Hepatoprotective Activity of Curcuma Vamana Ethanolic Rhizome Extract Against Paracetamol and CCl4 Induced Hepatotoxicity in Albino Rats

Introduction

Liver is one of the biggest organs in human body and the leading site for intense biotransformation and elimination. So it has a remarkable role in the maintenance, act and monitor homeostasis of the body. It is sophisticated with approximately all the biochemical pathways to growth, fight against disorders, nutrient supply, energy provision and reproduction.¹ The important physiology of the liver is carbohydrates, proteins and fat biotransformation, detoxification and elimination of bile and storage of vitamin. So, to maintain a healthy liver is a crucial factor for overall health and well being. But it is adNauseam and variedly manifests to environmental toxins, impact by poor drug habits, alcohol, prescribed and over the counter drug which can eventually lead to various liver ailments like hepatitis, cirrhosis and alcoholic liver disease.² Thus, liver disorders are some of the calamitous disease in the world today.

The liver diseases are one of the world complications. Even though its persistent occurrence, high morbidity and high mortality, its medical prevention is currently in adequate, so far not yet any therapy has successfully prevented the progression of hepatic disorders, despite freshly developed drugs have been used to treat chronic liver diseases, these drugs have persistent side effects. Therefore, that is a required innovation about suitable classical drugs that could replace the chemical ones.³ Liver injury due to chemicals (or) infectious agents may lead to progressive liver fibrosis and ultimately cirrhosis and liver failure.⁴ Anyhow, no effective treatment that delays disorders progression and problems has yet been found. Various instant studies indicate that classical herbs and micronutrients such as carotenoids and selenium may be useful for this purpose.^5,6 Paracetamol is a widely used medication which has a good safety profile^7-9 although large doses may lead to severe hepatic necrosis and fatal hepatic failure.¹⁰ If treated early enough, paracetamol overdose can be adequately treated with intravenous N-acetylcystein, but if hepatic encephalopathy develops, the risks of complications and death rise dramatically.^11-12. After a paracetamol overdose, orthotopic liver transplantation (OLT) is a treatment option for liver failure.¹³ Curcuma vamana plant has been identified in India western ghats of Kerala. The plant roots are used in the treatment of jaundice and liver disoder.¹⁴ The crude ethanol extract of Curcuma vamanat rhizomes of plant has been reported for its flavonoids which is responsible for the hepatoprotective activities.¹⁵ It is important to define prognostic factors for outcome in order to develop clear criteria for referral to specialized units. For the experimental development of liver necrosis, carbon tetrachloride (CCl4) is widely used.¹⁶ The main causes of CCl4 induced liver damage is LPO and decreased activities of enzymes and generation of free radicals and responsible for the causing hepatotoxicity.^17,18

Drug-induced hepatotoxicity, a well-known cause of liver abuse, presents clinicians with a huge issue.¹⁹ Clinically liver diseases are normally classified as, hepatocellular, such as viral hepatitis or alcoholic liver diseases. In these hepatocellular disorders indicates the liver injury, inflammation and necrosis more seen. Cholestatic (obstructive), such as gall stone or malignant obstruction, primary biliary cirrhosis and some drug induced liver disorders, features of inhibition of bile flow take precedence Mixed, features of both hepatocellular and cholestatic injury are existing. Several medicinal plants have been used to treat for various diseases in all over the world. Instant days, Indian medicinal plants are belonging to about 46 families were investigated as liver protective drugs.²⁰ Curcuma vamana commonly known as “Curcuma peethapushpa” is a tropical plant belonging to the family Zingiberaceae.

Materials and Methods

Chemicals and drugs

2-Thiobarbituric acid(TBA), Trichloroacetic acid(TCA), 5, 5'-Dithiobis-2-nitrobenzoic acid(DTNB), 1,1-diphenyl -2- picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl or DPPH), Epinephrine, Griess reagent were obtained from Sigma-Aldrich Corporation(St. Louis, MO, USA), Sodium nitroprusside(Qualigens., Mumbai), Ascorbic acid (Burgoyne Burbidge’s and Co., Mumbai), Mumbai. Serum ALT, AST, ALP, Total Bilirubin and LDL-cholesterol, HDL-cholesterol, Total cholesterol, Glucose enzyme test kits were obtained from Erba Diagnostic, Germany. Carbon tetrachloride(CCl4) (Nice Chemicals, Cochin), Standard Silymarin free sample from Himalaya Drug Co., Bombay and Paracetamol(HiMedia Lab. Pvt. Ltd, Mumbai). All further reagents and solvents are used in the experiment were of analytical grade.  

Plant material and preparation of plant extract

2-Thiobarbituric acid(TBA), Trichloroacetic acid(TCA), 5, 5'-Dithiobis-2-nitrobenzoic acid(DTNB), 1,1-diphenyl -2- picrylhydrazyl (α,α-diphenyl-β-picrylhydrazyl or DPPH), Epinephrine, Griess reagent were obtained from Sigma-Aldrich Corporation(St. Louis, MO, USA), Sodium nitroprusside(Qualigens., Mumbai), Ascorbic acid (Burgoyne Burbidge’s and Co., Mumbai), Mumbai. Serum ALT, AST, ALP, Total Bilirubin and LDL-cholesterol, HDL-cholesterol, Total cholesterol, Glucose enzyme test kits were obtained from Erba Diagnostic, Germany. Carbon tetrachloride(CCl4) (Nice Chemicals, Cochin), Standard Silymarin free sample from Himalaya Drug Co., Bombay and Paracetamol(HiMedia Lab. Pvt. Ltd, Mumbai). All further reagents and solvents are used in the experiment were of analytical grade.

Plant material and preparation of plant extract

The fresh rhizome was collected in the month of July 2018 from the Thiruvananthapuram district of Kerala. Kappali S. A. verified and authenticated it. Botanist and Professor, Department of Botony, Basaveshwara Science College, Bagalkot-587101, Karnataka, India. A specimen voucher (No: B.Sc/Bot/2019/76) was deposited for further reference. The rhizome was cleaned, air-dried and then subjected to coarse powdering and passed through a sieve #44 to get uniform powder size. The collected powder was successively extracted first with petroleum ether to defat and then with ethanol (60–65 °C) for 24 h by using a Soxhlet apparatus. After extraction, the solvent was distilled to yield a concentrated residue that was completely dried by lyophilization and stored in an airtight container at 2–8 °C. The extract of the obtained suspension (Yield=0.34%) was used for protective activity of the liver.

Animals

The Albino rats of either sex (190–250 g) were obtained from the central animal house of H.S.K. College of Pharmacy, Bagalkot-587101. The animals were housed at room temperature (21–28 °C) with 65±10% relative humidity for Twelve hour light and dark cycle and given laboratory feed of standard quality (Amruth, Sangli, Maharashtra). The study was approved and conducted as per the norms of the Institutional Animal Ethics C o m m i t t e e ( I A E C / H S K C O P / F e b r u a r y 2018/PG10).

Acute toxicity study

The acute oral toxicity of ethanol extract rhizome of Curcuma vamana was studied according to the OECD guidelines 425 21 and the female Swiss albino mice (25-30gm) were used for acute toxicity study. 2000mg/kg Limit dose was given per oral and afterwards behavioural and toxicological signs were observed for 4 hr and monitored upto 14 days and mortality was not occurred with higher limit dose. LD50 was calculated and it was found ˃2000 mg/kg.

Experimental protocol for paracetamol model

The animals were divided into six groups, each groups containing six animals. All the animals except normal group were intoxicated with paracetamol (1 g/kg, p.o.) every day for seven days animals in Group I were given merely a vehicle and acted as the control group. Group II served as paracetamol control and received paracetamol (1 g/kg, p.o.) for first seven days. Group III, Silymarine, P.O, served as the positive control and received paracetamol for first seven days and, Group, IV-100 mg/kg of EECV, V-EECV 200 mg/kg, VI-EECV 400mg/kg doses from 0^th day to 12^th day and same animals received paracetamol for first seven days. After 24hrs the last doses of extract administration, all the groups of rats were sacrificed. Serum and liver tissue homogenates of all experimental animals were collected and estimated for enzymatic levels and histopathological studies.

Experimental protocol for CCl₄ model

The animals were divided into six groups, each group containing six animals. On days 15th, 17th and 19th the rats of group II, III, IV, V, VI received 1 ml/kg body weight of carbon tetrachloride in liquid paraffin (1:1) orally with the respective assigned treatments. Rats of group III served as standard, received 200 mg/kg body weight of Silymarine and group IV, V and VI were received 100, 200 and 400 mg/kg b.wt of ethanol extract respectively of Curcuma vamana for a period of 19 days by oral administration. After 24hrs the last dose of extracts administration, all the groups of rats were sacrificed. Serum and liver tissue homogenates of all experimental animals were collected and estimated for enzymatic activity were measured by UV-spectroscopic method and histopathological studies carried out.

Assessment of biochemical parameters blood serum and liver homogenate

After the 24hrs of the last dose of extract with their respective assigned treatment, blood was collected from each animal by retro-orbital plexus and centrifuged (3000 rpm for 10 min) to separate serum for the estimation of AST²², ALT²³, Total Bilirubin²⁴, ALP²⁵, Total cholesterol²⁶, Total protein²⁷, HDL-cholesterol, LDL-cholesterol²⁹ and Glucose.³⁰

After collection of blood samples, the rats were sacrificed and their liver excised, rinsed in ice-cold normal saline, liver is perfused with ice cold normal saline to remove the traces of blood constituents then weighed and homogenized in cold phosphate buffer (0.1 M, pH 7.4). Centrifuged the homogenates at 10000 rpm for 10 min at 4 ºC(MPW-350R, Korea)and supernatant was used to estimation of Total Protein and Lipid peroxidation. The supernatant obtained centrifuged at 17000rpm for one hr at 4ºC. The supernatant obtained was used for further estimation of SOD, CAT, GSH and Total thiols.³¹

Lipid peroxidation (LPO)

Thiobarbituric acid reactive substances (TBARS) is add in the liver homogenate were estimated by the method Prabhakar et al. (2006). Briefly, 0.5 ml of the 10% homogenate was incubated with 15% TCA, 0.375% TBA, and 5 N HCl at 95 °C for 15 min, the mixture was cooled down, centrifuged, and the absorbance of the supernatant was measured at 512 nm in comparison to a suitable blank. The amount of lipid peroxidation was expressed as TBARS nmoles/mg of protein.³²

Superoxide dismutase(SOD)

SOD to inhibit the autooxidation of adrenaine to adrenochrome at basic pH.³³ Briefly, 25 ml of the supernatant obtained from liver homogenization was centrifuged was added to a mixture of epinephrine (0.1 mM) in carbonate solution (pH 10.2) for a total volume of 1 ml and subsequently measured at 295 nm to find the composition adrenochrome. The SOD(U/mg of protein) activity was calculated.

Catalase

The assay mixture consisted of 1.95ml phosphate buffer(0.05M, pH7.0), 1 ml H₂O₂ (0.019M), and 0.05ml homogenate(10%w/v) in a total volume of 3.0 ml. Absorption change was measured at 240 nm. CAT activity was calculated in terms of nM H₂O₂ consumed/min/mg protein.³⁴

Glutathione

GSH in liver tissue was quantified by the method described shortly here in, 5% tissue homogenate was prepared in 20 mM EDTA, pH 4.7, and 100 mL of homogenate or pure GSH was added to 0.2 M Tris-EDTA solution (1.0 mL, pH 8.2) as well as 20 mM EDTA, pH 4.7 (0.9 mL) followed by 20 mL of Ellman's reagent (10 mmol/L DTNB in methanol). After 30 min of incubation at room temperature, samples were centrifuged, and the absorbance was recorded at 412 nm.³⁶

Total thiols

This assay is based on the principle of formation of relatively stable yellow color by sulfhydryl groups with DTNB.³⁷ Briefly, 0.2 ml of brain homogenate was mixed with phosphate buffer (pH 8), 40 ml of 10mM DTNB, and 3.16 ml of methanol. This mixture was incubated for 10 min and the absorbance was measured at 412 nm against appropriate blanks.³⁸

Protein

Protein content in animal supernatant of homogenate was determined by method of Lowery et al.³⁹

Histopathological studies

The liver is taken from all groups were fixed with 10% formalin and embedded in paraffin wax and cut into longitudinal section of five µm thickness. These sections stained with H & E stains for histopathological assessment.

Statistical analysis

All values were expressed as mean±SEM. Results were analyzed statistically by using One Way Analysis of Variance (ANOVA) followed by multiple comparison Dunnet’s ‘t’ test. The p<0.05 was considered as significant.⁴⁰

Results

Biochemical estimation by using serum in paracetamol hepatotoxicity model

The different doses of ethanol extract of Curcuma vamana and standard group shows significant hepatoprotective action. The results summarized in tables (1) and (2). At the end of 12 days treatment, blood samples of control group animals showed significant (p<0.001) increase in the levels of AST, AST, ALP, total bilirubin, total cholesterols and LDL-cholesterols and decrease in the levels of glucose, HDL-cholesterols and total protein as compared to normal group animals, whereas blood samples from animals treated with standard drug and different doses (200, 400mg/kg) of extract showed significant (p<0.001) decrease in the levels of AST, ALT, ALP, Total bilirubin, Total cholesterols and LDL-cholesterols and increase in the levels of Glucose, HDL-cholesterols and Total protein, when compared with control group.

Enzymatic and Non-enzymatic estimation by using liver homogenate in paracetamol hepatotoxicity model

The enzymatic and non-enzymatic estimations summarized in table (3) reveal the potential hepatoprotective activity of Curcuma vamana ethanolic extracts. The liver homogenate of the control group showed significantly reduced activities of SOD, CAT, GSH and total thiols levels and increased lipid peroxidation level as compared to sham group. The EECV-administered groups standard group showed significant protection by preventing the increase of lipid peroxidation level and decrease of SOD, CAT, GSH, Total thiols levels increased significantly in 200 and 400 mg/kg EECV-treated groups as compared to the control group.

Biochemical estimation by using serum in CCl₄ induced hepatotoxicity Model

The different doses of ethanol extract of Curcuma vamana and standard group shows significant hepatoprotective action. The results were summarized in tables (4) and (5). After ninteen days treatment, control group animals blood samples showed significant (p<0.001) increase and decrease in the levels of serum biochemical parameters as compared to normal group animals, But animals treated with standard group and two different doses(200, 400mg/kg) of extract showed significant(p<0.001) decrease in the levels of AST, AST, ALP, Total cholesterols, Total bilirubin and LDL-cholesterols and increase in the levels of Glucose, HDL-cholesterols and Total protein, when compared with control group.  

Enzymatic and Non-enzymatic estimation by using liver homogenate in CCl₄ induced hepatotoxicity Model

The enzymatic and non-enzymatic estimations summarized in table (6) reveal the potential hepatoprotective activity of Curcuma vamana ethanol extract. The liver homogenate of the control group showed significantly reduced activities of SOD, CAT, GSH, and total thiols levels and increased lipid peroxidation level as compared to sham group. The EECV-administered groups standard group showed significant protection by preventing the increase of lipid peroxidation level and decrease of SOD, CAT, GSH, Total thiols levels increased significantly in 200 and 400 mg/kg EECV-treated groups as compared to the control group.

Histopathological studies

Histopathological observations of the normal group (Plate A) rat liver (Figure 1 and 2), showed normal hepatic architecture, absence of centrilobular necrosis and macrovesicular fatty changes and no dilation of portal vein. The control group rat liver (Plate B), exhibited intense centrilobular necrosis, vacuolization, macrovesicular fatty changes and distorted central vein architecture. The Standard group rat liver (Plate C) also restores the normal histopathological observations. However, the different doses of extract 200mg/kg treated (Plate E) and 400mg/kg treated groups (Plate F) showed normal hepatic architecture, absence of centrilobular necrosis and macrovesicular fatty changes, and no dilation of portal vein except some vacuolization and dilation of hepatic vein in case of 400mg/kg treated group rat liver.

All values of table were presented as a Mean ± SEM, n=6, One Way Analysis of Variance (ANOVA) followed by multiple Dunnet’s‘t’ test, by comparing different doses of extract treated groups with that of control (Paracetamol-treated) group. The minimum value of p<0.05 was considered as significant. *p<0.05, **p <0.01, ***p<0.001 as compared with control group and  ap<0.001 as compared with normal group. (EECV: Ethanol extract of Curcuma vamana).

All values of table were presented as a Mean ± SEM, n=6, One Way Analysis of Variance (ANOVA) followed by multiple Dunnet’s‘t’ test, by comparing different doses of extract treated groups with that of control (Paracetamol-treated) group. The minimum value of p<0.05 was considered as significant. *p<0.05, **p <0.01, ***p<0.001 as compared with control group and ap<0.001 and bp<0.01 as compared with normal group. (EECV: Ethanol extract of Curcuma vamana).

All values of table were presented as a Mean ± SEM, n=6, One Way Analysis of Variance (ANOVA) followed by multiple Dunnet’s ‘t’ test, by comparing different doses of extract treated groups with that of control (Paracetamol treated) group. The minimum value of p<0.05 was considered as significant. *p<0.05, **p<0.01, ***p<0.001 as compared with control group and ap<0.001 and bp<0.01 as compared with normal group. [EECV= Ethanolic extract of Curcuma vamana].

All values of table were presented as a Mean ± SEM, n=6, One Way Analysis of Variance (ANOVA) followed by multiple Dunnet’s‘t’ test. By comparing different doses of extract treated groups with that of control (CCl₄-treated) group. The minimum value of p<0.05 was considered as significant. *p<0.05, **p <0.01, ***p<0.001 as compared with control group and ap<0.001and bp<0.01 as compared with normal group. (EECV: Ethanol extract of Curcuma vamana).The DPPH free radical scavenging activity was calculated using the formula:

All values of table were presented as a Mean ± SEM, n=6, One Way Analysis of Variance (ANOVA) followed by multiple Dunnet’s‘t’ test. By comparing different doses of extract treated groups with that of control (CCl₄ -treated) group. The minimum value of p<0.05 was considered as significant. *p<0.05, **p <0.01, ***p<0.001 as compared with control group and ap<0.001and bp<0.01 as compared with normal group. (EECV: Ethanol extract of Curcuma vamana).  

All values of table were presented as a Mean ± SEM, n=6, One Way Analysis of Variance (ANOVA) followed by multiple Dunnet’s ‘t’ test, by comparing different doses of extract treated groups with that of control (CCl4-treated) group. The minimum value of p<0.05 was considered as significant. *p<0.05, **p <0.01, ***p<0.001 as compared with control group and ap<0.001 and bp<0.01 as compared with normal group. (EECV: Ethanol extract of Curcuma vamana).

Discussion 

Paracetamol and CCl₄ induced hepatic injuries are commonly used models for hepatoprotective drug screening⁴¹, the extent of hepatic damage is assessed by the serum and liver tissue homogenate level of increased cytoplasmic enzymes (AST, ALT, ALP, TB, LDL-cholesterol, Total cholesterols and LPO)⁴² and significantly decreases serum and liver homogenate HDL-cholesterol, Total protein, Glucose, GSH, CAT, SOD and total thiols. EECV when administered orally at the doses of 100, 200 and 400 mg/kg exhibited protection against paracetamol-induced lethality in rat suggesting hepatoprotective actions. The treatment of rat with CCl₄ is known to cause damage to drug metabolizing microsomal enzymes in hepatocytes which leads to a significant decrease in hepatic metabolizing capacity of drugs. Pretreatment of animals with EECV prevented the CCl₄ induced rise in serum level of transaminases and confirming a protective effect of EECV against CCl₄ induced damage to hepatocytes. Paracetamol is converted to a toxic reactive intermediate called N-acetyl-benzoquinone imine (NAPQI) following metabolism by a number of isozymes of cytochrome P-450 (CYPs), i.e. CYP 2E1⁴³, CYP 1A2⁴⁴, CYP 2A6⁴⁵, CYP 3A4 and CYP2D6.⁴⁶ The massive production of reactive species may lead to depletion of protective physiological moieties (glutathione and a- tocopherol, etc.), ensuing wide spread propagation of the alkylation as well as peroxidation, causing damage to the macromolecules in vital biomembranes⁴⁷.

The reactive species mediated hepatotoxicity can be effectively managed upon administration of agents possessing anti-oxidant⁴⁸, free radical scavenger⁴⁹ and anti-lipid per oxidant⁵⁰ activities. EECV treatment was able to ameliorate paracetamol induced hepatocellular damage as evidenced by prevention of increase in serum transaminase (AST, ALT, ALP, Total cholesterols etc) levels subsequent to its exposure. The inhibitors of CYPs are known to reduce the toxicity of paracetamol⁵¹ as well as CCl₄⁵², hence the reported inhibition of CYPs by EECV⁵³ might have contributed favourably toward the observed hepatoprotection.⁵⁴ When it comes to drug-induced acute hepatitis and leukotrienes, inflammation is key, the arachidonic acid metabolism's 5-lipoxygenase products have been implicated in a number of inflammatory diseases.^55-57

Conclusion

In conclusion, the results of the current study indicated that ethanol extract of Curcuma vamana showed hepatoprotective effects against paracetamol and carbon tetrachloride induced liver damage in albino rats. Future studies are required to evaluate the potential role of this plant extract in clinical conditions related with liver damage.

Acknowledgements

We are thankful to Principal, H.S.K. College of Pharmacy, Bagalkot, Karnataka, India, for providing necessary facilities during the course of this study.

Conflict of interest

The authors have no conflicts of interest.