International Journal of Health and Pharmaceutical Sciences ISSN 2278 - 0564
Research Paper
Vol 1, Issue 4, 2012
PRELIMINARY PHYTOCHEMICAL ANALYSIS AND ANTI BACTERIAL ACTIVITY OF TINOSPORA CORDIFOLIA LEAF EXTRACTS Venkanna Lunavath1, Raju Porika1, Sri Priya2 and Estari Mamidala* 1
Metabolic Disorders and Infectious Diseases Research Lab, Department of Zoology, Kakatiya University, Warangal-506009, (A.P), India 2 Department of Biotechnology, Dravidian University, Kuppam. (A.P), India
*
[email protected]
ABSTRACT: In India, different parts of several medicinal plants or their extracts are used for the treatment of various 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 some common bacterial species. Preliminary phytochemical screening revealed the presence of Alkaloids, Carohydrates, Glycosides, Saponins and Flavonoids. T. cordifolia showed high activity across Escherichia coli, Psuedomonas aeuruginosa and Staphylococcus aureus bacteria. 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 that Tinospora 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 pote ntial drugs and in recent years there has been anincreasing awareness about the importance of medicinal plants. Drugs from the plants ar e 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 years constitute the most obvious choice for examining the current search for therapeutically effective new drugs such as anticancer drugs, (Dewick, 1996), antimicr obial drugs (Phillipson and Wright, 1996)and
anti hepatotoxic compounds (Evans, 1996). About 80% of the world’s population depend s on herbal medicines and the governments of third world countries unable to sustaina com plete coverage with western type drugs have encouraged the rational development of tradit ional Treatments. Man has earlier discovered within his environment the wealth of importance of plants 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
www.ijhps.com
17
investigated scientifically for their medicinal purposes (Sanusi and Rabo, 2004). Researchers have reported that developing countries rely mainly on plants for the treatment of their prevailing ailments especially in areas where hospitals are not accessible (Lambo, 1979). Plant based drugs are being increasingly preferred in medical science. The curative parts of a medicinal plant are not simply its woody stem or its leaves, but the number of chemical compounds (phytochemicals) produced and uses for its own growth and development. The therapeutic 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 of glycosides, tannins, phenolic compounds, lipids, fixed and volatile oils, resins, various kinds of alkaloids etc. These phytochemicals are of immense importance to mankind. Phytochemical investigation of plants is an interesting area of research, leading to the isolation of several new compounds. In the present study, the hexane, chloroform, carbon tetrachloride and aqueous extract fractions of Tinospora cordifolia leaves were screened for phytochemical constituents and antimicrobial activity against Staphylococcus aureus 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 collected and 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), carbon tetrachloride (CT), and chloroform (CF) and aqueous fractions (AQ). All the four fractions (HXF, CTF, CFF and AQF) were evaporated 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 were screened according to the common phytochemical methods described by Kokate (1994) and Kokate et al (1995). Bacterial species selected for the study were the four pathogens, namely, two Gram-positive Staphylococcus aureus and Enterococcus faecalis and two Gramnegative Psuedomonas aeuruginosa and Escherichia 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 inhibitory concentration (MIC) for plant extracts using tetrazolium violet reduction as an indicator of growth. Residues of the different extracts were re-dissolved in methanol to a concentration of 1 mg/ml. for each of the four bacteria used, 100µl of each plant extract tested were two-fold serially diluted with 100 µl sterile distilled water in a sterile 96-well microtitre plates. A similar two- fold serial dilution of gentamicine (0.1mg/ml) was used as 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 37 0 C. To indicate bacterial growth 40 µl of 0.2 mg/ml p-iodonitrotetrazolium violet (INT) were added to each well and the plates incubated at 370 C for 30 minutes. Bacterial growth in the wells was indicated by a red colour, where as clear wells indicated inhibition of the bacterial growth by the plant extracts.
www.ijhps.com
18
RESULTS:
The yield of the methanol crude extract of Tinospora cordifolia was 45g (9%). The percentage yield of methanolic crude extract fractions of Tinospora S.No 1 2 3 4
cordifolia were showed in the table-1. The CFF fractions obtained highest yield (2.6%) when compared to other fractions. 0.7% yield obtained in CTF fraction which is lowest.
Fractions HXF CTF CFF AQF Table-1 Percentage of yield
In the present study, preliminary phy tochemical testing shows (Table‐2) the prese nce of high amountofglycosides, alkaloids, phenolics and other all the principal seconda ry metaboliteswere detected in different fractions of Tinospora cordifolia. Aqueous Extract of the drug shows the presence of alkaloids, saponins, phenolic compounds and flavonoids in high concentration and where as the glycosides, carbohydrates and oils are present in lower concentration.
Yield (%) 1.4 0.7 2.6 1.0
Alkaloids are present in all fractions of T. Cordifolia. Carbohydrates are moderately present in CFF. Protein, amino acids and oil are moderately present in CTF fraction of T. cordiofolia. Phenolic compounds and flavonoids are present in low concentration in HXF fraction of T. cordifolia. The presences of organic constituents are considered to be responsible for therapeutic action.
Table-2 Phytochemical screening for presence of different Phytoconstituents in Tinospora cordifolia leaves extract fractions Sl. No. I a. b. c. D II A B C D III a. B IV A V A B
Phytochemicals test Test for Alkaloids Mayer’s Test Wagner’s Test Hager’s Test Dragendorff’s Test Test for Carbohydrates Molish’s Test Fehling’s Test Barfoed’s Test Benedict’s Test Test for Glycosides Borntrager’s Test Legal’s Test Test for Saponin Foam Test Test for Proteins and Amino acids Millon’s Test Biuret’s Test
HXF
CTF
CFF
AQF
+ + ++ +
+ + ++ +
+ + ++ +
+ + ++ +
-
+ -
++ ++ ++ ++
+
-
+ +
+ +
+ +
+
+
+++
++
+
++ ++
+ -
-
www.ijhps.com
19
C VI A VII A B VIII A B C D E
Ninhydrin Test + Test for Phytosteroids Libermann – Burchard’s Test + Test for fixed oils and fats Spot Test + Saponification Test Tests for Phenolic Compounds and Flavanoides Ferric chloride Test + Gelatin Test + Lead acetate Test + Alkaline Rgt. Test + 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 the tested bacteria are presented in Table 3.T. cordifolia had better activity against all the tested organisms compared to other extract fractions. T. cordifolia AQF had MIC value of 0.08 mg/ml against S. aureus only. Plant species T. cordifolia HXF CTF CFF AQF Gentamicin (µg/ml)
In this study T .cordifolia had better activity against S. aureus. These substantial differences might be due to seasonal and geographical variations. Other conditions that might influence variation in biological activity includes genetic variability, plant age and growth climatic conditions.
Ec
Ef
Pa
Sa
0.08 0.15 0.15 0.25 8.0
0.15 0.08 0.15 0.15 1.6
0.08 0.08 0.15 0.15 0.2
0.08 0.15 0.30 0.08 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 a positive 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 of T. cordifolia and this is the first one. Presence of alkaloids, cyanogenetic glycosides, steroidal nucleus and reducing sugars were indicated to various 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 active compounds (Bopana and Saxena, 2007). In cases where mature trees or plants cannot be found, the younger ones suffice, which results in availability of inconsistent plant material of the same species (Von Ahlefeldat et al, 2003). All the test organisms were susceptible to T. cordifolia leaf extract
www.ijhps.com
20
fractions though to varying degrees. Alluri et al (2009) reported that the susceptibility of bacterial to plant extract fractions, on the basis of inhibition zone diameters varied according to strains and species, similar to the data obtained in this study. The result of the experiment showed that the leaf of Tinospora cordifolia may have some valuable anti-microbial activities against Gram positive and Gram negative microorganisms. This property tends to support the traditional medicinal stage in the treatment of bacterial infections. The result of the study justified the use of the plant in the treatment of diseases of microbial origin in herbal medicine. The demonstration of broad spectrum of antimicrobial activity by T. cordifolia may help to discover new chemical classes of antibiotic substances that could serve as selective agents for infectious disease chemotherapy and control. ACKNOWLEDGEMENT: We wish to express our profound gratitude to Prof. Raju, Department of Botany, Kakatiya University, Warangal (A.P) for identification of the plant. REFERENCES: 1. Alluri, V. Krishnaraju, and Chundi B .M. Rao. Antiinflammatory activity of vitex leucoxylon L. bark extracts against Freund’s complete adjuvant induced Arthritis in Sprague Dawley rat’, American Journal of infectious diseases, 2009; 5. 68‐73. 2. Bopana, N Saxeana, S. Asparagus racemosus – Ethnopharmacological evaluation and conservation needs. Journal of Ethnopharmacology, 2007; 110:1-15. 3. Eloff, J N, A sensitive ank quick method to determine the minimum inhibitory concentration of plant www.ijhps.com
extracts for bacterial. Plant Medica. 1998; 60: 1-8. 4. Evans, W C,Trease and Evans, Phar macognosy 14th ds, W B Sounders Company. London 1996; 612. 5. Irvine FR, Woody plants of Ghana (with special reference to their uses) Oxford University press. London, 1961; 285-286. 6. Kokate CK, Khandelwal KR, Pawar A.P and Gohale SB, Practical Pharmacognosy. Nirali Prakashan, Pune, 3rd edn. 1995; 137-139. 7. Kokate, C K, Practical Pharmacognosy, 4th ed, Vallabh Prakashan, New Delhi, India. 1994; 112-120. 8. Lambo JO, The healing power of herbs with special reference to obstetric and Gynecology in African medicinal (2nded). Ife press Nigeria pp1979; 24 – 27. 9. Mungole, A. J., Awati, A., Chaturvedi, A., Zanwar, P. Preliminary phytochemical screening of Ipomoea obscura (L) – A hepatoprotective medicinal plant. International Journal of Pharm Tech Research, 2010;2: 2307-2312. 10. Phillipson JD and Wright CW, Plants with anti protozoal activity In Trease and Evans, Pharmacognosy 1 4th eds., W.B.Saunders Company, L ondon. 1996; 426-433. 11. Sanusi SS, Rabo ET. An inventory of Medicinal plant the NigeriaSavannah Leviathan book Lagos. 2004; 21- 24.
21
