Hepatoprotective activity of Parmelia perlata ( Huds . ) Ach . against CCl 4 induced liver toxicity in Albino Wistar rats

Article history: Received on: 15/01/2014 Revised on: 12/02/2014 Accepted on: 24/02/2014 Available online: 27/02/2014 Many plants have been claimed to possess liver protecting activity. But, due to scarcity of potent modern medicine to treat severe liver diseases, many folk remedies of plant origin have been scientifically evaluated for their potential hepatoprotective activity in experimental animal models. Parmelia perlata (Huds.) Ach. (lichen, Parmeliaceae), commonly known as Dagad phool or stone flower in India, is mentioned in Indian Materia Medica as useful in treating a number of ailments. In the present study, hepatoprotective activity of aqueous slurry of P. perlata was investigated in CCl4 intoxicated Albino Wistar rats and the results were compared with, Silymarin. Intraperitoneal injection of CCl4, produced a marked elevation in the level of biochemical markers such as Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), bilirubin, cholesterol and triglycerides. Oral administration of P. perlata aqueous slurry at 0.7 g / kg and 1.0 g / kg in CCl4 intoxicated rats showed marked decrease in the level of biochemical markers and results were at par with the effect shown by Silymarin. The results of histopathological analysis were in compliance with the findings of blood biochemical parameter analysis. This study confirms that P. perlata aqueous slurry has phytocomponents with hepatoprotective potential.


INTRODUCTION
The prevalence of hepatopathy in the world indicates an increased incidence rate with severe consequences on the liver (Adefolaju et al., 2009).Herbal drugs are more widely used than allopathic drugs as hepatoprotectives because they are inexpensive, have better cultural acceptability, better compatibility with the human body and minimal side effects.Nearly 150 phytoconstituents from 101 plants have been claimed to possess liver protecting activity (Subramoniam and Pushpangadan, 1999).Therefore, many folk remedies from plant origin are being tested for their potential hepatoprotective activity in experimental animal models (Ganga Rao et al., 2012).One of the commonly used invivo models for investigating new hepatoprotective agents is liver injury induced by carbon tetrachloride (CCl 4 ), a chemical hepatotoxin that causes a free radical-mediated hepatic cellular damage (Chen et al., 2009;Roy et al., 2011).Several plants like Solanum xanthocarpum (Gupta et al., 2012), Spillanthes acmella ( Ganga Rao et al., 2012), Ardisia solanacea ( Samal, 2013 ), Adiantun incisum (Frank et al., 2012) etc. have been reported to possess hepatoprotective activity against CCl 4 induced hepatotoxicity.Similarly, some lichens like Usnea ghattensis have been reported to possess antioxidant and hepatoprotective activity against ethanol induced hepatotoxicity (Verma et al., 2008).
Parmelia perlata (Huds.)Ach.(lichen, Parmeliaceae), commonly known as Dagad phool or stone flower in India, is mentioned in Indian Materia Medica as useful in treating a number of ailments (Halama and Haluwin, 2004).It is collected and consumed in large quantities as a food supplement in India.P. perlata is used to treat wounds, infections, inflammation, skin diseases, diarrheoa, dysentery, cough, fever and renal calculi (Tay et al., 2004).
It has also been reported to possess antimicrobial (Thippeswamy, 2012), Anti-viral (Esimone et al., 2007), antioxidant activity (Patil et al., 2011) along with its analgesic, antipyretic (Muller, 2001) and antispasmodic potential (Correia, 1976).Thus, the present study is aimed to evaluate the hepatoprotective activity of aqueous slurry of P. perlata against CCl 4 induced hepatotoxicity in rats.The efficacy has been compared with the known hepatoprotectant, Silymarin.

Plant material
P. perlata was collected from local market (Thane, Maharashtra) and the herbarium of the sample was authenticated (AUTH/HRL/12-04).Sample was carefully segregated, cleaned and oven dried at 37° C to constant weight, powdered, sieved (BSS 85) and stored in airtight containers.

Drugs and chemicals
CCl 4 (GR grade, Batch no.: IG8G580178, Merck Specialties Pvt.Ltd) and Silybon tablets (Silymarin as silybin 70 mg, Batch no.SIAD0025, Micro Labs Limited) were procured from market.All other chemicals used were of analytical grade.

Quality control
The quality of the sample was evaluated for foreign matter, total ash, acid insoluble ash, alcohol soluble extractive and water-soluble extractive.The results were compared with those mentioned in API.

Animals
Adult albino Wistar rats (female 200-250 g) and Swiss Albino mice (18-22 g) procured from Haffkin Biopharmaceuticals, Parel, Mumbai were used in this study.The animals were maintained under standard laboratory conditions at an ambient temperature of 25 ± 2º C with 12-h light and dark cycle in an animal house with standard facilities under CPCSE approvals.They were fed with rat feed (AMRUT feed) and water ad libitum.

Preparation of aqueous slurry
Aqueous slurry of P. perlata was prepared by mixing appropriately weighed powdered material in distilled water.The animals were dosed individually as per their body weights.

Safety evaluation
Safety study of P. perlata aqueous slurry was conducted in mice as per OECD guidelines (No. 420, fixed dose procedure).The mice were fasted overnight for 10-14 hours and administered with aqueous slurry of P. perlata (2.0 g/kg) orally.The animals were observed individually during the first 30 min for all reflexes, periodically during the first 48 hours with special attention given during the first 4 hours (short-term toxicity) and daily thereafter for a total of 14 days (long-term toxicity) for alteration from general behavior and clinical symptoms like alteration of skin and fur texture, ptosis, excessive salivation, breathing problems, diarrhea etc. Daily body weight, food and water intake record was also maintained.The results were compared with control group (orally administered with DW).

Assessment of hepatoprotective activity against CCl 4 induced liver intoxication
Carbon tetrachloride intoxication in rats is an experimental model widely used to study necrosis and pathophysiological status of liver (Rao et al., 2012).In this study, albino Wistar rats were randomly divided into 6 groups with six animals in each.The group details are as follows:  Group I: Normal control  Group II: CCl 4 control (1.2 ml/kg)  Group III: CCl 4 control as natural recovery group  Group IV: CCl 4 control treated with daily dose of Silymarin (0.070 g/kg b.w.)  Group V: CCl 4 control treated with daily dose of P. perlata aqueous slurry (0.7 g/kg b.w. )  Group VI: CCl 4 control treated with daily dose of P. perlata aqueous slurry (1.0 g/kg b.w. ) The animals were fasted overnight before the initiation of the study.Animals from Group I received an intraperitoneal injection of 0.5 ml liquid paraffin/animal on the first day of the study and were treated as normal control.Those from Groups II, III, IV, V and VI received an intraperitoneal injection of 1.2 ml/kg CCl 4 (Koh et al., 2011;Nayak et al., 2008) in 0.5 ml liquid paraffin/animal on the first day of the study.The animals from Groups I, II and III received an oral dose of 2 ml of distilled water (D/W) once daily.A dose of 0.07 g / kg Silymarin (Silymarin clinical update, 1995) (in the form of Silybon tablets) suspended in 2 ml of D/W was administered orally to each animal of Group IV once daily and one hour post induction on the first day.The animals from Group V and VI received an oral dose of P. perlata aqueous slurry (0.7 g / kg and 1.0 g / kg respectively) / animal daily and one hour post induction on the first day.The animals from Groups I, II, IV, V and VI were sacrificed on the fourth day (72 hr after dosing) and those from Group III were sacrificed on seventh day of the study for comparative evaluation of the natural recovery in the study.Daily record of body weight and food and water intake was also maintained.
Prior to the sacrifice, 2 ml blood was collected from retro orbital plexus in non-heparinized vials.Blood was centrifuged at 5000 rpm for 8 mins and serum was separated.Biochemical parameters like Serum Glutamate Oxaloacetate Transaminase (SGOT), Serum Glutamate Pyruvate Transaminase (SGPT), Total Bilirubin, Cholesterol and Triglycerides were analyzed from the serum samples obtained.During autopsy, liver was excised, rinsed in saline, blotted and weighed.A small piece of liver tissue from the largest lobe was cut and fixed into bouin's fixative.Fixed tissues were processed for routine haematoxylin and eosin staining and evaluated.The extent of liver recovery was compared with known hepatoprotectant, Silymarin.Percent protection in individual biochemical parameters from their elevated values caused by the hepatoprotection was calculated as 100 × (values of CCl 4 Control -values of sample) / (values of CCl 4 control -values of vehicle) (Ganga Rao et al., 2012).

Statistical analysis
All values were expressed as mean ± S.E and statistically analyzed for significance using Dunnett's test, comparing group I with the other groups using graph pad prism 5 software.P≤0.05 was considered as statistically significant.

RESULTS AND DISCUSSION
Medicinal plants and their derivatives have been used since ancient time in various forms for the treatment of liver disorders.The medicinal use of lichens can be traced back to the 18 th dynasty (1700-1800 BC) when Evernia furfuracea was first used as a drug (Launert, 1981).Herbal medicinal texts included account of several species of lichen including Cladonia, Evernia, Lobaria, Parmelia, Peltigera, Pertusaria, Physica, Rocella, Usnea and Xanthoria (Perez -Llano, 1944).Many such lichens have been scientifically evaluated for various therapeutic activities such as antimicrobial, anti-tumor, antioxidant, immunomodulatory, tyrosinase-inhibitory etc (Malhotra et al., 2008).Hepatoprotective activity of the lichen Usnea ghattensis has been established in vitro (Verma et al., 2008).Though P. perlata has been reported to possess therapeutic activities such as antioxidant, antimicrobial etc, there exist no scientific evidence on its in vivo hepatoprotective activity.Thus, in the present work, aqueous slurry of the lichen P. perlata was evaluated as an alternative cure on CCl 4 induced hepatotoxicity in rats.The quality of P.perlata was found to be as per the prescribed limits in API for parameters like foreign matter, total ash, acid insoluble ash, alcohol soluble extractive and water soluble extractive.The safety of the drug was established by acute oral toxicity study carried out on rats at 2.0 g / kg body weight.The lichen was found to be safe as it showed no abnormal fluctuation in body weights and food and water intake of the animals.Clinical symptoms of toxicity were also found to be absent during the period of the study and no mortality was recorded.The safety study of the lichen revealed that the in form of aqueous slurry, it can be considered safe with a wide margin for oral use.CCl 4 induced hepatotoxicity has been chosen as the experimental model since the changes associated with the CCl 4 induced liver damage are similar to those of viral hepatitis (Contran et al., 1994).
Carbon tetrachloride is commonly used for inducing liver damage because it causes peroxidative degeneration in adipose tissue and is metabolized to trichloromethyl radical and trichloromethyl peroxy radicals which are involved in pathogenesis of liver (Frank et al., 2012;Recknagel, 1983).CCl 4 metabolites react with polyunsaturated fatty acids and form covalent adducts with lipids and proteins.These events lead to lipid peroxidation and destruction of cell membranes with the consequent liver injury (Clawson, 1989;Recknagel et al., 1989).Hepatic damage induced by CCl 4 results in an increase in the level of biochemical parameters like SGOT, SGPT, TG, CHO and TB (Rubea et al., 2011;Frank et al., 2012;Shailajan et al., 2005;2007).CCl 4 induction also causes classical fatty liver as indicated by significant increase in cholesterol.Group II showed marked increase in all biochemical parameters analyzed and showed severe loss of hepatic architecture with intense peripheral and central vein necrosis, fatty changes and crowing of the central vein histopathologically [Fig 1(b)].The established standard drug silymarin was used as modern control.Group IV animals were treated with Silymarin, at a dose of 0.07 g/kg.Silymarin treated rats showed marked recovery in biochemical parameters (SGOT, SGPT, TB, CHO and TG) and offered percentage protection of 64.74%, 58.42 %, 87.92%, 90.04% and 83.58% respectively.Histopathological findings supported the biochemical data as the treated animals showed near normal hepatic architecture with mild degree of necrosis that signifies recovery and protective effect of the drug [Fig 1(d)].
Animals from the groups V and VI were treated with aqueous slurry of P. perlata orally at doses of 0.7 g/kg and 1.0 g/kg respectively.The percentage protection offered by the slurry in terms of the reduction in the level of SGOT, SGPT, TB, CHO and TG levels was found to be 37.27%, 44.56%, 75.83%, 77.00% and 66.04% 0.7 g/kg and 53.38%, 64.79%, 106.61%, 99.45% and 72.10% at 1.0 g/kg.The histopathological results also showed marked recovery in the hepatic architecture and reduction in liver damage and cellular necrosis.P. perlata slurry at higher dose showed better results in terms of percent protection and histological findings when compared with the lower dose [Fig 1 (e  and f)].
Reduction in food and water consumption coupled with the decrease in body weight after CCl 4 treatment indicates toxic response, whereas any significant increase observed in these parameters after plant treatment indicates its protective action.Elevated levels were observed in the CCl 4 induction group as per the results obtained from biochemical parameters (Table 1), which indicate CCl 4 induced damage to the liver.The significant reductions in the levels of SGPT, SGOT, BIL, CHO and TG in case of Silymarin control and in the groups treated with the aqueous slurry of P. perlata suggest possible stabilization of the plasma membrane and the repair and recovery of hepatic tissue damage caused by CCl 4 intoxication (Thabrew et al.,1987).This may also imply that the serum levels of transaminases return to normal with the healing of hepatic parenchyma and the regeneration of hepatocytes (Anusha et al., 2011) All the groups except group III (Natural recovery) were sacrificed on day 4. Group III was sacrificed on 7 th day of the study.Percent reduction in biochemical parameters (SGOT, SGPT, BIL, CHO and TG) was found to be 20.59%,15.40%, 43.96%, 52.82% and 6.28% respectively.
Liver sections of the animals in this group showed similar results as to induction group [Fig 1(c)].From the observations of recovery group, it can be interpreted that as animals did not show significant reduction in biochemical parameters even after the extended recovery period of 7 days, the recovery rate is enhanced significantly by the administration of silymarin and P. perlata.Thus, the present study compares the activity of aqueous slurry of P. perlata at 0.7 g/kg and 1.0 g/kg with the established drug silymarin in CCl 4 intoxicated rats.The results confer that P. perlata shows hepatoprotective activity in a dose dependant manner and the protection shown by the aqueous slurry is at par with that of Silymarin.

Table . 1
: Biochemical estimation from serum: All values are Mean ± SEM.