INTRODUCTION:

Liver is the major organ which plays an important role in regulation of physiological processes, which includes vital functions like metabolism, secretion and storage, it also play a major role in detoxification and excretion of various exogenous and endogenous compounds like drugs xenobiotics etc. Any impairment of liver functions may lead to various implications on one’s health^[1],[2]. The diseases related to liver come under most serious ailments which may be classified as acute or chronic hepatitis (inflammatory liver diseases), cirrhosis (fibrosis of liver), and hepatosis (non-inflammatory diseases).

Toxic chemicals, drugs like paracetamol, antitubercular, anticancer agents or alcohol mainly cause liver diseases. These hepatotoxic chemicals mainly damage liver cells by lipid peroxidation or other oxidative stress induced cell damage^[3].

The drugs and xenobiotics in concentrated form arrives the liver through blood which is directly coming from gastrointestinal organs and spleen via portal vein. A number of mechanisms are responsible for inducing hepatic injury or worsening the damage process. Several chemicals damage mitochondria and intracellular organelle which release energy and its dysfunction produces excessive amount of oxidants, this in turn injure hepatic cells. Activation of certain enzymes like CYP2E1 in cytochrome P-450 system leads to oxidative stress and injury to hepatocyte, bile duct cells leading to accumulation of bile acid inside liver and this promotes further liver damage^[4].

Conventional drugs used in the treatment of liver diseases are often inadequate. Therefore it is necessary to search alternative drugs for treatment of liver diseases and to replace the currently used drugs which are doubtful of its efficacy and safety^[5].

Barleria cuspidate L is a perinneal herb belonging to the family Acanthaceae which is commonly named as Pido-Kanta-Shediyo. In Tamil and Sanskrit it is called Vellaimuli and Kurantaka respectively. The whole plant is known to possess various chemical constituents like alkaloids, carbohydrates, flavonoids, flavones, glycosides, steroids, tannins, phenols etc. In previous studies it has been reported that this plant has showed antihelminthic activity and also has wound healing potential. It is also used as folk medicine^[6]. There are no reports on hepatoprotective activity of this plant. Hence the present study was carried out to evaluate the hepatoprotective activity of Barleria cuspidate against CCl₄induced liver toxicity in experimental rats.

MATERIALS AND METHODS:

Collection and identification of Plant material:

Fresh samples of Barleria cuspidata were collected from local areas of Tirupathi, Andhra Pradesh. The botanical identity was confirmed at the department of botany, SV University Tirupathi, by a plant taxonomist.

Preparation of extract:

The dried leaves of Barleria cuspidata were taken, powdered in a grinder-mixer to obtain a coarse powder and then passed through 40 mesh sieve. About 200gms of powder was extracted by using 70% methanol by maceration process up to 24hrs. The solution was filtered through Whatmann filter paper and the resultant filtrate was distilled under reduced pressure for recovery of solvent. The dried extract thus obtained was kept in desiccators and used for further experiments.

Experimental animals:

Wistar albino rats of either sex (150-230gm) procured from Raghavendra enterprises (Bangalore), were used in the present study. The animals were housed in the clean propylene cages and maintained under standard conditions (25±2ºC, relative humidity 44 - 56% and 12 hours light and dark cycles respectively) and fed with standard rat diet (Mysore feeds, Bangalore) and purified drinking water ad libitum for 1 week before and during the experiments. Animals were handled with human care.

Institutional Animal Ethical Committee (IAEC) of P. Rami Reddy Memorial College of Pharmacy (1423/PO/Re/S/11/CPCSEA/110/01/2018) approved the present study.

Drugs and chemicals:

Silymarin (Sigma), carbon tetrachloride and all other reagents used were of analytical grade. Diagnostic kits used in this study were procured from Span Diagnostic Ltd., India and Excel diagnostic Ltd., India.

Acute toxicity studies:

Acute oral toxicity study was performed as per OECD-423 guidelines^[67]. The extract was administered orally at the dose level of 2000mg/kg body weight by gastric intubation and observed for 24 hours. If mortality was not observed, the procedure was repeated for further higher dose such as 4000mg/kg of body weight.

The mortality and morbidity was observed after 24 hours.

Experimental design:

The experimental design used to carry out the hepatoprotective activity of methanolic extract of B.cuspidata was CCl₄ induced hepatic damage model.

The animals were grouped into five groups with six animals in each. Except the normal group all the other groups received carbon tetrachloride (CCl₄) 50% v/v in coconut oil at a dose of 0.1ml/kg b.w intraperitoneally for 14 days. Normal groups received plain coconut oil orally. The standard group received silymarin 50mg/kg orally. Test groups received MEBC 200mg/kg and 400mg/kg b.w orally. On the 28^th day, blood was collected from each animal for serum analysis.^[7].

Assessment of serum marker enzymes:

The blood samples were collected in EDTA-free vials on day 0, 7, 14, 21, 28. The collected blood samples were centrifuged under cooling condition at 4000RPM for 10-15 minutes to separate plasma and serum. The separated serum was used for the estimation of biochemical parameters like SGOT, SGPT, ALP, total cholesterol, total albumin, total protein and TB using commercial available diagnostic kit (Span Diagnostic, Ltd)^[8].

Histopathological studies:

One representative animal from the each group was utilized for this purpose. The liver specimens obtained from the treated and control groups of animals were mounted in 10% buffered formalin solution for 24 hr. The formalin fixed liver samples were stained with haematoxylin-eosin for photo microscopic observations of the liver histopathological architecture.

Statistical Analysis:

All parameters are expressed as a mean value ± SEM. Differences between the mean value of tests and control groups were evaluated statistically by using the one way analysis of variance (ANOVA), Student’s t-test.

RESULTS:

Acute toxicity studies:

Acute oral toxicity study was performed as per OECD-423 guidelines. The methanolic extract of B.cuspidata was found to be safe since no animal died even at the maximum single dose of 4000mg/kg when administered orally. The animal did not show any gross behavioural changes.

Hepatoprotective activity:

Administration of CCl₄ induced a significant increase (p<0.05) in serum SGOT, SGPT, ALP, Total bilirubin, total cholesterol levels on 28^th day when compared to normal group. While a significant (p<0.05) reduction was observed in serum levels of above parameters on 28^th day in rats treated with standard drug silymarin when compared to control group. The trend was same with that of both the groups (IV and V) receiving methanolic extract of B.cuspidata (200mg/kg and 400mg/kg) witnessing a significant fall in above biochemical parameters on 28^th day when compared to control group as shown in the table no.01.

The serum levels of total protein and albumin were significantly decreased in rats up on administration of CCl₄ when compared to normal. Silymarin treated groups showed significant rise (p<0.05) in serum levels of total protein and albumin compared to control group. The MEBC (200mg/kg and 400mg/kg) treated groups also showed significant increase (p<0.05) in total protein and albumin levels compared to control group as shown in (table no.01).

Histopathological Studies:

Along with the levels of various biochemical parameters the histopathological observations after CCl₄ administration is assessed to find out the extent of hepatic damage caused. When compared to normal hepatic architecture of normal group animals, the control group animals showed severe hepatic damage manifested by intense steatosis, centrilobular necrosis, ballooning degeneration, nodal formation and fibrosis. Treatment of methanolic extract of B.cuspidata showed remarkable healing of damaged parenchyma resulting in regenerative effects as shown in (figure no.01).

Table.no 1: Effect of Barleria cuspidata on serum biochemical parameters

S. No	Group	SGOT (IU/L)	SGPT (IU/L)	Alk.P (IU/L)	TB (mg/dL)	TP (g/dL)	TC (mg/dL)	ALB (g/Dl)
I	Normal	70.5 ± 0.76	39.83 ± 1.4	153.7 ± 2.52	0.33 ± 0.04	6.41 ± 0.17	56.17 ± 2.07	3.83 ± 0.06
II	CCl₄ treated	232.8 ±2.24^###	186.7 ± 2.07^###	295.3 ± 3.18^###	1.7 ± 0.15^###	5.83 ± 0.08^##	147.8 ± 2.49^###	3.10 ± 0.09^###
III	Silymarin	139.3 ±1.47^***	116.2 ± 1.64^***	144.5 ± 5.27^***	0.63 ± 0.04^***	9.16 ± 0.12^***	89.67 ± 1.35^***	4.15 ± 0.10^***
IV	Test 1 (200 mg/kg PO)	190.5 ±1.43^***	160.3 ± 0.88^***	177.5 ± 1.25^***	1.46 ± 0.12^ns	4.08 ± 0.09^***	102.5 ± 1.17^***	3.80 ± 0.07^***
V	Test 2 (400 mg/kg PO)	175.3 ±1.45^***	136.3 ± 1.82^***	167.7 ± 2.49^***	0.76 ± 0.04^***	6.15 ± 0.11^**	100.5 ± 2.02^***	4.13 ± 0.05^***

All values were shown as mean ± SEM and n=6, ### indicates p < 0.001 when compared to normal group, *** indicates p < 0.001 when compared to control group

Figure 1: Histopathological observations

DISCUSSION:

The commonly used hepatotoxin for experimental study of liver diseases is carbon tetrachloride^[9]. CCl₄ upon administration causes acute liver damage that mimics natural causes, mediating some changes in liver function which finally leads to destruction of hepatocellular membrane. CCl₄ is activated to form various free radicals like trichloromethyl, Cl₃C-CCl₃ (hexachloroethane), COCl₂ (phosgene), etc by CYP 450. These free radicals are involved in the pathogenesis of liver damage in chain reaction which results in lipid peroxidation, covalent binding of macromolecules, discruption of metabolic mechanisms in mitochondria, decreasing levels of phospholipids, increasing triglyceride levels, inhibition of calcium pumps of microsomes that finally results to liver necrosis^[10].

The acute toxicity study revealed the absence of lethality among the tested animals when the extract was administered as a single dose. There were no signs of any gross behavioural changes indicating the safe usage of the extract at a dose of 200mg/kg.

The enzymes are released in to the circulation due to necrosis or membrane damage is measured in the serum. The increased serum levels of enzymes in CCl₄-induced liver damage can be reversed by the extract may be due to the prevention of the leakage of intracellular enzymes by its membrane stabilizing activity. Amino tranferases are group of liver specific enzymes which are most commonly utilized and considered as very sensitive and specific indicators of hepato cellular necrosis. These enzymes are aspartate amino transferase (AST, formerly serum glutamate oxaloacetic transaminase, SGOT) and alanine amino transferase (ALT, formerly serum glutamate pyruvate transaminase, SGPT) catalyses the transfer of amino acids of aspartate and alanine respectively to the keto group of glutaric acid^[11].

In the present study the rats treated with methanolic extract of B.cuspidata significantly decreased the serum levels of SGPT and SGOT with the healing of hepatic parenchyma and the regeneration of hepatocytes indicating its hepatoprotective activity.

A membrane bound glycoprotein enzyme Alkaline phosphatase is present in higher concentration in endothelium and sinusoids. It reaches the liver mainly from bone and excreted into the bile. During hepatobiliary diseases like large bile duct obstruction, intrahepatic cholestasis, or infiltrative diseases of liver, the serum levels of ALP are increased^[12]. The results of the present study indicate that both Test groups probably stabilize the hepatic plasma membrane from CCl₄- induced damage.

The elevated levels of bilirubin are seen in case of liver toxicity. Diseases of hepatocytes, obstruction to biliary excretion in to duodenum, in haemolysis, defects of hepatic uptake and conjugation of bilirubin pigments results in increase in the levels of bilirubin.

In present study the animals treated with methanolic extract of B.cuspidata significantly decreased the levels of bilirubin in serum indicating its hepatoprotective activity.

The liver plays an important role in the serum protein synthesis as it is the source of plasma albumin, fibrinogen including other important compounds like α and β-globulin. Liver is also involved in the synthesis of γ- globulin. Hepatic diseases results in the depletion of serum albumin levels. The elevated serum enzyme levels of Alk.P, AST and ALT mediates the escape of both non-protein and protein nitrogenous substances from injured cells resulting in the impairment in the metabolic biotransformation of amino acids in liver by synthesis, transformation etc^[13]. The decreased total protein levels is due to the initial damage produced and localised in the endoplasmic reticulum that results in the loss of CYP 450 leading to its functional failure followed by reduction in protein synthesis and accumulation of triglycerides leading fatty liver^[14].

In the current study the groups IV and V receiving methanolic extracts of B.cuspidata considerably raised the synthesis of TP by accelerating the regeneration process and protecting the liver cells. The enhanced levels of total protein in serum are indicative of the hepatoprotective activity.

The synthesis of bile acids from cholesterol which is obtained from plasma lipids or synthesized in liver is inhibited during CCl₄ intoxification resulting in increased cholesterol level. The marked decrease in cholesterol levels seen in rats treated with methanolic extracts of B.cuspidata indicates that the inhibition of bile acid synthesis is reversed.

Along with the levels of various biochemical parameters the histopathological observations after CCl₄ administration is assessed to find out the extent of hepatic damage caused. The histological evaluation showed severe damage in hepatocytes, basically supporting the alterations that are regarded in biochemical analysis.

When compared to normal hepatic architecture of normal group animals, the control group animals showed severe hepatic damage manifested by intense steatosis, centrilobular necrosis, ballooning degeneration, nodal formation and fibrosis. Treatment of methanolic extract of B.cuspidata showed remarkable healing of damaged parenchyma resulting in regenerative effects.

The potential of any hepatoprotective drug is determined by its ability of either decreasing the deleterious effect or restoring the normal hepatic physiology which has been altered by hepatotoxin. Both the test groups i.e. IV and V groups exhibited hepatoprotective activity. Both the test groups containing methanolic plant extract alone showed an improvement in the liver activity clearly indicating the hepatoprotective potential of the plant “B.cuspidata” might be due to presence of antioxidant potential. Thus the drastic healing of liver functions may be due to additive effect of its antioxidant potential.

The hepatoprotective activity is mainly due to the presence of chemical constituents in it. Phytochemical constituents like flavonoids, alkaloids, flavones and phenols are known to possess hepatoprotective activity. In addition to these compounds, the antioxidant and pro-oxidant like Vit C, α-tocopherol, ferulic acid, betalain alkaloid pigments and carotenoids in our plant i.e. B.cuspidata might be responsible for its antioxidant and thus hepatoprotective activity.

In summary, this study suggests that the oral administration of B.cuspidata alone significantly improve CCl₄ induced hepatotoxicity in rats. The extract may be protecting the liver by free radical scavenging activity and thus preventing peroxidation of lipids of the endoplasmic reticulum. And this may be due to the presence of flavonoids and alkaloidal pigments in our extract. However, the possibility that B.cuspidata might suppress the cytochrome P-450 mediated metabolic activation of CCl₄ cannot be ruled out.

CONCLUSION:

Our work aims to study the therapeutic activity of the methanolic extract of the plant Barleria cuspidata by examining the prevention of CCl₄ induced hepatotoxicity in rats. Our biochemical results demonstrated that both the test groups used i.e. Test 1 group (containing 200mg/kg MEBC) and Test 2 group (containing 400mg/kg MEBC) prevented CCl₄ induced hepatotoxicity in rats. This was evident from the results obtained from the serum biochemical parameters and tissue antioxidant studies.

In support to this study, histopathological results also show significant activity of the plant. In toxicant treated animals there will be severe disturbances in the cytoarchitecture of the liver. The same is observed in case of the humans who are suffering from major liver disorders. But in methanolic extract of Barleria cuspidata treated group animals exhibited minimal hepatic dearrangements and intact cytoarchitecture of the liver was maintained. In addition to this there is regeneration of hepatocytes also observed, which indicates hepatoprotective activity.

Finally based on improvement in serum marker enzyme levels, physical parameters, functional parameters and histopathological studies, it is concluded that the methanolic extract of Barleria cuspidata possess hepatoprotective activity and thus supports the traditional application of the same under the light of modern science.

ACKNOWLEDGEMENT:

The authors are grateful to P. Rami Reddy Memorial College of Pharmacy, Kadapa, Andhra Pradesh, India for providing necessary facilities to carry out this work.

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Received on 04.07.2019 Modified on 12.08.2019

Research J. Pharm. and Tech 2020; 13(2):538-542.

DOI: 10.5958/0974-360X.2020.00101.8