Advanced quality assessment of Sanshishi (Gardenia jasminoides Ellis) and Kampo medicines using a monoclonal antibody against geniposide.

Gardenia jasminoides Ellis, a plant widely used in traditional medicine, is known for its array of biological activities. A key bioactive compound, geniposide (GE), an iridoid glycoside, significantly contributes to the medicinal properties of the plant, with potential side effects. Thus, a reliable and efficient method for GE detection is required to ensure the quality of medicinal-grade G. jasminoides Ellis. This study developed such a method by first synthesizing GE-bovine serum albumin conjugates to function as immunizing agents in mice. This led to the production of a monoclonal antibody (mAb 3A6) against GE from the fusion of splenocytes from immunized mice with myeloma cells (P3U1), resulting in a hybridoma that produces mAb 3A6. Thereafter, we developed a mAb 3A6-based indirect competitive enzyme-linked immunosorbent assay (icELISA). The icELISA exhibited satisfactory sensitivity (0.391-12.5 µg/ml) and repeatability (coefficients of variation <10%). The accuracy of this method was validated through a spike-recovery assay (recovery of 101-112%). Furthermore, the icELISA was employed to determine the GE content in plant and Kampo medicine samples. The GE content positively correlated with those determined by high-performance liquid chromatography-ultraviolet. The proposed icELISA is rapid, cost-effective, and reliable for high-throughput GE detection in G. jasminoides Ellis, thereby contributing to the improved quality control and standardization of this valuable medicinal plant.

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

INTRODUCTION: 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. OBJECTIVES: 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). METHOD: 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. RESULTS: 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). CONCLUSION: 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.

The context of transcription start site regions is crucial for transcription of a plant tRNA(Lys)(UUU) gene group both in vitro and in vivo.

Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNA(Lys)(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNA(Lys)(UUU) coding sequences with their individual 5' and 3' flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5' flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNA(Lys) genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a ß-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNA(Lys)(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes.