used as apositive control. HXF=n-hexane fraction, CTF=Carbon tetra chloride fraction, CFF=Chloroform
fraction and AQF=Aqueous fraction.
DISCUSSION:Literature has no report on the
antibacterial activity ofT. cordifolia and thisis the first one. Presence of alkaloids,
cyanogenetic glycosides, steroidal nucleus
and reducing sugars were indicated tovarious extends in the plant material
screened for secondary metabolites. This is
not suprising for plants of the
Menispermaceae family (Mungole et al,2010). Cardiac glycosides and tannins were
not detected in the present studies (Table 2).
Plants harvested from the wild generally
vary in quality and consistency of activecompounds (Bopana and Saxena, 2007). In
cases where mature trees or plants cannot be
found, the younger ones suffice, whichresults in availability of inconsistent plant
material of the same species (Von
Ahlefeldatet al, 2003).
All the test organisms weresusceptible to T. cordifolia leaf extract
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fractions though to varying degrees. Alluri
et al (2009) reported that the susceptibilityof bacterial to plant extract fractions, on the
basis of inhibition zone diameters varied
according to strains and species, similar tothe data obtained in this study. The result of
the experiment showed that the leaf of
Tinospora cordifolia may have somevaluable anti-microbial activities againstGram positive and Gram negative
microorganisms. This property tends to
support the traditional medicinal stage in thetreatment of bacterial infections. The result
of the study justified the use of the plant in
the treatment of diseases of microbial originin herbal medicine. The demonstration of
broad spectrum of antimicrobial activity
by T. cordifolia may help to discover
new chemical classes of antibioticsubstances that could serve as selective
agents for infectious disease chemotherapy
and control.
ACKNOWLEDGEMENT:
We wish to express our profound gratitudeto Prof. Raju, Department of Botany,
Kakatiya University, Warangal (A.P) for
identification of the plant.
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