Rhamnosyl Icariside II is a rare secondary flavonoid glycoside isolated from Epimedium L. plants. It has better stability and physiological activity than the primary flavonoid glycosides of Epimedium L., therefore, conversion of the primary flavonoid glycoside into Rhamnosyl Icariside II would be desirable. In this study, a method for the enzymatic production of Rhamnosyl Icariside II from the total flavonoids of Epimedium wushanense was established, and the conditions were optimized. Six commercial enzymes were screened, and the reaction conditions for the best enzyme were optimized. Snailase was the most effective hydrolase, and the highest yield was obtained under the optimized conditions. To facilitate industrial production of Rhamnosyl Icariside II, a scaled-up pilot test was performed. The reaction solution was extracted with n-butanol to obtain the Rhamnosyl Icariside II crude product, which was then subjected to silica gel column chromatography and preparative chromatography. Finally, a product of Rhamnosyl Icariside II with purity of 99.1 % was achieved, in a total yield of 46.8 %. Compared to direct extraction and acid hydrolysis, this method improves the product yield and purity, which is of great significance for the large-scale production of Rhamnosyl Icariside II. This study provides a basis for the physiological activity study of Rhamnosyl Icariside II, and offers possibilities for future applications in the healthcare sector.

Two quality control standards, total flavonoid glycosides of Epimedii Folium and epimedin C of Epimedii Wushanensis Folium, were used to systematically evaluate the quality of Epimedium wushanense T. S. Ying, so as to provide reference for its germplasm screening and resource utilization. Seven representative populations of E. wushanense covering its main distribution areas were uniformly sampled during the flowering period. There were significant quality differences among the populations of E. wushanense. According to the quality standard of total flavonoid glycosides, all populations were superior to the quality standard of the Chinese Pharmacopoeia for Epimedii Folium, with more than 1.5 % total flavonoid glycosides. The variation ranges of epimedin A, epimedin B, epimedin C, icariin and total flavonoid glycosides were 0.40-0.76 %, 0.51-0.83 %, 1.70-9.31 %, 0.40-1.23 % and 3.05-10.61 %, respectively. According to the quality standard of epimedin C, all populations were better than the quality standard of the Chinese Pharmacopoeia for Epimedii Wushanensis Folium, with more than 1.0 % epimedin C. The variation range of epimedin C was 2.22-10.06 %. When comparing the results of the two methods, a trend of slightly lower mean values was found for total flavonoid glycosides, except for the HBXW population. The quality of E. wushanense was superior to both the quality standard of Epimedii Folium and Epimedii Wushanense Folium in the Chinese Pharmacopoeia. Epimedin C was the most abundant component. Among the investigated populations, HBXW and HBGK exhibited the highest quality, and may provide excellent genetic resources for standardized cultivation. In addition, the habitat of these populations can also serve a reference for cultivation conditions.

Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A-D (1-4) and two new prenyl flavonoid derivatives wushepimedoside E-F (5-6), and one know analog epimedkoreside B (7) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1-7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3β-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.

Epimedium wushanense is a well-known medicinal plant in Berberidaceae in China. In this study, we sequenced the complete chloroplast (cp) genome of E. wushanense. The results showed that the cp genome of E. wushanense was 157,283 bp in length, which is composed of a large single-copy region (LSC, 88,579 bp) and a small single-copy region (SSC, 17,082 bp) that were separated by a pair of inverted repeat regions (IRa and IRb, 25,811 bp). The chloroplast genome of E. wushanense contains 112 unique genes, of which are 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. The overall GC content was 38.78%. The phylogenetic tree analysis showed that E. wushanense was closely related to E. pseudowushanense, E. lishihchenii, and E. sagittatum.