Analysis of ginkgolides and bilobalide in Ginkgo biloba L. extract injections by high-performance liquid chromatography with evaporative light scattering detection

A RP-HPLC method with evaporative light scattering detection (ELSD) was developed for the determination of ginkgolides and bilobalide in Ginkgo biloba L. extract injections. The samples were extracted with ethyl acetate and the resulting extract was purified by aluminum oxide column. The resultant solution was determined by HPLC on a C(18) column with methanol-water (33:67, v/v) as eluent. The optimum ELSD parameters were set. The recovery of the method was between 98.3 and 102.1%. The method is suitable for routine quantitation of terpenes in Ginkgo biloba L. extract injections.     

HPLC-RI法测定银杏叶提取物中4种内酯的含量

目的:建立高效液相色谱法测定银杏叶提取物中4种内酯(银杏内酯 A、银杏内酯 B、银杏内酯 C、白果内酯)的含量。方法:采用 Symmetryshield 型 C_(18)色谱柱(3.9 mm×150 mm,5 μm),以甲醇-水(30:70)为流动相,流速为1.0 mL·min~~(-1),用外标法测定,示差折光检测器检测。结果:4种内酯浓度在0.02～0.24 mg·mL~(-1)范围内,与色谱峰面积呈良好线性关系,相关系数在0.9984～0.9998之间,检测限在0.025～0.035 μg之间,回收率在99.0%～100.2%之间。结论:本方法简单、准确、可靠、灵敏,适用于实验室和企业常规质量控制。     

Two new ginkgolides from the leaves of Ginkgo biloba

Two new diterpenoid compounds, ginkgolide P(1) and ginkgolide Q(2), were isolated from the leaves of Ginkgo biloba L. Their structures were elucidated by various spectroscopic methods, and the structure of 1 was further confirmed by X-ray crystallographic analysis. The activities of the compounds were evaluated against platelet aggregation induced by platelet activating factor (PAF), and the preliminary structure-activity relationship was also discussed.     

Bioavailability of ginkgolides and bilobalide from extracts of ginkgo biloba using GC/MS

The bioavailability of ginkgolides A, B and bilobalide was studied in rats after single oral administration of 30, 55 and 100 mg/kg Ginkgo extract EGb 761. The plasma levels of the terpene lactones were measured by a specific GC/MS method. The pharmacokinetics of the mentioned substances were found to be dose-linear. For the lowest dose maximum concentrations were 68, 40 and 159 ng/ml and half-lives 1.7, 2.0 and 2.2 h for ginkgolide A, B and bilobalide, respectively. Clearance values ranged from 24.2 to 37.6 ml/min/kg.     

Validation of capillary gas chromatographic method for determination of bilobalide and ginkgolides A, B, C in Ginkgo biloba dry and liquid extracts

A method for identification and quantitative determination of ginkgolides A, B, and C and bilobalide in liquid and dry extracts of Ginkgo biloba extracts has been developed. Determinations made by employing capillary gas chromatography technique with FID detection were preceded by derivatization using BSTFA with TMCS addition at 120 degrees C. Cholesterol was used as an internal standard. Validation of the method shows no interferences with concurrent constituents; average resolution (R), controlled for peaks of cholesterol and ginkgolide A was 1.53 (SD = 0.06). In the temperature program used (from 50 degrees C to 300 degrees C) the analyte retention times range from 11.2 min. (bilobalide) to 13.8 min. (ginkgolide C) and are of high repeatability of relative values (RRT): RSD = 0.05% / 0.07% for ginkgolides. High correlation coefficients (r), detector signal linearity: from 0.99962 for ginkgolide C to 0.99985 for ginkgolide A were obtained within the concentration range under investigation. The method is of high sensitivity: limits of detection and limits of determination are 35 pg and 44 pg for bilobalide, respectively, while for ginkgolides (Gk) are: 78 pg and 92 pg for GkA, 57 pg and 68 pg for GkB, and 213 pg and 320 pg for GkC.     

Analysis of ginkgolides and bilobalide in food products using LC-APCI-MS

A method was developed for the extraction and quantification of five marker compounds characteristic of Ginkgo biloba. Five ginkgo terpene trilactones: bilobalide and ginkgolides A, B, C, and J, were selected as marker compounds for this study. Initial studies produced a simple methanol extraction method for determination of gingko markers in solid dietary supplements. Five dietary supplements were analyzed and the results were later compared to the concentrations detected in the analysis of beverages. Beverage samples were prepared by extracting the ginkgo terpene trilactones using an optimized solid phase extraction (SPE) method. The extracts were analyzed using LC–atmospheric pressure chemical ionization (APCI)–MS in the negative ionization mode. The limits of detection of the extraction method ranged from 6.8 to 3.2 ng mL⁻¹. Using the optimized method, 14 drinks and 3 tea products were analyzed. Concentrations of total marker compounds in drinks ranged between 1685 and 21.4 ng mL⁻¹ with individual ginkgo terpene trilactones being detected at ppb concentrations. Analysis of brewed tea products presented much higher total marker compound concentrations ranging from 8.12 and 16.6 μg mL⁻¹. Analytical results reproducibility data, and recovery of the SPE method are presented.     

Ontogenic Aspects of Ginkgolide Production in Ginkgo biloba

The ontogenic aspects of ginkgolide production were studied by using GINKGO BILOBA seedlings, greenhouse plants, young trees, mature trees cuttings, and plant tissue cultures. Ginkgolide yield appeared to be increased with the age of the plants when the plants were grown under the same environmental conditions. Ginkgolide content in the leaves was increased when seedlings, young plants, and young trees were treated with fluridone, a carotenoid biosynthesis inhibitor. Ginkgolides appeared to be independently biosynthesized in leaves and roots of the GINKGO and stored in root bark and stem as more hydroxylated forms such as ginkgolide B or ginkgolide C.     

In vitro and in vivo effects of an extract of Ginkgo biloba (EGb 761), ginkgolide B,and bilobalide on apoptosis in primary cultures of rat hippocampal neurons

Primary cultures of rat hippocampal neurons were prepared and exposed to increasing concentrations of a peroxyl radical-generator, 2,2′-azobis 2 amidinopropane (AAPH). Addition of AAPH (20 or 50 mM) to the medium caused a decrease in cell viability and an increase in the number of apoptotic cells. Values for the number of nucleosomes were obtained using an ELISA technique. “Factor F,” an indicator of enrichment in nucleosomes, was found to be directly proportional to the number of neuronal apoptoses. Addition of an extract of Ginkgo biloba (Egb 761; 5–20 μg/ml) or ginkgolide B (one of its terpenoid constituents; 0.2 or 0.4 μg/ml) to the culture medium in vitro led to increases in cell viability and decreases in the number of hippocampal cells undergoing AAPH-induced apoptosis, whereas addition of bilobalide (another terpenoid constituent of Egb 761; 0.1–1.0 μg/ml) was ineffective. These in vitro results were corroborated and extended when these same substances were administered to rats in vivo. Oral administration of Egb 761 (50 mg/kg/day) for 8 days caused a significant increase in cell viability and a highly significant decrease in the numbers of both spontaneously occurring and AAPH-induced apoptoses. Similar protective effects were observed with ginkgolide B (2 mg/kg/day, p.o.), whereas bilobalide (2 mg/kg/day, p.o.) was ineffective. As AAPH enhances the production of peroxyl radicals, the protective actions of subacute in vivo treatments with Egb 761 and ginkgolide B appear to be associated with an anti-lipoperoxidative effect of these substances. Drug Dev. Res. 45:23–29, 1998. © 1998 Wiley-Liss, Inc.     

Production of antineoplastic agents by plant tissue cultures

A variety of callus tissues were induced from antineoplastic agent-producing plants such as PUTTERLICKIA VERRUCOSA, CEPHALOTAXUS HARRINGTONIA and TRIPTERYGIUM WILFORDII. Environmental conditions to optimize cultivation of the callus tissues and the suspension cultures were examined. The cell lines tested were recognized to produce cytotoxic substances except HELIOTROPIUM INDICUM. Among them, T. WILFORDII cultured cells produced a higher level of tripdiolide than the mother plant. Other products were preliminarily identified as expected antineoplastic agents.     

高效液相色谱法测定银杏叶片剂中银杏黄酮含量

目的建立高效液相色谱法测定银杏叶制剂天保宁片剂中银杏黄酮的含量。方法色谱柱为YWGC18-ODS柱(250mm×4.6mm),流动相由磷酸缓冲液(pH2)-四氢呋喃-甲醇-异丙醇(60:15:10:15)组成,流速0.5mL·min-1,紫外检测波长380nm。结果槲皮素和山柰酚在2.5-50mg·L-1,异鼠李素在1.362-27.38mg·L-1的浓度范围内线性关系良好,回归方程为槲皮素:y=145642x-314528,r=0.9994;山柰酚:y=94650x-197111,r=0.9993;异鼠李素:y-62031x-20056,r=0.9997;槲皮素、山柰酚、异鼠李素平均回收率分别为99.06%,99.89%,99.06%,RSD分别为1.33%,2.21%,1.85%。检测限槲皮素和山柰酚均为0.125mg·L-1,异鼠李素0.068mg·L-1。结论本法简便,快速,灵敏,准确,重现性好,可作为控制银杏黄酮制荆质量标准的依据。     

Inhibition of cGMP-phosphodiesterase-5 by biflavones of Ginkgo biloba

Ginkgo biloba dimeric flavonoids (GBDF) were shown to inhibit cAMP phosphodiesterase activity and to promote vasorelaxation. In particular, amentoflavone exhibited endothelium-dependent relaxation of rat aorta rings via enhanced generation and/or increased biological activity of nitric oxide, leading to elevated cGMP levels. The aim of this study was to investigate whether GBDF were able to inhibit cGMP-specific phosphodiesterase-5 (PDE5) as well. Human recombinant PDE5A1 was prepared by expression of the full-length cDNA of PDE5A1 in COS-7 cells. The PDE activity was determined in the presence of biflavones at 0.1-100 microM. All biflavones inhibited PDE5A1 in a concentration-dependent fashion, ginkgetin being the most potent (IC50 = 0.59 microM). The ability to inhibit the enzyme followed this order: ginkgetin > bilobetin > sciadopitysin > amentoflavone > sequoiaflavone. These data suggest that GBDF could exert a vasodilating effect through a mechanism independent of NO release.     

HPLC测定银杏叶中聚戊烯醇的含量

目的建立测定银杏叶中聚戊烯醇含量的方法。方法采用HPLC法,色谱柱为Kromasil ODS柱(250 mm×4.6 mm,5μm),流动相为异丙醇-甲醇-正己烷-水(50∶25∶15∶2),流速0.9 ml.min-1,柱温27℃,检测波长210 nm。结果在1.08~2.88μg.ml-1范围内,聚戊烯醇浓度与峰面积呈良好的线性关系(r=0.9979)。平均回收率为96.8%(RSD<2%,n=3)。结论所建方法快速、准确、稳定,为银杏叶中聚戊烯醇含量测定提供了可靠的方法。     

Ginkgolide A contributes to the potentiation of acetaminophen toxicity by Ginkgo biloba extract in primary cultures of rat hepatocytes

The present cell culture study investigated the effect of Ginkgo biloba extract pretreatment on acetaminophen toxicity and assessed the role of ginkgolide A and cytochrome P450 3A (CYP3A) in hepatocytes isolated from adult male Long-Evans rats provided ad libitum with a standard diet. Acetaminophen (7.5-25 mM for 24 h) conferred hepatocyte toxicity, as determined by the lactate dehydrogenase (LDH) assay. G. biloba extract alone increased LDH leakage in hepatocytes at concentrations > or =75 mug/ml and > or =750 mug/ml after a 72 h and 24 h treatment period, respectively. G. biloba extract (25 or 50 mug/ml once every 24 h for 72 h) potentiated LDH leakage by acetaminophen (10 mM for 24 h; added at 48 h after initiation of extract pretreatment). The effect was confirmed by a decrease in [(14)C]-leucine incorporation. At the level present in a modulating concentration (50 mug/ml) of the extract, ginkgolide A (0.55 mug/ml), which increased CYP3A23 mRNA levels and CYP3A-mediated enzyme activity, accounted for part but not all of the potentiating effect of the extract on acetaminophen toxicity. This occurred as a result of CYP3A induction by ginkgolide A because triacetyloleandomycin (TAO), a specific inhibitor of CYP3A catalytic activity, completely blocked the effect of ginkgolide A. Ginkgolide B, ginkgolide C, ginkgolide J, quercetin, kaempferol, isorhamnetin, and isorhamnetin-3-O-rutinoside did not alter the extent of LDH leakage by acetaminophen. In summary, G. biloba pretreatment potentiated acetaminophen toxicity in cultured rat hepatocytes and ginkgolide A contributed to this novel effect of the extract by inducing CYP3A.     

HPLC-ELSD法测定银杏叶茶中总内酯的含量

目的：采用RP-HPLC法测定低酚酸银杏叶保健茶中总内酯的含量。方法：采用AgilentC18（4.6mm×250mm,5μm）色谱柱,以甲醇-四氢呋喃-水（25：10：65）为流动相,柱温为30℃,蒸发光散射检测器检测。结果：银杏叶保健茶中银杏内酯A、银杏内酯B、银叶内酯C和白果内酯的进样量的对数与峰面积的对数线性关系较好,线性范围分别为0.485～9.701μg、0.460～9.204μg、0.501～11.899μg、0.935～18.703μg;银杏内酯A、银杏内酯B、银叶内酯C和白果内酯加样回收率分别为100.45%（RSD为2.79%）、98.29%（RSD为2.90%）、98.22%（RSD为2.53%）、101.32%（RSD为1.81%）。结论：本方法简便、准确、分离效果好,适合于银杏叶茶总内酯的含量测定。