Na2co3

The total polyphenol content of
vegetable methanol extracts was determined by the Folin–Denis
method with minor modifications (Folin et al., 1912). Briefly, a Folin
& Ciocalteu’s phenol reagent (20 μL) (Wako Pure Chemical
Industries, Ltd., Osaka, Japan), an aqueous solution of 10% Na₂CO₃ (40 μL) (Wako Pure Chemical Industries,
Ltd., Osaka, Japan), and distilled water (120 μL) were added
to 20 μL of the methanol extract and the mixture was incubated
at room temperature for 1 h under dark conditions. The absorbance
was measured at 750 nm with a spectrophotometer. An aqueous solution
of gallic acid was used as a standard.
Polyphenols in the hosoba-wadan
and nishi-yomogi methanol extracts were determined by HPLC using a
Mightysil RP-18 GP II (5 μm) column (4.6 × 250 mm²; Kanto Chemical Co., Inc., Japan) at a flow rate of 1.0 mL/min.
The sample was injected, and elution was performed with a system composed
of solvent A (0.1% phosphoric acid (Wako Pure Chemical Industries,
Ltd., Osaka, Japan) in water) and solvent B (0.1% phosphoric acid
in acetonitrile/methanol solution mixed at the same volume). The elution
was detected by a UV–vis detector at 280 nm. Compounds corresponding
to each peak were identified by comparison with the retention time
of reference standards and our previous report.^{8 (link)}