Biomedical Research

Abstract

Hepatitis-C is a global public health problem in India, and responsible for major chunk of morbidity and mortality. The present study focuses on effect of phyllanthus amarus therapy for protection of liver in hepatitis-C through investigating liver profile enzymes, antioxidant enzymes, antioxidant vitamins and lipid peroxidation. The study consists of 50 clinical diag-nosed hepatitis-C patients ranging in between age group 25-60 years. The control group in-cludes 50 ages and sex matched normal healthy persons. Oxidative stress was assessed by es-timating lipid peroxidation [LPO]. The parameters like serum bilirubin, total proteins and activity of liver profile enzymes were done. Activity of enzymatic antioxidants, superoxide dismutase [SOD], glutathione peroxides [GPx], catalase and levels of non-enzymatic anti-oxidant vitamin E and vitamin C was measured in plasma or erythrocytes. Methods used in the study are mainly enzyme kinetics by autoanalyzer and by turbidimetry. Plasma LPO levels were significantly high but activity of SOD, GPx, catalase and levels of vitamin E and vitamin C were significantly lowered in hepatitis- C on comparison with controls. After phyllanthus amarus therapy for 5 weeks and 10 weeks plasma LPO levels were significantly decreased and activity of SOD, GPx, catalase and vitamin E and vitamin C were signifi-cantly increased in hepatitis-C. The present study concludes that, hepatitis-C increases oxi-dative stress and may be playing an important role in hepatic cell damage and pathogenesis of hepatitis-C. This study strongly suggests that the therapy with phyllanthus amarus in-creases antioxidants and reduces lipid peroxidation of hepatic cellular and intracellular membranes and protects liver damage due to free radicals in hepatitis-C.

Keywords

Hepatitis–C, Phyllanthus amarus, Oxidative stress, Antioxidants.

Introduction

Hepatitis–C virus is an important pathogen, not only be-cause of its high prevalence and worldwide burden but also because of potentially serious complications of per-sistent HCV (hepatitis-C virus). These complications in-cludes cirrhosis, hepatocellular carcinoma and end stage liver disease necessitating liver transplantations [1,2,3].

Worldwide three different epidemiological patterns of HCV infection have emerged [3]. The seroprevalence of HCV infection based on detection of antibody to HCV is estimated to be 3% with more than 170 million people infected chronically. The rate is higher in person ages 30 to 49 years than in older or younger’s persons and higher in males than females [2].

The use of herbal drugs in the treatment of liver diseases has been a long tradition especially in esteem medicine [4,5]. The plant Phyllanthus amarus is used as ayurvedic medicine for over 2000 years and has a wide number of traditional uses. It is shown to be effective with other drugs in the treatment of jaundice due to infective hepati-tis [6].

The present study was planned to study the effect of phyl-lanthus amarus therapy in protection of liver in hepatitis-C with the help lipid peroxidation and antioxidant defense system, liver profile enzymes and vitamins.

Materials and Methods

The present study was carried out with 50 clinically diag-nosed hepatitis–C patients ranging in between age group 25 – 55 years from Government Medical College Hospital Miraj, Civil Hospital Sangli and Corporation Ayurvedic Hospital Sangli. Patients having obvious malignancy, he-patic diseases, associated renal disease, lung diseases, thyroid diseases, gastrointestinal diseases, alcoholic dis-eases, tobacco chewers, cardiac disease and addiction history of alcohol or smoking were excluded. The patients with co-existing other liver disorders were also excluded. Written consent was obtained from all enrolled patients as well as controls. For this study the institutions ethical committee approval was taken. All the patients were fol-lowed up every week for a period of ten weeks. At each follow up visit, any information about adverse events and a symptomatic evaluation was conducted, which was fol-lowed by through the clinical examination. The efficiency of the treatment was judged by the amelioration in subjec-tive symptoms physical and full medical examination and objective signs as well as the degree of improvement in various function tests.

Diagnosis of hepatitis – C was done by physicians and conformed with the help of medical history, physical, clinical, hematological and biochemical examinations included test for viral marker. Age and sex matched 50 normal healthy persons as per International Federation of Clinical Chemistry (IFCC) guidelines were included by excluding history of alcohol or smoking as control sub-jects.

Sample Collection

About 10 ml of fasting venous blood samples were col-lected under sterile condition from hepatitis- C patients before starting any therapy and from normal healthy sub-jects. After five weeks and ten weeks therapy interval 10 ml of fasting venous blood samples were collected from hepatitis- C patients. 5 ml blood was taken in sterile dry and acid washed ethylenediamine tetra acetic acid (EDTA) bulbs and 5 ml blood in plain bulbs. Plasma was separated by centrifuging the blood at 3000 rpm for 20 minutes at 40c. This plasma was used for estimation of malondialdehyde (MDA) and vitamin E. The packed cells were used for the analysis of vitamin C, SOD, catalase and GPx. The sera separated were used for the investiga-tion of parameters like total bilirubin, total proteins and activity of enzymes SGPT [7], SGOT [8], ALP [9], GGT [10], 5' NTP [11]. All these parameters were estimated in healthy subjects and all cases before and after the therapy with phyllanthus amarus. At the end of five weeks and ten weeks interval changes in these parameters from base line values were taken. MDA was determined as the measure of thiobarbituric acid reactive substances (TBARS) [12].

Erythrocytes ascorbic acid levels were estimated by the method of Tietz [13] Plasma separated was used for esti-mation of vitamin E by the method of Baker H et al [14]. SOD was determined in the hemolysate by the method of Mishra and Fridovich [15]. Catalase activity was meas-ured by the method of Beer & Seazer [16] and GPx activ-ity by Paglia and Valentine in erythrocytes [17].

Statistical Analysis

All values are presented as mean ± SD. Statistical signifi-cance was analyzed by Students‘t’ test.

Results

The levels of lipid peroxidation, vitamin E, vitamin C and activity of SOD, catalase and GPx are presented in the table-1. The levels of lipid peroxidation were significantly higher in hepatitis- C when compared with controls. The levels of vitamin E, vitamin C and activity of antioxidant enzymes SOD, GPx and catalase were significantly lower in hepatitis- C patients.

Table 1: Lipid peroxidation and antioxidants in hepatitis- C before and after therapy of Phyllanthus amarus.

The levels of total bilirubin and the activity of liver pro-file enzymes SGPT, SGOT, ALP, GGT and 5' NTP were significantly increased in hepatitis- C in comparison with control subject (Table-2).

Table 2: Changes in activity of liver profile enzymes before and after therapy in hepatitis- C patients.

Discussion

Thiobarbituric acid reactive substances, malondialdehyde (MDA) the indicator of lipid peroxidation were signifi-cantly elevated in hepatitis-C. There is association be-tween increased levels of MDA and hepatitis-C. Free radicals might be attack on unsaturated fatty acids in membrane and organelles to produce lipid peroxides. This free radical may cause loss of membrane and which may decrease in membrane permeability. Thus loss of mem-brane causes cellular damage and necrosis of liver in hepatitis-C.

The activities of liver enzymes were significantly in-creased in hepatitis-C. Elevated serum enzymes like SGPT, SGOT, ALP, 5' NTP and GGT are indicative to cellular damage and loss of functional integrity of cell membrane in liver. These enzymes are localized in the cell cytoplasm and cell mitochondria as well as found in bile. Damage of liver cells by hepatitis-B causes leakage of cellular enzymes into serum [18]. After five weeks and ten weeks therapy interval of Phyllanthus amarus there was significantly decreased activity of liver enzymes. This indicates that there is reduction in liver damage.

The increased concentration of bilirubin and significant raised activity of liver enzymes could be taken as an index of liver damage. After five weeks therapy interval there was significant decrease in LPO and it resumes to normal after ten weeks interval. Effect of hepatitis-C showed in-creased oxidative stress by decreasing antioxidant vitamin E and vitamin C and increasing LPO. This increased oxi-dative stress may increase consumption of vitamin E and C. Vitamin E traps free radicals and interrupts the chain reactions that damage the cell [19]. Thus in hepatitis-C, there is increased utilization of vitamin E and vitamin C due to oxidative stress.

SOD is an important antioxidant enzyme having scaveng-ing effect against superoxide anion and catalase is respon-sible for detoxification of H₂O₂ produced by action of superoxide dismutase and inhibits formation of superox-ide radicals [20]. Due to increased oxidative stress there may be increased utilization of enzymes in hepatitis-C to balance the decreased activity of antioxidant enzymes by oxidation through reactive free radicals. Thus the activity of SOD, GPx, and catalase may decrease in hepatitis-C. Decreased antioxidant enzymes and vitamin C and vita-min E might be causing oxiradical mediated injury and thus may contribute to liver damage. After ten weeks therapy with phyllanthus amarus in hepatitis-C decreased levels of vitamin E and vitamin C has been comes to near normal. This indicates that the utilization of vitamin E and vitamin C is decreased and this may be responsible for raised levels of Vitamin E and vitamin C. Increased activity of antioxidant enzymes and vitamins in hepatitis-C indicate that there might be regeneration of liver cells after therapy, which helps in curing hepatitis-C.

Phyllanthus amarus can detoxify the free radicals and has an antioxidant activity [21]. It has been shown to increase protein biosynthesis [22] and increase the rate of regen-eration of necrosed cells [23].

This study concludes that hepatitis-C increase oxidative stress and may be playing an important role in hepatic cell damage and play a role in pathogenesis of hepatitis. The phyllanthus amarus therapy is found to be equally effec-tive in patients of hepatitis-C. The study strongly suggests that the therapy with phyllanthus amarus increases vari- ous antioxidants and reduces lipid peroxidation of hepatic cellular and intracellular membranes. Thus phyllanthus amarus have role to protect liver damage due to free radi-cals in hepatitis- C.

Acknowledgement

The authors thank to Medical Social Worker (MSW) of Corporation hospital Sangli, Civil hospital Sangli and Government Medical College hospital Miraj for constant counseling of hepatitis-C patients to make this study pos-sible.

References

1. Fredman M, Fredman L, Brandt L. Gastrointestinal and Liver diseases.Alocholic Liver Diseases. 8th edition published by Saunders Elsevier 2006; 2:1543-1790.
2. Wastey A, Alter MJ. Epidemiology of hepatitis C; Geographic difference and temporal trends. Sem. Liver Dis.2000; 20: 1- 4.
3. Armstrong GL, Alter MJ, McQuillan GM, Margolis HS. The post incidence of Hepatitis-C virus infection; implication for the feature Burdon of chronic liver dis- ease in the United States. Haematology: 2000; 31(2): 777-781.
4. Gupta S, Khatri RL, Shrinivas G. Theory of infectious hepatitis and other liver disorders. Probe1972; 2: 93- 110.
5. Flora KD, Rosen HR, Brenner KG. The use of naturo- pathic remedies for chronic liver disease. Am. J. Gas- troenterol.1996; 91: 2654-1665.
6. Tograjan SP, Jayram S, Villicammai T. Phyllanthus Amarus and Hepatitis-B. Lancet 1990; 336: 949-950.
7. Moss DW, Handerson AK. Clinical enzymology, In Tietz text book of clinical chemistry, 3rd edition. C.A. Burtis and E.R. Ashood, Eds. WB Saunders, Philadel- phia 1994; pp 617-721.
8. Henry JB. Clinical diagnosis and management by labo- ratory methods. WB Sounders Company. Philadelphia 1979; pp 365-370.
9. Tietz N.W. Fundamentals of Clinical Chemistry. 2nd edition WB Saunders Company. Philadelphia 1976;pp 622- 628.
10. Wenger WC, Lott JA. Enzymes in Clinical Chemistry, Theory, analysis and correlation, Lawren A. Kaplan and A. J. Pesce, Eds. CV Mosby. Toronto 1994; pp1079-1134.
11. Lawren Kaplan, Amadeo Pesce, Steven kazmierczak.Clinical Chemistry, Theory, Analysis, Correlation. 4th edition 2003; pp 503-509.
12. Buege JA, Aust AD. Microsomal lipid peroxidation. In methods, Enzymol Estbrook RW, Pullman ME. New York: Academic Press 1987; pp 302-10.
13. Tietz NW. In textbook of Clinical Chemistry. Edited by NW Tietz, WB Saunders Company, Philadelphia. London Tornto. 1986; pp 960-62.
14. Baker H, Frank D, Winley NC. In clinical vitaminology methods and Interpretation. Interscience, Publisher New York. 1968; pp 169-76.
15. Mishra HP, Fridovech I. The role of superoxide anion in the auto oxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972; 247: 3170-3175.
16. Beer RF and Sizer TW. A spectroscopic method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 1952; 195:133-40.
17. Paglia De, Valentine WW. Studies on the quantitative,qualitative characterization of erythrocyte glutathione peroxidase. Journal of Lab Clin Med 1967; 70: 158- 159.
18. Subirkumar Das, Hiran K R, Sukesh Mukherjee andD.M. Vasudevan. Oxidative stress is the primary event: effect of ethanol consumption in brain. Ind J of Clin Biochem. 2007; 22 (1):99-104.
19. Nikam SV, Nikam PS, Ahaley SK. Role of free radical and antioxidant imbalance in pathogenesis of Parkin- son’s disease. Biomed Res 2009; 20 (1) : 55-58.
20. Wendel A. Glutathione peroxidase, In enzymatic basis of detoxification. W. B. Jokby ed; New York, 1980; pp 333-348.
21. Tagrajan SP, Jayaram S, Villicammai T. Phyllanthus amarus and hepatitis-B. Lancet 1990; 336: 949-950.
22. Tagrajan SP, Subramanian S. Thirunalasunder. Effect of phyllanthus amarus on chronic carrier of hepatitis B-virus. Lancet 1998; 2 (8614) : 764-766.
23. Oudhia P, Tripthi RS. Prospects of cultivation of medical plants in Chhattisgarh India. Recent progress in medical plants vol. 5 crop improvement production technology, trade and commerce. Sci Thch Pub USA. 2002; pp 211-236.