Chemicals
All the chemicals were procured from Sisco Research Laboratories Pvt. Ltd. (Mumbai, India) unless otherwise indicated. Silymarin was obtained from Sigma-Aldrich (USA). Fetal bovine serum (FBS), RPMI-1640, antibiotics and EZcount™ MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) Cell Assay Kit were purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Albumin, γ-glutamyl transferase (GGT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), bilirubin, protein, aspartate transaminase (AST), acid phosphatase (ACP), alanine transaminase (ALT), glucose, urea and cholesterol estimation kits were obtained from Crest Biosystems (Goa, India). TNF-α ELISA kit was procured from Ray Bio (Georgia, United States) and Thiobarbituric acid reactive substances (TBARS) assay kit was purchased from Cayman chemical company (USA). Milli-Q ultrapure water from the departmental central facility was used in the experiments.
Preparation of plant extract
Stem and root samples of white flowered variety of oleander were collected from the garden of University of North Bengal (26.71°N, 88.35°S), India. The plant materials were identified by senior plant taxonomist Prof. Abhaya Prasad Das of Department of Botany, University of North Bengal. The voucher specimen was stored at the herbarium of Department of Botany, University of North Bengal with an accession number of 09618.
The stem and root samples were washed properly with double distilled water to remove any dust and foreign materials. The samples were then chopped to 0.5 cm pieces and shade dried at laboratory temperature (25 °C). After 20 days, 70 % hydro-methanolic extract of Oleander stem and root were prepared according to the previous method [11]. A schematic representation of the extract preparation method is provided in the Additional file 1. The lyophilized extracts were stored at −20 °C until further use. The final yield of oleander stem (NOSE) and root (NORE) extracts were 11.82 and 15.22 % of dry weight (DW).
Animal maintenance
Swiss albino mice (6–8 weeks, 20–25 g) of both sex (3 male and 3 female per group) were maintained inside cage bins (Tarson, India) with rice husk bedding in the animal house of the Department of Zoology, University of North Bengal at a constant 12 h photoperiod (temperature 25 ± 2 °C; humidity 55 ± 5 %) with food and water ad libitum. All the experiments were approved by the ethical committee, University of North Bengal (No. 840/ac/04/CPCSEA) and conducted in accordance with the legislation for the protection of animals used for scientific purposes.
Acute toxicity study
OECD guidelines (test 423: Acute oral toxicity – Acute toxic class method, 2002) were followed to study the acute toxicity of the extracts [18]. Mice were divided into eight groups (n = 6) and fasted overnight prior to the experiment. NOSE and NORE were administered orally at 250, 500, 1000 and 2000 mg/kg body weight (bw) dose. Each groups were carefully observed at 30 min and then 2, 4, 8, 24 and 48 h for development of any clinical or toxicological symptoms such as tremors, convulsions, salivation, diarrhoea, lethargy, sleep, coma and alteration in respiratory patterns, skin colour, behaviour pattern etc.
Experimental design: in vivo
Animals were divided into 7 separate groups (n = 6) and following treatments were done once each day for 10 consecutive days: Control group received normal saline; CCl4 group received 1:1 (v/v) CCl4 in olive oil; Silymarin group received 1:1 (v/v) CCl4 in olive oil and 100 mg/kg bw silymarin; NOSE low group received 1:1 (v/v) CCl4 in olive oil and 50 mg/kg bw NOSE; NOSE high group received 1:1 (v/v) CCl4 in olive oil and 200 mg/kg bw NOSE; NORE low group received 1:1 (v/v) CCl4 in olive oil and 50 mg/kg bw NORE; NOSE high group received 1:1 (v/v) CCl4 in olive oil and 200 mg/kg bw NORE.
On 11th day i.e., 24 h after the last treatment, under anaesthesia (2 % ether), blood was collected from the treated animals by cardiac puncture and finally the animals were sacrificed. Blood was allowed to clot for 60 min at room temperature (25 °C) and then serum was collected by centrifuging at 1000 rpm for 5 min. The straw coloured serum was used to study liver marker enzymes. Liver samples were collected and washed with double distilled water to remove blood and used for antioxidant enzymatic assays. Liver tissue required for histological study were collected in Bouin’s solution.
Liver function test: in vivo
Serum samples from each group were used to study ACP, albumin, globulin, glucose, ALP, bilirubin, cholesterol, LDH, GGT, AST, ALT, total protein and urea levels using commercially available kits (Crest Biosystems).
Estimation of peroxidase and catalase activities
Peroxidase activity in the liver samples were estimated by measuring the oxidation of guiacol according to a standard method [19]. Catalase activity was measured by degradation of substrate H2O2 by catalase in the liver tissue samples following the standard method described by Luck [20].
Experimental design: in vitro
The in vitro hepatoprotective capacity of NOSE and NORE were studied according to previously standardized methods with minor modifications [14, 21, 22]. Briefly, seven groups of primary explant culture of mice hepatocytes were prepared in RPMI-1640 medium (containing 50 U/ml penicillin, 50 U/ml streptomycin and 50 U/ml nystatin) supplemented with 10 % FBS. After 48 h of the culture, the following treatments were done: Control had no separate treatment; CCl4 group received 25 μl/ml CCl4; Silymarin group received 25 μl/ml CCl4 and 100 μg/ml silymarin; NOSE low group received 25 μl/ml CCl4 and 25 μg/ml NOSE; NOSE high group received 25 μl/ml CCl4 and 100 μg/ml NOSE; NORE low group received 25 μl/ml CCl4 and 25 μg/ml NORE and NORE high group received 25 μl/ml CCl4 and 100 μg/ml NORE.
The plates were incubated for 2 h and then culture supernatants were collected by centrifugation (5000 rpm for 10 min).
Liver function test: in vitro
Culture supernatants from each group were analysed for ACP, ALP, bilirubin, LDH, AST, ALT and total protein levels using commercially available kits (Crest Biosystems).
Measurement of lipid peroxidation
The extent of lipid peroxidation was measured in six sets using TBARS assay kit (Cayman) according to the manufacturer’s instructions. In brief, 100 μl serum samples of different groups were mixed with 100 μl sodium dodecyl sulphate (SDS) solution. The tubes were placed on boiling water bath after addition of 4 ml colour reagent. After 60 min incubation, the tubes were kept on ice for 10 min to stop the reaction. Then the solutions were centrifuged (1600 g) for 10 min at 4 °C and the absorbance of the supernatants were recorded at 340 nm.
Measurement of TNF-α
The amount of TNF-α released in culture supernatants were measured using TNF-α ELISA kit (Ray Bio) according to the manufacturer’s instructions. Absorbance was immediately measured at 450 nm using Bio-Rad iMark™ microplate absorbance reader. Standard was run in parallel to the samples.
Measurement of inhibition of NO
Culture supernatants were used to determine the NO level using the Griess reagent method [23] with some modifications. Briefly, culture supernatants (60 μl) from each group was mixed with 240 μl of Griess reagent (1 % sulfanilamide and 0.1 % N-(1-naphthyl) ethylenediamine hydrochloride in 2.5 % H3PO4) in a 96-well plate and incubated for 20 min at room temperature. The purple azo-dye formed, was detected at 540 nm.
Measurement of cell viability
Hepatocyte necrosis results due to CCl4 toxicity. Therefore, MTT assay was performed to measure the protection rendered by NOSE and NORE against CCl4 mediated toxicity. Hepatocytes were cultured as described under the in vitro experimental section. The cell viability assay was performed in six sets using EZcount™ MTT Cell Assay Kit (HiMedia) according to the manufacturer’s instructions.
Histopathological studies
Liver samples were removed from the animals of the in vivo experiments after collection of blood and were fixed overnight in 10 % buffered formalin. The samples were subjected to dehydration and then embedded in paraffin blocks. Thick sections (4 μm) of the paraffin embedded livers were cut in a microtome and then dewaxed in xylene, rehydrated in a series of different grades of alcohol and then washed with distilled water for 5 min. Subsequently, the sections were stained with haematoxylin for 40 s and counterstained with eosin for 20 s. The sections were dehydrated in graded alcohol series and washed in xylene. The slides were observed (100× and 400×) for signs hepatic injury using Nikon ECLIPS E200 microscope.
Fourier Transform Infrared (FTIR) spectroscopy analysis
FTIR spectrophotometry was used to identify the characteristic functional groups in NOSE and NORE. Small quantity (<10 mg) of dried extracts were taken in CaF2 vessel and placed in the sample cup of a diffuse reflectance accessory. The IR spectrum was obtained using Shimadzu 8300 FTIR spectrophotometer at ambient temperature. Background correction was made by taking IR spectrum of de-ionized water as the reference in identical condition. The sample was scanned from 400 to 4000 cm−1 for 16 times to increase the signal to noise ratio.
GC-MS analysis
NOSE and NORE were initially bi-fractionated by dissolving in dichloromethane and n-hexane separately. The mixtures were centrifuged thrice (12,000 rpm) for 15 min. The clear supernatant was used for GC-MS analysis using Agilent 5975C GC-MS system (Agilent Technologies, USA) attached with HP-5 ms Capillary Column (30 m × 0.25 mm i.d. × 0.25 μm film thickness). The machine was equipped with inert MSD triple axis mass detector conditioned at ion trap 200 °C, transfer line 280 °C, electron energy 70 eV (vacuum pressure- 2.21e–0.5 torr) was used for analysis. Helium was used as carrier gas (1 ml/min). Sample volume was 2 μl and injected in a splitless mode. The column temperature was kept at 60 °C for 1 min followed by 5 °C/min up to 250 °C. The major and essential compounds present in samples were identified by their retention times and mass fragmentation patterns using Agilent Chem Station Integrator and the database of National Institute Standard and Technology (NIST) with a MS library version 2010.
Statistical analysis
All data are reported as mean ± SD of six measurements. Statistical analysis was performed and graphs were prepared using KyPlot Data Analysis and Visualization software version 5.0.2 (32 bit). Comparison between groups were performed using one-way analysis of variance (ANOVA). p < 0.05 was considered significant.