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| | | Year : 2009 | Volume : 3 | Issue : 2 | Page : 125-127 | | | Hepatoprotective activity of the methanolic extract of Tylophora indica (Burm. f.) Merill. leaves | | | M Mujeeb1, V Aeri1, P Bagri1, SA Khan2
1 Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi - 110 062, India
2 Department of HIT, Jeddah Community College, King Abdulaziz University, Jeddah, KSA, Saudi Arabia
Click here for correspondence address and email | Date of Submission | 28-Aug-2008 | | Date of Acceptance | 07-Feb-2009 | | Date of Web Publication | 6-Aug-2009 | | |      | |  Abstract | | | The methanolic extract of Tylophora indica leaves was screened for hepatoprotective activity in carbon tetrachloride induced hepatotoxicity in albino rats. The degree of protection was measured by estimating biochemical parameters like Serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, total protein and level of serum bilirubin (both total and direct). Hepatoprotective activity of methanolic extract at a dose of 200 mg/kg and 300 mg/kg body weight, i.p., was compared with Silymarin (25 mg/kg, i.p.) treated animals. Tylophora indica leaves (200 and 300 mg/kg) exhibited significant reduction in serum hepatic enzymes when compared to rats treated with carbon tetrachloride alone. Furthermore, histopathological studies were also done to support the study. Keywords: Carbon tetrachloride, hepatoprotective activity, Silymarin, Tylophora indica How to cite this article:
Mujeeb M, Aeri V, Bagri P, Khan S A. Hepatoprotective activity of the methanolic extract of Tylophora indica (Burm. f.) Merill. leaves. Int J Green Pharm 2009;3:125-7 |
How to cite this URL:
Mujeeb M, Aeri V, Bagri P, Khan S A. Hepatoprotective activity of the methanolic extract of Tylophora indica (Burm. f.) Merill. leaves. Int J Green Pharm [serial online] 2009 [cited 2014 Mar 11];3:125-7. Available from: http://www.greenpharmacy.info/text.asp?2009/3/2/125/54901 |
| Introduction | |  |
Tylophora indica (Burm. f.) Merill. (Family; Asclepidaceae) commonly known as Antamool in Ayurveda is a twining perennial plant distributed throughout southern and eastern part of India in plains, forests, and hilly places. [1] The plant has been reported to contain 0.2-0.46% alkaloids viz. Tylophorine, tylophorinine, [2],[3] tylophorinidine, (+)-septicine, isotylocrebrine, [4] tylophorinicine, sterols, flavonoids, [5] wax, resins and tannins.
In Ayurveda, the plant has been used in treatment of asthma, dermatitis, and rheumatism. [1],[6] The other reported activities include immunomodulator activity, [7] anti-inflammatory activity, [8] anticancer activity, [9] and antiamoebic activity. [10]
In the present study, the hepatoprotective effects of methanolic extract (flavonoid rich fraction) are investigated in a scientific manner to validate its use as alternative and complimentary herbal medicine.
| Materials And Methods | | |
Plant Material
Tylophora indica leaves were collected from herbal garden, Jamia Hamdard and authenticated by the taxonomist of Department of Botany, Faculty of Science, Hamdard University. The voucher specimen was deposited in the herbarium of university for future reference.
Preparation of Extract
The air-dried and coarsely powdered leaves of plant (1 kg) were Soxhlet extracted with methanol for 72 h and the methanolic extract was concentrated on a water bath and dried under reduced pressure to get a dark brown mass (80 g).
HPTLC Fingerprint and Preliminary Phytochemical Screening
Methanolic extract of plant leaves (1 g/ml) was applied (2 ml) on silica gel G F 254 High Performance Thin Layer Chromatography (HPTLC) plate in duplicate with bandwidth of 5 mm using Linomat V applicating device (Camag, Switzerland). The chromatogram was developed in Twin trough chamber using solvent system Toluene: Chloroform: Ethyl acetate (1:5:3) saturated with 10% acetic acid and scanned in scanner III at 366 nm wavelength using mercury lamp in fluorescence mode. On preliminary phytochemical screening of leaves of T. indica , extract showed positive tests for alkaloids, glycosides, steroids, and flavonoids.
Animals
The hepatoprotective activity was carried out on Wistar albino rats of either sex (120-150 g), supplied by Central animal house facility of Jamia Hamdard, New Delhi (Registration no. 173/CPCSEA). They were maintained in a 12 h light/dark cycle at 25 ± 2°C. They were allowed free access to standard pellet diet (Amrut Laboratory Rat Feed, Navamaharashtra, Pune, India) and water ad libitum . The study was approved by ethics committee CPCSEA and ethical norms were strictly followed during all experimental procedures.
Drugs and Dosing Schedule
Animals were divided into five groups; Group I (control), Group II (CCl 4 treated), Group III (CCl 4 + Silymarin treated), Group IV and V (CCl 4 + extract). Animals of group II, III, IV, and V were administered 50% (v/v) CCl 4 in olive oil at a single dose of 2 ml/kg body weight per day for 4 days by Subcutaneous (S.C.) route. Simultaneously but at different hours of the day, animals of group III, IV, and V were fed with Silymarin suspension (25 mg/kg body weight, i.p.), methanolic extract at a dose of 200 mg/kg and 300 mg/kg body weight, i.p. for 4 days, respectively. Animals of group I was given distilled water in a volume of 10 ml/kg body weight.
Serum Analysis and Histopathological Examination
On 5 th day, after treatment period the animals of all groups were anaesthetized and sacrificed. Blood was withdrawn from heart and serum was separated by centrifugation at 3000 rpm at 30°C for 15 min and analyzed for various biochemical parameters; Serum transaminases viz. Serum glutamate oxaloacetate transaminase (SGOT), [11] serum glutamate pyruvate transaminase (SGPT), [11] total protein and bilirubin (direct and total). [12] The histopathological studies were also carried out by reported method. [13] Liver was removed from sacrificed animal, sliced, and washed in normal saline. Liver pieces were processed in 10% formaldehyde solution. The pieces of liver were processed and embedded in paraffin wax. Sections made were 4-6 mm in thickness. They were stained with hematoxylin and eosin and lastly photographed.
Statistical Analysis
Results of biochemical parameters are reported as mean±S.E.M. statistical significance was determined by one way analysis of variance (ANOVA) followed by Dunnet's t -test. [14] P value <0.05 was considered statistically significant.
| Results | | |
High Performance Thin Layer Chromatography fingerprints of methanolic extract of T. indica leaves showed presence of 20 spots, confirming the presence of different class of phytoconstituents as revealed by preliminary phytochemical screening.
Administration of CCl 4 led to significant hepatocellular damage as evident from increase in serum activities of Serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) (268.39 U/ ml, 262.2 U/ml), and bilirubin (total; 3.36 mg/dl and direct; 2.16 mg/dl, respectively) concentration as compared to normal control group (46.23 U/ ml, 34 U/ml, 0.68 mg/ dl and 0.20 mg/dl, respectively). Treatment of rats with methanolic extract of leaves at a dose of 200 mg/ kg and 300 mg/kg body weight, i.p. exhibited significant reduction ( P <0.05) in CCl 4 induced elevation of serum glutamate oxaloacetate transaminase (SGOT) (129.27-150.26 U/ml), serum glutamate pyruvate transaminase (SGPT) (127.6- 147.6 U/ml) and bilirubin (total;1.95-2.17 mg/ dl, direct; 1.28-1.44 mg/dl, respectively) and increased the level of TP (total protein) (3.69-4.2 g/ dl) [Table 1] in a dose dependant manner. Treatment with Silymarin also reversed the hepatotoxicity significantly. Histopathological studies of liver sections in control animals showed normal hepatic cells with prominent nucleus and central vein [Table 2]. In CCl 4 treated animals the sections showed hydropic changes in centrilobular hepatocytes with single cell necrosis, congestion of central vein and sinusoids were seen with acute and chronic inflammatory cells mainly in central zone. Pretreatment of the animals with Silymarin and methanolic extract exhibited a significant recovery of hepatocytes in different sections of the liver.
| Discussion | | |
CCl 4 is one of the most commonly used hepatotoxin in the experimental study of liver diseases. [15] The hepatotoxic effects of CCl 4 are largely due to its active metabolite, trichloro methyl radical. [16] These activated radicals bind covalently to the macromolecules and induce peroxidative degradation of membrane lipids of endoplasmic reticulum rich in polyunsaturated fatty acids. This leads to the formation of lipid peroxides, which in turn give products like malondialdehyde (MDA) that cause damage to the membrane. This is evidenced by an elevation in the serum marker enzymes. The increase in the levels of serum bilirubin reflected the level of jaundice and increase of transaminases was the clear indication of cellular leakage and loss of functional integrity of cell membrane. [17] Methanolic extract has significantly reduced these liver enzyme levels and has increased the level of total protein in the serum in a dose dependant manner, which indicates hepatoprotection. Furthermore, results of hepatocellular damage caused by CCl 4 and its recovery by methanolic extract suggest that the drug might be considered a potential source of natural hepatoprotective agents, which could be related to free radical scavenging properties of flavonoids present in the high concentration in the methanolic extract of the plant.
Histopathological studies revealed that CCl 4 caused steatosis and hydropic degeneration of the liver tissue. T. indica pretreatment exhibited protection, which confirmed the result of biochemical studies. Also all the effects of methanolic extract of T. indica were comparable with those of Silymarin, a proven hepatoprotective drug. Further isolation of active principles responsible for hepatoprotective activity is currently under progress in our lab.
| Acknowledgment | | |
The authors are thankful to the Head, Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard, New Delhi, India for providing the necessary facilities to carry out the research work.
| References | | |
| 1. | Anonymous. The Wealth of India. National Institute of Science and Communication,CSIR, New Delhi, India, 1978. p398 - 399.  | | 2. | Phillipson JD, Tezcan I, Hylands PJ. Alkaloids of Tylophora species from Sri Lanka. Planta Med 1974;25:301-9. [PUBMED] | | 3. | Gopalkrishan C, Shakaranarayana D, Kameswaran L. Pharmacological investigation of Tylophorine, a major alkaloid of Tylophora indica . Indian J Med Res 1979;69:513-20. | | 4. | Govindchari TR, Viswanathan N, Radhakrishnan J. Tylophora alkaloids. J Ind Chem Soc 1973; Vol C; 1-9. | | 5. | Rastogi RP, Mehrotra BN. Compendium of Indian Medicinal Plants. Vol. 1. Luknow: CSIR New Delhi and CDRI;1995. p. 423-4. | | 6. | Chopra RN, Nayar SC, Chopra IC. Glossary of Indian medicinal plants. New Delhi: CSIR publication; 1956. p. 250-1. | | 7. | Ganguli T, Sainis KB. Inhibition of cellular immune responses by Tylophora indica in experimental models. Phytomedicine 2001;895:348-55. | | 8. | Gangluli T, Khar A. Induction of apoptosis in a human erythroleukemic cell lines K- 562 by Tylophora alkaloid involves release of cytochrome C and activation of Caspase 3. Phytomedicine 2002;9:288-95. | | 9. | Gopalkrishnan C, Shakaranarayana D, Nazimudin SK and Kameswaran L. Effect of Tylophorine, a major alkaloid of Tylophora indica on immunopathological and inflammatory reactions. Ind J Med Res 1980;71:940-8. | | 10. | Bhutani KK, Sharma GL, Ali M. Plant based antiamoebic drugs part I: Anti amoebic activity of Phenanthroindolizidine alkaloids; common structural determinants of activity with emetine. Planta med 1987;53(6):532-6. | | 11. | Rietman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Am J Clin. Pathol 1957;28:56-63. | | 12. | Malloy HT, Evelyn KA.The determination of bilirubin with the photometric colorimeter. J Biol Chem 1937;119:481-90. | | 13. | Luna LG. Manual of histology. Staining methods of Armed Force Institute of Pathology. 3rd ed. Mc Graw Hill Book Co. Ltd: 2002. | | 14. | Woolson RF. Statistical methods for the analysis of biomedical data. New York: John Wiley and Sons Inc; 1987. | | 15. | Johnson DE, Kroening C. Mechanism of early Carbon tetrachloride toxicity in cultured rat hepatocytes. Pharmacol Toxicol 1988;83: 231-9. | | 16. | Srivastava SP, Chen NO, Hotlzman JL. The in vitro NADPH dependant inhibition by CCl 4 of the ATP dependant Calcium uptake of hepatic microsomes from male rats. Studies on the mechanism o inactivation of the hepatic microsomal calcium pump by CCl 3 . radical. J Biol Chem 1990;265:8392-9. | | 17. | Saraswat B, Visen PK, Patnaik GK, Anticholestatic effect of picroliv, active hepatoprotective principle of Picrorhiza kurrooa, against carbon tetrachloride induced cholestasis. India J Exp Biol 1993;31:316-8. |
Correspondence Address:
M Mujeeb
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi -110 062
India
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DOI: 10.4103/0973-8258.54901
[Table 1], [Table 2] | |
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