with methanol (1:10) using Soxhlet apparatus. Finally, the air-dried sample was
stored at À80
C for further analysis.
5.2.6 Determination of Phenolic and Flavonoid Content
The phenolic compounds were analyzed using Folin-Ciocalteu method (FCM), and
the total phenolic content was calculated as
Gallic acid equivalent ¼ T ¼ C Â V=M:
The total flavonoid was analyzed using aluminum chloride colorimetric method,
and it was calculated as follows:
Quercetin equivalent ¼ T ¼ C Â V=M:
5.2.7 Determination of DPPH and FRAP Activity
The DPPH free radical scavenging assay was measured using DPPH free radical
test. The FRAP scavenging assay was measured by the method of Yildirim
et al. (2001).
5.2.8 High-Performance Liquid Chromatography
(HPLC)-Pulsed Amperometric 109 Detectors
Preparation of standards: 1 mg of each standard compounds (ascorbic acid (ASA),
gallic acid (GA), benzoic acid (BA), tannic acid (TA), quercetin (QN), resorcinol
(RL), catechol (CAT)) was dissolved in 1 mL of HPLC grade methanol. It was
sonicated and filtered by Whatman Nylon Membrane Filter (0.45 μm and 47 mm
diameter) before injecting into the column.
Specification of the HPLC-PAD instrument: Analysis of all the standard samples
was performed using 515 HPLC pumps and 2489 UV/VIS detectors of Waters
company, USA, having reverse phase water guard Column: Symmetry C18 (5 μm,
4.6Ã250 mm) and Hamilton microliter syringe using an injection volume of 20 μL.
The data analysis was done using Empower software.
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S. Sugumar et al.
stored at À80
C for further analysis.
5.2.6 Determination of Phenolic and Flavonoid Content
The phenolic compounds were analyzed using Folin-Ciocalteu method (FCM), and
the total phenolic content was calculated as
Gallic acid equivalent ¼ T ¼ C Â V=M:
The total flavonoid was analyzed using aluminum chloride colorimetric method,
and it was calculated as follows:
Quercetin equivalent ¼ T ¼ C Â V=M:
5.2.7 Determination of DPPH and FRAP Activity
The DPPH free radical scavenging assay was measured using DPPH free radical
test. The FRAP scavenging assay was measured by the method of Yildirim
et al. (2001).
5.2.8 High-Performance Liquid Chromatography
(HPLC)-Pulsed Amperometric 109 Detectors
Preparation of standards: 1 mg of each standard compounds (ascorbic acid (ASA),
gallic acid (GA), benzoic acid (BA), tannic acid (TA), quercetin (QN), resorcinol
(RL), catechol (CAT)) was dissolved in 1 mL of HPLC grade methanol. It was
sonicated and filtered by Whatman Nylon Membrane Filter (0.45 μm and 47 mm
diameter) before injecting into the column.
Specification of the HPLC-PAD instrument: Analysis of all the standard samples
was performed using 515 HPLC pumps and 2489 UV/VIS detectors of Waters
company, USA, having reverse phase water guard Column: Symmetry C18 (5 μm,
4.6Ã250 mm) and Hamilton microliter syringe using an injection volume of 20 μL.
The data analysis was done using Empower software.
72
S. Sugumar et al.