**2. Materials and methods**

#### **2.1. Chemicals**

Anti TB drug *i.e,* Rifampicin, Isoniazid, Pyrazinamide as anti TB kit was generously obtained from Government TB hospital, Gwalior (M.P.). All the other chemicals used in this study were of analytical grade and were procured from Sigma–Aldrich (USA), E Merck (Germa‐ ny), Ranbaxy Pvt. Ltd. and BDH Company (India).

#### **2.2. Preparation of plant extract**

Whole plant of *Phyllanthus amarus* was obtained from the authenticated ayurvedic dealer and was identified by the experts of Botany Department, Jiwaji University Gwalior, India. The shade dried plant was pulverized and extracted with 75% alcohol for 10 days with concomitant shaking and then filtered. The filtrate was evaporated in vacuum to yield a brownish powder (P. amarus) powder it was stored in refrigerator at 4°C and adminis‐ tered orally according to body weight of animals at different concentration.

#### **2.3. Animal maintenance and feeding**

Female albino rats of *Sprague Dawley* strain (160±10 g, b.w.) were used for the present investigation. Animals were housed in polypropylene cages under standard conditions (25±2°C temperature, 60%-70% relative humidity and 14 h light and 10 h dark) and were fed on standard pellet diet (Pranav Agro Industries Ltd., New Delhi, India) and drinking water *ad libitum*. Animals used in this study were treated and cared for in accordance with the guidelines recommended by the Committee for the Purpose of Control and Supervi‐ sion of Experiments on Animals (CPCSEA), Government of India, Ministry of Culture, Chennai.

#### **2.4. Experimental design**

Animals were divided into various groups of six animals each. *P.amarus* and anti TB drugs were given alternatively for 8 weeks (3 days/week). The groups were treated as follows:

Group I: Control (normal saline, 0.9%).

care system in India and have been found to be effective in the treatment of different diseases and are the beacon of the therapeutic sources for curing diseases from times immemorial (Merina *et al.,* 2012). *Phyllanthus amarus* Linn. is commonly known as *bhumi amla* (Joshi&Parle2007). It is reported to possess antiviral (Lee *et al.,* 1996), anticancer (Rajeshkumar *et al.,* 2002). But there is no scientific evidence for its hepatoprotective activity against ATD induced liver injury. Hence the present study was undertaken to explore the key behind the use of *P.amarus* as a hepatoprotective formulation against xenobiotics

Anti TB drug *i.e,* Rifampicin, Isoniazid, Pyrazinamide as anti TB kit was generously obtained from Government TB hospital, Gwalior (M.P.). All the other chemicals used in this study were of analytical grade and were procured from Sigma–Aldrich (USA), E Merck (Germa‐

Whole plant of *Phyllanthus amarus* was obtained from the authenticated ayurvedic dealer and was identified by the experts of Botany Department, Jiwaji University Gwalior, India. The shade dried plant was pulverized and extracted with 75% alcohol for 10 days with concomitant shaking and then filtered. The filtrate was evaporated in vacuum to yield a brownish powder (P. amarus) powder it was stored in refrigerator at 4°C and adminis‐

Female albino rats of *Sprague Dawley* strain (160±10 g, b.w.) were used for the present investigation. Animals were housed in polypropylene cages under standard conditions (25±2°C temperature, 60%-70% relative humidity and 14 h light and 10 h dark) and were fed on standard pellet diet (Pranav Agro Industries Ltd., New Delhi, India) and drinking water *ad libitum*. Animals used in this study were treated and cared for in accordance with the guidelines recommended by the Committee for the Purpose of Control and Supervi‐ sion of Experiments on Animals (CPCSEA), Government of India, Ministry of Culture,

Animals were divided into various groups of six animals each. *P.amarus* and anti TB drugs were given alternatively for 8 weeks (3 days/week). The groups were treated as follows:

tered orally according to body weight of animals at different concentration.

induced hepatic adverse effect.

**2. Materials and methods**

**2.2. Preparation of plant extract**

**2.3. Animal maintenance and feeding**

ny), Ranbaxy Pvt. Ltd. and BDH Company (India).

284 Pharmacology and Nutritional Intervention in the Treatment of Disease

**2.1. Chemicals**

Chennai.

**2.4. Experimental design**

Group II: *Per se* - 400mg/kg, b.w., *P.amarus* (p.o.), daily.

Group III: Experimental control-RIF+INH+PZA+ETH at 52, 70,175,140 mg/kg, b.w., (p.o.), 3days/week.

Group VI- VII: INH+RIF+PZA (as in group III) + *P.amarus* at 100, 200, 300 and 400 mg/kg, b.w., (p.o) 3days/week.

Group VIII: Positive control-Silymerin -50mg/kg b.w. (p.o.), daily.

All the animals were euthanized 24hours after the last treatment to perform various biochem‐ ical and histological analysis.

#### *2.4.1. Blood biochemical investigations*

Blood was drawn from retro-orbital venous sinus and serum was isolated. Serum Urea, Uric acid and Creatinine determined by commercially prepared kit method (E-Merck, Germany). Serum AST and ALT activity were determined with Reitman and Frankel, 1957.

#### *2.4.2. Tissue biochemical investigations*

The tissues viz., liver and kidney were quickly excised, washed in ice cold, normal saline and blotted individually freed from extraneous material on ash-free filter paper. The tissues were then homogenized separately in hypotonic buffer (0.008% NaHCO3), pH 7.4, using a Potter-Elvejham homogenizer at 600-1000 rpm in ice cold conditions.

The crude tissue homogenate was centrifuged at 2000 rpm for 15 min (0-4°C). The supernatant was collected and stored at- 20°C until used for estimating tissue biochemi‐ cal parameters. Lipid Peroxidation (LPO) was determined by measuring thiobarbituric acid reactive substances (TBARS) in tissues according to Sharma and Krishnamurthy, 1968.SOD and Catalase activity were determined according to Misra and Fridovich, 1972 and Aebi, 1974 respectively.

#### *2.4.3. Histopathological investigations*

Tissues were fixed with Bouin's solution. They were later sectioned using a microtome, dehydrated in graded alcohol, embedded in paraffin section, and stained with hemotoxylin and Eosin (H & E).

#### **2.5. Statistical analysis**

Results are presented as mean ± S.E. of six animals used in each group. Data were subjected to statistical analysis through one-way analysis of variance (ANOVA) at 5% significance level followed by Student's t-test at p≤ 0.05( Snedecor and Cochran,1989).


**Treatment Urea (mg/dl) Uric acid (mg/dl) Creatinine (mg/dl) Group I** 19.5±1.08 5.3± 0.292 0.4± 0.022 **Group II** 24±1.33 5.6± 0.309 0.5±0.027 **Group III** 52.6±2.91# 10±0.552# 0.8± 0.044#

**ANOVA F Value 32.005@ 20.642@ 16.887@** Data are mean ± S.E., n=6.ANOVA (F Values at 5% level).# P≤0.05 vs. Control,\*P ≤0.05 vs. ATD,@ Significant

**Group I** 0.31±0.017 0.3± 0.016 7.97± 0.441 7.5± 0.441 **Group II** 0.341±0.018 0.33± 0.018 8.136±0.449 7.78± 0.449 **Group III** 1.94±0.107# 1.28±0.071# 6.8± 0.376# 6.17± 0.376#

**Table 3.** *P.amarus* efficiency in reducing Anti TB drugs induced renal alterations.

47±2.59 7.6± 0.420\* 0.7± 0.038\* (16.91%) (51.06%) (25%)

Hepatoprotective effect of *Phyllanthus amarus*

http://dx.doi.org/10.5772/57373

287

44.1±2.44\* 6.22± 0.343\* 0.66± 0.036\* (25.67%) (80.42%) (35%)

35.7±1.97\* 6.1± 0.337\* 0.6± 0.033\* (51.05%) (82.97%) (66%)

30.7±1.70\* 6± 0.331\* 0.575± 0.032\* (66.16%) (88.90%) (75%)

27.8±1.544\* 5.8± 0.320\* 0.55± 0.030\* (74.92%) (89.36%) (83%)

**Lipid Peroxidation Reduced glutathione**

**Liver Kidney Liver Kidney**

0.69±0.038\* 1.096± 0.061\* 7.08± 0.391 6.69± 0.391 (76.68%) (18.77%) (23.93%) (39.09%)

0.57±0.034\* 1.096± 0.061\* 7.18± 0.396 7.0± 0.397 (84.04%) (18.77%) (32.47%) (62.40%)

0.44±0.0243\* 0.737± 0.041\* 7.4± 0.409 7.25± 0.409 (92.02%) (55.47%) (51.28%) (81.20%)

0.431±0.0238\* 0.721± 0.039\* 7.75± 0.428 7.33± 0.428 (92.57%) (57.04%) (81.19%) (87.21%)

0.335±0.018\* 0.665± 0.037\* 7.85± 0.434 7.34± 0.434 (98.46%) (62.75%) (89.74%) (87.96%)

**Value 174.79@ 70.42@ 1.566 1.971** Data are mean ± S.E., n=6.ANOVA (F Values at 5% level).# P≤0.05 vs. Control,\*P ≤0.05 vs. ATD,@ Significant

**Table 4.** Effect of *P.amarus* against AT drugs treated animals in tissue biochemical estimations.

**(n mole of TBARS/mg protein) (µ mole / g)**

**Group IV**

**Group V**

**Group VI**

**Group VII**

**Group VIII**

**Treatment**

**Group IV**

**Group V**

**Group VI**

**Group VII**

**Group VIII**

**ANOVA F**
