Animals
Wistar albino rats weighing 150–200 g were purchased from Sainath agencies, Hyderabad, Telangana, India with a prior permission from our institutional animal ethical committee (1820/GO/Re/S/15/CPCSEA, Date:01-09-2015) and used for the studies. The animals were caged under constant environmental and nutritional conditions (12:12 h light and dark cycle; at an ambient temperature of 25 ± 5 °C; 35–60% of relative humidity). They had free access to food and water ad libutum.
Collection and preparation of extracts
The whole plants of Lindernia ciliata were collected in August 2015, from cotton fields of Bhayyaram, Warangal, Telangana state, India. The plant was authenticated by Prof. V.S. Raju, taxonomist, Kakatiya University, Warangal. A voucher specimen of the plant (KU/UCPSC/54) is being maintained in the herbarium of Department of Pharmacognosy and Phytochemistry, University College of Pharmaceutical Sciences, Kakatiya University, Warangal.
The whole plants were dried thoroughly under shade, powdered coarsely and macerated with methanol in a round bottomed flask for 7 days with stirring at regular intervals and filtered after 7 days. It is then concentrated under reduced pressure using Rotavapour evaporator, (Buchi Rotavapour, Switzerland) to yield a semisolid mass (7.2%) and coded as LCME. A Pilot study was conducted on LCME to fractionate it with toluene, ethyl acetate, butanone and n-butyl alcohol in succession. Based on the yield and TLC profile of the fractions, the solvents- toluene and butanone were selected for fractionation of LCME.
60 g of LCME was suspended in 500 mL of water and fractionated with toluene and butanone in succession. The obtained fractions were concentrated under reduced pressure to yield corresponding extracts. They were given the codes, as TLF-LCME (Toluene fraction), BNF-LCME (butanone fraction) and AQF-LCME (Aqueous fraction- the residue left in the water after fractionation process).
Total phenolic content
The total phenolic content of the fractions of LCME was determined using the Folin–Ciocalteu colorimetric method [3]. The total phenolic content was expressed as Gallic acid equivalents (GAE) in mg per gram of extract.
Total flavonoid content
The total flavonoid content of the fractions of LCME was measured using the aluminium chloride colorimetric method [3]. It was expressed as Rutin equivalents (RE) in mg per gram of extract.
In vitro antioxidant studies
The fractions of LCME were screened to assess their antioxidant property by DPPH radical scavenging assay [4], superoxide scavenging activity [5], nitric oxide scavenging activity [6], hydroxyl radical scavenging activity [7], and reducing power assay [8].
Acute toxicity study
Acute toxicity study was carried out for all the fractions, TLF-LCME, BNF-LCME and AQF-LCME according to the OECD 423 guidelines [9] using female Wistar albino rats. They were observed for signs of toxicity and mortality for 72 h.
Assessment of hepatoprotective activity of fractions of LCME against paracetamol induced hepatotoxicity
The rats were divided into nine groups of six each, control, toxic, standard, and six test groups. The procedure was followed from Sabeena and Ajay, [10] with minor modifications.
Group I (Control group): Treated with vehicle, (1 ml/kg b.w. of 2% gum acacia in water) daily for 7 days.
Group II (Toxic group): Treated with vehicle (1 ml/kg b.w of 2% gum acacia in water) daily for 7 days followed by paracetamol on the eighth day.
Group III (Silymarin 100 mg/kg), Group IV (TLF-LCME 50 mg/kg), Group V (TLF-LCME 100 mg/kg), Group VI (BNF-LCME 50 mg/kg), Group VII (BNF-LCME 100 mg/kg), Group VIII (AQF-LCME 50 mg/kg) and Group IX (AQF-LCME 100 mg/kg) were treated with their respective standard or extracts followed by paracetamol on eighth day.
The blood and liver samples were collected from the animals of all groups 24 h after administration of paracetamol, for estimation of various serum biochemical parameters and histological studies respectively.
Assessment of antihepatotoxic activity of bioactive fraction against D-Galactosamine induced hepatotoxicity in rats
It was done according to Karan et al., [11] The rats were divided into five groups of six animals each. Group I served as normal and is given the vehicle i.e., 2% gum acacia in water (1 mL/kg b.w p.o) for 3 days. Toxic group (Group II), standard (Group III) and two test groups (Group IV, V) were given a single dose of D-Galactosamine (400 mg/kg intra peritoneal) on day 1 followed by vehicle (2% gum acacia 1 ml/kg b.w.p.o.), Silymarin (100 mg/kg b.w), BNF-LCME (50 mg/kg b.w) and BNF-LCME (100 mg/kg b.w) respectively for three times at the time point of 2 h, 24 h, 48 h after the administration of D-Galactosamine. The blood and liver samples were collected from the animals 1 h after the last treatment for determination of various serum biochemical parameters, in vivo antioxidant parameters and histological studies respectively.
Histological studies
The livers from all the animals were isolated and fixed in formalin solution and processed for histopathological examination.
Determination of prothrombin time (PT)
The prothrombin time was determined [12] by collecting blood from animals in normal capillary tubes and the capillaries were broken down into pieces until a thread was observed, and the clotting time was noted.
Development of qualitative analytical profile for the bioactive fraction
High performance liquid chromatography
The distinct chemo profile of the bioactive fraction was developed using HPLC. Shimadzu UFLC unit connected to Photo Diode Array detector was used. The column used was obtained from Grace smart (250 mm × 4.5 mm, Reverse phase C-8, 5 μm particle size). The details of the parameters maintained for the HPLC analysis are Mobile Phase: Methanol: Water: 10:90 (V/V); sample concentration: 100 μg/ml; volume of sample applied: 20 μl at detection wavelength: 220 nm.
Statistical analysis
The data obtained were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism version 3 (GraphPad Software, La Jolla California USA).
