Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka, 812-8582, Japan.
Faculty of Health Management, Nagasaki International University, 2825-7 Huis Ten Bosch, Sasebo, 859-3298, Japan.
J Nat Med. 2024 Jan;78(1):160-168. doi: 10.1007/s11418-023-01753-3. Epub 2023 Oct 7.
Saikosaponins are naturally occurring oleanane-type triterpenoids that are found in Bupleuri radix (root of Bupleurum falcatum) and exhibit a broad biological activity spectrum. Saikosaponin b2 (SSb2) is the main saikosaponin in Kampo medicine extracts and is a designated quality control marker for the same in the Japanese Pharmacopeia. Although some monoclonal antibodies (mAbs) against saikosaponins have been produced to evaluate the quality of Bupleuri radix and related products, anti-SSb2 mAbs have not been used to quantify SSb2 in Kampo medicines. To address this knowledge gap, we herein established a new hybridoma cell line secreting a highly specific anti-SSb2 mAb and developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) based on this mAb for the detection of SSb2 in Bupleuri radix-containing Kampo medicines. The generated SSb2-recognized mAb exhibited high specificity to SSb2 in icELISA. The developed assay featured high sensitivity (linearity range = 1.95-125 ng/ml), accuracy, precision and reproducibility (coefficient of variation < 5%), and the thus determined SSb2 contents were strongly correlated with those obtained using liquid chromatograph-mass spectrometer. These results suggest that the anti-SSb2 mAb-based icELISA method can be used for the quality control and standardization of Kampo medicines containing Bupleuri radix.
柴胡皂苷是一种天然存在的齐墩果烷型三萜皂苷，存在于柴胡根（北柴胡的根）中，具有广泛的生物活性谱。柴胡皂苷 b2（SSb2）是汉方药提取物中的主要柴胡皂苷，也是日本药典中该成分的指定质量控制标志物。虽然已经生产了一些针对柴胡皂苷的单克隆抗体（mAbs）来评估柴胡根及其相关产品的质量，但尚未使用抗-SSb2 mAbs 来定量汉方药中的 SSb2。为了弥补这一知识空白，我们在此建立了一种分泌高特异性抗-SSb2 mAb 的新杂交瘤细胞系，并基于该 mAb 开发了一种间接竞争酶联免疫吸附测定法（icELISA），用于检测含柴胡根的汉方药中的 SSb2。生成的 SSb2 识别 mAb 在 icELISA 中对 SSb2 表现出高特异性。开发的测定法具有高灵敏度（线性范围=1.95-125ng/ml）、准确性、精密度和重现性（变异系数<5%），因此确定的 SSb2 含量与使用液相色谱-质谱法获得的含量密切相关。这些结果表明，基于抗-SSb2 mAb 的 icELISA 方法可用于含有柴胡根的汉方药的质量控制和标准化。
