Antioxidant and Hepatoprotective Activity of Piper retrofractum Against Paracetamol-Induced Hepatotoxicity in Sprague-Dawley Rat

: The ethanol extracts of Piper retrofractum Vahl were investigated for antioxidant and hepatoprotective activity. Hepatoprotective activity against paracetamol-induced acute hepatotoxicity was estimated in Sprague-Dawley rat. In DPPH free radical assay the root and stem extracts showed IC 50 values at 133 and 91µg/mL, respectively, while ascorbic acid at 14µg/mL. Extracts also exhibited hydroxyl radical scavenging activity and reducing power. HPLC-DAD analysis indicated the presence of some polyphenolic compounds. Treatment of extracts significantly reduced the elevated serum levels of GPT ( P < 0.01), GOT ( P < 0.01) and bilirubin ( P < 0.001). Both extracts restored the reduced level of total proteins and albumin. A significant increase in HDL-c but decrease in LDL-c level was observed compared to induced control. In histopathological study of liver sections, both extracts showed minimal to mild multifocal and diffuse granular degeneration and mild to moderate lobular disarray compared to control group. Results suggest that both extracts can prevent paracetamol induced hepatotoxicity.


SUPPLEMENTARY MATERIAL
Supplementary material relating to this article is available online, alongside Figures S1-S4.

Plant materials
P. retrofractum plant was collected from Jessore, Bangladesh and identified by Bangladesh National Herbarium (BNH) located in Mirpur-1, Dhaka-1216, (Accession number: DACB-40675) and a voucher specimen was deposited there. After collection, two different parts (root and stem) were separated from undesirable materials and washed with tap water. After that shade drying was used to dry the separated parts to facilitate grinding and grinding was performed using a suitable grinder (Capacitor start motor, Wuhu motor factory, China). The powdered plant materials were macerated with 95 % ethanol for four days at room temperature (~25 °C) and then filtered. The obtained filtrates were dried with the help of a rotary vacuum evaporator at the temperature of 50°C to get crude extracts. The gummy concentrates were designated as EtPCS (ethanol stem extract of P. retrofractum) and EtPCR (ethanol root extract of P. retrofractum). The extracts were stored in refrigerator at 4°C and were diluted with normal saline prior to pharmacological screening.

Animals
For the present study Adult Sprague-Dawley strain male rats weighing 100-150 g were collected from the Pharmacy Department of Jahangirnagar University, Savar, Dhaka-1342.The animals were randomly selected and the divided primarily into two groups, normal and experimental groups. The research was carried out according to the rules that govern laboratory animals' use and the experimental protocol was approved by the Animal Ethics Committee, Khulna University.

DPPH free radical scavenging assay
Plant extracts and ascorbic acid (standard drug), were weighed 3 times and dissolved in ethanol to make the required concentrations by dilution technique. From the stock solution different concentrations (1, 2, 4, 8, 16, 32, 64. 128, 256 and 512µg/mL) of standard and sample were prepared. From each concentration one ml of both plant extracts and standard was taken in each volumetric flask followed by the addition of 3 ml of 0.004 % DPPH solution. After 30-minute incubation at room temperature, absorbance was taken at 517nm against blank using UV Spectrophotometer 1650 Shimadzu, Japan. All the observations were made in triplicate and the average values were recorded. The following formula was used to calculate the Percentage of scavenging activity: Scavenging activity = (A 0 -A 1 )/A0× 100 % where, A 0 is the absorbance of control and A 1 is the absorbance of sample or standard. The IC 50 value was calculated from the % inhibition vs. log concentration graph (Sharma & Bhat 2009;Hossain et al. 2016).

Hydroxyl radical activity test
In this test, 0.5ml 2-deoxy 2-ribose solution (2.8mM) was mixed with 12.5µL of different concentrations (6.25, 12.5, 25, 50, 100, 200, 400 & 800 mg/L) of sample extracts or standard. Then 1mL of 200 µM FeCl 3 , 1mL of 1.04mM EDTA, 0.5mL of 1mM H 2 O 2 and 0.5mL of 1Mm of ascorbic acid were added to prepare the reaction mixture. After 1-hour incubation at 37 0 C, 3.75mL of 2.8% TCA and 3.75mL of 1% TBA were added and kept at 100 0 C for 20 minutes. The absorbance was taken at 530nm. Blank was prepared simultaneously containing all the reagents except extract and standard. The percentage of hydroxyl radical scavenged by the extracts and standard compounds was calculated as follows: % Where A 0 is the absorbance of the control and A 1 is the absorbance in the presence of the sample of extract and standard (Halliwell et al. 1987;Sumi et al. 2016).

Reducing power assay
Sample was prepared at the concentrations of 500, 250,125, 62.5, 31.25, and 15.62mg/L by serial dilution of stock solution. 1 mL of the sample solution of each concentration was mixed with, 2.5mL potassium ferricyanide (K 3 Fe(CN) 6 , 1%) and 2.5mL phosphate buffer (200mmol/L, pH 6.6) having continuous shaking. The mixture was incubated for 20 minutes at 50°C to allow reactions to occur. Then 2.5mL trichloroacetic acid (CCl 3 COOH, 10%) was added and the mixture was centrifuged for 10 min at 1006×g. after that, 2.5mL supernatant was mixed with 0.5mL ferric chloride (FeCl 3 , 0.1%). After five minutes, absorbance was measured at 700nm. Extracts' reducing power were compared with standard by drawing absorbance versus concentration curve (Oyaizu 1986).

HPLC-DAD analysis of phenolic compounds
This analysis was performed for the detection and quantification of particular phenolic compounds present in ethanol extracts of P. retrofractum following a modified method (Jahan et al. 2014). It was accomplished on a Dionex UltiMate 3000 system equipped with a quaternary rapid separation pump (LPG-3400RS) and photodiode array detector (DAD-3000RS). Separation was performed at 30ºC using Acclaim® C18 (5µm) Dionex column (4.6 x 250 mm) where the flow rate and the injection volume were

Hepatoprotective activity test
Animals were selected in an arbitrary way and separated into five distinct groups and each group containing six rats. Plant extracts, paracetamol suspension and silymarin as a standard drug were given by an intra-gastric tube. A dose of 400mg/kg of plant extracts was taken according to pilot study. Group I was maintained as control receiving 0.1% Tween-80 in distilled water as a daily dose for about nine days.
Group II was for induced control and on 8 th day rats received paracetamol suspension (2g/kg single dose) In group III, rats received silymarin (100mg/kg) simultaneously for nine days + paracetamol single dose on 8th day. Rats from group IV and V received the same concentrations of EtPCS and EtPCR (400mg/kg) respectively, for nine days + paracetamol single dose on 8th day. After 48 hours of paracetamol administration, rats of all the groups were anaesthetized by ketamine injection and blood samples were collected from post vena cava. Serum was separated from the collected blood immediately after centrifugation at 3000 rpm for 10 min. Serum collected from each group were then examined for GPT, GOT, total bilirubin, total protein, albumin, as well as total cholesterol, HDL and LDL cholesterol.
Biochemical analysis was performed by Dimension RXL (Max)/vittros-250 auto analyzer using SPAN diagnostics kit. Whole livers were carefully dissected after sacrificing the animal and fixed in 10% formalin for histopathological examination. The tissues were entrenched and sectioned in a paraffin, having stained with hematoxylin and eosin and were inspected under light microscope (Baheti et al. 2006;Arsad 2014) The histopathological assessments were performed by a pathologist. A photomicroscope (Motic, Canada) provided with Motic Images Plus 2.0 software was used to take the photomicrographs of the microscopic sections.