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 Table of Contents  
ORIGINAL ARTICLE
Year : 2013  |  Volume : 1  |  Issue : 2  |  Page : 120-125

Hepatoprotective activity of Clearliv a polyherbal formulation in Wistar rats


1 Department of Pharmacology, J.S.S College of Pharmacy, Ootacamundl, Malaysia
2 Department of Pharmacology, J.S.S College of Pharmacy, Ootacamund, Malaysia
3 TIFAC-CORE, J.S.S College of Pharmacy, Ootacamund, Malaysia
4 Department of Pharmacology, J.S.S College of Pharmacy, Ootacamundl; Pharmacology Unit, Faculty of Pharmacy, AIMST University, Semeling, Bedong - 08100, Kedah, Malaysia
5 Green Milk Concepts, Herbal Division of Apex Laboratories, Chennai, Tamil Nadu, India

Date of Web Publication13-Dec-2013

Correspondence Address:
E P Kumar
Department of Pharmacology, Karpagam College of Pharmacy, Coimbatore - 641 032, Tamil Nadu, India

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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2321-4848.123023

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  Abstract 

Objective: To evaluate the hepatoprotective activity of Clearliv a polyherbal formulation in Wistar rats. Materials and Methods: The hepatoprotective potential of Clearliv was evaluated in thioacetamide-induced liver necrosis, DL-galactosamine [GalN]-induced liver injury, and carbon tetrachloride [CCl 4 ]-induced hepatitis models in Wistar rats. In all the models, Clearliv (at the dose levels of 800, and 1000 mg/kg) was administered for 3 days orally followed by single intraperitoneal administration of the hepatotoxicant on the last day after one hour of Clearliv administration. After 24 h of toxicant administration blood sample was collected by sino-orbital puncture in sodium EDTA tubes. The efficacy of Clearliv was evaluated by plasma biochemical parameters (AST, ALT, and ALP), and antioxidant enzyme (lipid peroxidation, catalase, superoxide dismutase, glutathione peroxidase, and hydroxyproline) levels. Results: In thioacetamide-induced necrosis, and GalN-induced liver injury models, Clearliv at 1000 mg/kg showed significant reduction in the elevated plasma liver markers, and elevated antioxidants levels. In CCl 4 -induced hepatitis, the Clearliv had favorable hepatoprotective effect, but the results were not significant. Conclusion: Clearliv 800, and 1000 mg/kg showed significant hepatoprotective effect against thioacetamide- and GalN-induced liver necrosis and injury, respectively.

Keywords: Antioxidant, carbon tetrachloride, Clearliv, DL-galactosamine, hepatoprotection, polyherbal formulation, thioacetamide


How to cite this article:
Kumar E P, Rajan VR, Kumar AD, Parasuraman S, Emerson S F. Hepatoprotective activity of Clearliv a polyherbal formulation in Wistar rats. Arch Med Health Sci 2013;1:120-5

How to cite this URL:
Kumar E P, Rajan VR, Kumar AD, Parasuraman S, Emerson S F. Hepatoprotective activity of Clearliv a polyherbal formulation in Wistar rats. Arch Med Health Sci [serial online] 2013 [cited 2019 Dec 8];1:120-5. Available from: http://www.amhsjournal.org/text.asp?2013/1/2/120/123023


  Introduction Top


In the recent years, due to vast amount of scientific ethno-pharmacological work carried out on herbs with claims of curative properties by folklore or traditional or by indigenous system of medicine, interest on the efficacy, and safety of the medicinal herbs has generated tremendous interest in the scientific community, pharmaceutical companies, and in general by the common man. [1] Due to this, several polyherbal products have come into the market claiming to have ingredients formulated based on the indigenous system of medicine in India. Ayurvedic system of medicine is a traditional Indian system of medicine, and it has been practiced for several centuries. Ayurveda system of medicine has been gaining acceptance in the Western countries because of the side-effects at the time associated with allopathic drugs. [2] In recent years, several pharmaceutical companies are involving in ayurvedic research to develop effective ayurvedic formulations. Clearliv is a marketed polyherbal formulation which claims that it can be used for the treatment of hepatic necrosis, hepatitis, and liver injuries. Hence, the present study was undertaken to determine the hepatoprotective effect Clearliv using chemical (thioacetamide, DL-galactosamine [GalN], and carbon tetrachloride [CCl 4 ])-induced hepatic disorders in Wistar rats. [3],[4],[5],[6]


  Materials and Methods Top


Polyherbal formulation

Clearliv is a marketed soft gelatin capsule obtained from Apex Laboratories Ltd., Chennai (Batch No.: 1385002, Mfg date: Jun, 2005), and it is formulated using aqueous extracts of Phyllanthus niruri, Eclipta alba, Boerhaavia diffusa, Tinospora cordifolia, Tribulus terrestris, Tephrosia purpurea, Indigofera tinctoria, Aconitum heterophyllum, Andrographis paniculata, Rubia cordifolia, Terminalia chebula, Curcuma longa, and Ricinus cummunis.

Animals

Healthy, adult female albino Wistar rats (180-220 g) were used for the study. The animals were obtained from central animal house, JSS college of Pharmacy, Ootacamund, and they were housed in large, spacious, hygienic polypropylene cages, and they were maintained under standard conditions of temperature (15 ± 5°C), relative humidity (40 ± 10°C), and 12/12 h light/dark cycle. The animals were fed with normal rat pellet (supplied by M/s Hindustan Lever Ltd., Bangalore, India), and water ad libitum. The study was approved by Institute Animal Ethics Committee, and all the animal experiments were carried out as per the recommendations of Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) guidelines, India.

Preparation of the drug solution

Tioacetamide and GalN at a dose of 500 mg/kg and 400 mg/kg, respectively, were prepared using normal saline. CCl 4 50 μl/kg was prepared using olive oil.

Effect of Clearliv on thioacetamide-induced necrosis in female Wistar rats

The rats were divided into five groups of six animals each as follows.

Group-I: Normal control

Group-II: Thioacetamide control

Group-III: Liv-52 (20 ml/kg)

Group-IV: Clearliv 800 mg/kg

Group-V: Clearliv 1000 mg/kg

The test substances, and standard drug were dissolved in distilled water, and they were administered once daily (p.o.) for 3 days. The normal and thioacetamide control animals were received distilled water at the dose of 1 ml/kg. Group-III to V animals were received Liv-52 (20 ml/kg), Clearliv 800 mg/kg, and Clearliv 1000 mg/kg, respectively. After one hour of last dose of vehicle/ drug administration, the animals of groups II to V were administered a single intraperitoneal injection of thioacetamide at a dose of 500 mg/kg. After 24 h of thioacetamide injection, the blood was drawn from all groups through sino-orbital puncture in sodium EDTA tubes under light diethyl ether anesthesia. [7] The plasma was separated by centrifuging the blood sample at 2,000 RPM for 20 min, and the plasma was used for estimation of aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein, and albumin. At the end of the experiment, the animals were sacrificed by cervical dislocation, and the liver was excised out, and stored in -70°C deep freezer until estimation of lipid peroxidation, and enzymatic antioxidants such as catalase, superoxide dismutase, glutathione peroxidase, and hydroxyproline. [8]

Effect of Clearliv on GalN-induced liver injury in female Wistar rats

The rats were divided into five groups of six animals each as follows.

Group-I: Normal control

Group-II: GalN control

Group-III: Liv-52 20 ml/kg

Group-IV: Clearliv 800 mg/kg

Group-V: Clearliv 1000 mg/kg

The test substances, and standard drug were dissolved in distilled water, and they were administered once daily (p.o.) for 3 days. The normal and GalN control animals were received distilled water at the dose of 1 ml/kg. Group-III to V animals received Liv-52 (20 ml/kg), Clearliv 800 mg/kg, and Clearliv 1000 mg/kg respectively. After one hour of last dose of vehicle/ drug administration, the animals of groups II to V were administered a single intraperitoneal injection of GalN at a dose of 400 mg/kg. After 24 h of GalN injection, the animals were anesthetized, and blood sample was collected in sodium EDTA tubes, and plasma was separated. [7] The plasma sample was used for analysis of biochemical parameters such as AST and ALT. At the end of the experiment, the animals were sacrificed by cervical dislocation, and the liver was excised out and stored in -70°C deep freezer until estimation of lipid peroxidation and catalase. [9],[10]

Effect of Clearliv on carbon tetrachloride-induced hepatitis in female Wistar rats

The rats were divided into five groups of six animals each as follows.

Group-I: Normal control

Group-II: CCl 4 control

Group-III: Liv-52 (20 ml/kg)

Group-IV: Clearliv 800 mg/kg

Group-V: Clearliv 1000 mg/kg

The test substances, and standard drug were dissolved in distilled water, and they were administered once daily (p.o.) for 3 days. The normal and CCl 4 control animals were received distilled water at the dose of 1 ml/kg. Group-III to V animals received Liv-52 (20 ml/kg), Clearliv 800 mg/kg, and Clearliv 1000 mg/kg respectively. After one hour of last dose of vehicle/ drug administration, group II to V animals were administered a single intraperitoneal injection of CCl 4 at a dose of 50 μl/kg. After 24 h of CCl 4 injection, the animals were anesthetized, and blood sample was collected in sodium EDTA tubes, and plasma was separated. [7] The plasma sample was used for analysis of biochemical parameters such as AST, and ALT. [11],[12]

Estimation of enzyme levels in plasma

The plasma liver markers such as AST, ALT, ALP, total protein, and albumin were estimated using semi-autoanalyzer (M/s Vital Scientific N.V., The Netherlands). The biochemical kits for AST, ALT, ALP, total protein, and albumin were obtained from E. Merck (India) Pvt. Ltd.

Estimation of biological parameters of liver tissue

The liver tissue was washed thoroughly in ice-cold saline to remove the blood after thawing, blotted the saline gently using filter paper, and homogenized with 50 mM tris-HCl. [13]

The lipid peroxidation levels in liver tissue were estimated using modified Ohkawa method. [14] To 0.2 ml liver tissue homogenate, 0.3 ml of 0.005% butylated hydroxytoluene (BHT) in methanol, and 2.1 ml of thiobarbituric acid were added. The mixture was incubated at 90°C for 45-60 min. Later on, the mixture was centrifuged at 4000 rpm for 10 min. The red chromogen supernatant layer was measured at 532 nm. The levels were expressed as μmoles of MDA/g of tissue. Tetra ethoxy propane was used as external standard for lipid peroxidation assay.

The liver catalase activity was estimated using available literate elsewhere. [15] To 100 μl liver homogenate, 2.25 ml of potassium phosphate buffer, and hydrogen peroxidase (catalyst) was added. The mixture was incubated at 25°C for 30 min. The change in absorbance at 240 nm was measured for 2-3 min in UV-visible spectrophotometer. The dy/dx for every 30 second for each assay was calculated, and the results were expressed as catalase units of protein. Catalase in 100 μl of sample was calculated using formula (dy/dx × 0.0003)/ (38.3956 × 10 6 ).

The liver glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were estimated using kits (Randox Diagnostic Kit, Ranbaxy Laboratories, India) according to the manufacturers' instructions. Results of GSH-Px, and SOD levels were expressed as mU/mg of protein, and SOD Units/ mg, respectively.

Hydroxyproline in the liver homogenate was estimated using available literate elsewhere. [16] To 100 μl of liver homogenate, 1900 μl of assay buffer (composition: citric acid, glacial acetic acid, sodium acetate, sodium hydroxide, and toluene as preservative), 1 ml of chloramine-T was added, and incubated at room temperature for 20 min. In that reaction, mixture 1 ml of 3,3'-Diaminobenzidine (DAB) was added, and again incubated at 60°C for 15 min. Later on, the reaction mixture was allowed to cool, and the absorbance was measured at 550 nm. The results was expressed as μg of Hydroxyproline/ g of tissue.

Total protein was estimated by the procedure described by Lowry et al. [17] To 50 μl of liver homogenate, 450 μl of 0.5N sodium hydroxide, and 500 μl of 10% trichloroacetic acid was added. It was centrifuged at 4000 RPM for 10 min, and supernatant was discarded. The sediment was reconstituted with 500 μl of 0.5N sodium hydroxide and incubated at 60°C for 10 min. Later on, 2 ml of alkaline cuprate tartarate, and 250 μl 2N Folins reagent were added. The blue color formed was measured at 540 nm, and the results were expressed as mg of protein/ g of tissue. The protein calibration curve was prepared using bovine serum albumin (10 mg/ml stock).

Statistical analysis

All the vales are mean ± SEM. Significant difference between groups were determined using one-way ANOVA followed by Tukey's multiple comparison test. The statistical analysis was calculated using GraphPad Instat 3.06 (GraphPad Software, CA). A P value less than 0.05 was considered to be significant.


  Results Top


In thioacetamide-, GalN-, CCl 4 - induced hepatotoxicity models, toxins are not having any influence on alterations in body weight. No significant alterations in body weight were observed in both treatment, and toxin-administered groups when compared to normal control group. The toxin-administered groups showed significant increases in biochemical and liver tissue homogenate oxidative parameters (P < 0.01) compared to normal control group. The effects of Clearliv on liver enzymes and oxidative parameters were summarized in [Table 1], [Table 2], [Table 3], [Table 4].
Table 1: Effect of Clearliv on plasma biochemical parameters in thioacetamide-intoxicated rats


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Table 2: Effect of Clearliv on liver tissue homogenate oxidative parameters in thioacetamide-intoxicated rats


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Table 3: Effect of Clearliv on plasma biochemical and liver tissue homogenate oxidative parameters in D-L-Galactosamine-intoxicated rats


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Table 4: Effect of Clearliv on plasma biochemical parameters in carbon tetrachloride-intoxicated rats


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In thioacetamide-induced necrosis, Clearliv at a dose of 1000 mg/kg showed significant reduction in the elevated plasma AST, ALT, ALP levels, and liver lipid peroxidation levels compared to thioacetamide control group. Thioacetamide-administered group showed significant increase in hyroxyproline levels, and decreased SOD, catalase, and GSH-Px levels compared to all other treatment group. Effect of Clearliv on plasma biochemical and liver tissue homogenate oxidative parameters in thioacetamide-induced necrosis rat was presented in [Table 1] and [Table 2].

In GalN-induced necrosis, Clearliv 1000 mg/kg significantly reduced the plasma AST, ALT levels, liver lipid peroxidation level and catalase levels compared to GalN control group. Effect of Clearliv on plasma biochemical and liver tissue homogenate oxidative parameters in GalN-induced necrosis was presented in [Table 3].

In CCl 4 -induced hepatitis, Clearliv 1000 mg/kg showed significant decrease in ALT levels. But Clearliv 800 and 1000 mg/kg does not reverse the CCl 4 -induced increase in AST levels. Effect of Clearliv on plasma biochemical parameters in CCl 4 -induced necrosis was presented in [Table 4]. The biochemical alterations concluded that the Clearliv does not reverse the CCl 4 -induced hepatitis in rats.


  Discussion Top


Clearliv at the dose levels of 800 and 1000 mg/kg showed a significant hepatoprotective effect against thioacetamide- and GalN-induced hepatotoxicity in rats. The preliminary acute toxicity (fixed dose procedure) study was carried out to determine the dose of Clearliv for pharmacological study. [18] A single oral dose toxicity study of Clearliv did not showed any toxic signs upto 2000 mg/kg in female rats.

Clearliv 1000 mg/kg significantly reduced the elevated levels of AST, ALT, and ALP produced by a single dose administration of thioacetamide. With respect to its antioxidant levels, Clearliv 1000 mg/kg showed a significant reduction in lipid peroxidation level, and elevation of glutathione peroxidase, superoxide dismutase, and catalase levels. Thus, it could be suggested that Clearliv with its free radical scavenging property has hepatoprotective activity in this model, which was further supported by reduced levels of hydroxyproline in liver tissue homogenate. Free radicals generated by thioacetamide oxidation also lead to cell death; it could be suggested that Clearliv may reduce the cell permeability, and thus offering protection against thioacetamide.

In GalN model, Clearliv 1000 mg/kg significantly reduced the levels of AST, ALT produced by single administration of GalN with respect to its antioxidant levels. Clearliv at 1000 mg/kg showed a significant reduction in lipid peroxidation, and elevation of catalase. Thus, it could be suggested that Clearliv has hepatoprotective activity in this model by stabilization of the cell membrane from oxidative stress.

In CCl 4 -induced hepatitis, Clearliv at 1000 mg/kg showed significant reduction in ALT levels, but did not produce significant reduction of AST levels.

Thioacetamide is used as a fungicide, and also it is a potent hepatotoxicant. [17] In thioacetamide-induced liver necrosis is initiated by biotransformation of thioacetamide by the microsomal FAD monooxygenase system. [19] Thioacetamide is bioactivated by CYP2E1 to thioacetamide sulfoxide and thioacetamide sulfodioxide, which are the penultimate and ultimate reactive metabolite that are responsible for [20] the liver injury by oxidative stress, lipoperoxidation, [21] increase in the cytosolic calcium and changes in DNA ploidy and distribution in the cell cycle phases, thereby leading to centrilobular necrosis. [22]

GalN-induced hepatotoxicity is one of the most widely employed models for studying liver injury. Single dose of GalN (500 mg/kg. i.p.) produces liver injury. [9] The metabolite of D-GaLN namely uridine phosphogalactosamine may deplete several uracil nucleotides such as UDP- galactose, UDP- glucose, and UTP causing reduction of mRNA and glycoprotein synthesis (reduction of ATP and glycogen synthesis), which leads to cellular membrane alteration. [23]

CCl 4 mainly acts by the generation of free radicals and increases the lipid peroxidation disturbing membrane integrity and function and thus producing liver damage. [24] Clearliv fails to produce hepatoprotective activity in CCl 4 model; this may be due to its poor capability in preventing the lipid peroxidation. This may not be the case of GalN- or thioacetamide-induced liver injury and necrosis, as the damage caused by the agents are most probably due to perturbation of Ca ++ and inhibition of mRNA and protein synthesis. In liver tissues, AST, and ALT are found in higher concentrations in cytoplasm and AST, in particular, also exists in mitochondria. In liver injury, membrane damage is caused to the plasma membrane of mitochondria and endoplasmic reticulum, which leads to the leakage of AST, ALT, and reduction in protein transportation, thereby decreasing the antioxidants, which further aggravates the liver damage impeding their regulatory metabolic function. [25] It could be suggested that Clearliv affords hepatoprotective activity by a consequence of stabilizing membranes of mitochondrial, endoplasmic reticulum, and by its moderate antioxidant activity.

The individual herb of Phyllanthus niruri, Eclipta alba, Boerhaavia diffusa, Andrographis paniculata, and Tinospora cordifolia have been evaluated for its hepatoprotective activity in various animal models. Eclipta alba at a dose of 50 mg/kg was studied in CCl 4 -, paracetamol-, and aflatoxin- induced hepatic injury, and has shown hepatoprotective activity by reducing the serum AST, and ALT. [26] Another study showed that Phyllanthus niruri at a dose of 50, 100 mg/kg reversed the biochemical and histopathological changes against paracetamol-induced liver damage in mice. [27] However, it was noted that the hepatoprotective activity of Clearliv was seen only at the doses of 800 and 1000 mg/kg, which could be considered as a high therapeutic dose levels, including polyherbal formulations like HD-03. [9] The reason could be that the stability, efficacy, and potency of various ingredients in polyherbal formulation depend on several factors such as time of collection of plants, storage period, self-life, and adequate standardization, which could affect efficacy of polyherbal formulation.

Therefore, it could be suggested that phytochemical studies could be performed based on the known markers for the raw material, and latter extended to the formulation, which may provide an insight to the lack of efficacy of this polyherbal formulation. Synergy is considered to be a key word with respect to the therapeutic activity of herbs. [28] This aspect is to be proved or studied in depth, which is beyond the scope of the present study. Nevertheless, the present study reveals the potential of Clearliv with respect to its hepatoprotective activity, especially in thioacetamide-induced necrosis model, and GalN-induced liver dysfunction.


  Conclusion Top


Clearliv at 800 and 1000 mg/kg showed a significant hepatoprotective action against various hepatotoxicant-induced liver necrosis, and injury.

 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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