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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 15  |  Issue : 1  |  Page : 114-117

Influence of combination of docosahexaenoic acid supplement and a polyherbal formulation (Liv. 52) on carbon tetrachloride-induced hepatic injury: A preclinical study


1 Department of Pharmacology, Melaka Manipal Medical College (Manipal Campus), Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Pharmacology, RAK College of Medical Sciences, RAK Medical and Health Sciences University, Ras al Khaimah, UAE
3 Melaka Manipal Medical College, Jalan Padang Jambu, Bukit Baru, 75150 Melaka, Malaysia
4 Department of Pharmacology, Raipur Institute of Medical Sciences, Raipur, Chhattisgarh, India

Date of Submission12-Dec-2019
Date of Decision18-Dec-2019
Date of Acceptance31-Dec-2019
Date of Web Publication13-Oct-2020

Correspondence Address:
Dr. Shakta Mani Satyam
Melaka Manipal Medical College (Manipal Campus), Manipal Academy of Higher Education, Manipal - 576 104, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_219_19

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  Abstract 


Background: Docosahexaenoic acid (DHA) has diverse functions in normal metabolism and health and are widely used as a nutritional supplement. Liv. 52 is a polyherbal formulation used in hepatic dysfunction. The present study was undertaken to investigate the influence of oral supplementation of Liv. 52 and DHA alone and their combination against carbon tetrachloride (CCl4) induced hepatic injury in Wistar rats. Materials and Methods: Hepatotoxicity was induced by administering 1:1 mixture of CCl4and olive oil; 1 ml/kg/72 h i.p. A total of 54 adult female Wistar (150–200 g) rats were divided into nine groups of six rats each as follows-Group 1-Normal healthy control (1 ml/kg/day of 2% gum acacia), Group 2: Negative control (CCl4 +1 ml/kg/day of 2% gum acacia), Group 3: Positive control (CCl4+ Silymarin 50 mg/kg/day), Group 4-9: CCl4+ Liv. 52–225 mg/kg/day, 450 mg/kg/day, DHA-300 mg/kg/day, 600 mg/kg/day alone and their combination. The treatment duration was 7 days. Hepatoprotective potential was studied by the estimation of serum alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) in experimental rats. Results: Serum ALT, AST, and ALP were significantly increased (P < 0.05) in hepatotoxic control rats compared to normal healthy control rats. There was statistically significant change (P < 0.05) in serum levels of ALT, AST, and ALP among Silymarin, Liv. 52 and DHA treated rats in comparison to hepatotoxic control rats.Conclusions: The present study revealed that Liv. 52 and DHA alone and in combination ameliorates the hepatic injury induced by CCl4in Wistar rats.

Keywords: Antioxidant, hepatoprotective, pro-oxidant, polyunsaturated fatty acids


How to cite this article:
Satyam SM, Bairy LK, Ern OT, Yen YG, Kanasin A, Muthaiah T, Ratnam US, Yadav K. Influence of combination of docosahexaenoic acid supplement and a polyherbal formulation (Liv. 52) on carbon tetrachloride-induced hepatic injury: A preclinical study. J Datta Meghe Inst Med Sci Univ 2020;15:114-7

How to cite this URL:
Satyam SM, Bairy LK, Ern OT, Yen YG, Kanasin A, Muthaiah T, Ratnam US, Yadav K. Influence of combination of docosahexaenoic acid supplement and a polyherbal formulation (Liv. 52) on carbon tetrachloride-induced hepatic injury: A preclinical study. J Datta Meghe Inst Med Sci Univ [serial online] 2020 [cited 2020 Oct 28];15:114-7. Available from: http://www.journaldmims.com/text.asp?2020/15/1/114/297980




  Introduction Top


The liver is one of the vital organs associated with the metabolism of endogenous substances as well as xenobiotics and is the first target for various toxic insults.[1] Nonalcoholic fatty liver disease is principally a disease of middle and old age. Nowadays, in spite of remarkable advances in the arena of modern medicine, hepatic diseases remain a foremost global concern.

The interaction among free radicals, antioxidants, and cofactors is important in upholding health, aging, and age-related diseases including hepatic dysfunction. Carbon tetrachloride (CCl4) is a potent, lipid-soluble hepatotoxic agent. It produces peroxidative degeneration of many tissues when bound to lipid and protein.[2],[3],[4] The fundamental structure of the liver of rats and humans is similar to the administration of CCl4 to rats is an accepted experimental model to produce hepatic damage.[5]

The 21st century is steering in a new era of nutritional science, demonstrating the astounding power of nutrition to benefit human health, particularly elderly people. To attain healthy aging, elderly people should be encouraged to acquire healthy lifestyles which should include diets rich in antioxidants. The screening of food supplements with hepatoprotective potential is an essential practice to combat the ever-increasing trends of hepatotoxins in the polluted environment. The antioxidant activity or the inhibition of the generation of free radicals is essential in providing protection against hepatic damage.[6] It is well documented that treatment with antioxidants such as Vitamins C and E can ameliorate the toxic effects of CCl4 on the liver and kidneys.[7]

Traditional medicines are effective in certain disorders and are based on experience in the use of plant products in the amelioration of common diseases. Currently, the use of herbal medicines for the prevention and control of liver diseases is in the focus of attention for both the physicians and the patients. The reasons for such shift toward the use of herbals include the expensive cost of conventional drugs, adverse drug reactions, and their inefficacy.

Liv. 52 is an ayurvedic polyherbal formulation developed by Himalaya Herbal Healthcare, Bengaluru (India). Liv. 52 is widely used in various hepatic disorders.[8],[9],[10] This polyherbal formulation contains mainly Mandur Basma, Tamarix gallica and herbal extracts of Capparis spinosa, Cichorium intybus, Solanum nigrum, Terminalia arjuna, and Achillea millefolium. Sandhir and Gill reported the hepatoprotective effects of Liv. 52 on ethanol-induced liver damage in rats.[11] However, very less scientific data regarding the identification and effectiveness of these herbs are available.

Many studies suggest that omega-3 polyunsaturated fatty acids (n-3 PUFA) have beneficial effects on metabolic disorders, including atherosclerosis, coronary heart disease, diabetes, and Alzheimer's disease, by modulating cell proliferation and suppressing inflammatory response and oxidative stress.[12],[13],[14] N-3 PUFA, including docosahexaenoic acid (DHA) and eicosapentaenoic acid, are well documented to ameliorate ethanol-induced fatty liver by regulating fatty acid metabolism, inhibiting lipogenesis, and suppressing inflammation and oxidative stress.[15],[16],[17],[18]

The objective of this study was to investigate the influence of oral supplementation of Liv. 52 and DHA alone and their combination against CCl4-induced hepatic injury in Wistar rats.


  Materials and Methods Top


Drugs and reagents

Liv. 52 tablets and DHA capsules were acquired from the pharmacy of Kasturba Hospital, Manipal. Silymarin and CCl4 were purchased from Sigma Aldrich, Bangalore. All the reagent kits were obtained from ASPEN Laboratories, New Delhi.

Animals

Fifty-four young female albino Wistar rats weighing 50–80 g were housed in separate polypropylene cages and maintained under standard conditions with temperature (22°C –24°C), 12 h light/12 h dark cycle and with relative air humidity of 40%–60%. The animals were acclimatized to the laboratory conditions for 1 week before the start of the experiment. The animals were provided with a normal pellet diet (VRK Nutritional Solutions, Pune, Maharashtra, India) and water ad libitum. Animals were described as fasted or deprived of food for 12 h but had allowed free access to water. The experiment was conducted after getting approval from the Institutional Animal Ethics Committee (IAEC/KMC/47/2015) and according to the ethical norms approved by the Ministry of Social Justices and Empowerment, Government of India and Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA) guidelines.

Experimental design

In the experiment, 54 young female Wistar rats (50–80 g) were used. The rats were divided into nine groups containing six rats in each group. Hepatotoxicity was induced by administering 1:1 mixture of CCl4 and olive oil; (1 ml/kg; i.p. every 72 h) to all the experimental animals in the group II to IX.[19] Treatment was done for 7 days as follows:

  • Group I: Normal control rats were given 2% gum acacia (1 ml/kg/day; PO).
  • Group II: CCl4 intoxicated control rats-2% gum acacia (1 ml/kg/day; PO) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).
  • Group III: CCl4 intoxicated rats-Silymarin (50 mg/kg/day; PO) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).
  • Group IV: CCl4 intoxicated rats-Liv. 52 (225 mg/kg/day; PO) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).
  • Group V: CCl4 intoxicated rats-Liv. 52 (450 mg/kg/day; PO) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).
  • Group VI: CCl4 intoxicated rats-DHA (300 mg/kg/day; PO) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).
  • Group VII: CCl4 intoxicated rats-DHA (600 mg/kg; PO) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).
  • Group VIII: CCl4 intoxicated rats-Liv. 52 (225 mg/kg/day; PO) + DHA (300 mg/kg/day) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).
  • Group IX: CCl4 intoxicated rats-Liv. 52 (450 mg/kg/day; PO) + DHA (600 mg/kg/day) and simultaneously administered CCl4: Olive oil (1:1); (1 ml/kg; i.p. every 72 h).


Collection of blood samples

At the end of the experimental period, the animals were anesthetized using a single dose of ketamine 80 mg/kg; i.p. following a 12 h fast and blood samples were collected from retro-orbital plexus by retro-orbital puncture. Thereafter, animals were euthanized by the cervical dislocation according to the annexure 6 of euthanasia of laboratory animals in the CPCSEA guidelines for the Laboratory Animal Facility. Following the collection of blood in microcentrifuge tubes and its clot formation, serum was obtained by centrifugation of blood at 3000 rpm for 20 min at 4°C using a refrigerated centrifuge (MIKRO 22R, Andreas Hettich GmbH and Co. KG, Germany).

Biochemical estimation

Serum was analyzed for assay of alanine transaminase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) as per the standard protocol given along with commercially available kits (ASPEN laboratories, New Delhi, India).

Statistical analysis

Using the Statistical Package for the Social Sciences (SPSS version 16.0; SPSS Inc., Chicago, USA), data were expressed as mean ± standard deviation and analyzed using the one-way analysis of variance followed by post hoc Tukey test. A level for P ≤ 0.05 was considered to be statistically significant.


  Results Top


In the present study, CCl4 intoxication caused a significant increase in serum ALT, AST, and ALP levels when compared to normal control rats (P < 0.001). Treatment with Liv. 52 and DHA alone and in combination at all the three above-mentioned doses have significantly decreased ALT and ALP when compared to CCl4 intoxicated control group (P < 0.001) and Silymarin treated (P < 0.001) hepatotoxic rats. Interestingly, beyond our expectation, there was a significant increase in the AST level in the group of rats treated with alone DHA-600 mg/kg (P < 0.001) and the combination of Liv. 52–225 mg/kg + DHA-300 mg/kg (P < 0.001) as well as the combination of Liv. 52–450 mg/kg + DHA-600 mg/kg (P < 0.001) when compared to CCl4 intoxicated control rats. Furthermore, AST level was significantly increased in the group treated with the combination of Liv. 52–225 mg/kg + DHA-300 mg/kg (P = 0.022) and Liv. 52–450 mg/kg + DHA-600 mg/kg (P < 0.001) in comparison with CCl4 intoxicated rats who were treated with Silymarin 50 mg/kg [Figure 1], [Figure 2], [Figure 3].
Figure 1: Effect of Liv. 52 and docosahexaenoic acid on serum aspartate transaminase levels

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Figure 2: Effect of Liv. 52 and docosahexaenoic acid on serum alanine transaminase levels

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Figure 3: Effect of Liv. 52 and docosahexaenoic acid on serum alkaline phosphatase levels

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  Discussion Top


CCl4 is commonly used in experimental animals to induce oxidative damage specifically in the liver. It causes lipid peroxidation associated with antioxidants depletion and subsequent tissue damage. This causes leakage of cellular enzymes AST, ALT, and ALP causing their rise in serum level.[19]

The present study revealed the hepatoprotective effect of Liv. 52 and DHA (alone and in combination) against CCl4-induced hepatic injury in Wistar rats. There was a significant rise in AST, ALT, and ALP in CCl4 intoxicated control rats compared to the treatment groups. ALT and ALP levels were significantly decreased in rats treated with Liv. 52 and DHA (alone and in combination), indicating their hepatoprotective potential. Hepatoprotective activity of Liv. 52 and DHA might be attributed to its direct antiperoxidative effect, membrane-stabilizing action on hepatocytes or may be its ability to restore the activity of antioxidants, superoxide dismutase, and glutathione.[11],[20],[21] The antioxidant effect and resultant hepatoprotective potential of Liv. 52 and DHA might be due to presence of flavonoids, omega 3 fatty acids, carotenoids, and Vitamin E and C.[11],[21] One of the studies demonstrated that Liv. 52 enhances acetaldehyde elimination and also prevents binding of acetaldehyde to cellular proteins and thereby exerts its protective effects.[22]

Our study demonstrated that DHA at the dose of 300 mg/kg decreases AST, ALT, and ALP levels, which is similar to the reports of Meganathan et al.[21] However, in our study, there was an attention-grabbing finding that there was a significant increase in AST level in the group of rats treated with alone DHA-600 mg/kg and combination of Liv. 52–225 mg/kg + DHA-300 mg/kg as well as combination of Liv. 52–450 mg/kg + DHA-600 mg/kg when compared to CCl4 intoxicated control rats. Furthermore, AST level was significantly increased in the group treated with the combination of Liv. 52–225 mg/kg + DHA-300 mg/kg and Liv. 52–450 mg/kg + DHA-600 mg/kg in comparison with CCl4 intoxicated rats who were treated with Silymarin 50 mg/kg. This is probably because an enormous amount of antioxidants eventually harm the organ systems. Young and Woodside reported that in certain circumstances, an antioxidant may even act as a pro-oxidant, for example, it can generate toxic ROS/RNS.[23] AST is not only present in the liver but also in the heart, skeletal muscles, kidney, erythrocytes, etc., whereas ALT is a very specific biomarker for liver function test, which came down to normal level with DHA treatment. Henceforth, we can convince the hepatoprotective potential of DHA alone and in combination with Liv. 52.


  Conclusions Top


Our study revealed that DHA and Liv. 52 alone and in combination, significantly ameliorates the hepatic injury induced by CCl4 in Wistar rats. Treatment with a high dose of DHA-600 mg/kg alone and in combination with Liv. 52 might adversely affect other organ systems, which were evident in the present study in the form of elevated AST levels. Influence of a high dose of DHA-600 mg/kg needs to be investigated on other organs such as the heart (where AST is present) with specific biomarkers to rule out its toxic effects if any. The study opens the perspective for further mechanistic studies.

Acknowledgment

We would like to acknowledge Melaka Manipal Medical College, Manipal Academy of Higher Education, Manipal, Karnataka (India) for their support toward the accomplishment of this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Cabré M, Camps J, Paternáin JL, Ferré N, Joven J. Time-course of changes in hepatic lipid peroxidation and glutathione metabolism in rats with carbon tetrachloride-induced cirrhosis. Clin Exp Pharmacol Physiol 2000;27:694-9.  Back to cited text no. 3
    
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Mandal JN, Roy BK. Studies with Liv. 52 in the treatment of infective hepatitis, chronic active hepatitis and cirrhosis of the liver. Probe 1983;22:217-42.  Back to cited text no. 8
    
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Karandikar SM, Joglekar GV, Chitale GK, Balwani JH. Protection by indigenous drugs against hepatotoxic effects of carbon tetrachloride – A long term study. Acta Pharmacol Toxicol (Copenh) 1963;20:274-80.  Back to cited text no. 10
    
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Lebbadi M, Julien C, Phivilay A, Tremblay C, Emond V, Kang JX, et al. Endogenous conversion of omega-6 into omega-3 fatty acids improves neuropathology in an animal model of Alzheimer's disease. J Alzheimers Dis 2011;27:853-69.  Back to cited text no. 14
    
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Huang LL, Wan JB, Wang B, He CW, Ma H, Li TW, et al. Suppression of acute ethanol-induced hepatic steatosis by docosahexaenoic acid is associated with downregulation of stearoyl-CoA desaturase 1 and inflammatory cytokines. Prostaglandins Leukot Essent Fatty Acids 2013;88:347-53.  Back to cited text no. 15
    
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Huang W, Wang B, Li X, Kang JX. Endogenously elevated n-3 polyunsaturated fatty acids alleviate acute ethanol-induced liver steatosis. Biofactors 2015;41:453-62.  Back to cited text no. 18
    
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Satyam SM, Bairy KL, Pirasanthan R, Mohandas RKG, Nath M. Grape seed extract and Zinc containing multivitamin-mineral nutritional food supplement ameliorates hepatic injury. Jokull J 2014;64:184-95.  Back to cited text no. 19
    
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Barbosa AM, Chagas TR, Nunes EA. Fatty liver and n-3 fatty acids ingestion: New mechanisms and perspectives from pre-clinical animal models. Liver Pancreat Sci 2016;1:1-4.  Back to cited text no. 20
    
21.
Meganathan M, Madhana GK, Sasikala P, Mohan J, Gowdhaman N, Balamurugan K, et al. Effect of dietary fish oil (omega-3-fatty acid) against oxidative stress in isoproterenol induced myocardial injury in albino Wistar rats. Global J Pharmacol 2011;5:50-3.  Back to cited text no. 21
    
22.
Chauhan BL, Kulkarni RD. Effect of Liv. 52, a herbal preparation, on absorption and metabolism of ethanol in humans. Eur J Clin Pharmacol 1991;40:189-91.  Back to cited text no. 22
    
23.
Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol 2001;54:176-86.  Back to cited text no. 23
    


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