for 1 h and absorbance was recorded

for 1 h and absorbance was recorded at 725 nm in the UV–Vis
spectrophotometer (Opron 3000 Hansan Tech. Co. Ltd., Korea). A
gallic acid standard curve was obtained for the calculation of phenolic content.
2.5. Total flavonoid content assay
The total flavonoid content of CBP was determined by the method ofZhuang et al. (1992)with some modifications. A 0.5 mL aliquot of sample solution was mixed with 2 mL of distilled water
and subsequently with 0.15 mL of a 5% NaNO2 solution. After
6 min, 0.15 mL of a 10% AlCl3solution was added and allowed to
stand further 6 min, thereafter, 2 mL of 4% NaOH solution was
added to the mixture. Immediately, distilled water was added to
bring the final volume to 5 mL. Then the mixture was properly
mixed and allowed to stand for 15 min. Absorbance of the mixture
was taken at 510 nm. Rutin was used as standard for the quantification of total flavonoid.
2.6. HPLC determination of flavonoid constituents in citrus byproducts
The constituents and the amount of flavonoids in CBP were analyzed by SPD-M20A (Shimadzu Co., Japan). For identification of the
flavonoid constituents in CBP, the retention time was compared
with authentic standards (11 flavonoids used as standards).
A Sihm pack VP-ODS (C18) column (2504.6 mm i.d.) used for
all chromatographic separation. The mobile phase was (A) water/