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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 2  |  Issue : 1  |  Page : 44-49

Hepatoprotective evaluation of Arogyavardhini Rasa against paracetamol-induced liver damage in rats


1 Department of Rasashastra and Bhaishajya Kalpana, IPGT & RA, Jamnagar, Gujarat, India
2 Pharmacology Laboratory, IPGT & RA, Gujarat Ayurved University, Jamnagar, Gujarat, India
3 Department of Rasashastra & Bhaishajya Kalpana, All Institute of Ayurveda, New Delhi, India

Date of Submission15-Aug-2016
Date of Acceptance12-Dec-2016
Date of Web Publication1-Jun-2017

Correspondence Address:
Yuga Raj Sapkota
Department of Rasashastra & Bhaishajya Kalpana, IPGT & RA, Gujarat Ayurved University, Jamnagar - 361 008, Gujarat
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/2468-838X.207421

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  Abstract 

Introduction: Liver is termed as Yakrut in ayurvedic classical literature, is one of the major organs for maintaining homeostasis, and is involved more or less with all the biochemical pathways in the body. Arogyavardhini Rasa (AVR) is one of the widely practicing ayurvedic herbo-mineral formulations in liver disorders. It has been used for the management of diverse types of Jvara (fever), Kushtha (skin disorders), Medoroga (altered lipid profiles associated with obesity), and other Yakrit vikara (liver disorders). In this study, AVR was prepared as per the 13th-century classical text Rasaratna Samuchaya. On the other hand, heavy metals causing toxicity, especially mercury present in this formulation, are an issue of concern.
Aim of the Study: Hepatoprotective effects of formulation were evaluated by paracetamol (PCM)-induced liver damage in rats to substantiate the role of metal mineral in the classical AVR formulation.
Materials and Methods: Effects of formulation were assessed on serum and liver tissue biochemical parameters and histopathological studies.
Results: PCM produced significantly impaired the liver and kidney functions as assessed through an increase in liver and kidney marker enzymes. Arogyavardhini-treated group significantly (P = 0.05) prevented this hepatotoxicity and strongly supported by histopathological examinations that revealed AVR shows the protection of liver tissue from PCM-induced hepatotoxicity.
Conclusion: The observation of the present study has stalwartly supported the hepatoprotective action of AVR against PCM-induced hepatotoxicity in rats.

Keywords: Bhasma, liver, mercury, organometallic, Rasaushadhi, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase


How to cite this article:
Sapkota YR, Bedarkar P, Nariya MB, Prajapati PK. Hepatoprotective evaluation of Arogyavardhini Rasa against paracetamol-induced liver damage in rats. BLDE Univ J Health Sci 2017;2:44-9

How to cite this URL:
Sapkota YR, Bedarkar P, Nariya MB, Prajapati PK. Hepatoprotective evaluation of Arogyavardhini Rasa against paracetamol-induced liver damage in rats. BLDE Univ J Health Sci [serial online] 2017 [cited 2019 Jul 17];2:44-9. Available from: http://www.bldeujournalhs.in/text.asp?2017/2/1/44/207421

In most of the ayurvedic literature, function of the liver is explained in relation to dosa (vital biological forces - humor), dhatu (body tissue), mamsa (muscle tissue), and mala (excreta).[1] In spite of incredible scientific innovation in the field of hepatology, liver disorders are still a worldwide health problem on the rise. Hepatotoxicity, jaundice, and hepatitis are the major hepatic disorders that account for a high death rate.[2] Conventional or synthetic drugs for the treatment of liver diseases are inadequate and can have serious side effects.[3] On the contrary, in the absence of a reliable liver protective drug in modern medicine, there are a number of medicinal preparations in Ayurveda, recommended for the treatment of liver disorders.[4]

Arogyavardhini Rasa (AVR) is one of such Kharaliya Rasaushadhi (formulations containing metallominerals and mercurials)used for the treatment of different types of Jvara (fever), Kushtha (skin disorders), Medoroga (obesity), and other Yakrit vikara (liver disorders).[5] It has been described in the 13th-century text by Rasa Vagbhata.[6] and Ayurvedic Formulary of India.[7]Rasaushadhi is the important formulation in ayurvedic therapeutics due to lesser therapeutic doses, enhancement of action of other ingredients of formulation, quicker action, and palatability [8] as compared to formulations prepared from drugs of plant or animal origin.[9] It is also used as a rejuvenator and to provide longevity.[6] Previous study suggests the role of AVR in acute viral hepatitis and possesses significant effects on cytoprotection and recovery of the liver function [10] and hepatoprotective effect against CCl4-induced liver injury.[11] It has been proven safe on liver, kidney, and brain through earlier studies.[12],[13]

Although many studies had been carried out, the issues are time and again raised because of mercury present in this formulation. Articles pertaining to the alarming level of heavy metals present in ayurvedic formulation had created lots of controversy regarding their efficacy and safety.[14] It is the need of time to explore the role of metallic drugs in Rasaushadhi formulations used in Ayurveda. In the present study, hepatoprotective effect was evaluated along with kidney function test and tissue homogenate parameter against paracetamol (PCM)-induced liver damage in rats to substantiate the impact of metallominerals and mercurial in AVR.


  Materials and Methods Top


Test drug

AVR as per the classical reference [Table 1] was prepared in the laboratory department.
Table 1: Ingredients used for the Preparation of Formulation

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Drugs and chemicals

Silymarin (Batch No. SIAD0036, Micro Labs Ltd., Solan) and PCM (Batch No. RG614, Glaxo Smith Kline Pharmaceuticals Ltd.) were procured for experimental study. Other chemicals and solvents used were of analytical grade.

Animals

Wistar strain albino rats (200 ± 20 g) of either sex were obtained from the animal house attached to the pharmacology laboratory. The animals were maintained on light/dark cycles with ideal laboratory conditions. Animals were fed ad libitum with Amrut brand rat pellet feed supplied by Pranav Agro Industries and tap water. All the protocols were approved by the Institutional Animal Ethics Committee (IAEC/17/2015/14) as per the guidelines of CPCSEA, India.

Experimental design

The hepatoprotective activity was carried out as per protocol designed in earlier works with slight modification.[15] Wistar albino rats of either sex were selected and divided into five groups each with six animals. Group I (normal control) and Group II (PCM control) received 1% carboxymethylcellulose in a dose of 10 ml/kg orally. Group III received a suspension of test drug AVR (90 mg/kg, PO), whereas Group IV (reference standard) received silymarin 100 mg/kg orally.

AVRand reference drug silymarin were administered for 7 consecutive days to the respective groups. Two doses of the toxicant (PCM 3 g/kg, PO) were administered orally to Group II on alternate days, i.e., 3rd and 5th day, and 1 h after administration of drugs into Groups III and IV. Animals fasted overnight, and on the 7th day, 1 h after drug administration, from the retro-orbital sinus, blood sample was withdrawn, and for biochemical estimation, serum was separated (liver and kidney function test). Afterward, animals were sacrificed and important organs were dissected, i.e., liver, heart, kidney, and spleen. For histopathological evaluation, a portion of the liver was preserved in 10% formalin.[16] A required amount of liver tissue was homogenized to estimate different tissue biochemical parameters.

Parameters

The blood was collected from supraorbital plexus in capillary tubes for serum biochemical investigations, including total cholesterol,[17] triglycerides,[18] high-density lipoprotein (HDL) cholesterol,[19] serum glutamic pyruvic transaminase (SGPT),[20] serum glutamic oxaloacetic transaminase (SGOT), and alkaline phosphatase (ALP),[21] total and direct bilirubin,[22] blood urea,[23] serum creatinine,[24] and uric acid.[25] These parameters were analyzed using fully automated biochemical random access analyzer (BS-200, Lilac Medicare Pvt. Ltd., Mumbai). The weighed liver tissue was taken and homogenized (2.5%) in ice-cold normal saline for estimation of different tissue biochemical parameters such as lipid peroxides [26] and superoxide dismutase.[27]

Statistical analysis

The data obtained have been expressed as mean ± standard error of the mean. Statistical comparison was done by paired and unpaired Student's t-test using Sigma stat software (version 3.5, Systat Software, Inc.), with the level of significance set at P < 0.05 for all the treated groups.


  Results Top


Effects on serum biochemical parameters of albino rats

PCM-treated group exhibited a significant rise in serum cholesterol, triglycerides, blood urea, serum transaminase, alkaline phosphate, total and direct bilirubin, and total and uric acid levels in comparison to a normal control group. AVR significantly reduced the serum triglyceride and blood urea level while produced nonsignificant decrease in the level of SGOT, SGPT, serum uric acid, and creatinine in comparison to the PCM control group. Above all, AVR produced significant decrease in total and direct bilirubin in comparison to PCM control group as shown in [Table 2],[Table 3],[Table 4].
Table 2: Effect of test drugs on Liver function Test biochemical parameters

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Table 3: Effect of test drugs on Lipid profile biochemical parameters

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Table 4: Effect of test drugs on Kidney function biochemical parameters

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Tissue homogenate biochemical parameter

PCM-treated rats exhibited increase in lipid peroxidation (LPO) in liver tissue. AVR produced nonsignificant decrease in LPO with concomitant increase in the level of superoxide dismutase (SOD) in groups treated with drug as compared to PCM control group [Table 5].
Table 5 : Effect of test drugs on liver tissue homogenate parameter

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Histopathological examination of liver

Sections from normal control rats exhibited normal cytoarchitecture [Figure 1]a and [Figure 1]b, whereas PCM administered rats showed chronic cell infiltration, fatty degenerative changes, and inflammation [Figure 1]c and [Figure 1]d. Sections of the liver from drug-treated groups showed mild to moderate fatty changes. AVR [Figure 1]e and [Figure 1]f and standard treated group [Figure 1]g and [Figure 1]h showed a better result than that of PCM-treated group.
Figure 1: Histopathological photomicrographs of representative sections of rat liver, (a) (1 × 100), (b) (1 × 400) normal control group showing normal cytoarchitecture, (c) (1 × 100), (d) (1 × 400) paracetamol control group at (3 g/kg PO) dose level showing chronic cell infiltration, fatty degenerative changes and inflammation, (e) (1 × 100), (f) (1 × 400) Arogyavardhini Rasa group at (90 mg/kg PO) dose level showing mild fatty degenerative changes, (g) (1 × 100), (h) (1 × 400) STD group at (100 mg/kg PO) dose level showing mild to moderate fatty degenerative changes

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  Discussion Top


In ayurvedic formulation where heavy metals are added as per the ayurvedic classical text, the presence of some heavy metals is not a contamination but is a part of any ayurvedic herbometallic formulation. Ayurvedic classical textbooks emphasize the role of Dhatu (metallic drug) in proper functioning of the human body. For that reason, a part is deliberately added and processed with drugs of plant origin to form herbometallic drugs.[28] Expert of Ayurveda has projected that near about 37% of about 600 medicinal formulations in the ayurvedic formulary contains at least one metal; thus, presence of heavy metals in these ayurvedic metallic minerals based preparation can still be a thousand-fold higher.[29] Metals and minerals used in Rasaaushadhi undergo huge change during processing (Shodhana, Marana, etc.). The mere less existence of a chemical amalgam compound of metallic source has nothing to do with the toxicity of finished product. These metallic formulations are used after various in-house process (samskaras) with numerous organic and inorganic resources and which is finally responsible for pharmacological activity in the body.

Effect of test drugs on liver function test

SGPT, a cytosolic enzyme, presents in the liver whose proportion in the liver is greater than that in the heart and skeletal muscle. SGOT, a mitochondrial enzyme, presents in huge quantity in the liver, heart, skeletal muscle, and kidney. SGOT, SGPT and ALP are elevated in most of the liver disorder condition causing extensive hepatic necrosis where the highest elevation is found ; such as severe acute viral hepatitis, toxic hepatitis or prolonged circulatory collapse. Here, in this study, similar substantial elevation in SGPT and SGOT level was observed after administration of PCM suggested the liver damage and inflammation of the liver in rats [Table 2]. This elevation was markedly reversed by AVR and silymarin. Hepatoprotection was observed more pronounced in the group treated with AVR. In case of serum ALP, nearly two-fold elevation in the PCM control group was found when compared to normal control. This elevation was markedly reversed by the standard drug, but not by test drugs. Total and direct bilirubin were markedly elevated in PCM control groups, while AVR produced a significant decline in the level of total and direct bilirubin and hence showed pronounced effect on bilirubin metabolism as compared to silymarin.

Effect of drug on lipid profile

The PCM control group showed a significant increase in triglyceride and cholesterol level when compared to a normal control group. This may be indicative of toxicant-induced cholestasis. This rise in serum cholesterol was markedly inhibited by AVR and standard group while triglyceride level was significantly decreased by test drug AVR. There was nonsignificant rise in the HDL-cholesterol level in the PCM group, which may be due to a gross increase in cholesterol level which was grossly reversed by AVR [Table 3]. Inflammation is known to reduce HDL levels and AVR has anti-inflammatory action. Hereafter, this might be the cause for elevated HDL level.[30] The current study also proved lipid lowering effect of test drug and reversed PCM-induced increments of lipids probably due to cholestasis secondary to hepatic cellular inflammation.

Effect of drug on kidney function

Serum creatinine is considered as a marker of kidney function. The toxicant PCM increases the creatinine level significantly in serum may be due to increase in muscle mass and/or impairment in kidney function. In the present study, AVR and standard drug produced nonsignificant decrease in serum creatinine level as compared to PCM, which may be due to early nephropathy. Urea is synthesized in the liver and excreted in kidneys through urine and is associated with acute renal disorders. Here, in this study, noticeable rise in the level of serum urea was found in PCM, whereas AVR-treated group showed the highest reversal effect as compared to PCM. In case of serum uric acid, a significant raise in the level of uric acid found in PCM group may be due to nephrotoxicity which was markedly attenuated by the trial drugs and reference standard drug [Table 4]. In a study, it was found that treatment with HgCl2 causes a considerable raised in serum creatinine and urea level, indicating an impaired renal function.[31] Hence, these reduced renal function parameters substantiate that trial drugs also exhibit nephroprotective properties although it contains mercury as one of its ingredients.

Effect of drug on tissue homogenate biochemical parameters

In case of lipid peroxide,[32] the concentration of MDA which was found to be increased in homogenate treated with PCM alone, which suggests enhanced LPO leading to cause tissue damage, failure of the antioxidant defense mechanism to keep away from development of excessive free radical. In this study, group treated with AVR nonsignificantly reverse these changes. SOD [33] was found to be reduced in homogenate treated with only PCM, which may be due to oxidative damage by PCM in rat liver. AVR with concentration of 90 mg/kg significantly increased action, therefore diminished reactive free radical-induced oxidative damage to the liver [Table 5]. Antioxidant study of Arogyavardhini vati at concentrations of 1% and 2% showed significant reduction of MDA concentration and showed significant improvement in glutathione and superoxide dismutase and they concluded that AVR possesses better free radical scavenging and antioxidant activities as that of Vitamin C.[34]

Effect of drug on histopathology of the liver

Microscopic examination of the liver sections from the AVR-treated group showed only mild fatty degenerative changes in comparison to the PCM control group. This clearly demonstrated protection of the liver by test drugs and more better with AVR from PCM-induced hepatotoxicity in rat's liver than the standard control group.

There may be several mechanisms, by which AVR exerted the beneficial effects in hepatoprotective action other than demonstrated which may have resulted in the pharmacological activity of the drug. Tamra Bhasma one of the ingredients has been proven as hepatoprotective [35]andalso beneficial in yakrut-pleeha vriddhi (hepato-spleen enlargement)[36] and in antihyperlipidemic.[37]Abhraka Bhasma[38] and Lauha Bhasma[39],[40] are proven therapeutic agents for hepatoprotective action in animal experiment. Picrorhiza kurroa, a major component of Arogyavardhini vati, has choleretic effects.[41]Katuki has an effective role in the treatment of Yakrit Gata Dosa (chronic hepatitis) and Kumbha Kamala. Katuki is mentioned as Pitta virechana. Pistachio, which is compared with gall bladder, is a part of hepatobiliary system. Deepana, Pachana, and Jvaraghna are among major therapeutic actions of AVR. Hence, it is postulated that it helps in the maintenance of the physiology and structure of yakrut (liver) as it is said to possess Rasayana (rejuvenating) property. Quercetine, Vitamin E are the strong antioxidants present in Neem leaf which may be responsible for hepatoprotective activity. Another study shows that methanol extract of Plumbago zeylanica significantly lowered the level of serum enzyme against liver damage by CCl4 in rats. The protective effect of AVR appears to rest on certain properties as that of standard drug silymarin, and it has the ability to increase the cellular content of SOD to regulate membrane permeability and increase membrane stability and against LPO as a result of free radical scavenging activity.

The study closely resembles acute hepatotoxicity; hence, the formulation may be tried to model mimicking chronic toxicity, as well as the duration of treatment with the drug under study (AVR) may be increased to exhibit comparatively more hepatoprotective effect.


  Conclusion Top


In this present study, AVR produces a good hepatoprotective action as well as nephroprotective in spite of its metallic content, especially mercury, which suggests that formulations containing metallominerals have the potential role in classical preparation, i.e., in ayurvedic formulation like AVR.

Acknowledgment

We are thankful to the Pharmacy, Gujarat Ayurved University, Jamnagar, for their help in procurement of raw materials, Dr. Harisha CR., Head of the Pharmacognosy Laboratory, for raw drugs authentication and staffs of Pharmacology Laboratory IPGT and RA for their help in animal studies and also like to acknowledge Prof. B. J. Patgiri, HOD, and Dr. Galib, Asst. Prof., Department of Rasashastra and Bhaishajya Kalpana, including Drug Research Institute for Postgraduate Research and Teaching in Ayurveda, Jamnagar, for their constant help and support to carry out the study.

Financial support and sponsorship

The study was supported by the Department of Rasashastra and Bhaishajya Kalpana including Drug Research Institute for Postgraduate Research and Teaching in Ayurveda, Jamnagar, Gujarat, India.

Conflicts of interest

There are no conflicts of interest.



 
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