Hepatoprotective activity of Thymus linearis against paracetamol and carbon tetrachloride-induced hepatotoxicity in albino mice

In the present study, the hepatoprotective activity of aqueous and ether extracts of Thymus linearis (250 and 500 mg/kg orally) was evaluated against carbon tetrachloride- and paracetamol-induced hepatic damage in mice. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) were assessed. Antioxidant activity of both the extracts was also determined using 1-1-diphenyl-2-picryl hydrazine (DPPH) scavenging method. The results indicated that both the extracts significantly produce a dose dependent reduction in serum levels of ALT, AST, ALP when compared to carbon tetrachloride-and paracetamol-treated groups. The maximum effect in all the parameters was observed at a dose of 500 mg/kg. The extracts also demonstrated a significant antioxidant activity. LD 50 of both extracts was found to be a 1050 and 900 mg/kg respectively. It is conceivable that the hepatoprotective activity of T. linearis might be due to the presence of certain pharmacologically active compounds.


Introduction
Many synthetic drugs are used to treat liver diseases but unfortunately these drugs have serious side effects (Guntupalli et al., 2006).The role of traditional medicines in solving health problems is valuable globally.This is even more impressive considering the fact that about 80% of people living in less developed countries rely exclusively on traditional medicines for their health care needs.Natural plants have a very important role in treatment of liver diseases.There are many natural plants in world which have hepatoprotective activity (Gite et al., 2010) like Carissa spinarum (Hegde and Joshi, 2010), Cocculus hirsutus (Thakare et al., 2009), Convolvulus arvensis (Ali et al., 2013), Dodonaea viscosa (Khan et al., 2013), Khamira Gaozaban Ambri Jadwar Ood Saleeb Wala (Akhtar et al., 2013), Ipomoea staphylina (Bag and Mumtaz, 2013), Suaeda fruticosa (Rehman et al., 2013), Trianthema decandra (Balamurugan and Muthusamy, 2008) and Trichodesma sedgwickianum (Saboo et al., 2013) showed hepatoprotective effect.
Pakistan is granted with large number of medicinally important plants and has ancient herbal treatment method.The local communities of different regions of Pakistan have centuries old knowledge about traditional uses of plants occurring in their areas.Several indigenous plants are used to treat liver disorders (Ivvora et al., 1989).
Thymus linearis Benth belongs to the Labiatae family which is one of the largest and most diverse family of medicinal plants.It is mostly used in home remedies.Plant is aromatic, antiseptic, diaphoretic, analgesic, diuretic, carminative and stimulant.It is also used in rheumatism gout and sciatica.The herbal decoction may assist to prevent hair loss (Sharma et al., 2005).It is also given in weak vision, complaints of liver, suppression of urine and mensturation (Qureshi, 2007).
Hence, this study was undertaken to investigate the hepatoprotective effects of extract on paracetamol and carbon tetrachloride (CCl4)-induced albino mice hepatic injury.

Chemicals
CCl4 (Merck Darmstadt, Germany), paracetamol (Zenith Pharma Karachi, Pakistan), silymarin, diagnostic kits for the estimation of ALT, AST and ALP (Diasys, Germany) were used.Chemicals and reagents used in these experiments were of analytical grade and prepared in distilled water.

Plant material used
The selected plant was collected from the mountains of the village Shikiyote; district Gilgit, Gilgit-Baltistan.Plant was identified and authenticated by Dr. Shair Wali, Department of Botany, Karakoram International University, Gilgit-Baltistan.Plant material was shade dried and powdered with a Chinese herbal grinder.The powdered material was stored in well closed cellophane bags at 4ºC in refrigerator.

Preparation of plant extracts
The powdered plant was extracted by method of cold maceration.One kg of plant powder was extracted by using solvents; ether and distilled water (Aghel et al., 2011).For extraction with each solvent, the plant powder was soaked for 72 hours with occasional shaking.It was passed through muslin cloth and then filtered through filter paper.The aqueous and ether extracts were prepared with the help of rotary evaporator.

Animals used
Adult healthy albino mice (25-30 g) of either sex were used in the study and kept under natural light and dark cycle at a temperature of 28 ± 4ºC, given a standard pellet diet and water ad libitum.

Hepatoprotective activity against paracetamolinduced toxicity in mice
After an overnight fasting, mice were divided into seven groups of five animals each.Group 1 was control group which received single dose of 5% tween 80 daily for 4 days orally and on 3 rd day single dose of 40% sucrose solution.Group 2 was paracetamol group and it received single dose of 5% tween 80 daily for 4 days orally and on 3 rd day single dose of paracetamol suspension (250 mg/10 mL water).Group 3 and 4 were treated daily 250 and 500 mg/kg of ether extract, while Group 5 and 6 received 250 and 500 mg/kg of aqueous extract of T. linearis respectively for 4 days orally and on 3 rd day single dose of paracetamol suspension.Group 7 served as silymarin treated which received silymarin for four days and on 3 rd day single dose of paracetamol.
Animals were sacrificed on 5 th day, and their blood was collected for biochemical study (Maheswari et al., 2008).

Hepatoprotective activity in CCl4-induced toxicity in mice
After an overnight fasting, mice were divided into seven groups of five mice each.Group 1 served as negative control and was treated with normal saline (0.9%) for four days.Group 2 was treated with normal saline and CCl4, on 1 st and 4 th day, and on 2 nd and 3 rd day respectively.Group 3 and 4 were treated daily 250 and 500 mg/kg of ether extract, while group 5 and 6 received 250 and 500 mg/kg of aqueous extract of T. linearis respectively for 4 days orally and on the 2 nd and 3 rd day CCl4 was given half an hour after the administration of extract dose.Group 7 served as silymarin treated and was given CCl4 and silymarin on 1 st and 4 th day, and on 2 nd and 3 rd day respectively.Animals were sacrificed on 5 th day, and their blood was collected for biochemical tests (Aghel et al., 2011).

Anti-oxidant activity
Solution of 1-1-diphenyl-2picryl hydrazine (DPPH) was prepared by dissolving 4.3 mg of DPPH in 3.3 mL of methanol and covered with aluminum foil to protect from light.150 µL DPPH solution was added to 3 mL of methanol and its absorbance was measured at 516 nm as control reading.Different concentrations (10,20,40,60,80, 100 µg/mL) of aqueous and ether extracts of plant were made and the volume was made uniformly to 150 µL using methanol.Each of the samples was then further diluted with methanol up to 3 mL and to each, 150 µL DPPH solution was added.After 15 min absorbance was measured at 516 nm by using methanol as blank at UV visible spectrometer.The capability to scavenge the DPPH radicals was calculated using the following formula: DPPH scavenged (%) = (Acontrol -Asample) / Acontrol × 100 Where, Acontrol = The absorbance of the control reaction mixture and ASample = the absorbance of sample at different concentrations (Molyneux, 2004)

Acute toxicity test
The aim of this experiment was to determine the LD50 of crude extract.Adult healthy mice of either sex, weighing 25-30 g were randomly divided into nine groups of 2 animals each.
Group 1 served as control and received normal saline 10 mL/kg.Group 2, 3, 4, 5, 6, 7, 8 and 9 were given different doses of aqueous and ether extracts of T. linearis in an ascending order i.e.100, 300, 600, 900 mg/ kg respectively.The mortality rate was observed for 24 hours.Since no mortality occurred so another nine groups of mice were taken.They were again treated with various doses of aqueous and ether extracts in an ascending order i.e. 1200, 1500, 1800, 2000 mg/kg respectively.All the doses were administered by intraperitoneal route.The highest dose which did not kill any mice, and the lowest dose which killed only one mice was noted.LD50 was calculated from the mean of these two doses (Shetty et al., 2007).

Preliminary phytochemical analysis
The aqueous and ether extracts of T. linearis were analyzed for the presence of different phytochemical constituents such as flavonoids, reducing sugars, tannins, phenolic compounds, saponins, alkaloids and cardiac glycosides by using standard methods (Khandelwal, 2006).

Statistical analysis
The data were expressed as mean ± standard error of mean (SEM) and analyzed by using t-test.P<0.05 was considered as significant.

Results
There was a significant increase in the level of liver enzymes in paracetamol-treated group compared to normal control group.Ether extract significantly (p<0.05)decreased the level of liver enzymes in 250 mg/kg and 500 mg/kg extract treated group.Aqueous extract also significantly (p<0.01)decreased the level of liver enzymes in 250 mg/kg and 500 mg/kg treated group.There was highly significant (p<0.001)decrease in the liver enzymes (ALT, AST, ALP) in silymarintreated group compared to paracetamol treated group (Table I).
In extract treated group with the dose of 250 mg/kg there was a significant (p<0.05)decrease in liver enzymes and with the dose of 500 mg/kg there was a significant (p<0.001),(p<0.05),(p< 0.01) decrease in the level of ALT, AST and ALP respectively.However, there was highly significant (p<0.001)decrease in the liver enzymes in silymarin-treated group compared to CCl4 -treated group (Table II).
In CCl4 treated group there was a significant increase in the level of liver enzymes compared to normal control group.Ether extract with a dose of 250 mg/kg significantly (p<0.05),(p<0.001),(p<0.05)decreased the level of ALT, AST, ALP respectively.There was a highly significant decrease in the liver enzymes in 500 mg/kg and silymarin-treated group compared to CCl4-treated group (Table II).
Preliminary phytochemical analysis revealed the presence of alkaloids, flavonoids.The other secondary metabolites like tannins, saponins, steroids, cardiac gly-   Data are expressed as mean ± SEM; Where, a p<0.05, b p<0.01 and c p<0.001 as compared to CCl4 treated group cosides, and reducing sugar were also present (Table III).

Discussion
The use of medicinal plants has been increased during the last few decades.However, the use of these medicinal plants into the modern therapy requires their toxicological and pharmacological evaluation on scientific basis.So, the current study was carried out to evaluate the hepatoprotective effect of T. linearis in paracetamol-and CCl4-induced hepatotoxic mice.In the present study aqueous and ether extract of the plant showed significant hepatoprotective effect.The results were in agreement with the previous study (Ahsan et al., 2009).
In our current study, the levels of ALT, AST and ALP were increased in paracetamol and treated mice which is in accordance with the previous findings (Mittal et al., 2010).All these enzymes metabolize in liver and damage to the liver causes the leakage of these enzymes into the blood circulation and their level increases (Wakchaure et al., 2011).
Treatment with T. linearis extracts restored the elevated values of liver enzymes near to normal level, which is a clear manifestation of antihepatotoxic effect of T. linearis extracts.The hepatoprotective activity of the extracts may be due to their stabilizing effect on plasma membrane as reported earlier by Nadeem et al. (2011).
The inhibition of lipid peroxidation by the active constituents of the extracts might also be involved in hepatoprotective effect which is duly supported by the study of Kanchana et al. (2011).The oxidative stress is considered an important cause of liver injury, induced by hepatotoxic agents.The free radicals especially reactive oxygen species (ROS) are involved in hepatic tissue damage.In our study, aqueous and ether extracts demonstrated the significant anti-oxidant activity in DPPH scavenging assay which is compared with standard anti-oxidant ascorbic acid.The extracts may produce the hepatoprotective effect by neutralizing the effect of free radical species.The results are in complete agreement with the investigation of Pourmorad et al. (2006).Increased protein synthesis, improved lipoprotein metabolism and presence of phytochemical constituents may also be related to the hepatoprotective effect of extracts (Ahsan et al., 2009).
Extracts in a dose of 500 mg/kg demonstrated more significant effects as compared to 250 mg/kg dose.So the aqueous and ether extracts produced the hepatoprotective effect in dose-dependent manner.Silymarin is used as standard hepatoprotective agent and has protective effect on hepatocytes (Kshirsagar et al., 2011).Aqueous and ether extracts of T. linearis attenuated significantly the decreased level of these enzymes and caused a subsequent recovery towards normalization almost like that of silymarin treatment.This study calls for further activity-oriented fractionation of these extracts to isolate the active principle (s) and elucidate exact mechanism of action.

Conclusion
The aqueous and ether extract of T. linearis have dosedependent hepatoprotective activity and it may be due to the presence of active constituent(s) in the extracts.
as mean ± SEM; Where, a p<0.05, b p<0.01 and c p<0.001, as compared to paracetamol control group

Figure 1 :
Figure 1: Percent inhibition of ascorbic acid, aqueous and ether extract of Thymus linearis