Ginkgo Biloba, Ginkgo Biloba Extract And Its Injection Chemical Components And Phenolic Acids Content In The Research

It is well known that Ginkgo biloba Leaves (GbL).have certain pharmacological effects and clinical value. The quality control of GbL and its products mainly focus on terpene trilactones, flavonol glycosides and alkyl phenols. Nevertheless about70%chemical components have been hardly studied, especially for the high polar compounds. It is also difficult to analyze primary flavonol glycosides because of lack of continuous supplier of reference substances. In order to obtain more comprehensive understanding of its components and provide scientific basis for establishing quality standard, an isolation research had been carried out in this thesis. Five hydroxybenzoic acids were isolated from the high polar part of GbL extract, and4of them were measured by HPLC in GbL, GbL extract and GbL injection. Four flavonol glycosides were enriched and calibrated reference substances. Four biflavones were also isolated and confirmed in the fingerprinting of GbL injection.1The separation and purified of componentsBy using silica gel, Sephadex LH-20, C18, MCI, prep-HPLC and other chromatography technologies,14components were isolated. On the basis of NMR and MS experiments, the structures were elucidated as5carboxylic acids:p-hydroxybenzoic acid,p-hydroxybenzoic acid, protocatechuic acid, gallic acid and6-hydroxykynurenic acid (6-HKA), gallic acid and m-hydroxybenzoic acid were isolated from this plant for the first time; five flavonols: quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, kaempferol-3-O-rhamnoside-2-glucoside, quercetin, the quantity of four flavonol glycosides were more than300mg; four bisflavones:ginkgetin, isoginkgetin, bilobetin and sciadopitysin.2The determination of4hydroxybenzoic acids in GbL, GbL extract and GbL injection.To establish an HPLC method for the determination of4hydroxybenzoic acids (gallic acid, protocatechuic acid,p-hydroxybenzoic acid,p-hydroxybenzoic acid) in GbL, GbL extract and its injection. The analyses was performed on a Thermo Syncronis C18column (250mm×4.6mm,5μm), the mobile phase was water containing0.1%phosphoric acid-acetonitrile (A) with gradient elution mode (0～8min,5.7%A;9～-45min,6.7%A) at the flow rate of1mL·min-1. The detection was set at260nm (0～30min) and234nm (30-45min), the column temperature was35℃. The standard curves showed good linearity. The results showed that the method had good precision, repeatability and stability, RSD values were less than1.95%. The average recovery were between98.45%～102.11%, RSD value to GbL was less than2.10%, and1.87%to ginkgo leaves extract and its injection. Using the optimized method, the contents of4hydroxybenzoic acids in ginkgo biloba extract and the injection supplied by many manufacturers were analyzed.3The calibration of six flavonol glycosides Four flavonol glycosides which were isolated according to the above works and the other two in hand were calibrated. On the basis of UV, NMR andMS, the structures were elucidated. Silica gel and polyamide thin-layer chromatography were set up to test the impurity. The samples were analyzed on Inertsil ODS-3(250mm×4.6mm,5μm) and Phenomenex Gemini C18(250mm×4.6mm,5μm) with diode array detector. Chromatography peak area normalization method was used to examine the purity. The purity of quercetin-3-O-glucoside, kaempferol-3-O-rutinoside, isorhamnetin-3-O-rutinoside, kaempferol-3-O-rhamnoside-2-glucoside, quercetin-3-O-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside, kaempferol-3-O-rhamnopyranosyl-2-O-(6-O-p-coumaroyl)-glucoside were95.6,98.4,96.6,97.5,96.6,94.5%.4Improvement to the fingerprinting of GbL injectionThe carboxylic acids and bisflavones in the fingerprinting of GbL injection were identified by their retention times.The analyses was perfonned on a Agilent Zorbax Eclipse Plus C18column (50mm×4.6mm,1.8μm), the mobile phase was water containing0.4%phosphoric acid-acetonitrile (A) with gradient elution mode (0～12min,14%A;17min,16%A;24min,20%A,26～40min,21%A) at the flow rate of0.6mL·min-1. The detection was set at360nm, the column temperature was40℃. Under this method, the retention time of carboxylic acids were in3min. Bisflavones, meanwhile, were after36min. The tlavonol glycosides had been confirmed. The qualitative investigation of carboxylic acids and bisflavones was used to improve the fingerprinting of GbL.In this research, the study of GbL and its praeparatum was enriched, the quality control of GbL products was expanded and promoted, scientific foundation for the quality assurance of GbL and its praeparatum was also provided.