Investigations on Hepatoprotective Potential of Centratherum Anthelminticum in Paracetamol and Carbon Tetrachloride (CCI4) - Induced Liver Injury

Abstract

A vast range of liver diseases are increasing day by day that also enhancing the rate of hospitalization and death in the world. Beside genetic, acquired causes including viruses, chemicals, alcohol, toxins, pollutants and drugs are uplifting this scale severely especially in developing or low income countries. Pakistan is also a big victim of this health hazard. Liver is one of the vital organs of the body, equipped with enzymes that are actively involved in all metabolism related to the synthesis, storage, detoxification and excretion of substances. However, it is at high risk of dysfunction and inflammation when it is exposed with the acquired factors of liver problems especially chemicals used in professional environment and daily use of high doses of analgesics. These reversible liver problems may turn into irreversible damages like fibrosis and cirrhosis if neglected. Interestingly, there are few medicines available for the treatment of liver problems, and majority of them are plant origin, though these are in clinical practice but not found as effective as they expected in the regeneration of liver cells. Therefore, researchers are finding new medicinal plant having improved hepatoprotective activity with the aim for development of new medicine in this regard. Centratherum anthelminticum (Wild) Kuntz (family Asteraceae), its seeds are commonly called as kali zeri or black cumin. These seeds are not only well famous for their culinary uses in Pakistan and neighbor countries but also for number of medicinal purposes especially anticancer, antidiabetic, and antihyperlipidemic. However, its antihepatotoxic activity was not reported. Therefore, in the present work, the hepatoprotective action of organic solvent extract of C.anthelminticum seeds in carbon tetrachloride (CCl4) and paracetamol (PCM)-induced hepatotoxic rats models was investigated and reported. In PCM-induced hepatotoxic model (PIH), except normal control group (distilled water 1 ml; group I), all other experimental rats were made hepatotoxic by administering PCM (1 gm/kg/day) orally and divided into PIH control (distilled water 1 ml; group II), positive control group (silyamrin 100 mg/kg; group III) and three test groups (IV, V & VI) treated with 200, 400 and 600 mg/kg of ESEt of C. anthelminticum separately for consecutive 9 days. After that, rats of all groups (6 rats/ group) were decapitated for the evaluation of hematological, biochemical and antioxidant parameters. Whereas physical parameter including percent body weight change (PBWC) was calculated by assessing the body weights of each rat of each group on first and last day of each treatment. In addition, weights of liver tissues (LW) of each group of rats were measured and histopathological studies of liver tissues have also been done. The results stated that the dosage of ESEt (200, 400 & 600 mg/kg) found efficient in decreasing the percent loss in body weights of rats in all tests in compared to group intoxicated with only PCM. Plus the weights of liver tissues of test groups were also found almost as same as observed in normal control group. Similarly, the same doses of extract was found normalizing the levels of liver-specific biomakers such as aminotransferases (both alanine; ALT & aspartate; AST), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), total bilirubin (TBR) especially indirect one (IDBR), total protein (TP) especially albumin (ALB) and uric acid (UA) in their own test groups when compared to PCM hepatotoxic control group. Hematological parameters including hemoglobin (Hb), red & white blood cells (RBC & WBC), hematocrit (HCT) and platelets (PLT) were also found better in all three extract treated test groups. In addition, aspartate aminotransferase: platelate ratio index (APRI) was also calculated and found lower than 0.5 in test groups that showed the liver protective property of extract. Similarly, percent inhibition (PI) of antioxidant parameters including superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH) and lipid peroxidation (LPO) was calculated in liver homogenates of all rats of all groups and found that all ESEt treated test groups have low PI of SOD, CAT & GSH and high PI of LPO when compared with PCM control group. The betterment in all physical, biochemical, hematological and antioxidant parameters showed by ESEt in test groups was strongly evident by observing decrease in necrosis and inflammation in histological slides of liver tissues of same test groups as compared to liver tissue slides of hepatotoxic group. Before conducting CCl4-induced hepatotoxic model, hexane soluble fraction (HSF) of ESEt was prepared and separated. Then rats were divided into two main groups normal control (distilled water 1 ml; group I) and CCl4-induced hepatotoxic group which sub-divided into hepatotoxic control (distilled water 1 ml/kg; group II), positive control (silyamrin100 mg/kg; group III), and three test groups (IV& V) administered with ESEt in doses of 600 & 800 mg/kg and group VI with HSF 600 mg/kg for consecutive 5 days. CCl4 (3ml/kg; in 1:1 dilution with olive oil) was injected intra-peritoneally in groups II to VI on 3rd and 5th day of experiment after 1 hour of their allocated treatments. After 24 hours of last injection of CCl4, rats of all groups were decapitated to collect serum and liver tissues. Methodology includes the determination of physical [PBWC, LW & (liver index; LI)], biochemical [ALT, AST, ALP, GGT, TBR, DBR (direct bilirubin), IDBR, TP, ALB, UA, TG (triglycerides), TC (total cholesterol), VLDL-c, LDL-c & HDL-c (very low, low & high-density lipoprotein cholesterols)] and PI of antioxidant [CAT, SOD, GSH & LPO] parameters. The doses (600 and 800mg) of ESEt and HSF (600 mg) were significantly decreased the PBWC, LW and LI in their respective test groups which was also accompanied with much decreased levels of ALT, AST, ALP, GGT, TBR, IDBR, UA and increased levels of TP and ALB in these test groups when compared to CCl4 intoxicated group that displayed completely vice versa situation of all these parameters. Similarly, ESEt and HSF were also found beneficial in decreasing the levels of bad lipids (TG, TC, VLDL-c & LDL-c) in test groups. Moreover, antioxidant status of test groups was found improved by observing decreased in PI of CAT, SOD, GSH and increased in PI of LPO as compared to high level of oxidative stress was found in hepatotoxic group by observing high PI of CAT, SOD, GSH and less PI of LPO. The liver protective effect of ESEt and its HSF was clearly identified by observing gradually improved structure of liver tissues in rats belonged to test groups administered with doses (600 & 800 mg) of ESEt as compared to hepatotoxic group whose liver tissue slide displayed harmful effects of CCl4 including fatty accumulation, ruptured and inflamed hepatocytes around abnormally enlarged central vein. Whereas, the most amazing finding of present study is the recovery of complete normal structure of liver tissue dissected out from rat of test group treated with HSF (600 mg). iv Therefore, the present study concludes that ESEt and its HSF are strong hepatoprotective and liver regenerative agents not only by normalizing the liver function test parameters, lipid profile and uplifting the antioxidant parameters but also capable of reversing the adverse anatomical changes induced by PCM and CCl4 in liver tissues by completely healing up the same tissue upto the normal structure. The liver protective property of ESEt and HSF may be resides in polyphenol, flavonoid and steroidal contents of ESEt which were already reported in our previous studies and fatty acids, hydrocarbons or waxes which could be possibly present in HSF of ESEt of C.anthelminticum