Method Validation. The reference compounds, including P-1, P-
2, and P-4, were accurately weighed, dissolved in methanol, and
diluted with methanol to an appropriate concentration. The solutions
were brought to room temperature and filtered through a 0.22 μm
membrane, and an aliquot of 10 μL was injected into HPLC for
analysis. Calibration curves were plotted by the peak area versus at
least six appropriate concentrations in triplicate of each analyte. The
limits of detection (LOD) and quantification (LOQ) were determined
on the basis of signal-to-noise (S/N) ratios of 3 and 10, respectively.The intra- and interday precision assays were performed by analyzing
calibration samples during a single day and on three consecutive days,
respectively. To ensure the repeatability, six different working
solutions prepared from the same sample were assessed. Recoveries
of the quantified constituents were determined using the sample for
which respective chemical contents had been predetermined. Each
standard solute was spiked at a close concentration with the sample.
Then, recoveries were calculated on the basis of the difference between
the total amount determined in the spiked samples and the amount
observed in the nonspiked samples.
Determination of Total Flavonoids Content. The total
flavonoids of the sample were determined by UV spectrophotometry
at a wavelength of 510 nm after the extraction reacted with coloring
agents.28 Rutin was used as a standard, and results were expressed as
milligrams of flavonoids equivalent per gram of dry sample.