Sorbex rxc8

Chromatographic analysis was carried out by using HPLC-DAD (Agilent Germany) attached with Sorbex RX-C8 (Agilent USA) analytical column. Briefly, mobile phase A was acetonitrile-methanol-water acetic acid (5: 10: 85: 1) and mobile phase B was acetonitrile methanol- acetic acid (40: 60: 1). A gradient of time 0–20 min for 0 to 50 % B, 20–25 min for 50 to 100 % B, and then isocratic 100 % B till 40 min was used. The flow rate was 1 ml/min and injection volume was 20 μl. Rutin and gallic acid were analyzed at 257 nm, catechin at 279 nm, caffeic acid at 325 nm and quercetin, myricetin, kaempferol were analyzed at 368 nm. Every time column was reconditioned for 10 min before the next analysis. All the samples were assayed in triplicate at ambient temperature. Quantification was carried out by the integration of peak using the external standard method using following formula: $\mathrm{Conc}.\mathrm{of}\mathrm{S}\mathrm{C}\mathrm{in}\mathrm{sample}=\left[\frac{\mathrm{Peak}\mathrm{area}\left(\mathrm{m}\mathrm{A}\mathrm{U}\ast \mathrm{s}\right)\mathrm{of}\mathrm{S}\mathrm{C}\mathrm{in}\mathrm{sample}}{\mathrm{Peak}\mathrm{area}\left(\mathrm{m}\mathrm{A}\mathrm{U}\ast \mathrm{s}\right)\mathrm{of}\mathrm{S}\mathrm{C}}\right]\times \mathrm{Conc}.\mathrm{Of}\mathrm{S}\mathrm{C}$
SC is for standard compound
The concentration of standard compound in each fraction was expressed as μg/100 of dry plant powder.

On account of significant antioxidant activity for most of the in vitro assays the EDEW extract was selected for HPLC-DAD analysis. HPLC analysis of EDEW was carried out by using HPLC-DAD (Agilent Germany) equipment using Sorbex RXC8 (Agilent USA) analytical column with 5 μm particle size and 25 ml capacity. Mobile phase was consisted of eluent A, (acetonitrile-methanol-water-acetic acid /5: 10: 85: 1) and eluent B (acetonitrile-methanol-acetic acid/40: 60: 1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50 % B), 20–25 min (50 to 100 % B), and then isocratic 100 % B (25–40 min) at flow rate of 1 ml/min. The injection volume of the sample was 20 μl. Before the injection samples were filtered through 0.45 μm membrane filter. Among the standards rutin and gallic acid were analyzed at 257 nm, catechin at 279 nm, caffeic acid at 325 nm and quercetin, myricetin, kampferol were analyzed at 368 nm [24 (link)]. Each time the column was reconditioned for 10 min before the next analysis. All samples were assayed in triplicates. Quantification was carried out by the integration of the peak using the external standard method. All chromatographic operations were carried out at an ambient temperature.

HPLC analysis of AHM was carried out by using HPLC-DAD (Agilent Germany) equipment using Sorbex RX-C8 (Agilent USA) analytical column with 5 μm particle size. Mobile phase consisted of eluent A, (acetonitrile–methanol–water–acetic acid /5: 10: 85: 1) and eluent B (acetonitrile-methanol-acetic acid/40: 60: 1). The gradient (A: B) utilized was the following: 0–20 min (0 to 50% B), 20–25 min (50 to 100% B), and then isocratic 100% B (25-40 min) at flow rate of 1 ml/min, and injection volume was 20 μl. Rutin and Gallic acid were analyzed at 257 nm, catechin and apigenin at 279 nm, caffeic acid at 325 nm and quercitin, myricetin, kaempherol were analyzed at 368 nm. Every time column was reconditioned for 10 min before the next analysis. All samples were assayed in triplicate. Quantification was carried out by the integration of the peak using the external standard method. All chromatographic operations were carried out at ambient temperature.