Study On The Resource Chemistry Of Ginkgo Biloba Leaves From Different Origin In China

This work was supported by the National Science and Technology Support Program of China (2011BAI04B03).2009’ Program for New Century Excellent Talents by the Ministry of Education (NCET-09-0163), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2010), and Construction Project for Jiangsu Engineering Center of Innovative Drug from Blood-conditioning TCM Formulae.This dissertation was divided into four chapters.In Chapter1, a detailed review was given about the latest research progress of Ginkgo biloba leaves. Besides, chemical composition, pharmacological effects and clinical applications of G. biloba leaves was also summarized.In Chapter2, the resource chemistry evaluation of G. biloba leaves were performed, and the results were summarized in eight sections.In Section1, chemical constituents of fallen Leaves of Ginkgo biloba were studied. Twenty-two compounds were isolated from the fallen leaves of Ginkgo biloba by silica gel and Sephadex LH-20column chromatography. The structures were elucidated by physico-chemical and spectroscopic methods as ginnol (1), octacosanoic acid (2), palmitinic acid (3), heptatriacontane (4), tetracosane (5),4,10-nonacosanediol (6),β-sitosterol (7), daucosterol (8), ginkgolide A (9), ginkgolide B (10), ginkgolide C (11), bilobalide (12), p-hydroxybenzoic acid (13), shikimic acid (14), genkwanin (15), apigenin (16), ginkgetin (17), isoginkgetin (18), sciadopitysin (19), bilobetin (20), rutin (21) and mannitol (22). Among them, compounds2,4～6and22were isolated from this plant for the first time. The results are helpful for the comprehensive utilization of ginkgo tree resources.In Section2, the total flavonol glycosides and terpene lactones in different ages, cultivation sources and gender of G. biloba leaves collected in November were determined by using the validated HPLC-ELSD and HPLC-PDA methods. The results showed that the contents of total terpene lactones and flavonol glycosides in the leaves of young ginkgo tree are higher than that in old tree, and they were higher in male trees than those in female. And geographical factors appear to have significant influence on their contents. All the results will provide good basis for comprehensive utilization of G. biloba leaves, especially the leaves from fruit cultivars.In Section3, the change of total ginkgolic acids was investigated in Ginkgo biloba leaves of different tree-age and cultivation sources for using fruit. The results showed that their contents varied with different ages and cultivation sources, and the contents in tree-age from10to30years were lower than that in50and100years. In terms of cultivation sources, the samples from Jiangsu, Shandong, Anhui and Zhejiang provinces had comparatively low contents of total ginkgolic acids in the leaves, which were also lower than the reported data in Ginkgo biloba leaves for the standard GBE, and which showed a certain regional distribution. These results provided scientific basis for the comprehensive utilization and development of G. biloba resources in our country.In Section4, to determine the content of free amino acids, a reliable method has been established by using hydrophilic interaction ultra-high performance liquid chromatography coupled with electrospray ionization mass.20free amino acids were simultaneously determined without derivatization in12min. The proposed method was fully validated in terms of linearity, sensitivity, repeatability as well as recovery. Furthermore, the principal component analysis was applied to the different G. biloba leaves collected in November (after fruit harvest season), which revealed that the samples from different production areas exhibited regional disparity in different clusters on the basis of their various hydrophilic interaction liquid chromatography coupled with mass profiles. The established approach could be helpful for evaluation of the potential values as dietary supplements and the quality control of G. biloba leaves, and might be utilized for the investigation of other medicinal herbs containing amino acids.In Section5, inorganic elements were determined by ICP-AES using microwave digestion, and principal components analysis (PCA) was used to analyze. The result showed that Ginkgo biloba leaves contained more than22inorganic elements, Fe、Zn、Cu、Mn、Cr、Co、Ni、Sr、 B、Si and Ni were essential trace elements, and Ca、P、K、Na、Mg were essential macro elements. PCA with seven factors (F1, F2, F3, F4, F5, F6, F7) selected could be used to evaluate the quality of G. biloba leaves. The function was following F=0.23017F1+0.12239F2+0.07967F3+0.07897F4+0.06525F5+0.06203F6+0.05671F7. The sample from Taixing, Shijiazhuang and Taian were the top three which indicated the quality in those cultivation sources were better based on inorganic elements. The large number of Ca, Mg and other beneficial elements may play a synergistic role in the prevention and treatment of cardiovascular and cerebrovascular diseases. All the results will provide good basis for comprehensive utilization of G. biloba leaves, especially the leaves from fruit cultivars.In Section6, the total contents of neutral and acidic polysaccharides in the samples of different cultivars from different regions were determined using UV-Vis spectrophotometry. The results showed that their contents varied with different ages and cultivation sources, and the contents in tree of300years is highest(4.62%), of10years is lowest(3.12%). The contents of the leaves of old ginkgo tree is higher than that of the youngs.These results provided scientific basis for the comprehensive utilization and development of G. biloba resources in our country.In Section7, an ultra performance liquid chromatography coupled with photodiode array detector and time-of-flight mass spectrometry (UPLC-PDA-TOF/MS) was proposed and validated for rapidly analyzing and evaluating Ginkgo biloba leaves from different origin by using multivariate statistical analysis. Two different kinds of G. biloba leaves, namely L-GBL the leaves which are collected from the tree of4-7years within August and September for preparing commercial extract of G. biloba leaves, and F-GBL:the leaves from fruit cultivars. Batches of these two kinds of G. biloba leaves were subjected to UPLC-PDA-TOF/MS analysis, the datasets of retention time (RT)-m/z pairs, ion intensities and sample codes were further processed with orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the difference between these G. biloba leaves, and to generate an S-plot. Those compounds correlated to the points at the two ends of "S" were regarded as the most differentiating components. By comparing the mass/UV spectra and retention times with those of reference compounds and/or tentatively assigned by matching empirical molecular formulae with those of the known compounds published in the literatures, these differentiating components between L-GBL and F-GBL were finally characterized as kaempferol3-0-[2-0-(6-O-p-hydroxy-trans-cinnamoyl)-β-D-glucosyl)-α-L-rhamnoside], kaempferol3-O-[2-O-(β-D-glucosyl)-α-L-rhamnoside], kaempferol3-O-[2-O,6-O-bis(α-L-rhamnosyl)-β-D-glucoside], ginkgolide C, and bilobetin. These compounds would be the potential chemical markers for the two kinds of leaves. The results suggested that this newly established approach could be used to rapidly evaluate the quality of herbs from different origin, and to provide good strategy for further rectify and standardize the herb market.In Section8, on the basis of liquid chromatography coupled with triple quadrupole mass spectrometry working in multiple reaction monitoring mode, an analytical method has been established to simultaneously determine flavonol glycosides, terpene lactones, biflavones, proanthocyanidins, and ginkgolic acids in Ginkgo biloba leaves. Chromatographic separation was carried out on an Acquity BEH C18column (100mm×2.1mm,1.7μm) with gradient elution of acetonitrile and0.10%formic acid (v/v) at a flow rate of0.4mL/min; column temperature,30℃. The developed method was validated in terms of linearity, accuracy, precision, stability, and sensitivity. The optimized method was successfully applied to analyze twenty-two G. biloba leaf samples of fruit cultivars collected from different places in China. Furthermore, hierarchical clustering analysis (HCA) was performed to evaluate and classify the samples according to the contents of the twenty-four chemical constituents. All of the results demonstrated that the developed method was useful for overall evaluation of the quality of G. biloba leaves, and this study was also helpful for the comprehensive utilization and development of G. biloba resources.In Section1of Chapter3, considering the merits of the established extraction processes of Ginkgo biloba, these methods were combined with absorption resin techinique in the present work. A technique based on ethanol-resin absorption method was extablished to extract Ginkgolides, terpene lactones and flavornoids. It is demonstrated that this extraction process possed merits of low cost, high recovery, and low retained level of organic solvent.In Section2of Chapter3, compare and evaluate pharmacological activity of EGB and standard EGB. The antioxidant activities of EGB and standard EGB were assessed by three vitro experiments of eliminating DPPH, eliminating OH·and deoxidizing Fe3+; the influence of action of PA in rabbits was observed by PA test induced by PAF; the protecting effection of neuronal damage induced by LPS was observed by neuronal damage rat models induced by LPS. EGB and standard EGB both possess strong activities of antioxidantion, abilities of inhibiting platelet aggregation, effection of protecting neuron cells, but all of the above is a little worse of EGB than the standard EGB. But no significant difference between the two groups. EGB has a bright prospect of development and application.In Section1of Chapter4, a sensitive and accurate ultra-high performance liquid chromatography coupled with triple quadrupole mass (UPLC-TQ-MS) method was developed for the determination of quercetin-3-O-β-D-glucopyranoside-(4�'1)-α-L-rhamnoside (QGR) in rat plasma using rutin as internal standard. Chromatographic separation was achieved on a Acquity BEH C18column (100mm×2.1mm,1.7μm) with gradient elution of acetonitrile and0.10%formic acid (v/v) at a flow rate of0.4mL/min. QGR and chloramphenicol were detected using electrospray negative ionization mass spectrometry in the multiple reaction monitoring (MRM) mode. The method demonstrated good linearity and did not show any endogenous interference with the QGR and chloramphenicol peaks. This method was successfully applied to a pharmacokinetic study of QGR in rats after intravenous (20mg/kg) and oral (40mg/kg) administration. After intravenous administration at the dose of20mg/kg, the elimination half-life (t1/2) value was estimated to be118.89±5.65min, and the mean area under the plasma concentration-time curve from time zero to the last measurable plasma concentration point AUC(0-t) and the mean area under the plasma concentration-time curve from time zero to time infinity AUC(0-∞)values were1775.96±36.92and1790.24±37.53ug/mL×min, respectively. After oral administration at the dose of40mg/kg, QGR was rapidly absorbed, reaching mean Cmax of495.69±58.36ng/ml at Tmax of20min. The mean AU(0-t) and AUC(0-∞)values were120.81±11.38and122.14±16.15ug/mL×min, respectively. The oral bioavailability (F) of QGR was calculated to be6.82±1.21%with an elimination half-life (t1/2) value of236.87±28.59min. The potential hydrolysis in the gastrointestinal tract, poor permeability through the intestinal epithelial membrane and first-pass effect in the liver might be responsible for the low bioavailability of these compounds including QGR.In Section2of Chapter4, ultraperformance liquid chromatography/quadrupole-time-of- flight mass spectrometry (UPLC-Q-TOF/MS) and the MetabolynxTM software combined with mass defect filtering were applied to identity the metabolites of QGR in rat after intravenous administration. MSE was used for simultaneous acquisition of precursor ion information and fragment ion data at high and low collision energy in one analytical run, which facilitated the fast structural characterization of8metabolites in rat plasma, urine and bile. The results indicated that methylation and glucuronidation were the major metabolic pathways of QGR in vivo. The present study provided important information about the metabolism of QGR which will be helpful for fully understanding the mechanism of this compound’s action. Furthermore, this work demonstrated the potential of the UPLC-Q-TOF/MS approach using Metabolynx for fast and automated identification of metabolites of natural product.