Study On Extraction And Analysis Of Active Costituents In Crotalaria Sessiliflora L.

Crotalaria sessiliflora L. is the dry herba of Crotalaria plant in Fabaceous genuses,and it is mainly used to the treat the disease of dysentery, sore furuncle, tinnitus and deafness, dizziness and various cancers such as squamous cell skin cancer, esophageal cancer, and cervical cancer and so on. The current study shows that Crotalaria sessiliflora L. contains active constituents including alkaloids and flavonoids. In this paper, the methods to intensify extraction of alkaloids and flavonoids were developed by using microwave-assisted technique. The optimum experimental conditions for the methods were systematically investigated. Using High performance liquid chromatography (HPLC), spectrophotometry and High performance liquid chromatography–mass spectrometry,the analytical methods and the fingerprint methods for alkaloids and flavonoids extracted from Crotalaria sessiliflora L. were also established respectively in order to make better use of this plant and its active constituents. The systematic research in this paper was carried out as following.1. The extraction methods of alkaloids and flavonoids from Crotalaria sessiliflora L. were developed by using Solvent reflux extraction (SRE), microwave-irradiated solvent reflux extraction (MISRE) and focused microwave-assisted extraction (FMAE). The extraction yields of alkaloids and flavonoids were significantly improved by MISRE and FMAE compared with SRE, and in which the FMAE is the most effective methods. So microwave can strengthen the extraction process of the alkaloids and flavonoids.2. The spectrophotometric methods of total alkaloids and total flavonoids were established by using bromothymol blue and vitexin as colour developing reagent and reference substance respectively. Monocrotaline, vitexin and isovitexin were determined by HPLC. These analytical methods were successfully applied to determine above active constituents in Crotalaria sessiliflora L. from the different regions with the satisfactory results, respectively.3. The flavonoids and alkaloids from Crotalaria Sessiliflora L. were analysed by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). The extracted from Crotalaria Sessiliflora L. contains oriention, isooriention, vitexin, isovitexin and monocrotaline by primary data of mass spectrometry, secondary data of tandem mass spectrometry (MS/MS) and reference substances. The chemical structures of other three compounds were deduced based on MS/MS data.4. The fingerprint methods of the alkaloid and flavonoid extracted from Crotalaria sessiliflora L. were studied and established by using HPLC. In fingerprint analysis of the extracted, the peaks of nine flavonoids and the peaks of the four alkaloids were determined as the common peaks respectively. Similarities of 10 batches of samples from the different regions were greater than 0.9 by using the "similarity of chromatographic fingerprints of Chinese medicine evaluation system". The analytical results of the fingerprint methods showed that 10 batches of samples had better similarities and the more significant fingerprint characteristics of Crotalaria Sessiliflora L..