Chemicals
Standards used in HPLC analysis were purchased from Sigma Aldrich. Iron, sodium nitroprusside, DPPH, ammonium molybdate and 1,10-phenanthroline were purchased from Biochemicals (Lahore, Pakistan).
Preparation of fruit extract
The fruits of plant were locally purchased, identified by a botanist and a voucher specimen was deposited at the Herbarium of University of Poonch, Department of Botany (Ref. No. BOT/2017/51).
Finely grounded fruit material of the plant (25 g) was placed for 15 min in boiling water (500 ml) was cooled and filtered with filter paper No. 1 (Pore size, 11 μM). The solvent was evaporated by rotary evaporator (45 °C) producing 3 g (12% w/w) extract.
In vitro lipid peroxidation assay
The anti-lipid peroxidative properties of aqueous extracts were studied by a method [13]. In brief the egg yolk was weighed to 1 g and diluted to 100 ml with 100 mM Tris-HCl, pH 7.4 and used as homogenate. The homogenate was incubated with Fe (II) or sodium nitroprussside with or without the extract and colour reaction was carried out by adding 600 μl of TBA and 600 μl of acetic acid (pH 3.4) for 1 h. The tubes were cooled and 2 ml of n-butanol was finally added and centrifuged. The absorbance was read at spectrophotometer at 532 nm.
DPPH radical scavenging activity
The scavenging of the DPPH radical was reported by the method [14]. Briefly, a 0.25 mM solution of the DPPH radical (0.5 mL) was added to a sample solution in ethanol (1 mL) at different concentrations (25–400 μg/mL) of the aqueous extracts. The mixture was shaken vigorously and left to stand for 30 min in the dark, then the absorbance was measured at 517 nm. The capacity to scavenge the DPPH radical was calculated using the equation:
$$ \left(\%\right)\ \mathrm{scavenging}=\left[\left(\mathrm{AoA}1\right)/\left.\mathrm{Ao}\right)\right]\times 100 $$
Where, Ao is the absorbance of the control reaction and A1 is the absorbance of the sample.
Metal chelating activity
The iron chelating ability of the aqueous extract was studied by the method [15]. Briefly 150 μL of freshly formed 2 mM FeSO4·7H2O was added in a mixture which have 168 μL of the 0.1 M tris HCl (pH 7.4), (218 μL) of saline and (25–200 μL/ml) concentration of plant extracts. The mixture of sample was incubate for 5 min before addition of 13 μL of 0.25% 1,10-phenanthroline (w/v). Absorbance was checked at 510 nm in spectrophotometer.
Antioxidant potential assay
The reducing ability of the aqueous extract was followed by phosphomolybdenum method [16]. The results were expressed as ascorbic acid equivalent. The assay was based on the reduction of molybdenum, Mo (VI)–Mo(V) by the extract and subsequent formation of a green phosphate/Mo(V) complex at acidic pH. The extract (0.1 mg/ml) was mixed with 3 ml of the reagent solution (0.6 M H2SO4, 28 mM sodium phosphate and 4 mM ammonium molybdate). The tubes were incubated at 95 C for 90 min. The mixture was cooled to room temperature and the absorbance of the solution was measured at 695 nm.
HPLC analysis of phenolics and flavonoids
T. chebula aqueous extract (1 mg/mL) was dissolved in HPLC grade methanol filtered and subjected for analysis by Shimadzu HPLC system as reported by Khaliq et al., [13].
Molecular docking
Human Rab8b Protein was used for the validation of compounds as anti-inflammation. The 3D structures of protein were downloaded from RCSB database. For docking PyRxvina docks tool were used.
Synergism of compounds
String is a free online software to analysis protein interactions. To check the synergism between compounds relevant protein of the compounds were analyzed.
Data analysis
The results were expressed as means± SD. The obtained data was analyzed by one way ANOVA and different group means were compared by Duncan Multiple Range Test (DMRT) wherever necessary; p < 0.05 was considered significant in all cases. Statistica (version 4.5; StatSoft Inc., Tulsa, OK, USA) was used as software package.
