Quality assessment method for Chinpi, dried Citrus spp. peel and its derived Kampo medicines using specific monoclonal antibody against hesperidin

Abstract

AbstractIntroductionHesperidin (hesperetin 7‐rutinoside, HP), a flavonoid glycoside found in Citrus unshiu Marcowicz or Citrus reticulata Blanco (Rutaceae), has been reported to exert a variety of pharmacological effects. As the efficacies and qualities of their dried peel, Chinpi and its derived Kampo medicines can be evaluated by their HP contents, a method for HP detection must be developed.ObjectivesTo produce a specific monoclonal antibody against HP (mAb 5D12) to detect the HP contents in Japanese traditional medicines via indirect competitive enzyme‐linked immunosorbent assay (icELISA).MethodBALB/c mice were immunised with many haptens of HP–bovine serum albumin (BSA) conjugates that were prepared using sodium periodate (NaIO4) to cause an immune response. In addition, conventional hybridoma techniques were utilised to generate mAb 5D12.ResultsThe detection range of HP by the mAb 5D12‐based icELISA was 1.56–25.0 ng/mL, with a detection limit of 1.12 ng/mL. The maximum coefficient of variation, as evaluated from the intra‐ and inter‐assays, was <10.0%, and the percentages of recovery, as determined by the spike‐recovery tests, were 105%–115%. Moreover, the HP content, which was obtained from the developed icELISA, correlated well with that obtained via high‐performance liquid chromatography–ultraviolet (HPLC‐UV).ConclusionThese validation analyses revealed that the established icELISA technique exhibited high precision and accuracy. Notably, this is the first report on the development of icELISA for the HP content‐based quality control of Chinpi and its derived Kampo medicines.