INTRODUCTION:

Liver injury may lead to fatal complications in the body since it plays a vital role in drug elimination and detoxification of exogenous and endogenous compounds. Despite the extraordinary regenerative capacity of this organ, continuous exposure to drugs or other non-infectiousagents leads to hepatotoxicity^1,2.

Synthetic drugs such as non-steroidal anti-inflammatory drugs are commonly employed in the management of many diseases as main course or as adjuvant therapy. Paracetamol (PCM) is most widely used antipyretic and analgesic drug of NSAID class. In most countries, Paracetamol hepatotoxicity is one of the major causes for drug induced liver damage. Paracetamol, which is a dose-dependent hepatotoxin produces clear evidence of liver injury within few hours or days³.

Modern medicine from plants have become a central focus of hepatoprotection today due to their less toxicity, easy accessibility, meagre cost and less side effects when compared to synthetic ones⁴. Traditional system of medicines which normally consists of herbs, are under active research worldwide and a number of traditional formulations are showing promising results in preventing and treating diseases⁵. Chooranam are fine powder of crude drugs and Aruvadha Churnam (AC), stated in the Siddha Formulary of India, contains a leaves of a herb called Aruvada (Ruta chalepensis) along with spices such as Fenugreek seeds, Cumin fruits, Black cumin seeds, Cinnamon bark, Liquorice root, Fennel fruits, Coriander fruits and sugar candy was selected for this study.The literature claims that it has the potential to treat liver related diseases, sore throat, loss of appetite, facial paralysis, eye disorders, insomnia, impotency in males and various other disorders⁶. The hepatoprotective activity of each individual component of this formulation has been reported earlier^7-13. Since the synergistic activity of all these components has not been reported, the present study aims at investigating the hepatoprotective effects of this traditional formulation on paracetamol induced liver damage.

MATERIALS AND METHODS:

Plant Materials:

Aruvadha Churnamformulation contains plant materials such as Ruta chalepensis(leaf), Cuminum cyminum (fruit), Glycyrrhiza glabra(root& rhizome),Trigonella foenum-graecum(seed), Cinnamomum verum (bark),Nigella sativa(seed), Foeniculum vulgare(fruit) and Coriandrum sativum (fruit)which were purchased from the local market and authenticated by Dr. E. Sasikala, Research officer, Department of Pharmacognosy, Siddha Central Research Institute, Chennai.

Preparation of Aruvadha Churnam:

The plant materials were shade dried, powdered and mixed in proportions as per Siddha Formulary of India to prepare AC formulation⁶. 10gms of the AC formulation consists of 0.625 gms of Ruta chalepensis, 0.625 gms of Cuminum cyminum, 0.625 gmsof Glycyrrhiza glabra, 0.625 gms of Trigonella foenum-graecum, 0.625 gms of Cinnamomum verum, 0.625 gms of Nigella sativa, 0.625 gms of Foeniculum vulgare, 3.75 gms of Coriandrum sativum and1.875 gms ofsugar candy.Luke warm water was used as a vehicle for administration of this formulation as mentioned in the Siddha Formulary of India.

Experimental Animals:

Wister rats weighing 150-200g were used in this study and were procured from TANUVAS, Chennai. All the animals were housed in polypropylene cages at standard husbandry conditions (Temperature: 23 ± 3.5ºC, Relative humidity: 55 ± 10%, 12h: 12h light /dark cycles, ventilation rate: 08-20 Air changes/hr) and were provided with standard pellets and water ad libitum.The Animals were handled and properly acclimatized for two weeks before the initiate the experiment.The experimental protocols were approved by the Institutional Animal Ethics Committee (IAEC ApprovalNo:121/PHARMA/SCRI,2013) of Siddha Central Research Institute, Chennai.

Chemicals:

Paracetamol and Silymarin were purchased from Sigma Aldrich. All the chemicals used in this experiment were of analytical grade and were obtained from Merck– India Pvt. Ltd. Diagnostic kits used in this study were procured from Siemens Diagnostics Ltd., India.

Experimental Design:

The animals were divided into six groups of six rats each and the treatment was scheduled as follows.

Group I : Normal Control (NC)

GroupII :Hepatotoxic control (HC) (PCM, 3g/kg bw,orally)

Group III: Standard Control (SC), Silymarin (50mg/kg bw.orally) + PCM (3g/kg bw,orally)

Group IV: Aruvadha Churnam(AC-1) (360mg/kg bw,orally) + PCM (3g/kg bw,orally)

Group V: Aruvadha Churnam(AC-2) (1800mg/kg bw,orally) + PCM (3g/kg bw,orally)

Group VI: Aruvadha Churnam (AC-3) (2400mg/kg bw,orally) + PCM(3g/kg bw,orally)

Group III was treated with Silymarin and Groups IV, V and VI were treated with AC for 8 days. Hepatotoxicity was induced by administering a single oral dose of PCM (3g/kg bw) to groups II, III, IV,V and VI on the 8^th day after an hour of Silymarin and AC treatment. 48 hours after the paracetamol administation, blood was collected from the retro-orbital plexus of each animaland serum was separated by centrifugation at 2000 g for 5 minutes at 4 °C and used for biochemical analysis¹⁴. The rats were then sacrificed by CO₂ euthanasia and the liver was dissected, cleaned of extraneous tissue, washed by ice cold saline, blotted with filter paper and weighed immediately. Sections of livers from each group were fixed immediately in 10% neutral formalin for a period of at least 24 h, dehydrated in graded (50–100%) alcohol and embedded in paraffin. Crosssections of the liver tissue (5–6 μM thickness) were prepared and stained with hematoxylineosin dye. These sections were subjected to microscopical examination.

Statistical analysis:

All the values were represented as Mean±SEM. Statistical significance between more than two groups was tested using two way ANOVA followed by the Tukey test using computer based fitting program (prism graph pad version 5.04.). Statistical significance was set at P < 0.05.

RESULTS:

Effect of Aruvadha Churnamon serum biochemical parameters:

In our study, there was a drastic and significant increase in the serum glutamate oxaloacetate transferase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin, serum creatinine, total cholesterol, triglycerides levels and decrease in total protein, albumin levels in the hepatotoxic group when compared to the normal group indicating paracetamol induced hepatotoxicity. Silymarin treated group showed a significant normalization of the above mentioned biochemical parameters when compared to hepatotoxicgroup. The trend was same with that of AC groups but the effect was augmented in AC-2 group compared to AC-1, AC-3 groups. [Table 1].

Effect of Aruvadha Churnamon Histopathology of Liver:

Histopathological studies also have confirmed that Aruvadha Churnamshowed a protective effect. At 1800 mg/kg dose, the Aruvadha Churnamtreated rat livers showed only minimal necrosis and inflammation which is comparable with that of standard [Figure 1].

DISCUSSION:

Paracetamol is an antipyretic and analgesic drug, which generates hepatic and renal tubular necrosis at toxic doses¹⁵. The experimental model of Paracetamol induced hepatotoxicity in rats serves as an adequate model to estimate the activity of hepatoprotective drugs¹⁶.In this study,the Paracetamol dose (3gm/kg bw) has induced a significant hepatocellular damage which is indicated by the serum biochemical parameters and liver histopathology.

Changes involved in the anatomy or functions of liver are characterized as liver disease¹⁷. Since liver is a port of a variety of enzymatic metabolic activity, any damage to the hepatic cells leads to the release of the serum marker enzymes into the blood stream. SGOT and SGPT which are available in higher concentrations in the cytoplasm (Hepatic cells) is found to be increased in the serum due to a liver injury which leads to the leakage of the plasma membrane^18,19. The decrease in levels of SGOT, SGPT in AC treated groups signifiesthat AC is effective in maintaining the liver function.The decrease was effective in AC-2 group compared to AC-1 and AC-3 groups.

Alkaline Phosphatase present in the cells lining biliary ducts is increased in response to cholestasis and biliary pressure. This explains the increase of ALP levels in HC whereas the damage in the AC-2is much lesser, owing to the restoration of stability of biliary function.Due to the damage of the hepatocytes, the unconjugated bilirubin was released into the blood stream which has occurred because of the inhibition of conjugation the blocked biliary tract^20,21. An increased levels of bilirubin in HC symbolizes hepatic injury. However the administration of the formulation has shown to decrease the levels of bilirubin in the AC treated groups, especially in AC-2.

In presence of severe hepatotoxicity that impedes further hepatic metabolism of the parent paracetamol, there may be 'spill over' of paracetamol to the kidney where it will be metabolized. Nephrotoxicity then results when there is insufficient glutathione in the renal parenchyma²². This condition can be diagnosed by measuring the serum creatinine levels. Rise in serum creatinine levels in HC group indicates acute renal injury due to paracetamol toxicity whereas in AC treated animals there were significant lower levels of serum creatinine compared to HC group animals. The effect was prominent in AC-2 group which was comparable to SC group.

Protein synthesis is affected due to liver damage, thus causing Hypoproteinemia²⁰. The reduction in total protein and albumin levels is attributed to the initial damage produced and localized in the endoplasmic reticulum, in the paracetamol induced HC group.These levels were raised in the AC treated groups indicating the stabilization of the endoplasmic reticulum which in turn has led to protein synthesis. Thus AC treated groupsrestored protein synthesis in hepatic cells and the effect was pronounced in AC-2 treated group. Paracetamol intoxication was found to alter the cholesterol biosynthesis mechanism and triglycerides secreting mechanism in hepatocytes which has led to the elevated cholesterol and triglyceride levels in HC group¹⁶. The reduction of these levels was observed in AC treated groups and the effect was more significant in AC-2 treated group. Histopathological studies also have confirmed the anti-hepatotoxic effect of Aruvadha Churnam in this study and protective effect was more profound at a dose of 1800 mg/kg bw.

Silymarin, being a potent anti-hepatotoxic agent have also reduced the elevated levels of SGOT, SGPT, ALP, bilirubin, creatinine, total cholesterol, triglycerides and also increased the levels of total protein, albumin in rats. AC-2 group has shown anti-hepatotoxic effect comparable to that of Silymarin group (SC).

From the biochemical and histopathological observations, it is evident AC treatment had caused restoration of liver function to normalcy. The hepatoprotective effect of AC is mainly due to the phytocomponents of the formulation which were individually reported to have hepatoprotective effect^7-13. AC formulation may also have scavenged liver by antioxidant mechanisms as it is evident that most of the herbs in the formulation were previously reported to have free radical scavenging effects^23-29.Also it was reported that compounds that block or retard the chain reaction of oxidation may prevent oxidative stress-induced hepatotoxicity ^30,31.

Thus AC may have shown the hepatoprotective effect by maintaining the structural integrity of hepatocellular membrane and hepatic cell architecture against paracetamol-induced damage.

The hepatoprotective effect of AC was dose dependent up to a dose of 1800 mg/kg b.w. but above this dose, ceiling effect was observed. Thus the degree of hepatic cellular damage is lesser in magnitude in AC-2 group when compared to AC-1 and AC-3 groups, thus proving its efficacy against paracetamol toxicity at medium dose. The evaluations of all these parameters proves that AC possesses potent hepatoprotective activity at intermediate dose i.e.,1800mg/kg bw.

Currently, the occurrence of drug associated hepatic damage is augmenting at an alarming rate and concurrently the use of paracetamol is becoming extensive and often abused. This study establishes the protective effect of Aruvadha Churnam, a Siddha formulation against paracetamol induced liver damage, which provides a support for its traditional use in treatment of liver disorders.

Table 1: Effect of Aruvadha Churnam on Serum Biochemical Parameters

Parameters	Group I Normal control (NC)	Group II Hepatotoxic Control (HC)	Group III Standard Control (SC) (50mg/kg bw)	Group IV AC-1 (360mg/kg bw)	Group V AC-2 (1800mg/kg bw)	Group VI AC-3 (2400mg/kg bw)
SGOT (IU/L)	96.56 ± 2.89	144.66 ± 8.37*	100.21 + 3.21**	116.42 ± 2.65**	102.33 ± 5.78**	103.66 ± 6.89**
SGPT (IU/L)	60.67 ± 6.49	107.67± 6.84*	64.54±4.44**	85.09 ± 5.29**	68.33 ± 7.84**	74.33 ± 7.25**
ALP (IU/L)	178.67 ± 8.17	254.33±8.67*	189.7±2.605**	214.67±5.04**	191.67±4.33**	199.33±9.92**
Bilirubin (mg/dL)	0.52 ± 0.03	0.83 ± 0.14*	0.58 ± 0.02**	0.75 ± 0.01**	0.62 ± 0.08**	0.69 ± 0.01**
Total Protein (gm%)	10.62 ± 0.15	7.2 ± 0.06*	10.27 ± 1.76**	8.4 ± 0.89**	9.88 ± 1.76**	8.1 ± 0.42**
Albumin (gm%)	3.97 ± 0.37	3.05 ± 0.13*	3.76±0.03**	3.40 ± 0.25**	3.67 ± 0.15**	3.46±0.03**
Creatinine (mg/dL)	0.56 ± 0.06	0.83 ± 0.03*	0.57±0.03**	0.71 ± 0.06**	0.60 ± 0.03**	0.68 ± 0.12**
Total Cholesterol (mg/dL)	68.33 ± 10.33	97.33 ± 12.39*	70.67±12.88**	84.33 ± 9.49**	74.67±11.88**	79.21 ± 10.16**
Triglycerides (mg/dL)	104.43 ± 9.31	167.22 ± 7.07*	116.66 ± 5.93**	140.66 ±8.64**	120.67 ±10.11**	136.66 ± 5.93**

All values are expressed as Mean ± SEM; N = 6. Significant levels: ^aindicate p<0.05, when compared to Normal Control, ^bindicate p<0.05, when compared to Hepatotoxic Control.

Figure 1: Photomicrographs of sections of Liver taken at 400X Magnification

a) Normal Cytoarchitecture (Normal control). b)The sections of liver obtained from the hepatotoxic control showed a focal necrosis and an intense inflammation. c) Liver section of silymarin (50 mg/kg) treated rats exhibit almost normal hepatic parenchyma with lesser degree of focal necrosis and inflammation. d-e) The low dose (360mg/kg b.w) and the intermediate dose groups (1800mg/kg b.w) showed almost very minimal necrosis and mild inflammation. f) The high dose group (2400mg/kg b.w) showed tiny focal necrosis and mild portal inflammation.

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Received on 16.04.2018 Modified on 30.06.2018

Research J. Pharm. and Tech 2018; 11(8): 3380-3384.

DOI: 10.5958/0974-360X.2018.00622.4