Fingerprint analysis of Ophiopogonis Radix by HPLC-UV-ELSD coupled with chemometrics methods

A new, simple and reliable method using HPLC-UV-ELSD was developed to generate the fingerprint of Ophiopogonis Radix. Homoisoflavonoids and steroidal saponins were determined simultaneously in a single run. A total of 27 Ophiopogonis Radix samples were analyzed, and 18 reference substances were used for the identification of the common peaks. The fingerprint was further analyzed by chemometrics methods including similarity analysis （SA）, hierarchical clustering analysis （HCA） and principal component analysis （PCA）. The results indicated that the combination of chromatographic fingerprint and chemometrics analysis could be used for the geographical differentiation and quality evaluation of Ophiopognnis Radix.

Cause and control of Radix Ophiopogonis browning during storage

In the storage of Radix Ophiopogonis, browning often happens to cause potential risk with regard to safety. Previously few reports investigate the browning of Radix Ophiopogonis. In this research, the causes and mechanisms of the browning of Radix Ophiopogonis were preliminarily elucidated. Content determination by high-performance liquid chromatogra- phy （HPLC） and spectrophotometry, enzyme activity determination by colorimetry, and morphological observation by electron mi- croscopy were performed in the present study. Uniform design and three-dimensional response surfaces were applied to investigate the relationship between browning and storage factors. The cortex cell wall of browned Radix Ophiopogonis was ruptured. Compared with the normal Radix Ophiopogonis, cellulase and polyphenol oxidase enzymes were activated, the levels of 5-hydroxymethylfurfural （5-HMF）, total sugars, and reducing sugars were increased, while the levels of polysaccharides and methylophiopogonanone A were de- creased in browned Radix Ophiopogonis. The relationship between the storage factors and degree of browning （Y） could be described by following correlation equation： Y= - 0.625 4 ＋ 0.020 84 × X3 ＋ 0.001 514×X1×x X2 - 0.000 964 4 × X2 × X3. Accompanied with browning under storage conditions, the chemical composition of Radix Ophiopogonis was altered. Following the activation of cellulase, the rupture of the cortex cell wall and the outflow of cell substances flowed out, which caused the Radix Ophiopogonis tissue to become soft and sticky. The main causes of the browning were the production of 5-HMF, the activation of polyphenol oxidase, Maillard reactions and enzymatic browning. Browning could be effectively prevented when the air relative humidity （HR）, temperature, and moisture content were under 25% RH, 12 ℃ and 18%, respectively.

A monoclonal antibody-based competitive ELISA for the determination of ruscogenin in Chinese traditional medicines and biological samples

A competitive enzyme-linked immunosorbent assay(ELISA) was developed to determine ruscogenin(RUS) by using the monoclonal antibody(McAb). The monoclonal antibody against RUS, secreted from the established hybridoma cell lines, was identified as being of the IgG1 isotype. The McAb exhibited high specificity to RUS, showing a very slight cross reactivity with diosgenin(15.7%), and no cross-reactivity to sarsasapogenin, diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1. The established ELISA, at an IC50 value of 157.55 ng.mL-1 and a detection limit(IC20) of 20.57 ng.mL-1, was compared with HPLC analyses, and a good correlation between ELISA and HPLC-ELSD analyses of RUS in the extract of Radix Ophiopogonis was obtained. The experimental data indicated that the ELISA method exhibits more advantages over HPLC-ELSD, such as low detection limit, high specificity, low background, and no requirement for sample pre-treatment, and is more suitable for the determination of natural components in Chinese traditional medicines and in biological samples for pharmacokinetic studies.