Home | About PM | Editorial board | Search | Ahead of print | Current Issue | Archives | Instructions | Subscribe | Advertise | Contact us |  Login 
Pharmacognosy Magazine
Search Article 
  
Advanced search 
 


 
RESEARCH ARTICLE
Year : 2009  |  Volume : 5  |  Issue : 19  |  Page : 232-237 Table of Contents     

Drug Induced Hepatotoxicity: Effect of Polyherbal Formulation


1 Smt. R. B. P. M. P. C, Kailash Nagar, Bhavanagar Road, Atkot-360040, India
2 M.S. University of Baroda, Vadodara, India

Date of Submission25-Nov-2008
Date of Decision05-Feb-2009
Date of Acceptance10-Apr-2009
Date of Web Publication29-Dec-2009

Correspondence Address:
Jitendra Vaghasiya
Smt. R. B. P. M. P. C, Kailash Nagar, Bhavanagar Road, Atkot-360040
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


Rights and PermissionsRights and Permissions
   Abstract 

To evaluate effect of Polyherbal Formulation (PHF) on drug induced hepatotoxicity in rats and assessment of any possibility of co administration of PHF along with such hepatotoxic drug. Hepatotoxicity in rat was induced by Methotrexate (1mg/kg p.o./week for 3 weeks) and protective effect of PHF (0.25ml/kg/p.o. and 0.5ml/kg/p.o. either along with drug or followed by inducing hepatotoxicity) was measured by estimating marker enzymes for liver function like Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase and . glutamic transpeptidase. Oxidative stress markers like lipid peroxidation, reduced glutathione, super oxide dismutase and catalase. Protein profile likes Total bilirubin, direct bilirubin, Total albumin and Total protein. Histopathological study was carried out to confirm hepatotoxicity. Methotrexate induced hepatotoxicity characterized by significant (P<0.001) increase in marker enzymes for liver function and oxidative stress. Protein profile significantly depleted. Administration of PHF either along with Methotrexate or followed by inducing hepatotoxicity significantly improved (P<0.001) the level of marker enzymes for liver function, oxidative stress and depleted proteins profile. The study suggests protective role of PHF in drug induced hepatotoxicity and it can be utilized to treat the hepatotoxicity with long-term clinically useful drugs.

Keywords: Hepatotoxicity; Methotrexate; Oxidative stress; Polyherbal formulation; Protein profile


How to cite this article:
Vaghasiya J, Bhalodia Y, Rathod S. Drug Induced Hepatotoxicity: Effect of Polyherbal Formulation. Phcog Mag 2009;5:232-7

How to cite this URL:
Vaghasiya J, Bhalodia Y, Rathod S. Drug Induced Hepatotoxicity: Effect of Polyherbal Formulation. Phcog Mag [serial online] 2009 [cited 2018 Nov 17];5:232-7. Available from: http://www.phcog.com/text.asp?2009/5/19/232/58166


   Introduction Top


Drug-induced Hepatotoxicity (DIH) account for 9.5% of all suspected adverse drug reaction [1] . Injury may be a direct toxic effect or immunological reaction to either of the drug or an active metabolite formed by bioactivation [2] . It is reported that 62% of withdrawn drugs having toxic metabolite [3] . Although, with the exception of rare cases, DIH subsides after cessation of treatment with the drug, this represents an important diagnostic and therapeutic challenge for physicians.

Methotrexate major activity results from binding to the enzyme dihydrofolic reductase, thus preventing conversion of folic acid into its active form, folinic acid. This in turn blocks the synthesis of nucleic acids, certain amino acids and indirectly proteins. This might lead to damage of organelles and plasma membranes of hepatic parenchymal cells interfering with their function and allowing leakage of enzymes. The hepatic malfunction is probably due to a direct toxic action of the methotrexate, since most reaction is dose dependent [4] . Methotrexate causes repression of enzymes involved in lipid peroxidation (acyl-CoA dehydrogenase, medium chain enoyl CoA hydratase and very long chain acyl-CoA synthatase) were associated with microvesicular lipidosis which leads to oxidative stress mediated liver necrosis, fibrosis and bile duct hyperplasia [4] .

Polyherbal formulation contains plant extract of Himsra, Kasani, Kakamachi, Arjuna, Kashmarda, Birranjshipa, Jharuka and processsed in 50mg herbs each in powder form of Bhringraja, Punarnava, Guduch, Daruharidra, Mulaka, Amalaki, Chitraka, Vidanga, Haritaki, and Parpata. Each having reported hepatoprotective, liver tonic, antioxidant and anti-inflammatory activity.

The main objective of present study was to assess hepatoprotective effect of Polyherbal formulation in drug induced hepatotoxicity and assessment of any possibility of co administration along with such hepatotoxic drug.


   Materials and Methods Top


Polyherbal formulation

Polyherbal Formulation (PHF) was procured as a gift sample from M/S Apostle Remedies, Vadodara.

Animals

Healthy adult male Wistar rats weighing 150-200g were used. Rats were housed in polypropylene cages, maintained under standardized condition (12-h light/dark cycle, 24°C, 35 to 60% humidity) and provided free access to palleted CHAKKAN diet (Nav Maharashtra Oil Mills Pvt. Ltd., Pune) and purified drinking water ad libitium. All experiments and protocols described in present study were approved by the Institutional Animal Ethics Committee (IAEC) of M. S. University, Baroda and with permission from Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Social Justice and Empowerment, Government of India.

Drug Induced Hepatotoxicity

Methotrexate a clinically useful drug was selected for inducing hepatotoxicity in rats. Drug induced hepatotoxicity in rats was produced by orally administration of methotrexate (1mg/kg/week) for 3 weeks [4] . Silymarin was used as reference standard.

Experimental protocol

Animals were divided into seven groups, each having 6 rats and treated accordingly. Group: 1 - rats received normal standard diet for 3 weeks, Group: 2 - rats received PHF (0.25ml/kg/p.o.) alone for 3 weeks, Group: 3- rats received methotrexate, Group: 4 - rats received methotrexate along with PHF (0.25ml/kg/p.o.) for 3 weeks, Group: 5 - rats received methotrexate along with PHF (0.5ml/kg/p.o.) for 3 weeks, Group: 6 - rats received methotrexate for 3 weeks followed by PHF (0.25ml/kg/p.o.) for 1 week. Group: 7 - rats received methotrexate along with Silymarin (SLM) (20 mg/kg/p.o.) for 3 weeks.

Collection of serum

Blood samples were withdrawn from retro-orbital plexus under light ether anesthesia without any anticoagulant and allowed for 10 minutes to clot at room temperature. It was centrifuged at 2500 rpm for 20 minutes. The serum obtained was kept at 4°C until estimation of liver function. All the animals were euthanasiously sacrificed after blood collection with spinal dislocation method and liver removed for study of oxidative stress markers and histopathology.

Estimation of liver function

Estimation of marker enzymes for liver function like Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) and Alkaline phosphatase (ALP) were done by using kit, Span Diagnostic Ltd, India and λ glutamic transpeptidase (λGTP) was done by using kit, Dade Behring Ltd., UK. Estimation of Lactate dehydrogenase (LDH) was done by using kit, Enzopak-Reckon diagnostics. Protein profile likes Total bilirubin, direct bilirubin; Total albumin and Total protein were done by using kit, Span Diagnostic Ltd, India.

Estimation of oxidative stress markers

Liver kept in cold conditions (precooled in inverted Petridish on ice). It was cross chopped with surgical scalpel into fine slices in chilled 0.25 M sucrose, quickly blotted on a filter paper. The tissue was minced and homogenized in 10 mM Tris-HCl buffer, pH 7.4 (10%w/v) with 25 strokes of tight teflon pestle of glass homogenizer at a speed of 2500 rpm. The clear supernatant was used for oxidative stress markers assays like lipid peroxidation [5] , Reduced Glutathione [6] , Super oxide dismutase [7] and Catalase [8] .

Histopathological study

Liver was collected after blotting free of blood and tissue fluids, it was kept in 5% formalin. 5-15µm thick section was serially cut on a leitz microtome in horizontal plane and mounted on glass slide with the help of egg albumin in glycerine solution (50% v/v). They were then stained with 10% hematoxylin for 3-5 minutes and placing in running water intensified the staining. The hematoxylin stained sections were stained with 10% eosin for 2 minutes. The sections were observed and desired areas were photographed in an Olympus photomicroscope. The sections were viewed under 40X magnifications.

Statistical analysis

All the values are expressed as mean ± S.E.M. Statistical significance between more than two groups was tested using one-way ANOVA followed by the Bonferroni multiple comparisons test using computer based fitting program (Prism, Graphpad 3.). Differences were considered to be statistically significant when p < 0.05.


   Results Top


Effect Polyherbal formulation on Marker Enzymes of Liver Function

Serum levels of ALT, AST, λGTP, ALP and LDH were significantly (P<0.001) increased after treatment with methotrexate compared to control. PHF (0.25ml/kg) perse has no effect on ALT, AST, λGTP, ALP and LDH but PHF (0.25ml/kg and 0.5 ml/kg) when administrated along with or after treatment with methotrexate produced significant (P<0.001) decreased in the levels of ALT, AST, λGTP, ALP and LDH. Silymarin treatment also produced significant (P<0.001) decreased in the levels these enzymes but was less than that of PHF. Correspondingly, there was significantly (P<0.001) increased in the ratio of ALT/AST after methotrexate, which was significantly (P<0.001) decreased after treatment with PHF and Silymarin [Table 1].

Effect Polyherbal formulation on Protein Profile

Methotrexate administration caused significant (P<0.001) increase in serum Total Bilirubin and significant (P<0.001) decrease in Direct Bilirubin, Total Protein and Albumin compared to control. There was no effect of PHF (0.25ml/kg) alone on serum Total Bilirubin, Direct Bilirubin, Total Protein and Albumin. PHF (0.25ml/kg and 0.5ml/kg) along with methotrexate, PHF (0.25ml/kg) followed by methotrexate and Silymarin showed significant (P<0.001) decreased in Total Bilirubin and increased in Direct Bilirubin. PHF (0.25ml/kg and 0.5ml/kg) along with methotrexate, Silymarin and PHF (0.25ml/kg) followed by methotrexate treatment were found to caused significant (P<0.001, P<0.05 respectively) increase in Total Protein and Albumin compared to methotrexate control. Correspondingly the changes were observed in the ratio of Albumin/Globulin [Table 2].

Effect Polyherbal formulation on Oxidative stress markers

Methotrexate significantly (P<0.001) increased lipid peroxidation and caused significant (P<0.001) decrease in reduced glutathione (GSH), Super oxide dismutase (SOD), Catalase (CAT) compared to control. There was no significant effect of PHF (0.25ml/kg) alone on lipid peroxidation, GSH, SOD and CAT. PHF (0.25ml/kg and 0.5ml/kg) along with methotrexate and PHF (0.25ml/kg) followed by methotrexate was found to caused significant (P<0.001) decrease in lipid peroxidation and significant (P<0.001) increase in GSH, SOD and CAT as compared to methotrexate control. Silymarin along with Methotrexate produced effect similar to PHF [Table 3].

Effect of Polyherbal formulation on Histopathological changes

Liver section of control rats revealed the normal hepatic hexagonal lobules and normal morphology. Liver tissue of methotrexate treated rats showed vacuolation, degeneration of hepatocyte, mild inflammation and piecemeal necrosis. PHF and Silymarin treatment improved structural integrity of liver cells [Figure 1].


   Discussion Top


ALT and AST are enzymes produced within the cells of the liver, as the cells are damaged, leaks into the bloodstream leading to a rise in the serum levels. ALP is an enzyme, which is associated with the biliary tract, and is elevated; biliary tract damage and inflammation should be considered. It is used often times to confirm that the alkaline phosphatase is of the hepatic etiology by λGTP. Mild to moderate elevation of ALT, AST, ALP (1-3 times) are usually seen in drug toxicity [9],[10],[11] .

Elevated serum ALT, AST, ALT/AST, .GTP, ALP, LDH and CPK levels in methotrexate treated animals compared to control animals is attributed as damage to the structural integrity of liver [12] , and presumptive markers of drug induced necrotic lesions in the hepatocyte. This decrease in elevated serum ALT, AST, ALT/AST, λGTP, ALP and LDH levels in PHF along with or followed by methotrexate treated animals in part may be due to the protective effect of PHF on liver cells following restoration of liver cell membrane permeability [13] .

This protective effect indicates reduction in enzymes present in the extra cellular milieu as liver cell. Protective effect of component of PHF has also been observed in several experimental studies [14],[15],[16] . It can be stated that PHF contains Tamarix gallica, and crude herbal extracts of Capparis spinosa, Cichorium intybus, Solanum nigrum, T. arjuna, and A. millefolium, these medicinal herbs alone or in combination can influence in restoration of the cellular functions and structural integrity of liver.

Methotrexate treated animals significantly increased Total Bilirubin and decreased in Direct Bilirubin, Total Protein and Albumin reflects liver cell damage or bile duct damage and synthetic function of the liver has been markedly diminished, indicated drug induced hepatotoxicity [17] .

PHF along with and followed by methotrexate significantly normalized Total Bilirubin, Total Protein, Direct Bilirubin and Albumin indicating liver curative effect. The curative and hepatoprotective effect Cassia occidentalis, Cichorium intybus and Solanum nigrum of PHF were observed against chemically induced liver damage in experimental animals [18] . The diuretic effect of T. arjuna and anti-inflammatory and anti-immunotoxicity effect of Cichorium intybus have been shown in clinical and experimental studies [19],[20] .

In present study increased in lipid peroxidation and depletion of antioxidant enzymes such as GSH, SOD and CAT in methotrexate treated animals compared to control animals indicated generation of oxidative stress. Methotrexate causes repression of enzymes involved in lipid peroxidation (acyl-CoA dehydrogenase, medium chain enoyl CoA hydratase and very long chain acyl-CoA synthatase) were associated with microvesicular lipidosis which leads to oxidative stress mediated liver necrosis, fibrosis and bile duct hyperplasia [4] .

PHF along with and followed by methotrexate significantly reduced lipid peroxidation and increased antioxidant enzymes such as GSH, SOD and CAT as compared to methotrexate treated animals indicated modification of oxidative stress by PHF. The protection of liver cells against toxic materials including drugs, lipid peroxidation and free radical injury may decrease inflammation [21] . Immune dysfunction is component of liver disease and thus immunomodulation by herbal therapy prevent oxidative stress, inflammation and strengthens the detoxifying power of liver cell [22] . The anti-oxidative property of esculetin and p-methoxybenzoic acid the main constituent of Cichorium intybus and Capparis spinosa, respectively, have been reported in chemically induced hepatotoxicity in experimental animals [23],[24] . Achillea millefolium, another component of PHF contains flavonoids and terpenoids with anti-oxidative and anti-inflammatory properties [25],[26] . Furthermore anti-oxidative property of flavonoid content of Tamarix gallica and inhibitory effect Solanum nigrum crude extracts on free radical-mediated DNA damage increase the hepatoprotective effect of PHF [27] . In addition, the antioxidative, anti-lipoproxidative and increase in glutathione content of the liver cells was observed with arjunolic acid and flavonoids present in T. arjuna [28]

Although there is insufficient information to establish the mechanism of action of PHF protection, this could be due to its anti-inflammatory, anti-oxidative, immunomodulating as well as restorative effects.


   Conclusion Top


The study suggest protective role of Polyherbal Formulation in drug induced hepatotoxicity and this effect may be due to its anti-inflammatory, anti-oxidative, immunomodulating as well as restorative effects. Polyherbal Formulation can be utilized to treat the hepatotoxicity with long term clinically useful drugs, which are at the risk of developing hepatotoxicity.


   Acknowledgement Top


We are deeply indebted to M/S Apostle Remedies, Vadodara. for providing gift samples of Polyherbal Formulation (Livpep).

 
   References Top

1.Zimmerman H. Hepatotoxicity: the adverse effects of drugs and other chemicals on the liver. 2nd ed. Philadelphia: Lippincott, Williams & Wilkins (1999).  Back to cited text no. 1      
2.Kaplowitz N. Biochemical and cellular mechanisms of toxic liver injury. Semin Liver Dis. 22 : 137-44 (2002).  Back to cited text no. 2      
3.Temple R.J. and Himmel M.H. Safety of newly approved drugs: implica­tions for prescribing. JAMA. 287 : 2273 - 2275 (2002).  Back to cited text no. 3      
4.Hung Q., Jin X. and Elias T. Gene expression profiling reveals multiple toxicity endpoint induced by hepatotoxicant. Muta Res. 147-167 (2004).  Back to cited text no. 4      
5.Slater T.F., Sawyer B.C. The stimulatory effects of carbon tetrachloride and other halogenoalkanes or peroxidative reactions in liver fractions in vitro. Biochem J. 123 : 805-814 (1971).  Back to cited text no. 5      
6.Moran M.S., Depierre J.W. and Mannervik B. Levels of glutathione, glu­tathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et Biophysica ACTA. 582 : 67 (1979).  Back to cited text no. 6      
7.Misra H.P. and Fridovich I. The role of superoxide anion in the autooxi­dation of epinephrine and a simple assay of SOD. J Biol.Chem. 247 : 3170 (1972).  Back to cited text no. 7      
8.Colowick S.P., Kaplan N.O. and Packer L. Methods in Enzymology. Aca­demic Press, London. 105 : 121(1984).  Back to cited text no. 8      
9.Rosen H.R. and Keefe E.B. Evaluation of abnormal liver enzymes, use of liver tests and the serology of viral hepatitis: Liver disease, diagnosis and management. 1st ed. New York; Churchill livingstone publishers 24-35 (2000).  Back to cited text no. 9      
10.Friedman SF, Martin P, Munoz JS. Laboratory evaluation of the patient with liver disease. Hepatology, a textbook of liver disease. Philedelphia; Saunders publication. 1 : 661-709 (2003).  Back to cited text no. 10      
11.SimkoV. Alkaline phosphatases in biology and medicine. Dig Dis. 9 : 189-193 (1991).  Back to cited text no. 11      
12.Chenoweth M.B. and Hake C.L. The smaller halogenated aliphatic hydro­carbons. Ann. Rev. Pharmacol. 2 : 363-398 (1962).  Back to cited text no. 12      
13.Kalab M. and Krechler T. The effect of the heptoprotective agent Liv-52 on liver damage. Cas. Lek. Cesk. 136 : 758-760 (1997).  Back to cited text no. 13      
14.Kataria M. and Singh L.N. Hepatoprotective effect of Liv-52 and kumary­asava on carbon tetrachloride induced hepatic damage in rats. Indian J. Exp. Biol. 35 : 655-657 (1997).  Back to cited text no. 14      
15.Sandhir R. and Gill K.D. Hepatoprotective effects of Liv-52 on ethanol induced liver damage in rats. Indian J. Exp. Biol. 37 : 762-766 (1999).  Back to cited text no. 15      
16.Mathur S. Role of Liv-52 in protection against beryllium intoxication. Biol. Trace Elem. Res. 41 : 201-215 (1994).  Back to cited text no. 16      
17.Green R.M. and Flamm S. AGA techinal review of evaluation of liver chemistry tests. Gastroenterology. 123 : 1367-1384 (2002).  Back to cited text no. 17      
18.Kanase A., Patil S. and Thorat B. Curative effects of mandur bhasma on liver and kidney of albino rats after induction of acute hepatitis by CCl4. Indian J. Exp. Biol 35 : 754-764 (1997).  Back to cited text no. 18      
19.Bharani A., Ganguly A. and Bhargava K.D. Salutary effect of Terminalia arjuna in patients with severe refractory heart failure. Int. J. Cardiol. 49 : 91-99 (1995).  Back to cited text no. 19      
20.Kim J.H., Mun Y.J., Woo W.H., Jeon K.S. and Park J.S. Effects of the etha­nol extract of Cichorium intybus on the immunotoxicity by ethanol in mice. Int. Immunopharmacol. 2 (6): 733-744 (2002).  Back to cited text no. 20      
21.Yang H., Chen Y., Xu R., Shen W. and Chen G. Clinical observation on the long-term therapeutic effects of traditional Chinese medicine for treat­ment of liver fibrosis. J. Tradit. China Med. 20 : 247-250 (2000).  Back to cited text no. 21      
22.Jiang W., Li S., Wang C. and Wang Y. Comparative study of effects of three kinds of herbal mixture decoctions on improving immune senescence and free radical metabolism. Chin. Med. J. 110 : 750-754 (1997).  Back to cited text no. 22      
23.Gilani A.H., Janbaz K.H., Shah B.H., Martin-Aragon S., Benedi J.M. and Villar A.M. Effects of the antioxidant (6,7-dihydroxycoumarin) esculetin on the glutathione system and lipid peroxidation in mice. Gerontology. 44 : 21-25 (1998).  Back to cited text no. 23      
24.Germano M.P., De Pasquale R., D'Angelo V., Catania S., Silvari V. and Cos­ta C. Evaluation of extracts and isolated fraction from Capparis spinosa L. buds as an antioxidant source. J. Agric. Food Chem. 27 : 1168-1171 (2002).  Back to cited text no. 24      
25.Glasl S., Mucaji P., Werner I., Presser A. and Jurenitsch J. Sesquiterpenes and flavonoid aglycones from a Hungarian taxon of the Achillea millefo­lium group. Z. Naturforsch. 57 (11-12): 976-982 (2002).  Back to cited text no. 25      
26.Goldberg A.S., Mueller E.C., Eigen E. and Desalva S.J. Isolation of the anti-inflammatory principles from Achillea millefolium (Compositae). J. Pharm. Sci. 58 (8): 938-941 (1969).  Back to cited text no. 26      
27.McPhail D.B., Hartley R.C., Gardner P.T. and Duthie G.G. Kinetic and stoichiometric assessment of the anti-oxidant activity of flavonoids by electron spin resonance spectroscopy. J. Agric. Food Chem. 12 : 1684-1690 (2003).  Back to cited text no. 27      
28.Sumitra M., Manikandan P., Kumar D.A., Arutselvan N., Balakrishna K., Manohar B.M. and Puvanakrishnan R. Experimental myocardial necrosis in rats: role of arjunolic acid on platelet aggregation, coagulation and anti­oxidant status. Mol. Cell. Biochem. 224 (1-2): 135-142 (2001).  Back to cited text no. 28      


    Figures

  [Figure 1]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
    Abstract
    Introduction
    Materials and Me...
    Results
    Discussion
    Conclusion
    Acknowledgement
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed2592    
    Printed187    
    Emailed2    
    PDF Downloaded61    
    Comments [Add]    

Recommend this journal