Identification And Evaluation Of The Quality Of Guizhou Epimedium

ObjectiveHerba Epimedii, as a perennial barrenwort in the family Berberidaceae, has a long history of application in Traditional Chinese Medicine (TCM). It was first included in Shen Nong Ben Cao Jing, one of the earliest Chinese Pharmacopedia. Herba Epimedii is effective in aphrodisiac, keeping strong bodies, and elimination of antirheumatics. China is the center of Herba Epimedii as approximately40species grow here. There are about15species of Herba Epimedii commercially developed for herb medicine. Herba Epimedii has rich chemical components and broad pharmacological effects, therefore, it has great potential in development. Many products have been developed such as Xianlinggubao products, Yishenling granule, and Yishenzhuanggu granule.’Qian yin yang huo’is included in Standards of Guizhou ethnic and Chinese Herb Medicine, a local standards of Guizhou province. The major five species of’Qian yin yang huo’ are E. acuminutum Franch, E. myrianthum Steam, E. leptorrhizum Steam, E. sagittatum Maxim. var. glabratum Ying, and E.coactum. These species are the major ones used as medicine in Guizhou and Miao, however, their standards are relatively simple. To further improve the standards of’Qian yin yang huo’, to regulate the medicine market, and to improve the quality of medicine, the qualitative and quantitative analysis of’Qian yin yang huo’was conducted in this research, using pharmacognosy, molecular biology, mass spectrometry technology and fingerprint techniques. The quality of different species of’Qian yin yang huo’was also evaluated.MethodsThe samples of’Qian yin yang huo’were collected and identified at the early stage of the project; the morphological characteristics of the leaves and the molecular techniques such as DNA barcoding was adopted to inspect the identification characteristics; Mass spectrometry was used to analyze the chemical compositions of’Qian yin yang huo’; Content determination of5flavonoids and establishment of fingerprint was conducted to evaluate the quality and of ’Qian yin yang huo’.ResultsIn this research,35samples of’Qian yin yang huo’(including three samples of stems, and two samples of E. pseudowushanense) were collected. Initial identification of species was conducted based on the morphological characteristics of the leaves. Molecular techniques such as DNA barcoding was adopted, in which sequences such as psbA-trnH and ITS were used to conduct NJ tree clustering analysis. It was found that there is some correlation between the genes of the plants and the location of samples.RRLC-DAD-ESI-MSn was used to analyze the chemical compositions for five species of ’Qian yin yang huo’. Combined molecule ion peaks with the fragmentation mechanism of characteristic fragment ions,17compounds are identified compared with standard reference substance or literature, including16prenylated flavonoids and one alkaloid.The fingerprints of35samples of ’Qian yin yang huo’ from5species are established and quantitative determination of five flavonoids (Epimedin A\B\C, Icariin, Baohuoside I) were conducted. It was found that the fingerprints of ’Qian yin yang huo’ samples can be greatly impacted by factors such as sampling seasons, location, and parts of the plants. The quantitative determination indicates that the quality of ’Qian yin yang huo’ is low and inappropriate for medicine use; samples of E. acuminutum FranchI and E. myrianthum Steam collected in spring and summer have higher quality; the prenylated flavonoids from the stem is low and should not be used together with leaves. Such findings provide reference in improving the standards of ’Qian yin yang huo’ used in Guizhou Chinese herb medicine.In this research, using Icariin as internal refenrence, the relative correction factor (RCF) between Icariin and other four components were established. The relative retention time was used to recognize target peaks. Quantitative Assay of Multi-components by Single-marker(QAMS) was conducted as well. The results by using this approach is very close to that measured using external reference method.ConclusionThis project made a primary research on ’Qian yin yang huo’ from four aspects,such as the morphological characteristics,the molecular level,the chemical compositions analyzation and the quatitative determination of index component.The expected experimental objective had been completed.And the theoretical foundation was provided as well for the further improvement of the quality standards of ’Qian yin yang huo’...