Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
Anal Chim Acta. 2013 Jun 27;785:104-10. doi: 10.1016/j.aca.2013.04.053. Epub 2013 May 4.
Pueraria candollei associated preparation is widely applied in folk Thai medicine for rejuvenating purpose in aged people, which correlated with its pharmacological activities reported by pre-clinical and clinical trials. Therefore, standardized products of this plant are needed by consumers and health care personnel. Miroestrol, a potent and stable phytoestrogen in P. candollei, exhibited potential to be biomarker for quality control of P. candollei samples in research or industrial levels. Indirect competitive enzyme-linked immunosorbant assay (ELISA) for miroestrol determination was developed and validated by using polyclonal antibody from rabbit immunization. The polyclonal antibody recognized specifically to miroestrol, which exhibited cross-reactivity to deoxymiroestrol and isomiroestrol with 6.68% and 1.05%, respectively. The linearity range of measurement was 0.73-3000 ng mL(-1), which coefficient of variation (CV) of both intra- and inter-plate determination was less than 5%. With spiked samples of known amount miroestrol, the percentages of recovery were 98.80-104.37% and 98.31-106.69% in P. candollei and its involved product samples, respectively. Validated ELISA was comparable with published HPLC method (R(2)=0.9996) (Yusakul et al.) in samples with various miroestrol contents. For application, the P. candollei involved preparations contained miroestrol 0.695±0.037-12.108±0.285 μg g(-1) dry wt. The developed ELISA was high performance for miroestrol determination, which could be applied for P. candollei quality control in research fields and industrial productions.
野葛相关制剂广泛应用于民间泰国医学中，用于老年人的抗衰老目的，这与其临床前和临床试验报道的药理学活性有关。因此，消费者和医务人员都需要这种植物的标准化产品。野葛中的有效且稳定的植物雌激素——米罗昔酚，有望成为研究或工业水平上野葛样品质量控制的生物标志物。通过用兔免疫产生的多克隆抗体，建立并验证了用于米罗昔酚测定的间接竞争酶联免疫吸附测定（ELISA）。该多克隆抗体特异性识别米罗昔酚，对去氧米罗昔酚和异米罗昔酚的交叉反应性分别为 6.68%和 1.05%。测量的线性范围为 0.73-3000ng/mL，板内和板间测定的变异系数（CV）均小于 5%。用已知量米罗昔酚添加的样品进行测定，回收率分别为 98.80-104.37%和 98.31-106.69%，在野葛及其相关产品样品中。验证后的 ELISA 与已发表的 HPLC 方法（R(2)=0.9996）（Yusakul 等人）在具有不同米罗昔酚含量的样品中具有可比性。在应用中，野葛相关制剂中米罗昔酚的含量为 0.695±0.037-12.108±0.285μg/g 干重。该 ELISA 法用于米罗昔酚的测定具有较高的性能，可用于研究领域和工业生产中野葛的质量控制。
