1.2Department of Veterinary Pharmacology and Toxicology, 3Department of Veterinary Pathology, 1N. T. R College of Veterinary Science, Gannavaram, 2,3College of Veterinary Science Tirupati, Andhrapradesh
Received: 22 Jan 2018, Revised and Accepted: 15 Apr 2018
Objective: To study the hepatoprotective activity of an aqueous and alcoholic extract of Acorus calamus rhizomes against the paracetamol induced hepatotoxicity in rats.
Methods: Hepatotoxicity was induced by oral administration of paracetamol and chemical parameters such as Glutathione peroxidase, Glutathione reductase, Glutathione, Catalase, lipid peroxidation and histopathological changes in liver was studied by comparing with Silymarin, a standard hepatoprotective drug.
Results: Treatment of rats with aqueous and alcoholic extract of Acoruscalamus rhizome after paracetamol administration normalized the altered levels of above parameters which may comparable with Silymarin and Vit-E. The hepatoprotective activity was confirmed by histopathological examination of the liver tissue of control and treated animals.
Conclusion: Based on the result it can be concluded that Acorus calamus rhizome possesses hepatoprotective effect against paracetamol-induced hepatotoxicity in rats.
Keywords: Acorus calamus rhizome, Paracetamol, Silymarin, Vit-E, Hepatoprotection
© 2018 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
Hepatotoxicity is one of the major adverse effects produced by many of the drugs. Hepatic disorders are the most common and serious health problems in animals and human beings. Drugs accounted for 50% of the liver failure out of which 39% is due to acetaminophen .
Acorus calamus is a semiaquatic perennial aromatic herb with creeping rhizomes belonging to the family Araceae. It is commonly called as vacha, vasa, sweet flag etc. It is commonly used to protect the children from kapha disorders and for the improvement of the intelligence and as a memory enhancer. The rhizome powder of A. calamus is used in the training of talking birds. The medicated oil of calamus roots is used externally for massages to relieve vata and kapha disorders .
The rhizomes of A. calamus possess spasmolytic , ectoparasiticide, insect repellant , anti-secretagogue, antiulcer and cytoprotective , antidiarrheal , hypolipidemic , anthelmintic and antibacterial , neuroprotective , larvicidal , antioxidant , bio pesticide , antiproliferative and immunosuppressive  anticonvulsant  and antifungal , properties.
However, a perusal of available literature revealed no reports on the antihepatotoxic potential of Acoruscalamus extract of the rhizome. Hence the present study is designed to screen the ethanolic and aqueous extract of the rhizome of Acoruscalamus for its hepatoprotective effect in male rats.
Paracetamol IP was procured from M/S Granules India as gift sample, Silymarin was procured from Microlabs Bangalore as gratis. All other chemicals used in the study were of analytical grade and procured from a standard supplier.
Collection and identification of plant material
Whole plant of Acoruscalamus was collected from the local market and surrounding areas of Tirupati, Andhra Pradesh, India. The plant was identified and authenticated by the herbarium specialist, Department of Botany, S. V. University, Tirupati.
Preparation of Alcoholic extract of Acoruscalamus rhizome
Acoruscalamus rhizomes were dried in shade, later they were powdered and extracted (1.5 kg) successively with 30 liters of 60% alcohol in a soxhalet extractor for 18-20 h. The extract was distilled and concentrated to dryness under reduced pressure and controlled temperature (40-50 °c) and finally freeze-dried. The ethanolic extract yielded a weight of 150 g (10% w/w).
Preparation of Aqueous extract of Acoruscalamus rhizome
The dried rhizomes of Acoruscalamuswere powdered and the powdered material was taken in a round bottom flask and was extracted with water for 48 h at room temperature. After 48 h, the solution was concentrated in a rotatory evaporator. Aqueous and alcoholic extract of Acoruscalamus was suspended in 0.5% CMC.
Table 1: Experimental design
|Group I||0.5% Carboxymethylcellulose p. o for ten days|
|Group II||Paracetamol @ 2g/kg p. o on Day one+0.5% Carboxymethylcellulose p. o for ten days|
|Group III||Paracetamol @ 2g/kg p. o on Day one+Ethanolic extract of Acoruscalamus rhizome(600 mg/kg) p. o for ten days|
|Group IV||Paracetamol @ 2g/kg p. o on Day one+Aqueous extract of Acoruscalamus rhizome (600 mg/kg) p. o for ten days|
|Group V||Paracetamol @ 2g/kg p. o on Day one+Silymarin @ 25 mg/kg p. o for ten days|
|Group VI||Paracetamol@ 2g/kg p. o on Day one+Vitamin E(30 mg/kg) p. o for ten days|
Male albino rats of wistar strain weighing 150-200g were obtained from Department of Laboratory Animal Medicine, TANUVAS, Madhavaram milk colony, Chennai. The animals were maintained under standard laboratory conditions with food and water adlibitum. Approval of the experimental protocol was obtained prior to the conduct of the experiment from the institutional animal ethics and biosafety committee. The experiment was conducted in Department of Pharmacology and Toxicology, College of Veterinary Science, Tirupati.
Twenty four hours after the last day of treatment blood was collected from all the experimental animals by retrobulbar plexus puncture under ether anesthesia and whole livers were collected after sacrificing the animals by decapitation. Blood samples were used for the estimation of RBC enzyme profile viz Glutathione Peroxidase (GPx) Activity: , Glutathione Reductase activity: Spectrophotometric method–, Erythrocytic Catalase activity: , Lipid peroxidation activity from hepatic tissue homogenate Thiobarbituric acid reactive substances The liver specimens obtained from control and treated groups were fixed in 10% formalin ad stained with H and E stain.
The data were subjected to statistical analysis by applying one way ANOVA as per the standard methods of Snedecor and Cochran . Differences between means were tested using Duncan’s multiple comparison tests and significance was set at P<0.05 and P<0.01.
Table 2: Effects of alcoholic and aqueous extracts of A. calamus rhizome on erythrocyte antioxidant system (Glutathione peroxidase (U/ml), Glutathione reductase (U/ml), catalase (moles/sec)
|S. No.||Title||Glutathione peroxidase (U ml-1)||Glutathione reductase (U ml-1)||Catalase (moles sec-1)|
Table 3: Effects of alcoholic and aqueous extracts of A. calamus rhizome on glutathione (mg/dl) levels in serum
|S. No.||Title||Glutathione (mg dl-1)|
One way ANOVA, the values are mean±SE, n=6, Different superscripts a, b, c, d are statistically significant at P<0.001 and P<0.005
Table 4: Effects of alcoholic and aqueous extracts of A. calamus rhizome on lipid peroxidation in hepatic tissue (nM MDA/g tissue)
|S. No.||Title||Lipid peroxidation (nmol g-1)|
One way ANOVA, the values are mean±SE, n=6, Different superscripts a, b, c, d are statistically significant at P<0.001 and P<0.005
Group 1: Normal architecture of hepatic cells, H and E x 70
Group 2: Perivascular infiltration of mononuclear cells, H and E x 70
Group 5: Normal architecture of hepatic cells, H and E x 70
Group 2: Section showing sinusoidal haemorrhages, H and E x 70
Group 6: Mild degenerative changes in liver parenchyma, H and E x 70
Group 3 and 4: Mild proliferation of bile duct, H and E x 70,
Fig. 1: Histoarchitecture of the liver in different treatment groups
In the present study, the levels of glutathione peroxidase were reduced in group 2 when compared to group 1 [20, 21]. Silymarin prevented the reduction of glutathione peroxidase by protecting the glutathione depletion. Acoruscalamus prevented the altered glutathione peroxidase levels due to its antioxidant activity .
Glutathione reductase activity was reduced in group 2 which are in accordance with the other research findings on hepatotoxicity studies . Vitamin E antagonized the glutathione reductase levels due to its antioxidant activity . Acoruscalamus attenuated the altered glutathione reductase level due to its antioxidant activity .
Catalase, primarily antioxidant defence component is a tetrameric hemoprotein present in peroxisomes and catalyzes the dismutation of H2O2 reduces methyl and ethyl hydroperoxides. The liver, kidney and RBC possess relatively high levels of catalase .
The catalase activity in the paracetamol treated group was reduced significantly (P<0.01) when compared to control group which is in correlation with the reported data [27, 28]. Silymarin normalized the altered catalase levels . Vitamin E increased catalase levels due to its antioxidant property . Acoruscalamus prevented the reduction in catalase activity. In the present study glutathione levels were reduced significantly (P<0.01) in paracetamol toxic group when compared to the control group [20, 27]. Silymarin antagonized the depletion of glutathione due to its free radical scavenging activity. Alcoholic extracts of Acoruscalamus showed marked protective effect than aqueous extract in glutathione depletion. Some of the studies reported that concurrent administration of Acoruscalamus extract with acrylamide significantly increased the glutathione levels in corpus striatum of rats . The protective action of Vitamin E may be due to its antioxidant activity and also due to its increased red cells level of reduced glutathione .
Lipid peroxidation serves as a marker of oxidative stress. Lipid peroxidation is oxidative deterioration of polyunsaturated lipids and it involves ROS and transition metal ions. It is a molecular mechanism of cell injury leading to the generation of peroxides and lipid peroxides, which can be decomposed to yield a wide range of cytotoxin products, most of which are aldehydes such as MDA. A substantial increase in hepatic lipid peroxidation was evident by elevated MDA level in liver homogenate of the paracetamol administered group [20, 21]. There is a direct correlation between glutathione depletion and lipid peroxidation. The elevated levels of MDA were suppressed by Silymarin. Silymarin reduced the MDA levels in tissue due to its antioxidant activity. Vit. E inhibits the lipid perodixation due to its antioxidant activity. Acoruscalamus reduced the paracetamol-induced lipid peroxidation.
The results of the present study revealed that treatment with aqueous and alcoholic extracts of Acoruscalamus normalized the activity of lipid peroxidation in liver tissue. The present study revealed the anti-lipid peroxidation activity of Acoruscalamus against paracetamol-induced lipid peroxidation in rats. Hence, the Acoruscalamus rhizomes can be used in the treatment of liver disorders after a detailed investigation of active compounds and the exact mechanism involved in the hepatoprotective activity.
In the present study, histopathological findings such as degeneration of hepatic cells sinusoidal haemorrhages and bile duct proliferation were noticed in group II [20, 31]. Treatment with silymarin (group V), Vit. E (group VI) and Acoruscalamus (Group III and IV) preserved the structural integrity of the hepatic cellular architecture.
The present study demonstrates the antioxidative capacity of Acoruscalamus. The antioxidative effect inturn could have protected the decrease of GSH and other enzymes restoring the free radical scavenger’s enzymatic activity. Thus, the use of Acoruscalamusmay be recommended for lifestyle-related diseases including hepatic diseases and also to improve the general health condition in animals including human beings. Extensive studies in target species should be preceeded before the clinical application of Acoruscalamus as an antioxidant agent.
All the author have contributed equally
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