Identification of a naturally-occurring 8-[α-D-glucopyranosyl-( 1→6)-β-D-glucopyranosyl] daidzein from cultivated kudzu root

Introduction - Kudzu root (Radix puerariae) is a rich source of isoflavones that are effective in preventing osteoporosis, heart disease and symptoms associated with menopause. The major isoflavonoids in kudzu root extracts were reported as puerarin, daidzin and daidzein. Recently, an unknown isoflavonoid (compound 1) was detected from one-year-old kudzu root cultivated in Vietnam. Objective - To identify a novel compound 1 in kudzu root extract and determine the structure of the compound by ESI⁺ TOF MS-MS, ¹H-, ¹³C-NMR and enzymatic hydrolysis. Methodology - Samples were prepared by extraction of one-year-old kudzu root with 50% ethanol and the isoflavonoids were purified using recycling preparative HPLC. Unknown compound 1 was detected using UV-light at 254 nm in TLC and HPLC analyses. The molecular weight of 1 was determined using a TOF mass spectrometer equipped with an electrospray ion source. The structure of 1 was determined from the ¹³C and ¹H NMR spectra recorded at 100.40 and 400.0 MHz, respectively. Results - ESI⁺ TOF MS-MS analysis shows that 1 is a puerarin diglycoside. The interglycosidic linkage of diglycoside determined by ¹H-, ¹³C-NMR, and enzymatic hydrolysis suggests that 1 has a glucosyl residue linked to puerarin by an α-1,6-glycosidic bond. This compound is the first naturally-occurring 8-[α-D-glucopyranosyl-(1→6) -β-D-glucopyranosyl]daidzein in kudzu root. The concentration of glucosyl-α-1,6-puerarin in kudzu root was 2.3 mg/g as determined by HPLC. Conclusion - The results indicate that puerarin diglycoside is one of the major isoflavonoids in kudzu root and has a significant impact on the preparation of highly water-soluble glycosylated puerarin.