Quality Study On Fritillariae Cirrhosae Bulbus

The underground bulbs of six genus Fritillaria in Lilium. listed as "Chuan-beimu" in the2010edition of the Chinese Pharmacopoeia, have been widely used for thousands of years in traditional Chinese medicine as antitussive, anti-asthmatic and expectorant agents. Fritillariae cirrhosae bulbus is considerated as the highest value in genus Fritillaria. Currently, the sharp increased market demand pushed wild plants to the brink of extinction and break the equilibrium in the market. In order to control quality of Fritillariae cirrhosae bulbus, several gene Fritillaria species were analyzed in some aspect, including quantification of fat-soluble and water-soluble components, determination of mycotoxins, analysis on adaptive cultivated area and biosynthesis of isosteroidal alkaloids.Fritillariae bulbus, Beimu in Chinese, derived from the bulbs of many Fritillaria species has been used as an antitussive and expectorant herb. Besides, the bulbs of more than30other species were utilized locally as substitutes for Fritillariae bulbus in the markert. Therefore, it is necessary to develop some methods to identify different Fritillaria species and their substitutes confused Fritillariae cirrhosae bulbus in market for the clinical safety of drugs. In this study,99patches Fritillariae cirrhosae bulbus from five Fritillaria species were analyzed by high-performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) and high-performance liquid chromatography coupled with a photodiode array detector (HPLC-DAD), respectively. Fingerprint analysis based on HPLC-ELSD was established. The result indicated that peimisine is a common constituent in all Fritillaria species investigated.10active nucleosides and nucleobases were detected in all samples, which could be useful for quality evaluation and control of all Fritillaria species in the future. In addition, take F. unibracteata for example. A simple and reliable method of ultra-performance liquid chromatography coupled with a photodiode array detector (UPLC-PAD) was developed for the quantitative analysis of10active nucleosides and nucleobases, including cytidine. uridine, guanosine, inosine. thymidine. cordycepin. adenosine. uracil, hypoxanthine and adenine, for quality control of F. unibracteata. The developed HPLC-ELSD for isosteroidal alkaloids and HPLC-DAD for nucleosides and nucleobases could be useful to control quality of Fritillariae cirrhosae bulbus.Mycotoxins are widely distributed toxic secondary metabolites produced by several molds. The growth of molds under diverse environmental conditions could contaminate Fritillariae cirrhosae bulbus. Several studies have provided strong evidence that some mycotoxins have carcinogenic, teratogenic and neurotoxic effects. In the work, an effective method was established for simultaneous determination of aflatoxin B1and ochratoxin A in Fritillariae cirrhosae bulbus by solid-phase extraction coupled with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). And a rapid method was developed for simultaneously screening Fritillariae cirrhosae bulbus contaminated by DON, NIV, ZEN and T-2using LC-MS. These approaches could be implemented for the routine analysis to investigate the contamination of Fritillariae cirrhosae bulbus.Owing to the increased demand for Fritillariae cirrhosae bulbus, wild Fritillaria species are threatened by overexploitation, habitat destruction and lack of proper cultivation practices. Thus, Ex-situ cultivation becomes an immediate action to sustain medicinal plant resources. The traditional Chinese medicine geographic information system (TCMGIS) was applied to predict the large-scale distribution of Fritillaria species. Adaptability analysis of producing area (with similarity rate>90%) was outputted, which gives insights into the discovery of potential habitats for Fritillaria species cultivation.F. cirrhosa D. Don is the primary source of Fritillariae cirrhosae bulbus, which has been used as a traditional Chinese medicine for thousands of years as an antitussive apophlegmatic and expectorant. Previous pharmacological investigations indicated that isosteroidal alkaloids were responsible for the therapeutic effects. However, few studies have investigated how isosteroidal alkaloids are synthesized in the plant. To investigate the biosynthetic pathway of the isosteroidal alkaloids in F. cirrhosa, transcriptomic analysis was developed in the present work. A total of1,205,646high-quality sequence reads were generated from the cDNA library of F. cirrhosa bulbs and assembled into45,073unigenes. Many genes involved in the biosynthesis of isosteroidal alkaloids were discovered, including all the genes encoding enzymes catalyzing multiple reactions from acetyl-CoA to the first cyclic backbone, cycloartenol, in this pathway. Some candidate genes encode tailoring enzymes responsible for the modification of cycloartenol into various isosteroidal alkaloids. This study will lay the foundation for the elucidation of the mechanism underlying the biosynthesis of the isosteroidal alkaloids in F. cirrhosa and will also provide an important opportunity to further identify the uncharacterized compounds in the pathway.