HEPATOPROTECTIVE ACTIVITY OF ASPARGUS RACEMOSUS ROOT EXTRACT ON LIPOPOLYSACCHARIDE INDUCED OXIDATIVE STRESS IN RATS

Lipopolysaccharide (endotoxin) produces an inflammatory condition leading to multiple organ failure. LPS most potent bacterial products is used for induction of host oxidative stress responses and liver injury. Present study was undertaken to investigate the effect of Asparagus racemosus Willd . root extract in lipopolysaccharide (LPS) induced oxidative stress in rats by measuring oxidative stress markers, nitric oxide, liver function test and cytokines. The obtained data showed that LPS administration significantly reduced glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT), total cholesterol (TC) and albumin (ALB). There was significant increase in malondialdehyde (MDA), cytokines activity, serum aspartate transaminase(AST), alanine transaminase(ALT), alkaline phosphate (ALP), total bilirubin (TB) and nitric oxide(NO). The methanolic extract of Asparagus racemosus (MEAR) administration significantly (P<0.05) reduced LPS-induced oxidative stress by normalizing liver GSH, SOD, CAT, MDA, NO, cytokines and liver function markers. MEAR significantly increased ALB and TC level. Our results suggest that MEAR protects the liver against liver toxicity induced by LPS. 5-6 . LPS after binding to immune cells initiate a cascade of events that up-regulate expression of the inflammatory cytokines including TNF-α, IL-6 and IL-1β. TNF- α and other cytokines stimulate the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs) by activated macrophages causing liver damage 7 . The oxidative stress generated induces a rapid alteration in the antioxidant systems by depleting the cellular stores of endogenous antioxidants such as GSH, SOD and CAT 8 . The liver plays very important role in the defense against LPS induced toxicity.


INTRODUCTION
LPS, gram-negative bacterial endotoxin induced hepatic failure has lead to high mortality. The severity of subsequent organ damage might depend on the difference between excess production of ROS and antioxidant defenses [1][2][3] .
LPS binds to liver proteins, producing oxygen free radicals and proinflammatory cytokines 4 .
Release of these toxic mediators is the contributing factor to most of LPS toxicity in the liver and in the systemic circulation [5][6] . LPS after binding to immune cells initiate a cascade of events that up-regulate expression of the inflammatory cytokines including TNF-α, IL-6 and IL-1β. TNF-α and other cytokines stimulate the production of reactive oxygen species (ROS) and reactive nitrogen intermediates (RNIs) by activated macrophages causing liver damage 7 . The oxidative stress generated induces a rapid alteration in the antioxidant systems by depleting the cellular stores of endogenous antioxidants such as GSH, SOD and CAT 8 . The liver plays very important role in the defense against LPS induced toxicity.

Treatments
Animals were randomly divided into seven groups as follows:- After six hours of LPS or saline injection blood was collected from tail vein for liver function test and all the animals were sacrificed, liver was removed, stored and homogenates was used for biochemical estimation.

Determination of liver MDA content
Suspension medium (1 ml) was taken from the 10% tissue homogenate. TCA (0.5 ml of 30%) was added to it, followed by TBA reagent (0.5 ml of 0.8%.) The tubes were then covered with aluminium foil and kept in shaking water bath for 30 minutes at 80 degree celsius. After half an hour tubes were taken out and kept in ice-cold water for further thirty minutes and ccentrifuged at 3000 rpm for 15 minutes 13 .
The absorbance of the supernatant was read at 540 nm at room temperature against appropriate blank.

Determination of liver glutathione
Liver tissue (300mg) was homogenized in EDTA (5 -8 ml of 0.02 M) and then cold distilled water (4 ml ) was added to it. After mixing it well, TCA (1 ml of 50 % ) was added and shaken intermittently for 10 minutes using a vortex mixer. After 10 minutes the contents were centrifuged at 6000 rpm for 15 minutes. Following centrifugation, supernatant (2 ml) was mixed with Tris buffer (4 ml of 0.4 M). The whole solution was mixed well and DTNB (0.1 ml of 0.01M) was added to it. The absorbance was read within 5 min of the addition of DTNB at 412 nm against a reagent 14 .

Determination of liver catalase
Liver tissue was homogenized in 50 mM/L potassium phosphate buffer with a ratio of 1:10 w/v.
The homogenate was centrifuged at 10,000 rpm at 4º C in a cooling centrifuge for 20 minutes.
Catalase activity was measured in supernatant obtained after centrifugation. Supernatant (50 µl) was added to cuvette containing 2.95 ml of 19 mM/L solution of H 2 O 2 prepared in potassium phosphate buffer. The change in absorbance was monitored at 240 nm wavelength at 1-minute interval for 3 minutes. Presence of catalase decomposes H 2 O 2 leading to decrease in absorbance 15 .

Determination of Superoxide dismutase
The supernatant was assayed for SOD activity by inhibiting pyrogallol autoxidation. Cytosolic supernatant (100 μl) was added to Tris HCl buffer (pH 8.5). The final volume of 3 ml was adjusted with the same buffer. pyrogallol (25 μl) of was added and changes in absorbance at 420 nm were recorded at 1 minute interval for 3 minutes. The increase in absorbance at 420 nm after the addition of pyrogallol was inhibited by the presence of SOD 16 .

Markers of liver Function
The activity of biochemical parameters such as AST and ALT were estimated by Reitman and Frankel method 17 , ALP and TB were estimated by King and Dangerfield method [18][19] ALB and TC level were estimated by the methods of Webster and Zlatkis, respectively 20-21 .

Determination of Nitric oxide (NO): Griess Reaction
After the experiment, animals were sacrificed and the tissues were washed with PBS (pH 7.4) and placed on ice as described earlier. Sample (50l) was added with Griess reagent (100l) and reaction mixture was incubated for about 5-10 minutes at room temperature.The optical density was measured at 540 nm in microplate reader according to the reagent manufacturer's protocol.
Calculations were done after generating a standard curve from sodium nitrite in the same buffer as used for preparation of homogenate 22 .

Enzyme-linked immunosorbent assay
Cytokines were measured from tissue samples using commercially available ELISAs for rat TNF-α, IL-1β and IL-6. The ELISAs were operated according to the manufacturer's instructions.
The intensity of the color measured is in proportion to the amount of rat cytokine bound in the initial steps. The sample values were then read off from the standard curve 23 .

Statistical Analysis
All results are expressed as mean ± SEM. Groups of data was compared with analysis of variance (ANOVA) followed by Tukey-kramer multiple comparison test. p<0.05 was considered statstically significant.

Acute toxicity
The extract from roots of Asparagus racemosus administered orally to rats up to dose of 2000 mg/kg showed no toxicity and animal death during the evaluated period thus suggesting low toxicity of the extract. One-tenth and one-twenty of the maximum tolerated dose of the extract tested (2000 mg/kg) for acute toxicity did not indicate mortality and were selected for evaluation of the effect of Asparagus racemosus i.e. 100 and 200 mg/kg.

Oxidative stress markers
The

Nitric oxide Activity
In the rats pretreated with Asparagus racemosus, the levels of NO significantly reduced compared to disease control ( Table 3). Dose of 200 mg /kg was more effective than that of 100 mg/ kg.

Cytokine Activity
In the rats pretreated with Asparagus racemosus, the levels of cytokines significantly reduced (p<.001) as compared to disease control (Table 3). There was dose dependent recovery on the LPS induced elevation of the cytokines level in rats.
In the present study, administration of LPS to rats resulted in development of oxidative stress which led to anxiogenic response and damage in liver tissue in rats. Therefore, it is more susceptible to oxidative stress than other tissues 26 .
In our study Asparagus racemosus root (MAER) significantly decreased liver cytokines level after 6 hr of LPS administration as compared to rats treated with disease control.
Root of Asparagus racemosus supplementation increased the levels of GSH, SOD, CAT and decreased the level of TBARS, cytokines and nitric oxide significantly in the LPS-challenged animals. In a study done by N Palanisamy and S Manian showed that A.
racemosus extract has hepatopotective activity by inhibiting production of free radical via inhibition of hepatic CYP2E1,increasing removal of free radical by induction of antioxidant enzyme and improving non-enzymatic thiol antioxidant GSH. Thus A. racemosus acts as a free radical scavenger 27 . In addition to its direct cytotoxic effects, it is able to induce chemokines macrophage chemotactic protein-1 and vascular cell adhesion molecule-1, which is the key to hyper inflammation and consequent liver damage.
Asparagus racemosus is a medicinal plant with well-known antioxidant property 27 . Scientific evaluation of this claim using experimental model of LPS induced oxidative stress in rats was ascertained in this study.

Conclusion
Oral administration of methanolic extract of Asparagus racemosus root (MEAR) protected rats from LPS induced liver injury. The protection may be due to the reduction of oxidative stress which occurs by alteration in levels of antioxidant enzymes in oxidative stress rats. These observations suggest that MEAR may be clinically viable protection against variety of conditions where cellular damage is a consequence of oxidative stress. In conclusion, the present study provides experimental evidence for MEAR as a hepato-protective agent.

Conflict of interest statement
We declare that we have no conflict of interest.