INH-RIF (Isoniazid – Rifampicin) administration causes a number of changes in vital organs and systems. The liver is one of the target organ affected by INH-RIF (Santhosh et al., 2006). Rifampicin is widely used in most antitubercular chemotherapeutic regimens (Snider et al., 1984). However, this drug is also well known as hepatotoxic agent at doses (Steele et al., 1991). In the present study, following an overdose of INH-RIF, the rise in liver marker enzymes level has been attributed to damaged structural integrity of the liver.

When the liver plasma membrane is damaged, a variety of enzymes located in cytosol are released into the blood stream and their estimation in the serum is a quantitative marker for the extent and type of hepatocellular damage (Ansari et al., 1991). As the liver has a wide variety of functional capabilities, no one single test can provide an accurate assessment of its function. In this present study, assessment of various hepatic marker enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and γ-glutamyl transferase) and liver function markers (total bilirubin and protein), renal function markers (Urea, Uric acid, Creatinine), The

medicinal properties of folk plants are mainly attributed to the presence of flavonoids, but may also be influenced by other organic and inorganic compounds (Repetto and Llesuy, 2002). Aporosa lindleyana is a much branched, evergreen, glabrous tree, grown in India and Sri Lanka. It possesses antioxidant activity (Badami et al., 2005) and hepato protective effect (Ramakrishnan S and Venkataraman R, 2010) and also showed anti hyperglycemic effect (Jayakar and Suresh, 2003). Aporosa lindleyana have much medicinal properties such as diuretic, antiviral and good analgesic activity. Roots are used to treat headache, fever and jaundice, seminal loss and excessive thirst (Anonymous, 1985, Chopra et al., 1992, Kirtikar and Basu, 1987). The silymarin with a optimal dose has provided a better inhibition of the elevated level of AST, ALT, ALP and serum bilirubin (Durairaj et al., 2008).

2. MATERIALS AND METHODS:

2.1. Collection and processing of plant material:

The roots of Aporosa lindleyana was collected from Keeriparai, Kanyakumari District, Tamilnadu during the month of January 2009. The specimen was identified by Dr. V. Chelladurai, Taxonomist, Department of Ayurvedic Sciences, Tirunelveli District. The roots were cleansed and shade dried for a weak and grounded into uniform powder. 1 g of plant material was added to 20 ml of aqueous ethanol (20% v/v) for 18 h at room temperature.

2.2. Experimental Animals:

Healthy adult male albino Wistar rats, bred and reared in Central Animal House, Department of Pharmacology, K.M College of Pharmacy, Uthangudi, Madurai, were used for the experiment. Weight matched animals (180-200g) were selected and housed in polypropylene cages layered with husk and kept in a semi-natural light/dark condition (12 h light/12 h dark). The animals were allowed free access to water and standard pellet diet (Amrut Laboratory Animal Feed, Pranav Agro Industries Ltd., Bangalore, India). Animal handling and experimental procedures were approved by the Institutional Animal Ethics Committee (IAEC Registration Number: 661/02/c/CPCSEA) and animals were cared in accordance with the “Guide for the care and use of laboratory animals” and “Committee for the purpose of control and supervision on experimental animals” (CPCSEA).

2.3. Experimental Design:

2.3.1. Preliminary Study:

Preliminary study was carried out to determine the optimum dose of EREAL by assessing serum hepatic marker enzyme activities in hepatotoxic rats. EREAL was given at different doses (i.e. 100, 200 and 300 mg/kg/BW) to different groups of animals. The animals were randomly divided into seven groups of six animals each. The EREAL and silymarin were dissolved in 2 mL of 1% CMC vehicle solution and fed by intubation. Among the three doses the 200 mg dose was more effective. The protective effect at the dose of 200 mg/kg BW was more pronounced than that of other two doses 100 and 300 mg dose was used for further study.

2.3.2. Experimental Protocol for Further Study:

The animals were randomly divided into five groups of six animals each. EREAL (200 mg/kg BW) and silymarin (70 mg/kg BW) was suspended in 2 mL of 1% CMC (vehicle solution) and fed by intragastric tube daily for 21 days. After 21 days of treatment, the animals were fasted for 12 h, and sacrificed by cervical dislocation. Blood was collected in tubes with a mixture of potassium oxalate and sodium fluoride (1:3) for the estimation of various biochemical parameters. Tissue (liver and kidney) were surgically removed, washed with cold physiological saline, cleared off adherent lipids and immediately transferred to ice-cold containers. Erythrocytes were also prepared for the estimation of various biochemical preparations

2.3.4. Serum preparation:

Blood was collected in a dry test tube and allowed to coagulate at ambient temperature for 40 min. Serum was separated by centrifugation at 2000 rpm for 10 min.

2.3.5. Tissue sampling for histological study:

For histological study, three rats from each group were perfused with cold physiological saline, followed by formalin (10% formaldehyde). The liver and kidney were excised immediately and fixed in 10% formalin. Then dehydrated on treatment with a serious of different concentration of ethanol and embedded in paraffin wax. 3-5µm thick sections were cut using a microtome and stained with hemotoxyin and eosin. The specimens were evaluated with light microscope. All histopathological changes were examined by pathologist.

2.4. Estimation of hepatic markers:

2.4.1 Assay of Aspartate Aminotransferase (Ast, Ec 2.6.1.1):

Serum aspartate aminotransferase was assayed by using the diagnostic kit method (Reitman and Frankel, 1957). AST catalyses the transfer of amino group from L-aspartate to α-ketoglutarate with the formation of oxaloacetate and glutamate. The amount of oxaloacetate was measured by converting it into pyruvate by treating with aniline citrate and then reacting the pyruvate with 2, 4-dinitrophenylhydrazine to form 2, 4-dinitrophenyl hydrazone derivative which is brown colored in alkaline medium. The absorbance of this hydrazone derivative is correlated to AST activity.

2.4.2 Assay of Alanine Aminotransferase (Alt,

Ec 2.6.1.2:

Serum alanine aminotransferase was assayed by using the diagnostic kit based on the method (Reitman and Frankel, 1957). ALT catalyses the transfer of amino group from L-alanine to -ketoglutarate with the formation of pyruvate and glutamate. The pyruvate so formed, was allowed to react with 2, 4-dinitrophenylhydrazine to produce 2, 4-dinitrophenylhydrozone derivative which is brown colored in alkaline medium. The absorbance of this hydrazone derivative is correlated to ALT activity.

2.4.3 Estimation of Alkaline Phosphatase (Alp, Ec 3.1.2.3.1):

Plasma alkaline phosphatase was estimated by using the diagnostic kit method (Kind and Kings, 1954). ALP catalyses disodium phenyl phosphate into phenol and disodium hydrogen phosphate at pH 10. Phenol so formed reacts with 4-aminoantipyrine in alkaline medium in the presence of oxidizing agent potassium ferricyanide to form a red colored complex whose absorbance is proportional to the enzyme activity.

2.4.4. Estimation of γ-glutamyl transferase (ggt, ec 2.3.2.2):

The enzyme activity was assayed according to the method (Rosalki and Rau, 1972). GGT hydrolyses peptide bonds, in which a terminal glutamic acid residue is linked by its -carboxyl group to an amino group. The enzyme is of low specificity for non-glutamyl moiety, so that synthetic substrates such as -glutamyl-p-nitroanilide are acted upon by GGT which catalyses the simultaneous transfer of glutamyl residues to an amino acid or peptide acceptor, glycyl glycine is chosen for this purpose, to form a yellow product paranitroanilide.

2.4.5. Estimation of Serum Bilirubin:

Serum bilirubin was estimated by the method (Malloy and Evelyn, 1937). Serum was diluted with water, and methanol added in an amount insufficient to precipitate the proteins, yet sufficient to permit all the bilirubin to react with diazo reagent.

2.5. Estimation of renal function markers:

2.5.1. Estimation of urea:

Serum urea was estimated by using the diagnostic kit based on the method (Fawcett and Scott, 1960). Urea is hydrolysed in the presence of water and urease to produce ammonia and carbon dioxide. Under alkaline conditions, the ammonia so formed reacts with hypochlorite and sodium salicylate in the presence of sodium nitroprusside to form a green colored chromophore. The intensity of the color produced is proportional to the concentration of urea in the sample.

2.5.2. Estimation of Uric Acid:

Serum uric acid was estimated by using the diagnostic kit enzymic method (Caraway, 1955). Uric acid in the sample is oxidized by uricase to allantoin. In this reaction 1 mole of hydrogen peroxide is formed for every mole of uric acid oxidized. Hydrogen peroxide reacts with 3, 5-dichloro-2-hydroxybenzene sulfonic acid and 4-aminoantipyrine to give quinoneimine dye. Intensity of the color of this dye was proportional to the concentration of uric acid in the sample.

2.5.3. Estimation of Creatinine

Seum creatinine was estimated using the diagnostic kit method (Tietz, 1987 using Jaffe’s, 1886) color reaction. The assay of creatinine has been based on the reaction of creatinine with alkaline picrate. Most of the contaminants reacting with the Jaffe’s reagent produce a colour at a lower rate than does creatinine. The initial rate of colour formation is proportional to the concentration of creatinine in the sample.

2.5.4. Estimation of Tissue Protein:

Protein in the tissues was determined after trichloro acetic acid precipitation method (Lowry et al., 1951).

3. RESULTS AND DISCUSSION:

3.1 Effect of Ereal on Hepatic Marker Enzymes and Bilirubin in the Serum:

As the liver has a wide variety of functional capabilities, no one single test can provide an accurate assessment of its function. In this study, assessment of various hepatic marker enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and γ-glutamyl transferase) and liver function markers (total bilirubin and protein) were used to determine the effect of EREAL on INH-RIF induced hepatotoxicity.

Table 1 showed the activities of hepatic marker enzymes such as serum AST, ALT, ALP, GGT and bilirubin in INH-RIF induced hepatotoxic and control rats. Increased activities of AST, ALT, ALP, GGT and bilirubin levels and decreased total protein levels were observed in INH-RIF induced rats. Oral administration of EREAL (200mg/kg BW) and silymarin (70mg/kg BW) improved these parameters towards normal. The protective effect at the dose of 200 mg/kg BW was more pronounced than that of other two doses (100 and 300 mg/kg BW).

_{Table 1. Effect of EREAL on hepatic marker enzymes and bilirubin in the
serum}

Time	AST(IU/L)	ALT(IU/L)	ALP(IU/L)	GGT(IU/L)	Bilirubin(mg/L)	Total Protein (mg/dL)
Control rats received 1% CMC only	120.41±3.62a	346.62±1.14a	120.40±2.93 a	92.6.41±1.68 a	0.062.±0.05 a	0.50.41±0..08 a
Control +EREAL (300mg/kg BW)	125.6±4.51a	351.65±2.10a	128.51±4.32a	95.28±2.65a	0.68.±0.04a	0.54.41±0.06a
INH+RIF (50mg/kg BW)	432.8±10.23b	168.241±3.46b	340.0±6.88b	182.8±6.21b	2.02.±0.16b	0.30±0.02b
INH-RIF+EREAL (100mg/kg BW)	310.4±10.21c	110.85±4.32c	210.34±7.28c	165.40±2.86c	1.75±0.13c	0.34±0.02c
INH-RIF+EREAL (200mg/kg BW)	232.4±2.98d	64.8±2.01d	188.4±5.96d	119.4±4.22d	0.88±0.10d	0.40±0.08d
INH-RIF+EREAL (300mg/kg BW)	274.26±4.80e	86.34±3.28e	198.10±7.28e	145.40±3.25e	1.08±0.18e	0.38±0.06e
INH-RIF+Silymarin (70mg/kg BW)	170.4±1.98f	54.2±1.52f	172.8±5.21f	106.6±1.32f	0.74±0.06f	0.43±0.11f

Values are given as means SD for six rats in each group.

Values not sharing a common superscript differ significantly at p < 0.05. DMRT.

Serum aminotransferases activities have long been considered as sensitive indicators of hepatic injury. This injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells (Molander et al., 1955). Therefore, the abnormal high level of serum biomarker enzymes and bilirubin observed in this study are the consequence of INH-RIF induced liver dysfunction and denotes the damage to the hepatic cells. Oral administration of EREAL exhibited a significant reduction in to activities of serum AST, ALT, ALP, GGT, bilirubin and improved protein levels remarkably to the normal group that is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage and effective control of bilirubin level and ALP activity points towards an early improvement in the secretary mechanism of the hepatic cell.

The silymarin with a optimal dose has provided a better inhibition of the elevated level of AST, ALT, ALP and serum bilirubin (Durairaj et al., 2008). This results indicate that the hepatoprotective activity of the EREAL probably through the correction of cellular integrity of hepatic cell and its regeneration.

3.2. Effect of Ereal on Renal Function Markers in the Serum

The kidney plays a central role in the regulation of the balance of body salt and water, and then disordered regulation of renal functions is responsible for the altered balance of salt and water in pathophysiological states including hepatotoxicity.

The effect of EREAL on renal function markers are shown in Table 2. The levels of urea, uric acid and creatinine were significantly increased in INH-RIF induced rats and treatment with EREAL and silymarin significantly decreased the levels of urea, uric acid and creatinine.

Chronic is oftenly associated with a wide range of metabolic disorder including serum urea, uric acid and creatinine derangements (Adeneye et al., 2008). As such elevation in the serum concentration of these levels, particulary, serum urea, uric acid and creatinine are considered reliable, well documented parameters for investigating drug-induced nephrotoxicity in animals and man (Adelman et al., 1981). Treatment with EREAL significantly attenuated the elevated serum concentrations of these parameters, in dose related pattern. It shows that EREAL, to an extent, preserves the functional capacity of the kidney from the adverse effects of INH-RIF.

Table 2. Effect of EREAL on the levels of renal function markers in the serum:

Groups	_{Urea (mg/dL)}	_{Uric acid (mg/dL)}	_{Creatinine (mg/dL)}
_{Control rats received 1% CMC only}	_{23.37 ± 2.41a}	_{1.65 ± 0.07 a}	_{0.83 ± 0.05 a}
_{Control + EREAL (200mg/kg BW)}	_{21.18 ± 2.12 a}	_{1.53 ± 0.10 a}	_{0.87 ± 0.06 a}
_{INH-RIF – EREAL (50 mg/kg BW)}	_{45.75 ± 3.40 b}	_{2.86 ± 0.21 b}	_{1.97 ± 0.11 b}
_{INH-RIF + EREAL (200 mg/kg BW)}	_{30.59 ± 2.29 c}	_{1.86 ± 0.15 c}	_{1.23 ± 0.12 c}
_{INH-RIF + Silymarin (70 mg/kg BW)}	_{28.43 ± 2.12 c,d}	_{1.78 ± 0.09 d}	_{1.04 ± 0.05d}

Values are given as mean SD for six rats in each group.

Values not sharing a common superscript differ significantly at p < 0.05. (DMRT).

3.3. Histopathological changes in the liver and kidney:

Histopathological examination of INH-RIF induced hepatotoxic rat liver showed micro and macro vesicular fatty changes (Figure 1 and 2). Administration of EREAL and silymarin reversed the changes which was

Histopathological examination of INH-RIF induced hepatotoxic rats of kidney showed cloudy swelling of tubules and glomerulosclerosis. Administration of EREAL and silymarin showed mild condition of focal fatty infiltration of glomeruli and tubules whereas silymarin treatment showed normal glomeruli and focal area of haemorrage. Oral administration of EREAL and silymarin reduced the index of kidney pathology showed that the EREAL is not only a hepatoprotective but also protect the renal from the toxicity caused by INH-RIF.

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Received on 19.01.2020 Modified on 14.02.2020

Asian J. Res. Pharm. Sci. 2020; 10(1):11-16.

DOI: 10.5958/2231-5659.2020.00003.X