HPLC-CAD Analysis
HPLC detection and quantification of polyphenolic compounds
Detection and quantification of selected phenolic compounds in the ethanol extract were determined by HPLC-DAD analysis as described by Ismet et al. [13] with some modifications. It was carried out on a Dionex UltiMate 3000 system equipped with quaternary rapid separation pump (LPG-3400RS) and photodiode array detector (DAD-3000RS). Separation was performed using Acclaim® C18 (5µm) Dionex column (4.6 x 250 mm) at 30 ºC with a flow rate of 1 ml/min and an injection volume of 20 µl. For the preparation of calibration curve, a standard stock solution was prepared in methanol containing arbutin (AR), (-)-epicatechin (ECA) (5 µg/ml each), gallic acid (GA), hydroquinone (HQ), vanillic acid …show more content…
One unit of CAT activity was defined as an absorbance change of 0.01 as units/min.
Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. [15]. 1.0 ml sample of 10% homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 4ºC for 1 hr and then centrifuged at 1200 × g for 20 min at 4ºC. The total volume of 3.0 ml assay mixture composed of 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (5,5-dithiobis-2-nitrobenzoic acid), (100 mM). The yellow color of the mixture was developed, read immediately at 412 nm on a Smart SpecTM plus Spectrophotometer and expressed as ng/mg protein.
Histopathalogical determination
For microscopic evaluation liver tissues were fixed in neutral buffered formalin and embedded in paraffin, sectioned at 5 μm and subsequently stained with hematoxylin/eosin to see the architecture of hepatic tissue and inflammatory cell infiltration. Sirius red staining for fibrosis and Prussian blue staining for iron deposition were also done in liver sections. Sections were then studied and photographed under light microscope (Zeiss Axioscope) at 40 magnifications. Collagen deposition was semi quantitatively measured using NIH Image J free software (Version
Detection and quantification of selected phenolic compounds in the ethanol extract were determined by HPLC-DAD analysis as described by Ismet et al. [13] with some modifications. It was carried out on a Dionex UltiMate 3000 system equipped with quaternary rapid separation pump (LPG-3400RS) and photodiode array detector (DAD-3000RS). Separation was performed using Acclaim® C18 (5µm) Dionex column (4.6 x 250 mm) at 30 ºC with a flow rate of 1 ml/min and an injection volume of 20 µl. For the preparation of calibration curve, a standard stock solution was prepared in methanol containing arbutin (AR), (-)-epicatechin (ECA) (5 µg/ml each), gallic acid (GA), hydroquinone (HQ), vanillic acid …show more content…
One unit of CAT activity was defined as an absorbance change of 0.01 as units/min.
Reduced glutathione assay (GSH)
Reduced glutathione was estimated by the method of Jollow et al. [15]. 1.0 ml sample of 10% homogenate was precipitated with 1.0 ml of (4%) sulfosalicylic acid. The samples were kept at 4ºC for 1 hr and then centrifuged at 1200 × g for 20 min at 4ºC. The total volume of 3.0 ml assay mixture composed of 0.1 ml filtered aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml DTNB (5,5-dithiobis-2-nitrobenzoic acid), (100 mM). The yellow color of the mixture was developed, read immediately at 412 nm on a Smart SpecTM plus Spectrophotometer and expressed as ng/mg protein.
Histopathalogical determination
For microscopic evaluation liver tissues were fixed in neutral buffered formalin and embedded in paraffin, sectioned at 5 μm and subsequently stained with hematoxylin/eosin to see the architecture of hepatic tissue and inflammatory cell infiltration. Sirius red staining for fibrosis and Prussian blue staining for iron deposition were also done in liver sections. Sections were then studied and photographed under light microscope (Zeiss Axioscope) at 40 magnifications. Collagen deposition was semi quantitatively measured using NIH Image J free software (Version