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

Hesperidin (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.

To 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).

BALB/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.

The 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).

These 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.