Assessment of antihepatotoxic activity was done by determining the glutamyl pyruvate transaminase (SGPT) and glutamyl oxalacetic acid transaminase (SGOT) enzyme activity. The enzyme assay was carried out by Reagent Kits maintained by Biocon India Ltd Bangalore and the procedures were essentially those described in the literature available with kits. Estimations were made on Auto-analyser; Reitman and Frankel method (1957) was used for determining the enzyme activity in the supernatant of various groups.

On fifth day, animals were sacrificed and blood was collected directly through retro-orbital plexus. serum was separated after coagulating at 37 ◦C for 30 min and centrifuging at 1200–1500 rpm for 15–20 min. Serum was analyzed for various biochemical parameters, i.e. serum glutamyl oxalacetic acid transaminase (SGOT), serum glutamyl pyruvate transaminase (SGPT).¹² acid phosphatase (ACP) and alkaline phosphatase (ALP).¹³ serum bilirubin (SB).¹⁴ and total protein.¹⁵

Determination of antioxidant enzyme activity: The liver was perfused with 0.86% cold saline to remove all the red blood cells. Then it was suspended in 10% (w/v) ice cold 0.1M phosphate buffer (pH 7.4) cut into small pieces, and required quantity was weighed and homogenized using a Teflon homogenizer. The homogenate was used for estimation of enzymic and non-enzymic antioxidants like SOD, ¹⁶ CAT, ¹⁷ GPx, ¹⁸ and lipid peroxide level.¹⁹

Statistical analysis: All Data were expressed as mean S.D. and analyzed with one-way analysis of variance (ANOVA). Dunnett’s t test was used to calculate Statistical significance. P<0.05 and P<0.01 were considered statistical significance.

Results: Serum activities of transaminases, SGPT, SGOT and ACP, ALP, serum bilirubin and total protein are given in Table.1 where the single dose of APAP significantly elevated SGPT, SGOT activities when compared to normal animals. (Table.1).

Figure 1

Table.1: Effect of Sphaeranthus indicus flower heads on Paracetamol induced heptotoxicity in Rats

Treatment of AQS & MES (300mg/kg) extracts 1h prior to APAP administration significantly protected the elevation of marker enzymes, serum bilirubin and ACP and ALP activities. Reduced activities of enzymic and non-enzymic antioxidants and enhanced activities of lipid peroxidation were seen in the APAP-treated group (Table.2), whereas standard silymarin and the drug treated group showed significant (p<0.01) rise in antioxidant level with reduction in lipid peroxidation level when compared with the standard drug, silymarin (Table. 2).

Figure 2

Histopathology of Group II animals shows patches of liver cell necrosis with inflammatory collections around central vein where as drug treated group showed absence of cell necrosis but with minimal inflammatory conditions around the central vein. The MES (300mg/kg, p.o)-treated group showed minimal inflammatory conditions with near normal architecture possessing higher hepatoprotective action (Fig.3)

Discussion: In the present study the methanolic extract of Sphaeranthus indicus was observed to exhibit hepatoprotective effect as demonstrated by a significant decrease in liver function markers and also serum bilirubin concentrations and by preventing liver histopathological changes in rats induced with hepatotoxicity. Moreover, the methanolic extract of Sphaeranthus indicus enhanced the activities of antioxidant enzymes (SOD, CAT, GPx) and diminished the amount of lipid peroxides against paracetamol-induced hepatotoxicity in these animals, suggesting the reduction of oxidative stress in this scenario plays a role in mechanism of its hepatoprotective effect. Paracetamol at therapeutic doses is primarily metabolized and detoxified by glucuronidation and sulphation and subsequently followed by renal excretion.²⁰ However when paracetamol is taken in a toxic doses, the compound is converted to a toxic form N-acetyl-p-benzo-quinone imine (NAPQI). Which is an electrophilic intermediate, oxidized by cytochrome P₄₅₀ and converted to a highly reactive and toxic metabolite as in the case of paracetamol over dose.²¹ NAPQI can rapidly react with the glutathione (GSH) and lead to a 90% total hepatic GSH depletion in the cells and mitochondria, which can result in hepatocellular death and mitochondrial dysfunction.²² In addition NAPQI can increase the formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as superoxide anion, hydroxyl radical, and hydrogen peroxide, and nitro oxide and peroxynitrite, respectively. Excess level of ROS and RNS can attack biological molecules such as DNA, protein and phospholipids, which leads to lipid peroxidation, nitration of tyrosine, and depletion of anti oxidant enzymes (SOD, CAT, GPx) that further results in oxidative stress.²³ NAPQI can also induce DNA stand breaks and promote apoptosis and necrosis in paracetamol-induced hepatotoxicity.²⁴ In the present study, the data suggested that high dosage of paracetamol in the liver could lead to decreased level of anti oxidant enzymes (SOD, CAT, GPx) and present a significant level of hepatotoxicity in the course of treatment. However, the methanol extract of Sphaeranthus indicus could raise the level of SOD, CAT, and GPx against the Paracetamol-induced oxidative stress mediated by ROS and RNS. Furthermore, the level of MDA was increased in the group receiving paracetamol administration, but pretreatment with the methanol extract of Sphaeranthus indicus reduced the amount of MDA. This result indicated the decreasing the formation of lipid peroxidation is also one of the event of preventing the oxidative toxicity by paracetamol.

In conclusion the present study has demonstrated that methanolic extract of Sphaeranthus indicus has hepatoprotective effect against paracetamol-induced hepatotoxicity in rats. Interestingly the more active hepatoprotective compound of Sphaeranthus indicus appears to exist in methanolic extract and not more in aqueous fraction, which could include flavonol and flavonoid .The enhanced level of antioxidant enzymes and reduced amount of lipid peroxides are suggested to be the major mechanism of Sphaeranthus indicus methanolic extract in preventing the development of liver damage induced by paracetamol.

Figure 3

Figure: Histopathology studies of liver tissues

The author is grateful to secretary Mr. Prashant Jain Translam group of institutions for providing necessary facilities.