GC-MS测定家犬血浆中银杏内酯A、B的含量

银杏叶制剂作为治疗和预防动脉粥样硬化的有效药物,越来越为人们关注。银杏叶成分复杂,其中银杏黄酮具有扩张血管,降血脂,改善微循环的作用;银杏内酯是血小板活化因子强拮抗剂,对免疫系统,中枢神经系统,缺血损伤有保护作用,并有抗休克、抗过敏及抗炎作用。由于银杏内酯含量较低,对其进行的药动力学研究较少,且多采用的是非特异性的生物测定方法。我们参照中国药典有关药物制剂生物利用度和生物等效性试验的指导原则和相关实验方法,建立的GC-MS-SIM方法,可准确测定血浆中银杏内酯A(GA)和银杏内酯B(GB)的含量,为其药动力学的研究提供了可靠的分析手段。     

Chemical analysis and quality control of Ginkgo biloba leaves,extracts, and phytopharmaceuticals

The chemical analysis and quality control of Ginkgo leaves, extracts, phytopharmaceuticals and some herbal supplements is comprehensively reviewed. The review is an update of a similar, earlier review in this journal [T.A. van Beek, J. Chromatogr. A 967 (2002) 21–55]. Since 2001 over 3000 papers on Ginkgo biloba have appeared, and about 400 of them pertain to chemical analysis in a broad sense and are cited herein. The more important ones are discussed and, where relevant, compared with the best methods published prior to 2002. In the same period over 2500 patents were filed on Ginkgo and the very few related to analysis are mentioned as well. Important constituents include terpene trilactones, i.e. ginkgolide A, B, C, J and bilobalide, flavonol glycosides, biflavones, proanthocyanidins, alkylphenols, simple phenolic acids, 6-hydroxykynurenic acid, 4-O-methylpyridoxine and polyprenols. In the most common so-called “standardised” Ginkgo extracts and phytopharmaceuticals several of these classes are no longer present. About 130 new papers deal with the analysis of the terpene trilactones. They are mostly extracted with methanol or water or mixtures thereof. Supercritical fluid extraction and pressurised water extraction are also possible. Sample clean-up is mostly by liquid–liquid extraction with ethyl acetate although no sample clean-up at all in combination with LC/MS/MS is gaining in importance. Separation and detection can be routinely carried out by RP-HPLC with ELSD, RI or MS, or by GC/FID or GC/MS after silylation. Hydrolysis followed by LC/MS allows the simultaneous analysis of terpene trilactones and flavonol aglycones. No quantitative procedure for all major flavonol glycosides has yet been published because they are not commercially available. The quantitation of a few available glycosides has been carried out but does not serve a real purpose. After acidic hydrolysis to the aglycones quercetin, kaempferol and isorhamnetin and separation by HPLC, quantitation is straightforward and yields by recalculation an estimation of the original total flavonol glycoside content. A profile of the genuine flavonol glycosides can detect poor storage or adulteration. Although the toxicity of Ginkgo alkylphenols upon oral administration has never been undoubtedly proven, most suppliers limit their content in extracts to 5 ppm and dozens of papers on their analysis were published. One procedure in which a methanolic extract is directly injected on a C8 HPLC column appears superior in terms of sensitivity (<5 ppm), separation, simplicity and validation and will be incorporated in the European Pharmacopoeia. Alternatively GC/MS and ELISA methods can be used. A sharp contrast to the plethora of papers on terpene trilactones, flavonol glycosides, and ginkgolic acids forms the low number of papers on biflavones, proanthocyanidins, simple phenolics, simple acids, and other constituents that make up the remaining 70% of Ginkgo standardised extracts. More research in this direction is clearly needed. For the analysis of Ginkgo proanthocyanidins (7%) for instance, no reliable assays are yet existing. Finally the growing literature on pharmacokinetic and fingerprinting studies of Ginkgo is briefly summarised.     

Preparative isolation of terpene trilactones from Ginkgo biloba leaves

This study investigated and compared some techniques for the preparative isolation of terpene trilactones, including ginkgolides (GA and GB, etc.) and bilobalide (BB), from Ginkgo biloba leaves. The crude Ginkgo biloba L. extracts (GBE) were prepared using an extractor with solvent refluxing operated under an optimal extraction condition. The extraction yield was 20-23% and the purity of terpene trilactones was about 1.0-1.4 wt%. Before the isolation operations, the extracts were dissolved in de-ionized water. The isolation procedures included the method of liquid-liquid extraction and the method of column chromatography. For the method of liquid-liquid extraction using ethyl acetate as the organic solvent operated under the optimal extraction conditions, the purity, concentration ratio, and yield of terpene trilactones were 13.5-18.0%, 15-16, and >99%. For the method of column chromatography, XAD-7HP, XAD-4, and C-18 adsorbents with different polarities were used as the packing materials. Only for the XAD-7HP column, a part of more polar impurities was efficiently separated with the majority of terpene trilactones by a proper step-gradient elution, which resulted in an efficient isolation: the purity, concentration ratio, and yield of terpene trilactones were approximately 20, approximately 15, and approximately 80%. In comparison, the XAD-7HP column achieved the highest purity, but at the expense of the yield of terpene trilactones; on the contrary, the liquid-liquid extraction method, achieving the highest yield but with a slightly lower purity, was proved to be superior to the method of column chromatography in the current isolation stage.     

南雄银杏叶中内酯的高效液相色谱法测定

用高效液相法分析了广东省南雄市不同采收时间，不同树龄的银杏叶中的银杏内酯含量；采用Kromasil C18柱、甲醇－水－四氢呋喃流动相、流速1mL/min、差示折光检测器、柱温35℃、外标法定量，样品用聚酰胺柱纯化，获得了满意的分离效果；结果表明银杏内酯含量随树龄不同，采收时间不同而不同，南雄银杏叶内酯平均含量为0．23％（w）。     

Complete 1H NMR spectral analysis of ten chemical markers of Ginkgo biloba

The complete and unambiguous ¹H NMR assignments of ten marker constituents of Ginkgo biloba are described. The comprehensive ¹H NMR profiles (fingerprints) of ginkgolide A, ginkgolide B, ginkgolide C, ginkgolide J, bilobalide, quercetin, kaempferol, isorhamnetin, isoquercetin, and rutin in DMSO‐d₆ were obtained through the examination of 1D ¹H NMR and 2D ¹H,¹H‐COSY data, in combination with ¹H iterative full spin analysis (HiFSA). The computational analysis of discrete spin systems allowed a detailed characterization of all the ¹H NMR signals in terms of chemical shifts (δ_H) and spin‐spin coupling constants (J_HH), regardless of signal overlap and higher order coupling effects. The capability of the HiFSA‐generated ¹H fingerprints to reproduce experimental ¹H NMR spectra at different field strengths was also evaluated. As a result of this analysis, a revised set of ¹H NMR parameters for all ten phytoconstituents was assembled. Furthermore, precise ¹H NMR assignments of the sugar moieties of isoquercetin and rutin are reported for the first time. Copyright © 2012 John Wiley & Sons, Ltd.     

Regioselective thionocarbonation of ginkgolides: facile preparation of ginkgolide J

Ginkgolide J, a minor constituent of terpene trilactone mixture of Ginkgo biloba leaves extract, can be readily prepared from an abundant ginkgolide C in two steps: thionocarbonation and subsequent deoxygenation. Regioselectivity of the first thionocarbonation step can be controlled by a proper choice of the base and solvent combination.     

Analysis of terpenes fromGinkgo biloba L. extracts by reversed phase high-performance liquid chromatography

Summary The main terpenes ofGinkgo biloba L. extracts (bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) have been separated by isocratic elution on a 3 μm C₁₈ Spherical column using 2-propanol:water (10∶90) as eluent.     

大孔吸附树脂对银杏内酯和白果内酯吸附性能的研究

本文比较了ＡＢ－８、Ｄ４０２０、ＮＫＡ－９、ＡＢＤ－４和ＤＭ－１３０五种大孔吸附树脂对银杏内酯Ａ、Ｂ和白果内酯的吸附及解吸性能,并研究了相应的吸附动力学过程,实验结果表明ＡＢ－８树脂是一种较适宜的吸附剂。