Study On Fingerprint By HPLC And Biological Activity Of Guiyujin

Guiyujin is the dry root derived from Curcuma kwangsiensis S.G.Lee et C.F.Liang,which is one of the origins of Radix curcumae. Guangxi is the authentic region of Guiyujin, and where about60%Guiyujin Supplied around the whole nation. This paper based on Guiyujin in Guangxi province. High Performance Liquid Chromatography (HPLC) fingerprint and the pharmacological activity and fundamental substance were studied.1Main content is as follows:(1) In this paper, HPLC analysis was performed for10batches of authentic region in Guangxi province from curcuma kwangsiensis. The pattern analysis and similarity calculation were evaluated by using the national pharmacopoeia committee recommended " The computer aided similarity evaluation software of Traditional Chinese medicine (TCM) fingerprint ", the HPLC fingerprint of Guiyujin was established.(2) In this paper, a screening on drugs against spinal cord injuries was studied.Neurite outgrowth activity was evaluated for extracts of natural medicines by using primary cultured cortical neurons (SD rat, E17); Extention of axons and dendrites were quantified by immunocytochemistry using pNF-H antibody (axon marker) and MAP2a&2b (dendrite marker); microscopic analysis and image analysis were used for measure the length of axons and dendrites.Then the experiment of CSPG inhibitors was used to check on the preliminary screening results, and some nature medicine that have some active from the first screening were further separate by system solvent extraction methods, then test the active site. Effective parts were further isolated and purified by using the column chromatography and preparation chromatography, compounds were identified by using MS,1H-NMR,13C-NMR. HMQC, HMBC for the structure identification.(3)In this paper the effect of inhibiting proliferation of Guiyujin to Hela cells and MCF-7cells were detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method, the active sites were found out; the autophagy body were observed and analyzed by immunofluorescence staining, the expression of LC3protein was detected with western blotting; explored the influence of active extract of Guiyujin against autophagy; explored the possible mechanism to Hela cells.2The results of the study are as follows:(1) set up HPLC fingerprint of Guiyujin, and12feature peaks in fingerprints were identified, the relative retention time between samples was consistent, the resemblance was higher among10samples(>90%).The characteristic peak area from10sample of authentic region had good separation, characteristics and uniqueness.(2) The preliminary experiments indicated that:compare to the other three extracts, The water extracts of Guiyujin have effect to extend axons in cortical neurons. in CSPG experiment, Guiyujin water extract and the remain water extraction indicate strong activity.From Guiyujin water extract16compounds have been isolated, and4have been identified:Glucose, citric acid, fumaric acid, and the disaccharide composed of glucose.(3) In vitro experiments showed that:only petroleum ether and ethyl acetate parts had inhibiting proliferation to Hela cells among four kinds of extracts of Guiyujin, and the effect of inhibiting proliferation was dose-effect relationship, ethanol and water soluble parts had no inhibiting proliferation against this kinds of cancer cells. The petroleum ether part of Guiyujin increased the the number of autophagic vacuole in the Hela cells from immunofluorescence staining. And increased the expression of LC3-Ⅱ protein after24h.The petroleum ether part of Guiyujin elevated ROS levels in Hela cells through flow cytometry instrument, so this study preliminary confirmed Guiyujin petroleum ether part induced autophagy by improve the level of ROS in cervical cancer.3Conclusions:To sum up, this dissertation establishes the HPLC fingerprint of Guiyujin, at the first time the experiment of CSPG inhibitor spot was used on screening as vitro model for spinal cord injury. And at the first time the water part of Curcumae Radix is determined to be the active part which can promote spinal cord injury repair, and4compounds were identified from the active site. At the same time. the active site of Guiyujin and Preliminary mechanism of antitumor were studied, which provide a reference for the quality control and the establishment of evaluation standards of Guiyujin.