Ethanolic and aqueous extracts prepared from leaves of Coscinium fenestratum were evaluated for hepatotoxicity in cockerels. Acetaminophen was given @ 500 mg/kg b wt orally to induce hepatocellular damage. Ethanolic extract of C fenestratum @ 60 mg/kg b wt helped in restoration of Hb, PCV, TEC, TLC and lymphocytes and heterophils as well as total protein, albumin and globulin, glucose, cholesterol, bilirubin and activity of AST, ALT, ALP and LDH. Liver sections of treated animals clearly showed normal hepatic cells and central vein thereby confirming hepatoprotective activity. Phytochemical analysis of ethanolic extract showed presence of alkaloids, coumarin, flavonoids, glycosides, resins, sterol, tannin and triterpenes.
Liver constitutes undisputedly the lifeline system of animal which also plays a major role in the digestion, metabolism and utilization of feed nutrients. Hepatotoxicity is a common ailment resulting into various metabolic disorders or even mortality. Herbs play a major role in the management of various liver disorders along with other system associated diseases and some plants are reported to possess hepatoprotective property. Coscinium fenestratum has mainly been used for treating diabetes mellitus (Jittaprasatsin et al., 2005; Punitha et al., 2005; Shirwaikar et al., 2005). Its stem is used for dyspepsia and as a febrifuge while hypotensive (Singh et al., 1990) and hepatoprotective (Venukumar and Latha 2004) properties have also been reported. Antioxidant (Venukumar and Latha, 2002) and antibacterial (Nair et al., 2005) activities are also recorded. The present study was planned to record the hepatoprotective action of C. fenestratumextracts in experimental hepatotoxicity in cockerels.
Material and Methods
The leaves of C. fenestratum were procured from Medicinal Research and Development Center, Pantnagar and were shade dried and ground in a Willey Grinder at room temperature. For preparation of the ethanolic or aqueous extract, 100 gm powder of C. fenestratum was soaked in 1000 ml of absolute ethanol or water for 48 hr at 370C with continuous stirring. The contents were filtered and concentrated by evaporation at lower temperature (45-50°C) and reduced pressure using rotatory vacuum evaporator (Singh et al., 2001) and lyophilized to get the final extract residue and stored at 40C in for further use.
Ethanolic and aqueous extracts of C. fenestratum were analysed for major phytochemical groups, viz. alkaloids, anthraquinones, flavonoids, saponins, tannins, sterols, reducing sugars, glycosides, resins, triterpenes, proteins and coumarins by standard methods (Das et al., 1964, Harborne, 1973, Sofawara, 1982 and Arunadevi, 2003). One hundred cockerels belonging to same hatch and aged three months, were procured from Instructional Poultry Farm of University, Pantnagar and randomly divided into five groups I, II, III, IV and V of 20 each. All the groups had almost equal average body weight and maintained under standard deep litter managemental conditions. Gr I served as healthy control and Gr II received acetaminophen @ 500 mg/kg b wt orally for 7 days (Bhar et al., 2009) served as infected control. Gr III received silymarin (as a standard reference) along with acetaminophen for 7 days and thereafter only silymarin upto 35th day. In cockerels of Gr IV and Gr V ethanolic and aqueous extracts of C. fenestratum were given @ 60 mg/kg b wt along with acetaminophen for 7 days and thereafter only extract were given upto 35th day.
The blood samples were collected on day 0, 7, 15, 21, 28, 35 and 42 of treatment, for haematological (Hb, TEC, TLC, PCV and DLC) biochemical (glucose, total cholesterol, total protein, albumin, globulin, albumin: globulin ratio, blood urea nitrogen and serum bilirubin) and activities of liver enzymes (AST, ALT, ALP, LDH). Liver samples were collected in 10% buffered formalin for histopathological study and examined for any type of gross changes from each group on 7th, 21st and 35th day of treatment. The formalin fixed tissue pieces were serially dehydrated in alcohol and acetone, embedded in paraffin blocks and sections were cut and stained in hematoxyline and eosin stain for histopathological examination by standard procedures. The results were analysed as per the method described by Snedecor and Cochran (1994).
Results and Discussion
The ethanolic extract residue of C. fenestratum was yellowish dark brown in colour and oily in consistency with 6.58% yield, whereas its aqueous extract was yellowish brown in colour and solid, powder in consistency with 5.64% yield. On phytochemical analysis, ethanolic extract revealed the presence of alkaloids, anthraquinones, flavonoids, glycosides, reuducing sugar, resins and saponin. Aqueous extract also revealed presence of these groups with absence of resin and tannin and presence of protein. Significant decrease in Hb and PCV values were observed in Gr II as compared to Gr I, III, IV and V from 7th day onward up to the end of experiment (Table 1).
Table 1: The value of Hb, PCV, TEC, TLC, lymphocytes and heteropphils in cockerels treated with Coscinium fenestratum
|Haematological||0 day||7th day||28th day||42nd day|
Significant decrease in TEC and lymphocytic values and significant increase in TLC and heterophils values were observed from 7th day onward upto the end of experiment in Gr II as compared to other groups (Table 1). Ethanolic and aqueous extracts significantly restored these values to normalcy. The prominent reduction of Hb, PCV and TEC in acetaminophen treated birds in could be attributed to the toxic metabolite of acetaminophen, N – acetyl – p – benzoquinone, which was reported to cause oxidative stress and haemolysis of erythrocytes and hepatocytes leading to regenerative anaemia with methmoglobin and Heinz body formation (Vijayakumar and Latha, 2004). Disintegration of erythrocytes in the circulation might have resulted in reduction of Hb content of blood, which in turn was associated with decrease in PCV and TEC (Chauhan et al., 2008). It shows that extracts of C. fenestratum protects the disintegration of erythrocytes (Agusta, 2003). Increased TLC values might be due to stress coupled with inflammatory changes in body tissue, which is responsible for phagocytosis of toxic substances and neutrophilia was induced by tissue demand for phagocytic function (Duncan and Prasse, 1977). An increase in heterophil count and decrease in lymphocyte count was also reported by Hedau et al. (2008) as was observed in present study. Venukumar and Latha (2002) also found the restoration of TLC with the administration of C. fenestratum.
There was significant decrease in the levels of glucose, bilirubin and cholesterol levels in Gr III, IV and V (Table 2). Total proteins and albumin showed a marked reduction after induction of hepatopathy in untreated group from 7th day till end of experiment (Table 2). Significant increase in the globulin values was recorded in all the treated groups. Increase in glucose and cholesterol may be due to the degenerative hepatic lesions and the altered values in the cholesterol might be due to the interference with the metabolism of fat in the liver. Over dosage of acetaminophen leads to increased generation of reactive oxygen species (ROS) due to excessive production of NAPQI (James et al., 2003; Yen et al., 2007). The high level of ROS is known to cause impairment of antioxidant enzyme activities and substantially leads to oxidative stress. Oxidative stress in turn induces various deleterious actions including lipid peroxidation, which is characterised by oxidative degeneration of membrane (Yen et al., 2007). Chinnaiah (2002), Jittaprasatsin et al. (2005), Shriwaiker et al. (2005) also observed that C. fenestratum lowered the glucose, bilirubin and cholesterol level. Serum protein contents were markedly decreased after paracetamol administration, as shown in the current investigation. Such depletion may be due to be a direct oxidation of the thiol groups of contiguous amino acids with the formation of protein-protein disulphides (Kyle et al., 1990). Globulins are intermediate proteins which are involved in antibody formation. Its higher level in C. fenestratum fed group may be correlated with the immunomodulatory property of the herb (Shirode and Rao, 2008). Due to the damage of hepatocytes, there was decreased elimination of bilirubin and thus an increase was observed.
Table 2: The value of glucose, cholestrol, total protein, albumin, globulin and A : G ratio in cockerels treated with Coscinium fenestratum
|Biochemical||0 day||7th day||28th day||42nd day|
Increased activities of ALT, AST, ALP and LDH in Gr II reflect the damage of liver hepatocytes and impairment of liver functions. The largest pool of ALT is found in the hepatic parenchymal cells of the liver (Amacher, 1998). Extracts of C. fenestratum significantly reduced the elevation of these enzymes and it was also similar to the level of group treated with silymarin (Table 3). Hepatoprotective effect of C. fenestratum is evidenced by the improvement of ALT, AST, ALP and LDH levels. Manokaran et al. (2008) also reported hepatoprotective effect of this plant.A significant decrease in feed consumption and body weight was observed in Gr II as compared to Gr I, III, IV and V from 14th day onward till end of experiment. Body weight increased in Gr IV at 42nd day of treatment as compared to other groups which might be due to increase in function of hepatocyte and increased feed intake.
Table 3: The activities of AST, ALT, ALP and LDH in cockerels treated with Coscinium fenestratum
|Biochemical||0 day||7th day||28th day||42nd day|
The biochemical results were comparable to the histopathological analysis of liver sections in each group. The healthy control birds revealed normal cellular architecture with sinusoidal spaces and central veins while, intoxicated cockerels revealed centrilobular hepatic necrosis. The changes in the mild form of degeneration were characterized by the presence of edema, dilated sinusoids, swollen hepatocytes, thickness of hepatic arteriols associated with infiltration of hetrophils and mononuclear cells and peripheral lymphoctic infiltration. When C. fenestratumextract was given with acetaminophen, significant decreases in hepitocellular damage were observed. Similar observation was seen in silymarin treated group.
These results indicated that C. fenestratum has hepatoprotective action. It increases the Hb, PCV, TEC, lymphoctes, total protein, albumin, globulin and decreases glucose, total cholesterol, bilirubin, AST, ALT, ALP and LDH values to normalcy.
The authors are thankful to the Dean, College of Post Graduate Science, Dean, College of Veterinary and Animal Sciences and Director Experiment Station, G.B. Pant University of Agriculture and Technology for providing necessary facilities to carry out this research work.