HPLC separation of hesperidin and the C-2 epimer in commercial hesperidin samples and herbal medicines

Abstract

Hesperidin (2S-form), the flavanone 7-O-glycoside, is the main constituent of some Citrus species. The peels of two Citrus species are used as a crude drug, Aurantii nobilis pericarpium, in the Japanese Pharmacopoeia and as components in Kampo formulae. Thus, HPLC analysis of hesperidin as a marker compound is needed for quality control of medicines. Hesperidin was separated from the corresponding C-2 epimer by normal-phase HPLC using a chiral column. Moreover, narirutin and neohesperidin were also separated from the corresponding C-2 epimer. The analyses of commercial hesperidin samples revealed that they contained the C-2 epimer and that the relative ratio of hesperidin to the epimer ranged from 92:8 to 59:41. The HPLC application to Citrus extracts suggested that naturally occurring hesperidin in Citrus has the 2S configuration; however, the dry extracts of rikkunshito and chotosan, which are Kampo formulations containing Aurantii nobilis pericarpium, were found to contain a considerable amount of the (2R)-epimer. These data suggest that the decoction process of the formulae partly converts hesperidin to the epimer. Because diastereomers differ from each other in physicochemical and biological activities, HPLC to separate hesperidin from the C-2 epimer should be introduced into the letter of approval for herbal medicines.

Identification of natural epimeric flavanone glycosides by NMR spectroscopy

“…95%, Sigma-Aldrich). It is known that commercial flavanones and glycoside derivatives are mixtures of enantiomers or epimers (Caccamese, Manna, & Scivoli, 2003;Uchiyama, Kim, Kawahara, & Goda, 2005). When measuring the NMR spectra of commercial naringin the presence of an additional set of signals belonging to second flavanone was detected.…”

Methods of analysis and separation of chiral flavonoids

“…In freshly squeezed red grapefruit juice, 56% of the naringin was in the 2S form whereas lower percentage of the 2S epimer was found in commercial white and red grapefruit juice [95]. A Chiralpak IA column was also able to separate naringin directly under normal phase isocratic conditions although baseline resolution was not obtained [91]. Finally, separation of naringin and naringenin by capillary electrophoresis using sulfobutyl ether ␤-cyclodextrin as the selector was accomplished and grapefruit juice was determined to be essentially 50:50 in naringin epimers [96].…”

“…The amylose tris (3,5-dimethylphenylcarbmate) column Chiralpak IA has the advantage of being and immobilized chiral stationary phase instead of a silica gel supported stationary phase allowing to afford a wider range of solvents to be employed as the mobile phase. Furthermore, it has been shown to have the ability to resolve hesperidin, neohesperidin, narirutin, and naringin [91].…”

“…The Chiralcel OD-RH (tris-3,5-dimethylphenylcarbamate) column has demonstrated the ability to resolve naringenin enantiomers in isocratic reverse phase in a validated assay in biological matrices [90]. Chiralcel OD in normal phase has been utilized for the direct separation of epimers of the glycosides narirutin, hesperidin, neohesperidin, and naringin [91], and the aglycones naringenin, hesperetin, eriodictyol, and pinocembrin [58]. The 2,3,4-tris-O-(3,5-dimethylphenylcarbamoyl) CSP demonstrated the ability to resolve flavanone [69].…”

“…In orange/sour orange cross freshly squeezed juice, hesperidin was almost exclusively in the 2S epimer (92%) and in lemon juices (96%) [95]. In a recent study, hesperidin was separated using normal phase HPLC in commercial hesperidin and herbal medicine samples and although the 2S epimer predominated there was significant 2R hesperidin present in some samples [91]. Finally, baseline separation of hesperidin and hesperetin by capillary electrophoresis using sulfobutyl ether ␤-cyclodextrin as the selector was accomplished and 2S hesperidin predominated in lemon and orange juice [96].…”