International Journal of Drug Development and Research

Keywords

Hepatotoxicity, Galactosamine, Triphala, Antioxidant

INTRODUCTION

D-Galactosamine (D-GalN) is used as a model hepatotoxin to induce experimental liver injury. [1,2]It blocks transcription in hepatic cells and thus halts protein synthesis in them[2] .It has been known that D-GalN causes decrease in uracil nucleotides, which causes block in nucleic acid and protein synthesis and the loss of intracellular calcium homeostasis, leading to cell membrane damage [1, 3-5]. It results in spotty hepatocyte necrosis and parenchymal inflammation [1] and also causes imbalance in energy metabolism of hepatic cells [6].

Triphala is Indian ayurvedic herbal formulation consisting of the dried fruits of, Terminalia chebula, Phyllanthus emblica or Emblica officinalis and Terminalia bellerica. Triphala is used in formulation of many Ayurvedic medicines for the treatment of many diseases [7,8]. In ayurveda, it is known to be an important medicine and is believed to boost health, immunity and longevity [9-11] .It is rich in antioxidants and thus plays a crucial role in the treatment of a wide variety of conditions like infections, obesity, anemia, fatigue, constipation, tuberculosis, pneumonia and AIDS [12,13] .Triphala is known to contain Vitamin C, ellagic acid, gallic acid, chebulinic acid, bellericanin, ;-sitosterol and flavanoids. [14] Its components E.officinalis, T.belerica and T.chebula are known to possess anti-Inflammatory, antimutagenic, anti-oxidant, cytoprotective, gastroprotective, hepatoprotective, anti-bacterial and anti-cancer activity [12,15,16] .Triphala has been known to promote liver function. However, the hepatoprotective effect of Triphala against Dgalactosamine induced hepatotoxicity has not yet been reported to the best of our knowledge.

MATERIALS AND METHODS

Animals:- Male Swiss albino mice weighing about 25-30grams were purchased from the S.L.R &.T.C karigiri, Vellore, India. The animals were maintained under standard conditions of temperature (24±20C) with 12 hr. dark light cycle and were fed with commercial pelleted feed and water ad libitum. The animal studies were approved by the guidelines recommended by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of culture, Government of India, Chennai.

Drugs and Chemicals:-The commercially available Triphala capsule was obtained from the Himalaya Drug Company, Bangalore, India. Silymarin was obtained from the Microlabs, Goa, India and D-galactosamine was purchased from SISCO Research Laboratories, Bangalore. All other reagents and chemicals used were standard reagents of analytical grade from SRLs, SD fine and other chemical companies.

Experimental Protocol:- In this experiment, mice were randomly allocated into 5 groups, each group consisting of six animals. All animals were made to fast 24 hrs before the experiment. Group I, the control group, received saline (0.89% NaCl); Group II, galactosamine induced test group, hepatotoxicity was induced by a single dose of galactosamine (700mg/kg, i.p); Group III, drug treated group (Triphala + galactosamine), Triphala extract (1000mg/kg, i.p) was administrated after the single injection of galactosamine; Group IV, positive control group (Silymarin + galactosamine), Silymarin (25mg/kg, i.p) was administered after the single injection of galactosamine. Group V; Triphala extract group, received Triphala (1000mg/kg, i.p.) suspended in saline solution. The mice were decapitated after 18 hrs of galactosamine injection; blood was collected from the trunk and serum was separated and stored at -70º C. Tissue sample from the liver were processed for biochemical and histological analysis.

Biochemical Parameters:-The activities of serum aspartate transaminase (AST) or SGOT (Serum Glutamate Oxaloacetate Transferase) [17], alanine transaminase (ALT) or SGPT (Serum Glutamate Pyruvate Transferase) [17] ,alkaline phosphatase (ALP)[18], superoxide dismutase (SOD) [19], catalase (CAT) [20], glutathione reductase (GR) [21], glutathione peroxidase (GPx) [22], glutathione-s-transferase (GST) [23] , total reduced glutathione (GSH) [24], lipid peroxidation (LPO) [25] and TNF-H ( ELISA,cayman chemicals,USA) was estimated.

Histopathological Studies:- Immediately after the sacrifice, a portion of the liver was fixed in 10% formalin and then washed and dehydrated in descending grades of isopropanol and finally with xylene. The tissue was then embedded in molten paraffin wax. Sections were cut at 5 μm thicknesses, stained with haematoxylin and observed under microscope.

Statistical Analysis:- The data obtained was computed to calculate the mean, standard deviation (S.D) and ANOVA to find out the statistical significance between the control and experimental groups.

Units: LPO:nmol of MDAformed/mg protein;CAT:μmol of H2O2 consumed/min/mg protein; SOD: Units/mg protein(1 U=amount of enzyme that inhibits the autooxidation of pyrogallol by 50%);GPx: μg of GSH utilized/min/mg protein; GR: nmol of NADPH oxidized/min/mg protein; GST: nmol of 1-chloro-2,4-dinitrobenzene-GSH conjugate formed/min/mg protein; Total reduced glutathione glutathionenmol/ mg/protein. Values are expressed as mean ± S.D. of six animals. Comparisons made are as follows: a-control vs D-GalN; b-D-GalN vs Triphala; c- D-GalN vs Silymarin. The symbols represent statistical significance at: * p < 0.05.Statistical analysis was calculated by one way ANOVA followed by Student’s Newman-Keul’s test.

SGOT and SGPT levels in serum samples of the control, galactosamine, Triphala +Gal, Silymarin + Gal and Triphla extract groups. Each value represents the mean ± SD of six mice. The symbols represent statistical significance at: * p < 0.05.Statistical analysis was calculated by one way ANOVA followed by Student’s Newman- Keul’s test.

The ALP levels in serum samples of the control, galactosamine, Triphala +Gal, Silymarin + Gal and Triphala extract groups. Each value represents the mean ± SD of six mice. The symbols represent statistical significance at: * p < 0.05.Statistical analysis was calculated by one way ANOVA followed by Student’s Newman-Keul’s test.

The serum bilirubin levels in serum samples of the control, galactosamine, Triphala +Gal, Silymarin + Gal and Triphala groups. Each value represents the mean ± SD of six mice. The symbols represent statistical significance at: * p < 0.05.Statistical analysis was calculated by one way ANOVA followed by Student’s Newman-Keul’s test.

The TNF-H levels in serum samples of the control, galactosamine, Triphala +Gal, Silymarin + Gal and Triphala extract groups. Each value represents the mean ± SD of six mice. The symbols represent statistical significance at: * p < 0.05.Statistical analysis was calculated by one way ANOVA followed by Student’s Newman-Keul’s test.

RESULTS

The activity of alanine transaminase, aspartate transaminase and alkaline phosphatase in serum were significantly (p<0.05) increased in Dgalactosamine control group as compared to the normal group. The levels of the above enzymes were significantly reversed on treatment with Triphala extract (fig.1).

There was a significant (p<0.05) reduction in levels of SOD, Catalase, Glutathione peroxidase, GR, GST and Total Reduced Glutathione and increment in Lipid peroxidation level of D-GalN control group as compared to the normal control group. The results show that the activity of antioxidant enzymes (SOD, CAT, GPx, GR, GST, Total Reduced Glutathione) were increased (p<0.05) significantly at the dosage of 1000mg/kg of Triphala treated and also reduction in the lipid peroxidation levels in D-GalN induced intoxication was observed (fig.2).

Liver injury induced by D-GalN shows a significant increased level of bilirubin in the D-galactosamine treated group. Administration of Triphala (1000mg/kg) prevented the increase in bilirubin level. Also, Silymarin which is used as standard prevented the liver from elevating levels of serum bilirubin (fig.3).

The level of pro-inflammatory cytokine TNF-H in the serum of control and experimental animals was analyzed. The level of TNF- H in the D-galactosamine treated mice were systemically overproduced in the serum, while the elevated level of TNF-H was found to be decreased in Triphala extract administered mice treated with D-galactosamine (fig.4).

Photomicrographs of liver sections in fig.5 (A) Control(Group I) shows central vein surrounded by normal hepatocytes, (B) galactosamine(Group II) shows hepatocytes with enlarged nuclei with condensed and dispersed chromatin, (C)Acute galactosamine shows fatty change with enlarged cell, vacuolated cytoplasm and large nuclei with condensed chromatin, (D)silymarin + galactosamine (Group III)shows reactive hepatocytes(binucleate)and normal with degenerative hepatocytes, (E) Triphala+galactosamine(Group IV) shows ballooning degenerative cells with apoptotic hepatocytes, (F)Triphala (Group V)shows central vein with normal hepatocytes. (Haematoxylin & eosin stain, 400 X magnifications). (fig.5)

DISCUSSION

D-GalN- induced acute liver injury in mice has been used to mimic the sequences of events in viral hepatitis [26] .Other organs are not deteriorated by the effect of galactosamine. The metabolism of D-GalN depletes several uracil nucleotides including UDPglucose, UDP-galactose and UTP and forms uridinediphosphogalactosamine instead [27,28]. Accumulation of uridine-diphosphogalactosamine contributes to disturbance in protein metabolism [12,24,29-31] .

An evidence of hepatic injury is a leakage of cellular enzymes into the plasma [32] .When plasma membrane of liver cells is damaged, a variety of enzymes normally located in the cytosol are released into the blood stream which provides a quantitative index of the extent and type of hepatocellular damage [33,34].

Liver damage induced by D-GalN generally reflects disturbances of cellular metabolism which leads to characteristic changes in the serum enzyme activities. When rise in levels of ALT, AST and ALP occurs; it can be interpreted as a result of the liver cell destruction or change[36] in the membrane permeability indicating the severity of hepatocellular damage induced by D-GalN which is in accordance with previous reports [32,33,35-37] .These enzymes are characteristic of liver damage and therefore their release into the serum confirmed the GalN induced liver damage.

AST levels usually increase alongwith an increase in the levels of ALT. Significant (p<0.05) elevation in the transaminases level could be taken as an indicative of liver damage. In our study the rise in ALT, AST and ALP levels induced by Dgalactosamine administration was significantly(p<0.05) reduced by Triphala pretreatment suggesting that hepatoprotective activity might be due its protective effect against loss of functional integrity of the cell membrane in hepatocytes.

Glutathione is an important endogenous antioxidant system that is found mainly in high concentration in liver and it is known to have important role in protective processes. [38] The activities and levels of anti-oxidative enzymes (SOD, CAT, GPx, GST, glutathione reductase and total reduced glutathione) show significant decrease in the D-galactosamine induced mice. Recent study also suggested that a reduction in the activity of SOD is associated with the accumulation of highly reactive free radicals which are responsible for other deleterious effects [39,40] .Thus the modifying role of Triphala extract observed in our study may be due to the antiperoxidative action of its components that was reported earlier [41,12].

Determination of serum bilirubin is important for the assessment of liver function and unusual increase in the levels of bilirubin in the serum indicate severe abnormalities in normal hepatocyte activities [42,43] .An increased level of bilirubin in this study is in agreement with previous reports showing that DGalN induced hepatitis is characterized by increased levels of bilirubin in serum [44,45] .The Triphala mediated suppression of the increased bilirubin level suggests the possibility of the Triphala being able to stabilize biliary dysfunction.

Excessive production of free radicals results in the oxidative stress, which leads to damage of macromolecules and can induce lipid peroxidation in vivo [37,46,47] .In D- GalN treated mice, increase in MDA levels were found which is in consistent with the previous findings. Pretreatment of Triphala inhibited lipid peroxidation, suggesting that Triphala may exert a stabilizing action on liver cell membranes. Above results suggest that Triphala and silymarin possess significant protection against Dgalactosamine induced hepatotoxicity in mice.

TNF-H is the major mediator and leads to apoptotic liver injury [48,49] .A recent study has shown that ROS are involved in D-GalN induced liver damage in TNF- H mediated apoptosis of hepatocytes[50,51].Concomitant TNF-H exposure and ROS, either extrinsically generated by nonparenchymal cells or intrinsically generated in hepatocytes, may act in concern to promote apoptosis and liver injury[46] .A significant decrease in TNF-H level was observed in Triphala and Silymarin treated mice.

Histopathological observations confirmed the membrane stabilizing effect of Triphala in Dgalactosamine challenged mice. Hepatocyte necrosis induced by D-GalN was prevented by treatment of Triphala.

CONCLUSIONS

Triphala possess hepatoprotective property and this may in part be explained by the presence of important antioxidative factors. There is an alarming increase in the incidence liver injury caused by Hepatitis. The present study has thus demonstrated the protective properties of Indian ayurvedic herbal formulation Triphala against D-galactosamine induced hepatotoxicity in mice. Therefore, further studies along these lines would be worthwhile.

Declarations

Conflict of interest

The Author(s) declare(s) that they have no conflicts of interest to disclose.

Funding

This research received no specific grant from any funding agency in the public, commercial or not-forprofit sectors.

Tables at a glance

Table 1

Figures at a glance

Figure 1 Figure 2 Figure 3 Figure 4 Figure 5

References

1. Heekyoung Chung ,Hyun-Jun Kim,Ki-SeokJang,MingooKim,JungeunYang,Kyung-Sun Kang,Hyung-LaeKim,Byung-Il Yoon,Mi-OckLee,Byung-HoonLee,Ju Han Kim,Yong-Sung Lee, Gu Kong. Analysis of differential gene expression profiles on D-galactosamine-induced acute mouse liver injury and regeneration. Toxicology 2006; 227(1-2) :136–144.
2. Keppler D, Lesch R, Reutter W, Decker K. Experimental hepatitis induced by Dgalactosamine. ExpMolPathol1968 ;9(2):279–290.
3. Decker K, Keppler D. Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death. Rev PhysiolBiochemPharmacol 1974; 71: 77-105.
4. Decker K, Keppler D, Pausch J. The regulation of pyrimidine nucleotide level and its role in experimental hepatitis.Adv Enzyme Regul1973 ;7: 205-230.
5. S K El-Mofty, M C Scrutton, ASerroni, C Nicolini, J L Farber. Early reversible plasma membrane injury in galactosamine-induced liver cell death.American journal of Pathology 1975; 79:579-596.
6. Keppler D, Decker K. Studies on the mechanism of galactosamine hepatitis: accumulation of galactosamine-1-phosphate and its inhibition of UDP-glucose pyrophosphorylase. Eur J Biochem 1969; 10:219-25.
7. Awasthi L, Nath B. Chemical examination of TerminaliabellericaRoxb. Part I. A new cardiac glycosides. Indian chemical society 1986; 45: 913- 917.
8. Ram Chandra Reddy ,RamanaKumari V, Madhava Reddy SV, Azeem B, Prabhakar MA, Appa MC, Rao AVN. Cardiotonic activity of the fruits of TerminliaChebula. Fitoterapia 1990; 61:517-524.
9. Sandhyaa T, Mishra KP. Cytotoxic responsible of breast cancer cell lines, MCF 7 and T 47 D to Triphala and its modification by antioxidants.CancerLett 2006; 238: 304-313.
10. Yan Shi, Ravi PS ,Sanjay KS. Triphala inhibits both in vitro and in vivo xenograft growth of pancreatic tumor cells by inducing apoptosis. BMC Cancer 2008 ;8:294.
11. MuthusamySenthilKumar ,ShanmugamKirubanandan, RamasamySripriya, Praveen Kumar Sehgal P. Triphala Promotes Healing of Infected Full-Thickness Dermal Wound . Journal of Surgical Research 2008;144:94 –101.
12. Sabina EP, Rasool M. An in vivo and in vitro potential of Indian ayurvedic herbal formulation Triphala on experimental gouty arthritis in mice. Vascular Pharmacology 2008;48(1):14-20.
13. El-Mekkawey M, Merelhy M. Inhibitory effects of Egyptian folk medicines on human immunodeficiency virus (HIV) reverse transcriptase. Chemical Pharmaceutical Bulletin 1975;45:641-648.
14. Jagetia GC, Baliga MS, Malagi KJ, SethukumarKamath M. The evaluation of the radioprotective effect of Triphala (an ayurvedic rejuvenating drug) in the mice exposed to I-radiation. Phytomedicine 2002; 9: 99-108.
15. Pulok K Mukherjee, SujayRai, Sauvik Bhattacharyya, Pratip Kumar Debnath, TuhinKantiBiswas, Utpalendu Jana, SrikantaPandit, BishnuPadaSaha, Pradip K Paul. Clinical study of Triphala: A well knownPhytomedicine from India. Iran J Pharmacol Therapeutics 2006; 5: 51-54.
16. Jose K ,Kuttan R. Hepatoprotective activity of Emblicaofficinalis and Chyavanaprash. J Ethnopharmacol 2006; 72: 135-140.
17. King J. The transferases alanine and aspartate transaminases, In: Van D. eds,Nostrand,London,Practical clinical enzymology, 1965a, pp 121-138.
18. King J. The hydrolases – acid and alkaline phosphatases.In: Van D eds.,Nostrand Company Limited, London, Practical clinical enzymology, 1965b, pp191-208.
19. Marklund, Marklund. Involvement of superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase.Eur J Biochem 1974; 47:469-474.
20. Bellomo G, F Mirabelli, D DiMonte, P Richelmi, H Thor, C Orre-nius, S Orrenius. Formation and reduction of glutathione-mixed disulfides during oxidative stress.BiochemPharmacol 1987; 36:1313- 1320.
21. Rotruck JT, Pope AL, Ganther HE, Swanson AB,Hafeman DG, Hoekstra WG. Selenium, biochemical role as a component of glutathione peroxidase purification and assay.Science 1973; 179: 588-590.
22. Habig WH, Pabst MJ, JakobyWB.Glutathione-Stransferase. The first enzymatic step in mercapturic acid formation. J Bio Chem 2006; 249: 7130-7139.
23. Moron M, Depierre J, Mannervik B. Levels Of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochimica et BiophysicaActa (BBA) - General Subjects 1979;582(1): 67–78.
24. Ohkawa H. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction; Anal Biochem 1979; 95: 351-358.
25. Silverstein R. D-galactosamine lethality model: Scope and limitations. J Endotxin Res 2004; 10:147-162.
26. Frank Maley, Anthony L Tarentino, John F McGarrahan, Rudolph DelGiacco .The metabolism of D-galactosamine and N-acetyl-D-galactosamine in rat liver. Biochemical Journal 1968; 107:637-644.
27. Keppler DO, Rudigier JF, Bischoff E, Decker KF.The trapping of uridine phosphates by D-galactosamine, D-glucosamine and 2- deoxy-D-galactose. Eur J Biochem 1970; 17: 246–253.
28. Decker K, Keppler D. Galactosamine-induced liver injury. In: Popper, H., Schaffner, F.eds.Progress in Liver Disease, New York: Grune, 1972,pp 183-199.
29. Konishi Y, Shinozuka H, Farber JL. The inhibition of rat liver nuclear ribonucleic acid synthesis by galactosamine and its reversal by uridine.Laboratory Investigation 1974; 30: 751-756.
30. Endo Y, Kikuchi T, Nakamura M. Ornithine and histidine decarboxylase activities in mice sensitized to endotoxin, interleukine-1 or tumor necrosis factor by D-galactosamine. British Journal of Pharmacology 1992; 107:888-894.
31. Manabe A, Cheng CC, Egashira Y, Ohta T, SanadaH.Dietary wheat gluten alleviates the elevation of serum transaminase activities in D-galactosamineinjected rats. Journal of Nutritional Science and Vitaminology 1996; 42: 121-132.
32. MitraSK,Venkataranganna MV, Sundaram R, GopumadhavanS.Protective effect of HD-03, an herbal formulation, against various hepatotoxic agents in rats. Journal of Ethnopharmacology 1998; 63:181-186.
33. Ravikumar V, Shivashangari KS, DevakiT.Hepatoprotective activity of Tridaxprocumbens against D galactosamine/lipopolysaccharide induced hepatitis in rats. Journal of Ethnopharmacology 2005; 101(1–3): 55–60.
34. Hwa-Kyung Lim, Hack-Seang Kim Hong-SerckChoi,SeikwanOh,Choon-GonJang,JongwonChoi,Seung-Hwan Kim,Myung-JeiChang.Effects of acetylbergenin against D-galactosamine-induced hepatotoxicity in rats. Pharmacological Research 2000; 5: 471-474.
35. Rastogi, R, Saksena S, Tripathi SC, Hussaini FA, Shoeb A, Dhawan BN, Ram VJ. Hepatoprotective activity of 6-(4-bromohenyl)-4-methylthio-2-oxo- 2H-pyran-3-carbonitrile against hepatotoxicity induced by galactosamine and thioacetamide in rats. Medical Science Research 1998; 26: 729-731.
36. SreepriyaM ,Devaki T. Effect of Indigoferatictoria Linn. On liver antioxidant defense system during Dgalactosamine/ endotoxin-induced acute hepatitis in rodent. Indian Journal of Experimental Biology 2001; 39:181-184.
37. Abul K Najmi, K KPillai, S N Pal, M Aqil. Free radical scavenging and hepatoprotective activity of jigrine against galactosamine induced hepatopathy in rats. Journal of Ethnopharmacology 2005;97(3): 521–525
38. DrotmanRB ,Lawhorn GT. Serum enzymes are indicators of chemical induced liver damage. Drug and chemical Toxicology 1978;1:163-171.
39. SambrekarSudhir N, Patil PA, PatilSA.Hepatoprotective activity of Mussaendafrondosa Linn extract in ethanol treated mice. Int J Drug Res Tech 2012;2 (6): 446-453.
40. Jose K, Kuttan R. Inhibition of oxygen free radicals by Emblicaofficinalis extract and Chyavanprash. Amala Res Bull 1995; 15:46-52.
41. Martin P, Friedman LS. Assessment of liver function and diagnostic studies.In:Friedman,L.S., Keeffe,E.B.eds, Hand Book of Liver Disease, Churchill Livingstone: Philadelphia, 1992,pp 1-14.
42. GopiSudheer Kumar J, Bala Krishna RB, Vijay Kumar G, RanganayakuluD.Hepatoprotective and antioxidant activity of the alcoholic extract of Ipomoea turpetnm against anti-TB drugs induced hepatotoxicity in rats. Journal of Advances in Drug Research 2010;(1).
43. Sree Ramamurthy M ,Srinivasan M. Hepatoprotective effect of Tephrosiapurpurcea in experimental animals. Indian Journal of Pharmacology 1993; 25: 34-36.
44. Katsumi Maezono, Kazunori Mawatari, Kenta Kajiwara, AyakoShinkai, Toshio Maki. Effect of alanine on D-galactosamine induced acute liver failure in rats. Hepatology 1996; 24:1211- 1216.
45. SinclariAJ,Barnett AH, LunieJ.Free radical and auto-oxidant systems in health and disease. Journal of Applied Medicine 1991; 17: 409.
46. Anbarasu C, Rajkapoor B, Bhat KS, John Giridharan, A Arul Amuthan, Satish K. Protective effect of Pisoniaaculeata on thioacetamide induced hepatotoxicity in rats.Asian Pacific Journal of Tropical Biomedicine 2012;511-515.
47. Hua Wang De-Xiang Xu, Jin-Wei Lv,HuanNing,Wei Wei. Melatonin attenuates lipopolysaccharide (LPS)-induced apoptotic liver damage in D-galactosamine-sensitized mice.Toxicology 2007;237(1–3):49–57.
48. Tiegs G, Wolter M, WendelA.Tumor necrosis factor is a terminal mediator in galactosamine/endotoxininduced hepatitis in mice. BiochemPharmacol 1989; 38:627-631.
49. Osawa Y , Nagaki M, Banno Y, Yamada Y, Imose M, Nozawa Y, Moriwaki H, Nakashima S. Possible involvement of reactive oxygen species in Dgalactosamine- induced Sensitization against tumor necrosis factor-alpha-induced hepatocyte apoptosis.J Cell physiol 2001;187: 374-385.
50. Han D ,Hanawa N, Saberi B, KaplowitzN.Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNFinduced apoptosis. Free RadicBiol Med 2006; 41: 627-639.