Assessment of Hepatoprotective Activity of Rajata Bhasma in CCl¬4 Induced Hepatotoxicity Rats

Anand Prakash Rai; Shalini Tripathi; Om Prakash Tiwari

Rai A. P, Tripathi S, Tiwari O. P. Assessment of Hepatoprotective Activity of Rajata Bhasma in CCl4 Induced Hepatotoxicity Rats. Biomed Pharmacol J 2019;12(4).

Manuscript received on :08-Oct-2019
Manuscript accepted on :05-Nov-2019
Published online on: 22-11-2019

Plagiarism Check: Yes
Reviewed by: Hendry Irawan
Second Review by: Nicolas Padilla
Final Approval by: Dr Ian James Marti

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Assessment of Hepatoprotective Activity of Rajata Bhasma in CCl₄Induced Hepatotoxicity Rats

Anand Prakash Rai¹*, Shalini Tripathi² and Om Prakash Tiwari³

¹Institute of Pharmacy, H.C.P.G College, Bawan Begha, Varanasi (U.P.) Pin-221102

²Rameshwaram Institute of Technology and Management, Sitapur road, Lucknow (U.P.) Pin-227202

³Varanasi college of Pharmacy, Near Babatpur Airport, Varanasi (U.P.) Pin-221105

Corresponding Author E-mail: anandphd256@gmail.com

DOI : https://dx.doi.org/10.13005/bpj/1802

Abstract

Rajata Bhasma (RB) is an Ayurvedic formulation and used for the treatment of liver disorders. Till date scientific validation according to modern tool of RB has been not performed. Hence it was planned to evaluate the hepatoprotective activity of RB in CCl₄induced liver cirrhosis rats. The different formulation RB1 (9 puta) and RB2 (17 puta) were prepared by following classical methods. The SGOT, SGPT, ALP, ACP, total bilirubin and direct bilirubin in blood significantly enhance in CCl₄ treated rats compared to normal group rats. After treatment with the different doses of RB1 (50 mg/kg and 100 mg/kg) and RB2 (50 mg/kg and 100 mg/kg) significantly decreased the CCl₄ induced alteration in SGOT, SGPT, ALP, ACP, total bilirubin and direct bilirubin in blood. The RB1 and RB2 treated rats significantly increased the level of catalase, glutathione peroxidase, superoxide dismutase enzyme, whereas lipid peroxidation was decreased, when compared to CCl₄ treated group rats. Theses property confirmed the antioxidant properties of RB. The findings suggest that hepatoprotective activity of RB may be due to free radical scavenging property.

Keywords

Rajata Bhasma; hepatoprotective activity; antioxidant actiivit; CCl₄

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Introduction

Liver is the biggest organ in the vertebrate body and the site for exceptional digestion. Liver toxicity stay one of the genuine medical issues and are mostly brought about by toxic chemicals. In spite of the colossal advances made in allopathic prescription, no compelling hepatoprotective drug is accessible^1,2. The use of natural remedies for the treatment of liver diseases has a long history, starting with the Ayurvedic treatment, and extending to the Chinese, European and other systems of traditional medicines. The 21^st century has seen a change in outlook towards remedial assessment of Ayurvedic products in liver disease models via cautiously synergizing the qualities of the traditional systems of medicine with that of the modern concept of evidence-based medicinal evaluation, standardization and randomized placebo controlled clinical trials to support clinical efficacy³. A large number of Ayurvedic formulations have been claimed to have hepatoprotective activity.

Ayurveda, a traditional Indian System of Medicine, is accepted to in presence from days of yore. There are confirmations for the utilization of medications got from minerals, vegetable and animal products. It is accepted, that the teachings have been gotten from Lord Dhanvantari, which has taken its shape during the Vedic time and the substance referenced in Ayurvedic exemplary messages specifically Charaka Samhita and Susrutha Samhita. Charaka Samhita features the analysis of sickness, though Susrutha Samhita manages surgeries and careful devices. Aside from treating unpleasant maladies, Ayurvedic practices fit the prosperity of a person through Yoga practices and meditation⁴.

The bhasmas derived using several metals are known for their numerous therapeutic uses against dreadful diseases. For instance, Swarna bhasma (Gold based bhasma) has been indicated for many degenerative diseases. Tamara bhasma (Copper based bhasma) has been used to treat leucoderma, cardiac problems, liver and stomach related disorders. Rajata Bhasma (Silver based bhasma) has known for effect against diabetes, fever, anaemia and psychological disorders. Vanga bhasma (Tin based bhasma) is prescribed to patients suffering from diseases like diabetes mellitus, asthma, anaemia and gastric ulcers⁵. The present study was intended to evaluate hepatoprotective activity of two different samples acquired from Rajata Bhasma.

Material and Methods

Preparation of Rajata Bhasma

The Rajata Bhasma was prepared in two different form, RB1 prepared by 9 puta while RB2 prepared by 17 puta. The detailed procedure is given below⁶:

Rajat foil cut into small pieces and amalgam was formed with parade in mortar

Purified gandhaka was added to amalgam and triturated till formation of proper Kajjali

Followed by impreganation with kumara swarasa to preparation of Chakrikas (Pellets)

Dried chakrikas were placed in sharvana and laghu puta was given

After first puta, Rajata was in completely powder form

In subsequent two putas, half amount of kajjali was added, triturated with kumara swarasa and puta was given

From 4-9 puta, half part of gandhaka was added in place of kajjali (Considered as a RB1)

Remaining puta were followed without addition of Kajjali or Gandhaka

17 puta were given to obtain Rajat Bhasma that passing all classical parameter (Considered as a RB2)

Hepatoprotective activity of RB1 and RB2

Assessment of hepatoprotective activity was carried out on wistar albino rats. The animals were segregated into SIX groups of six animals each. Group I served as normal control receiving 5% CMC (10ml/kg). All other groups received CCl₄ (1ml /kg i.p.) with equal volume of olive oil (50% v/v) for two successive days. Group II animals were maintained as CCl₄ group, while Group III rats were administered RB1 (50 mg/kg body weight) orally, Group IV rats were administered RB1 (100 mg/kg body weight) orally, Group V rats were administered RB2 (50 mg/kg body weight) orally and Group VI rats were administered RB2 (100 mg/kg body weight) orally for seven days. After the drug treatment all the animals were sacrificed by cervical dislocation. Blood was collected from the carotid artery and was allowed to clot for 45 min at room temperature; serum was separated by centrifugation at 2500 rpm for 15 min, used for the estimation of various biochemical parameters.

Biochemical estimation

Biochemical parameters such as serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), acid phosphatase (ACP) and serum bilirubin were determined^7,8.

Analysis of antioxidant enzymes of liver tissue

The antioxidant activities in the rat liver homogenate were assayed for superoxide dismutase (SOD), Catalase (CAT) and Glutathione (GSH) and activity lipid peroxidation (LPO)^9-12.

Statistical analysis

The results are expressed as mean ± SD of six independent experiments. Statistical significance between the groups was evaluated by one-way analysis of variance (ANOVA) followed by Dunet’s test. A P < 0.05 value was considered as statistically significant.

Results and Discussions

Bhasma is sole of Ayurveda metal-based preparations made by following complex pharmaceutical processes incorporating herbs, converting them into a suitable form. They are preventive and complementary alternate medicine used in the Indian subcontinent since seventh century BC and widely recommended for treatment of a variety of chronic ailments. Conventional modern medicine is not always successful to control liver toxicity in all cases. Rajata Bhasma are safe to detoxify the liver toxicity.

Hepatoprotective activity of RB

Hepatoprotective activity of RB was determined in CC1₄induced hepatotoxicity rats. It was observed that the rats treated with CCl₄ significantly altered the biochemical parameters in serum compared to normal group of rats (Table 1). This indicates the severe damage of liver of rats.

The rats treated with different doses of RB1 (50 mg/kg and 100 mg/kg) and RB2 (50 mg/kg and 100 mg/kg) leads to significantly reduced the CCl₄ induced alteration in SGOT, SGPT, ALP, ACP, total bilirubin and direct bilirubin of blood. It was found that the RB1 and RB2 offer protection against toxin as evidenced by remarkable reduction in all biochemical parameters (P<0.05). The RB2 exhibited maximum hepatoprotective activity compared to RB1.

Table 1: Effect of RB1 and RB2 on liver function test for different parameters in animals treated with CCl₄

Treatment	SGOT (AST) (U/L)	SGPT (ALT) (U/L)	ALP (U/L)	ACP (U/L)	Bilirubin (mg/100 ml of blood)
Treatment	SGOT (AST) (U/L)	SGPT (ALT) (U/L)	ALP (U/L)	ACP (U/L)	Direct (mg/dl)	Total (mg/dl)
Normal rats	80.17±2.56	72.35±5.13	115.43±3.54	125.64±5.24	0.21±1.53	0.47±1.23
Control rats [CCl₄ (0.5 ml/kg i.p.)]	195.41±6.23*	223.18±4.18*	251.39±7.53*	242.69±6.24*	3.14±0.14*	5.47±1.19*
RB1 (50 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	91.36±6.32^a	92.13±2.65^a	162.53±4.23^a	156.24±6.54^a	0.51±1.63^a	0.71±0.18^a
RB1 (100 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	80.64±4.84^a	76.53±2.56^a	120.32±6.23^a	130.59±5.64^a	0.28±0.53^a	0.42±0.43^a
RB2 (50 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	96.72±4.51^a	89.12±7.22^a	141.75±5.74^a	147.24±6.31^a	0.45±0.62^a	0.63±1.34^a
RB2 (100 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	76.25±1.86^a	69.49±3.32^a	110.48±4.23^a	121.57±3.63^a	0.23±0.41^a	0.41±1.37^a

Values are expressed as mean ± SEM, n = 6 in each group. *P<0.05 when compared with normal group, ^aP<0.05 when compared with CCl₄ treated group considered as statistically significant.

Antioxidant activity

The findings of antioxidant study are shown in table 2. The administration of the RB1 (50 mg/kg and 100 mg/kg) and RB2 (50 mg/kg and 100 mg/kg) counteracted the CCl₄-induced free radical activity. The SOD, GPx and CAT enzyme levels were statistically significant increased, whereas lipid peroxidation was decreased, when compared to CCl₄ treated rats (P < 0.05). These results suggest that the RB1 and RB2 displays an antioxidant activity. The RB2 exhibited maximum antioxidant activity compared to RB1.

Table 2: Effect of RB1 and RB2 on oxidative stress induced by CCl₄ in the liver of experimental animals

Treatment	Enzymes involved in oxidative stress in liver
Treatment	LPO (Mole/gm)	SOD (U/gm)	GSH (μMole/gm)	Catalase (U/mg)
Normal rats	58.17±2.34	63.58±2.54	4.61±1.73	9.14±0.43
Control rats [CCl₄ (0.5 ml/kg i.p.)]	189.42±4.32*	12.51±6.24*	0.24±1.53*	0.92±0.96*
RB1 (50 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	88.73±2.46^a	40.58±1.32^a	3.71±1.86^a	7.15±1.35^a
RB1 (100 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	62.24±4.35^a	65.42±1.75^a	5.29±1.63^a	9.43±0.37^a
RB2 (50 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	82.36±1.23^a	42.79±3.74^a	3.89±0.32^a	8.11±0.75^a
RB2 (100 mg/kg)+ CCl₄ (0.5 ml/kg i.p.)	61.47±3.46^a	60.17±2.63^a	5.09±0.57^a	10.73±1.34^a

Values are expressed as mean ± SEM, n = 6 in each group. *P<0.05 when compared with normal group, ^aP<0.05 when compared with CCl₄ treated group considered as statistically significant

Liver participate in several metabolic activities, and in order to fulfill this role, release a wide variety of enzymes. Liver can be injured by many toxicants, as well as by chemicals or drugs. In our model, CCl₄ serves as a toxicant. CCl₄-related hepatotoxicity is associated with elevation in enzyme levels, which may be attributed to the generation of trichloromethyl free radical during metabolism by the hepatic microsomes, which in turn begin lipid peroxidation. Hepatocellular necrosis decreases SOD, CAT and GPx activities, and the increase of such activities into basal values, is a clear indication of plasma membrane stabilization and tissue repair as well. Such an effect is in agreement with the view that enzyme activities are restored into normal conditions and healing of the hepatic parenchyma, as well as hepatocyte regeneration, are observed. SOD, CAT, and GPx constitute an enzyme defense mechanism against oxidative damage. Under CCl₄ conditions such enzyme activities are decreased, but under RB1 and RB2 treated conditions, a significant increase in their activities is observed, which may serve as a biochemical strategy to reduce lipid peroxidation^1,2,8. The study revealed that the RB1 and RB2 under evaluation, at both studies doses, showed a hepatoprotective activity against CCl₄-induced liver damage. The present findings demonstrated that the RB1 and RB2 have free radical scavenging activity and were prominent in inhibiting the lipid peroxidation. The free radical scavenging properties may be one of the mechanism for protective effects of liver.

Conclusion

The RB1 and RB2 have resilient antioxidant activity and may confer a favorable effect against oxidative stress. The RB1 and RB2 exhibited significant hepatoprotective activity in rats intoxicated with CCl₄. Further the metal-based nanoparticles size of bhasma enhances the bioavailability of therapeutic efficacy. This properties assist the development of newer drugs in modern medicine of Ayurveda.

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Assessment of Hepatoprotective Activity of Rajata Bhasma in CCl­4 Induced Hepatotoxicity Rats

Assessment of Hepatoprotective Activity of Rajata Bhasma in CCl₄Induced Hepatotoxicity Rats