Preparation of extracts
The air-dried leaves of the plant were powdered with a mechanical grinder to obtain a coarse powder, which was then subjected to successive extraction in a soxhlet apparatus using hexane, ethyl acetate, ethanol and water. Each time before extracting with the next solvent, the material was dried in hot air oven at 40°C. Extracts were then filtered through a Whatman No.1 paper filter and concentrated to the dry mass with the aid of rotary evaporator. The extraction process was repeated three times at different time periods. It was observed that there was no significant difference in the percentage yield and content of phyto-constituents that are believed to play an important role in biological activities. The yield of each extract was measured and residues were stored in dark glass tubes for further analysis. The different extracts were designated as APH (for hexane extract), APE (for ethyl acetate extract), APA (for ethanol extract) and APW (for water extract). The dried extracts were dissolved in dimethyl sulfoxide (DMSO) as 20 mg/mL and diluted with phosphate-buffered saline (PBS, pH 7.4) to give final concentrations.
Antioxidant ability assays
The total antioxidant activity of extracts was evaluated by green phosphomolybdenum complex according to the method of Prieto et al.. An aliquot of 10 μL of sample solution was mixed with 1 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in micro centrifuge tube. Tubes were incubated in a dry thermal bath at 95°C for 90 min. After cooling, the absorbance of the mixture was measured at 695 nm against a blank. Ascorbic acid have been used (r2 = 0.964) for reference and the reducing capacities of the analyzed extracts were expressed as mg of ascorbic acid equivalents (mg AAE)/g of dw.
Ferric-reducing/antioxidant power (FRAP) assay
The Fe3+ reducing power of the extracts was determined by the method of Oyaizu  with slight modifications. Briefly, extracts and standard (ascorbic acid) in 1 mL of appropriate solvents were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%), and then mixture was incubated at 50°C for 30 min. Afterwards, 2.5 mL of trichloroacetic acid (10%) was added to the mixture, which was then centrifuged at 5000 rpm for 10 min. Finally, 2.5 mL of the upper layer solution was mixed with 2.5 mL of distilled water and 0.1 mL of FeCl3 (0.1%). The absorbance was measured at 700 nm and the reducing power of the extracts was presented as mg AAE/g of dw.
DPPH• radical scavenging activity
The DPPH free radical scavenging activity of leaf extracts of A. precatorius was measured in term of hydrogen donating or radical scavenging ability using the stable radical DPPH . Briefly, 0.004% w/v of DPPH radical solution was prepared in methanol and then 900 μL of this solution was mixed with 100 μL of extract solution containing 40–400 μg/mL of dried extract. The absorbance was measured at 517 nm after 30 min of incubation. Methanol (95%), DPPH solution and ascorbic acid were used as blank, control and reference respectively.
Hydroxyl radical scavenging activity
The ability of the extracts to inhibit site-specific hydroxyl radical-mediated peroxidation was carried out according to the method given by Hinneburg et al.  with some modifications. Briefly, the mixture containing FeCl3 (10 mM), ascorbic acid (1 mM), H2O2 (10 mM), deoxyribose (28 mM) and different concentrations of test samples (40–400 μg/mL) in 500 μL phosphate buffered saline (PBS, 20 mM, pH 7.4) was incubated for 30 min at 37°C. After adding 1 mL of trichloroacetic acid (10%, w/v) and 1 mL thiobarbituric acid (2.8% w/v; in 25 mM NaOH), the reaction mixture was boiled for 15 min. The extent of oxidation was estimated at 532 nm and the scavenging activity of test sample was expressed as the percentage inhibition of the deoxyribose degradation to malondialdehyde. Ascorbic acid was used as the positive control.
Hydrogen peroxide scavenging assay
The ability of plant extracts to scavenge hydrogen peroxide was determined according to Long et al. . A 40 mM of H2O2 solution was mixed with different concentrations of plant extracts (20–200 μg/mL) and incubated for 3.5 h at room temperature. After incubation, 90 μL of the H2O2-sample solution was mixed with 10 μL of HPLC-grade methanol and 0.9 mL of FOX reagent was added (prepared by mixing 9 volumes of 4.4 mM BHT in HPLC-grade methanol with 1 volume of 1 mM xylenol orange and 2.56 mM ammonium ferrous sulfate in 0.25 M H2SO4). The reaction mixture was vortexed and then incubated at room temperature for 30 min. The absorbance of ferric-xylenol orange complex was measured at 560 nm. Ascorbic acid was used as the reference compound.
Nitric oxide scavenging activity
The free radical scavenging potential of A. precatorius was further substantiated by scavenging of nitric oxide radical assayed by sodium nitroprusside method . The reaction solution (50 μL) containing 10 mM sodium nitroprusside in PBS (pH 7.0) was mixed with different concentration (40–400 μg/mL) of sample extracts, followed by incubation at 37°C for 20 min under light. After incubation, the samples were mixed with 300 μL of Griess reagent (1% sulfanilamide, 2% H3PO4). The samples were again incubated for 30 min at room temperature under light conditions followed by the addition of 0.1% N-(1-naphthyl) ethylenediamine dihydrochloride. The absorbance was recorded at 546 nm and the results were expressed as per cent of scavenged nitric oxide with respect to the negative control without addition of any antioxidant. Ascorbic acid was used as a positive control.
Superoxide radicals scavenging activity
The scavenging activity of the plant extracts towards superoxide anion radicals was measured by the nitro-blue tetrazolium (NBT) reduction method  with minor modifications. Superoxide anions were generated in a non-enzymatic phenazine methosulfate nicotinamide adenine dinucleotide (PMS-NADH) system through the reaction of PMS, NADH and oxygen. It was assayed by the reduction of nitroblue tetrazolium. In the experiment, the superoxide anion was generated in 2 mL of phosphate buffer (100 mM, pH 7.4) containing 500 μL of 156 μM nitroblue tetrazolium (NBT solution), 500 μL of 468 μM nicotinamide adenine dinucleotide (NADH) solution and 300 μL of different concentrations (40–400 μg/mL) of extracts. DMSO and L-ascorbic acid were used as solvent and positive control respectively. The reaction was initiated by adding 100 μL of 60 μM phenazine methosulfate (PMS) to the mixture. After 5 min of incubation at room temperature, the absorbance was measured at 560 nm against blank. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity.
Inhibition of lipid peroxidation assay
Fe2+ induced lipid peroxidation is one of the established system for assessing antioxidant action of different plant extracts. A modified thiobarbituric acid-reactive species (TBARS) assay  was used to measure the lipid peroxide formed using rat liver homogenate as lipid rich media. Malondialdehyde (MDA), a secondary end product of the oxidation of polyunsaturated fatty acids, reacts with two molecules of TBA yielding a pinkish red chromogen. Healthy albino rats of the wister strain (250 grams) were sacrificed (procedure described was reviewed and approved by the University of Hyderabad, School of Life Sciences’ animal ethics committee) and liver was perfused with 0.15 M KCl, homogenate was centrifuged at 800 g for 15 min at 4°C and the supernatant was used for thiobarbituric acid assay. The extracts of A. precatorius at different concentrations (40–400 μg/mL) were mixed with the liver microsome preparation and incubated at room temperature for 10 min. Then, 50 μL Fenton’s reagent (10 mM FeCl3; 10 μL of 2.5 mM H2O2; 0.1 M L-ascorbic acid) in phosphate buffer (0.2 M, pH 7.4) were added, and the volume was made to 1 mL. The tubes were then incubated for 30–45 min at 37°C to induce lipid peroxidation. Thereafter, 2 mL of ice-cold HCl (0.25 N) containing 15% trichloroacetic acid, 0.5% thiobarbituric acid and 0.5% butylated hydroxytoluene (BHT) were added in each sample followed by heating at 100°C for 15 min. The reaction mixture was put in an ice bath for 10 min. The mixture was centrifuged at 1000 rpm for 10 min and the extent of lipid peroxidation was subsequently monitored by formation of thiobarbituric acid reactive substances (TBARS) as pink chromogen in presence or absence of extracts and standard (L-ascorbic acid). The absorbance of the supernatant was measured spectrophotometrically at 532 nm and decline in formation of pink chromogen in pre-treated reactions was considered as inhibition of lipid peroxidation.
A panel of four human cell lines namely, (a): human colon adenocarcinoma cells - Colo-205, (b): human retinoblastoma cancer cells - Y79, (c): human hepatocellular carcinoma cells - HepG2 and (d): Leukaemia cells - SupT1 were used to study antiproliferative activity. The cell lines were obtained from National Centre for Cell Sciences (NCCS), Pune, India. The cell lines HepG2 & Colo-205 were cultured in Dulbecco’s modified Eagle’s medium (DMEM) and Y79 & SupT1 in RPMI 1640 containing 10% (v/v) FBS, 100 units/mL penicillin and 100 μg/mL streptomycin. Cells were maintained in a humidified incubator with 5% CO2 for 24 h at 37°C and seeded onto 75 cm2 culture flasks. In vitro response to extracts and standard drug was evaluated by means of a growth inhibition using the MTT assay . The cells were seeded at a density of ~5 × 103 per well using a brief trypsinization. Furthermore, Doxorubicin and sample extracts (25–200 μg/mL) dissolved in dimethylsulfoxide (DMSO; not exceeding the concentration of 2%), and further diluted in cell culture medium were added into a 96-well plate. After 48 h of incubation, 20 μL of MTT reagent (5 mg/mL) were added and mixtures were reincubated for 4 h. Finally the absorbance of formazan was measured at 550 nm. Doxorubicin was also assayed as a positive control at the concentration of 0.5–10 μg/mL. The resulting growth data represents the net outcome of cell proliferation and cell death. The cell viability (%) was obtained by comparing the absorbance between the samples and a negative control.
Assessment of extract toxicity in normal cells
To assess the toxicity of the plant extracts on primary cells (peritoneal murine macrophages), the MTT toxicity assay was used. Thioglycollate-elicited mouse peritoneal macrophages were harvested from female BALB/C mice . Experimental protocol was again undertaken in accordance with the ethical guidelines and the permission of the University of Hyderabad, School of Life Sciences’ animal ethics committee was obtained. Toxicity toward mouse peritoneal macrophages was assessed with cells plated in 96-well plates at a density of 2 × 106 cells per well (in 200 μL volume). After adherence, the medium was removed and replaced by one of the media containing the plant extracts. The plates were incubated for 72 hrs at 37°C in a humidified 5% CO2 incubator. Control cells were incubated with culture medium alone. Cell viability was determined by a colorimetric assay with the tetrazolium salt MTT . Absorbance of the formed formazan product was measured at a test wavelength of 540 nm. Results were expressed as percentage cellular viability of the extracts.
Calculations and statistical analysis
The percentage inhibitions of radicals, lipid peroxidation and cytotoxic activities of the extracts were calculated using the formula:
All results are expressed as mean ± standard deviation (SD) values average from 3 to 4 independent experiments performed in duplicate. IC
value (the concentration of the extracts required to scavenge 50% of radicals) was calculated for different extracts of A. precatorius.
Statistical differences between correlated samples were evaluated using Student’s t-test and composite treatments were compared using one-way analysis of variances (ANOVA) and considered significantly different where probability values were found to be equal to or less than 0.05. Statistical tests as well as mean and SD calculations and graphical representation of the results were performed using GraphPad Prism v5 and Sigmaplot v11.0 software’s.