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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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