| This work was supported by the National Science and Technology Support Program of China, the2009’Program for New Century Excellent Talents by the Ministry of Education, and the Priority Academic Program Development of Jiangsu Higher Education Institutions.This dissertation was divided into four major chapters.In Chapter1, a detailed review was given about the latest research progress of ginkgo seeds including history evaluation, resource chemistry, pharmacological effects, and toxicological research.In the second Chapter, the resource chemical constituents from ginkgo seed three parts including episperm, mesosperm, and kernel were studied. This chapter was divided into three sections.In Section1, chemical constituents from episperm were studied base on the ginkgo seeds comprehensive utilization and exploration. The compounds were isolated by silica gel, polyamide, and Sephadex LH-20gel column chromatography, and their structures were identified on the basis of spectral analysis and physicochemical data. As a result, twenty four compounds were obtained from the ethanol extract, and their structures were elucidated as (75,8R,11S)-nonacosanetriol (GBS-1),(10R,12R,15S)-nonacosanetriol (GBS-2), octacos-anoic acid (GBS-3), lignoceric acid (GBS-4), eicosanoic acid (GBS-5), ginnol (GBS-6), β-sitosterol (GBS-7), hydroginkgolic acid (GBS-8), ginkgolic acid (GBS-9),6-(10-heptadecene)-salicylic acid (GBS-10),2,3-dihydroxypropyl eicosate (GBS-11), apigenin (GBS-12), quercetin (GBS-13), kaempferol (GBS-14), sciadopitysin (GBS-15), ginkgetin (GBS-16), isoginkgetin (GBS-17), ginkgolide A (GBS-18), ginkgolide B (GBS-19), ginkgolide C (GBS-20), bilobalide (GBS-21), daucosterol (GBS-22), D-glucose (GBS-23), and sucrose (GBS-24). Among them, GBS-1and GBS-2were new compounds. Compound GBS-11was obtained from this plant for the first time.In Section2, chemical constituents from mesosperm of ginkgo seeds were studied. The compounds were isolated by silica gel and Sephadex LH-20gel column chromatography, and their structures were identified on the basis of spectral analysis and physicochemical data. As a result, eighteen compounds were obtained from the ethanol extract, and their structures were elucidated as stearine (GBM-1), palmitic acid (GBM-2), ginnol (GBM-3), β-sitosterol (GBM-4), n-dotriacontanol (GBM-5), n-docosanol (GBM-6), hydroginkgolic acid (GBM-7), ginkgolic acid (GBM-8), docosanoic acid2,3-dihydroxypropyl ester (GBM-9), eicosanoic acid2,3-dihydroxypropyl ester (GBM-10), hexadecanoic acid2,3-dihydroxypropyl ester (GBM-11), plamiticacid-1,3-dipropyl ester (GBM-12), dilinoleic acid-1,3-dipropyl ester (GBM-13), glyceryl tristearate (GBM-14), glycerol tripalmtate (GBM-15), ginkgolide B (GBM-16), ginkgolide C (GBM-17), and daucosterol (GBM-18). All these compounds were obtained from mesosperm of ginkgo seeds for the first time, and compounds GBM-5, GBM-6, and GBM-9～GBM-15were isolated from this plant for the first time.In Section3, chemical constituents from the ginkgo kernal were studied. The compounds were isolated by silica gel and Sephadex LH-20gel column chromatography, and their structures were identified on the basis of spectral analysis and physicochemical data. As a result, twenty compounds were obtained from the ethanol extract, and their structures were elucidated as hexacosanoic acid (GBK-1), palmitic acid (GBK-2), ginnol (GBK-3), β-sitosterol (GBK-4), hexadecanoic acid2,3-dihydroxypropyl ester (GBK-5), ursolic acid (GBK-6), sciadopitysin (GBK-7), ginkgetin (GBK-8), isoginkgetin (GBK-9), daucosterol (GBK-10), ginkgolide A (GBK-11), ginkgolide B (GBK-12), ginkgolide C (GBK-13), uracil (GBK-14), coniferin (GBK-15), daphnenoside (GBK-16), liquirtin (GBK-17), adenosine (GBK-18), D-glucose (GBK-19), and sucrose (GBK-20). Compounds GBK-1～GBK-5, GBK-7～GBK-10, and GBK-14were obtained from mesosperm of ginkgo seeds for the first time, and compounds GBK-6and GBK-15～GBK-18were isolated from this plant for the first time.In Chapter3, the resource chemistry evaluation on the ginkgo seeds were performed, and the results were summarized in six sections.Section1:resource chemistry evaluation of the nucleosides and nucleobases.In this study, a rapid, simple and sensitive analytical method was developed for the quantitative determination of nucleosides and nucleobases by using hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HILIC-UPLC-TQ/MS2). The developed method was successfully applied to ginkgo seed samples.1. The analysis of nucleosides and nucleobases in the different parts of ginkgo seedTo evaluate the effectiveness of the HILIC-UPLC-TQ/MS2method, it was applied for the analysis of five parts of ginkgo seeds (episperm, mesosperm, endopleura, embryoid and plumule) collected from three major production areas of G. biloba in China (Jiangsu, Shandong, and Guangxi provinces). The total content of all the target compounds ranged from64.1to3249.4μg/g, and the contents varied greatly among different parts. The data demonstrated that the distribution and determination of nucleosides and nucleobases in five parts of ginkgo seeds were:plumule> embryoid> episperm> endopleura> mesosperm. It was revealed that the contents of ribonucleosides in different parts of ginkgo seeds were obviously larger than that of deoxynucleosides. The contents of guanosine, uridine and their corresponding bases were extremely larger than any other nucleosides and nucleobases in embryoid and plumule.2. The analysis of nucleosides and nucleobases in the different tree ages of ginkgo seedTo further demonstrate the regularity of the nucleosides and nucleobases in ginkgo seeds, the proposed method was applied to analyze eleven edible parts of the ginkgo seeds materials of different plant-ages obtained from Tancheng in Shandong. The total content of these investigated compounds in sample24(100years) reached1930.3μg/g, whereas it was only642.8μg/g in sample23(50years). The results were shown that adenosine, guanosine, uridine,2’-deoxycytidine-5’-monophosphate, cytidine-5’-monophosphate were generally detected in many samples. In brief, levels of the nucleobases are evidently higher than those of the nucleosides in different samples.3. The analysis of nucleosides and nucleobases in the different geographical origins of ginkgo seedThe investigated compounds in samples27～48of ginkgo seeds collected from different places in China were simultaneously evaluated by HILIC-PLC-TQ/MS2. It was shown that guanosine and uridine were the major constituents, and their contents varied from388.7to522.7μg/g, and86.4to415.3μg/g, respectively. Remarkable differences were also found among the contents of the20nucleosides and nucleobases in ginkgo seeds of different cultivation regions. For example, guanine was found to be a predominant constituent in many samples, and its highest content was658.3μg/g, whereas it was as low as1.8μg/g in sample44. Furthermore, the content of hypoxanthine, xanthine, and five deoxynucleosides were observed to be lower than the others in all markers; In addition, cAMP,2’-deoxycytidine-5’-monophosphate, and cytidine-5’-monophosphate were also quantitatively determined, and they were detected in all samples. Total contents of the target compounds in the22batches of ginkgo seeds varied from772.8to1799.6μg/g.The contents of nucleosides and nucleobases in ginkgo seeds were determined for the different parts, habitats and plant-ages. The result showed that adenosine, guanosine, uridine, adenine, and guanine existed richly in all tested ginkgo seeds, especially in the plumule.Section2:resource chemistry evaluation of the free amino acids in ginkgo seed samples.This work presented a new analytical methodology based on hydrophilic interaction ultra-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry in multiple-reaction monitoring (MRM) mode for analysis of24underivatized free amino acids (FAAs) in functional foods.1. The analysis of free amino acids in the different parts of ginkgo seedThe validated HILIC-UPLC-TQ/MS2method was applied for the analysis of ginkgo seed five parts (episperm, mesosperm, endopleura, embryoid and plumule) collected from three major production areas of G. biloba in China (Jiangsu, Shandong and Guangxi provinces). The total content of all the target compounds ranged from341.2to13136.4μg/g and the contents varied greatly among different parts. Among the five parts, plumule, episperm and embryoid had higher FAAs levels, whereas mesosperm and endopleura had lower content. The data demonstrated that the distribution and determination of FAAs in ginkgo seeds five parts were arranged in a decreasing order as plumule> episperm> embryoid> endopleura> mesosperm. The major FAAs were GABA, Pro, Gin and Glu for the plumule, Gin and Glu for the episperm, endopleura and mesosperm, and Glu and GABA for the embryoid. In addition, the episperm were much different from the other four parts. For example, the total contents of essential amino acids and four non-protein amino acids (GABA, Hpro, Cit and Orn) were2380.49μg/g and11729.95μg/g in the episperm, respectively, and occupied approximately7.7%and38.2%of total amino acids, whereas they occupied approximately20.0-26.6%and10.7-26.9%in the other four parts.2. The analysis of free amino acids in the different tree ages of ginkgo seedTo further demonstrate the regularity of FAAs in ginkgo seeds, the proposed method was also applied to analyze the edible parts of eleven ginkgo seeds materials of different plant-ages obtained from the largest G biloba region in China (Tancheng, Shandong province). The content of24FAAs ranged between4330.4and8813.7μg/g. The highest content was of Glu followed by GABA, Arg, Pro and Gin. It is noticeable that both the acidic amino acid (Glu) and the basic amino acid (Arg) were among the dominant contributors. The non-protein amino acid GABA presented in almost quadruple amount than the other three non-protein amino acids. The content of FAAs in these investigated ginkgo seeds revealed remarkable results, among which the age-related changes was especially striking. Our data showed that the content of the FAAs in ginkgo seed edible parts changed with age. A steady increase is evident in the content of FAAs from8-year until about40-year age. The significant age-related changes plotted were derived from such total analyses on the series of ginkgo seed specimens aged8to600years. Besides, the results plotted also showed that the content of each FAA increases in the ginkgo seeds with different age, reaching a maximum in the sample22(40years).3. The analysis of free amino acids in the different geographical origins of ginkgo seedThe investigated compounds in22samples of ginkgo seed edible parts collected from different places in China were also simultaneously evaluated by the proposed HILIC-UPLC-TQ/MS2. The results showed that all of these ginkgo seed samples were rich in the FAAs, especially for essential amino acids such as Trp, Phe, Leu, Ile, Met, Val, Thr and Lys. Our study also revealed that FAAs exhibited a different concentration level in different localities, suggesting that the distribution of FAAs had geographical differentiation. Obviously, the contents of total and essential FAAs in sample28～31(the famed G. biloba growing area of Xuzhou and Taizhou, Jiangsu) and sample47(the largest G. biloba growing area of Tancheng, Shandong) were approximately1.5and3.5times higher than those in the other locality samples, respectively. As for the individual compounds determined in the experiments, GABA and Glu contributed the largest fractions to total FAAs. In particular, the non-protein amino acid GABA functions as an important neurotransmitter signal in humans, has blood pressure lowering activity, and has been used as component of pharmaceuticals and functional foods. Except for GABA, the content of three other non-protein amino acids in ginkgo seeds were at a trace level. Furthermore, the content of the rest12free Pro, Tyr, Cys, Ala, Gly, Gln, Ser, Asn, Asp, His, Arg and Lys amino acids in different sampling localities showed consistency and no variation at relate lower level (approximately100μg/g), indicating substantial and stable genetic diversity of these amino acid compositions in ginkgo seeds. From a nutritional and functional point of view, this research results indicated that ginkgo seeds were healthy and nutritional food for the supplement of FAAs, especially the essential amino acids and GABA. In addition, one obvious conclusion could be given that ginkgo seeds cultivated in major production regions (Jiangsu and Shandong province) have a high content of these nutritional and functional substances than other areas.Generally, ginkgo seeds were rich in FAAs, which could be used as nutritional substance to provide and/or complement the need of life activity.Section3:resource chemistry evaluation of the character components in ginkgo seeds, including flavones, ginkgolides, ginkgolic acids, and phenylpropanol.The character components in ginkgo seeds were analyzed using UPLC-TQ/MS2.1. The analysis of flavones, ginkgolides, ginkgolic acids, and phenylpropanol in the different parts of ginkgo seedGinkgolides and ginkgolic acids were rich in different parts of ginkgo seed, but the different composition contents varied greatly among different parts, especially ginkgolides. The results demonstrated that the distribution and determination of ginkgolide contents in ginkgo seed different parts were arranged in a decreasing order as ginkgolide C (GC)> ginkgolide A (GA)> ginkgolide B (GB) in episperm, mesosperm and endopleura. The decreasing order of ginkgolide contents was GC> GB> GA in the other parts namely embryoid and plumule. This result was according with the previous isolation. The data also showed that the higher contents of ginkgolide (GC, GA and GB) and ginkgolic acid were found in the plumule, ginkgolic acid and ginkgoneolic acid were found in the episperm. However, the less content of these target compounds were determinate in embryoid, mesosperm and endopleura.2. The analysis of flavones, ginkgolides, ginkgolic acids, and phenylpropanol in the different tree ages and geographical origins of ginkgo seedAll the analyzed compounds were accumulated in the different tree ages and geographical origins of ginkgo seed. The results showed that the content of the target compounds were too low in ginkgo seed edible parts. These compounds did not show significant various by tree age and geographical origin factors.Section4:resources chemistry evaluation of the ginkgolic polysaccharides in ginkgo seeds1. The analysis of ginkgolic polysaccharides in the different parts of ginkgo seedsThe compositions of polysaccharides in the different parts of ginkgo seeds were neutral and acidic polysaccharides. The ratio of neutral and acidic polysaccharides in ginkgo seeds ranged from213:1to1.32=1. The data indicated that neutral polysaccharide was the main part of total polysaccharides in different parts of ginkgo seed. The contents of neutral polysaccharide were higher than acidic polysaccharide in individual part of ginkgo seeds. The results demonstrated that the distribution and determination of neutral polysaccharide in ginkgo seeds five parts were arranged in a decreasing order as episperm> plumule> embryoid> endopleura> mesosperm, of acidic polysaccharides and total polysaccharides as episperm> embryoid> plumule> endopleura> mesosperm. The contents of total polysaccharide were much higher in episperm than other parts.2. The analysis of ginkgolic polysaccharides in the different tree ages of ginkgo seedA steady increase is evident in the content of ginkgo kernal total polysaccharides from8-year until about40-year age. The analysis results showed that the contents of neutral polysaccharides were not significance various in different tree-age ginkgo kernal. The contents of acidic polysaccharide increased a maximum in the sample (40years), which directly led to the total polysaccharide vary tendency in the ginkgo seeds with different age.3. The analysis of ginkgolic polysaccharides in the different geographical origins of ginkgo seedThe resulted indicated that the contents of total polysaccharides reached highest in Changting (Fujian,6.96%) and lowest in Jiangyan (Jiangsu,5.18%) sample. There was no significant various of the content of neutral and acidic polysaccharides in the different geographical origins of ginkgo kernal.Section5:resources chemistry evaluation of the inorganic elementsA comparative study on the29inorganic elements in the different regions, parts, and tree-ages of ginkgo seed was carried out by using ICP-AES. As the results, a characteristic distribution of high content of K and low content of Na was observed in all the samples. Among major elements, the average contents of K, Ca and Mg were relatively higher, whereas Fe and B were relatively higher among the trace elements.Section6:resources chemistry evaluation of the fatty acidsThe analysis of oil in ginkgo kernal was evaluated using GS-MS. The results exhibited that the content of oleinic, linoleic and many unsaturated fatty acids in ginkgo oil were higher than normal oil. Usually eating ginkgo seeds could prevent cardiovascular and cerebrovascular diseases, and the prospect was very broad in the development of ginkgo seeds.In chapter4, the material basement study of the efficacies in ginkgo seedsSection1:the effects of different exacts of ginkgo kernal and compounds using asthma mouse modelThe ginkgo kernal exacts and compounds were evaluated about astringing lung, eliminate sputum, and relieving asthma traditional effects using asthma mouse model. Bronchoalveolar lavage fluid (BALF), phlogocyte, and pathological section were selected as investigation targets. The results showed that the aqueous decoction exact of ginkgo kernal was the higher effects part among other exacts and compounds. Ginkgolides, ginkgo kernal character compounds, showed lower effects in astringing lung, eliminate sputum, and relieving asthma. So the further researches in ginkgo kernal effect material basement need to be done according recent study. The data indicated that the aqueous detection of ginkgo kernal should give considerable attention.Section2:the effects of anti-aging on drosophila by different exacts of ginkgo kernalTo study the possible anti-aging function of different ginkgo kernal exacts, the results showed that the life span of drosophila of the ginkgo kernal aqueous decoction exact was lengthenced than the control and other exact groups. So the aqueous detection of ginkgo kernal should give considerable attention.The main features and innovations in this dissertation are presented:1. Using column chromatographic method and spectral technology,40compounds were isolated from ginkgo seed. Among these compounds,2compounds were new compounds. Furthermore,14compounds were isolated from the ginkgo seed for the first time.2. The qualities of ginkgo seed from different parts, tree ages and cultivation regions were evaluated based on their contents of nucleosides, nucleobases, amino acids, flavones, ginkgolides, ginkgolic acids, phenylpropanols, polysaccharides, inorganic elements and fatty acids. This method was demonstrated to be very helpful in utilizing ginkgo seed resources efficiently. The new analytical methods were respectively developed on nucleosides, nucleobases and amino acids. The ultrasound extraction parameters which were used to extract ginkgo characteristic contents (flavones, ginkgolides, ginkgolic acids and phenylpropanols) were optimised, and the new anatical method was developed for simultaneously detection the above four types of characteristic compounds in ginkgo seed.3. The effects of astringing lung, relieving asthma and anti-aging were evaluated by different exacts of ginkgo kernal for the first time. The data indicated that water extract of ginkgo kernal showed good effects. |
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