Ginkgo Terpene Trilactones Research Articles

Substantial Differences in Proanthocyanidin Contents among Ginkgo biloba Leaf Extracts in Herbal Medicinal Products Obtained from the German Market.

Pharmacologic activity of proanthocyanidins in Ginkgo biloba leaf extract has recently been reported. The objective of the present study was to screen proanthocyanidin contents in herbal medicinal products containing Ginkgo extracts. A recently published HPLC method for quantification of proanthocyanidins in G. biloba leaf extract EGb 761 was adopted to also be suitable for finished herbal medicinal products. The method was applied to 14 products from the German market. For each product, a set of three individual batches was purchased and analyzed. Substantial differences in proanthocyanidins contents were found among distinct products, ranging from 0.30 to 5.86%. The batch-to-batch variability within each product was low. The highest concentrations are in a similar range as, for example, the amount of Ginkgo terpene trilactones specified in the monograph for G. biloba leaf extract in the European Pharmacopeia. Although it has not yet been established whether and to what extent proanthocyanidins contribute to the overall pharmacological or clinical efficacy of Ginkgo extracts, a potential impact on the purported benefits of different contents in proanthocyanidins cannot be ruled out. Quality assessment of different Ginkgo extracts in the future may include proanthocyanidins.

Insight into Ginkgo biloba L. Extract on the Improved Spatial Learning and Memory by Chemogenomics Knowledgebase, Molecular Docking, Molecular Dynamics Simulation, and Bioassay Validations.

Epilepsy is a common cause of serious cognitive disorders and is known to have impact on patients’ memory and executive functions. Therefore, the development of antiepileptic drugs for the improvement of spatial learning and memory in patients with epileptic cognitive dysfunction is important. In the present work, we systematically predicted and analyzed the potential effects of Ginkgo terpene trilactones (GTTL) on cognition and pathologic changes utilizing in silico and in vivo approaches. Based on our established chemogenomics knowledgebase, we first conducted the network systems pharmacology analysis to predict that ginkgolide A/B/C may target 5-HT 1A, 5-HT 1B, and 5-HT 2B. The detailed interactions were then further validated by molecular docking and molecular dynamics (MD) simulations. In addition, status epilepticus (SE) was induced by lithium–pilocarpine injection in adult Wistar male rats, and the results of enzyme-linked immunosorbent assay (ELISA) demonstrated that administration with GTTL can increase the expression of brain-derived neurotrophic factor (BDNF) when compared to the model group. Interestingly, recent studies suggest that the occurrence of a reciprocal involvement of 5-HT receptor activation along with the hippocampal BDNF-increased expression can significantly ameliorate neurologic changes and reverse behavioral deficits in status epilepticus rats while improving cognitive function and alleviating neuronal injury. Therefore, we evaluated the effects of GTTL (bilobalide, ginkgolide A, ginkgolide B, and ginkgolide C) on synergistic antiepileptic effect. Our experimental data showed that the spatial learning and memory abilities (e.g., electroencephalography analysis and Morris water maze test for behavioral assessment) of rats administrated with GTTL were significantly improved under the middle dose (80 mg/kg, GTTL) and high dose (160 mg/kg, GTTL). Moreover, the number of neurons in the hippocampus of the GTTL group increased when compared to the model group. Our studies showed that GTTL not only protected rat cerebral hippocampal neurons against epilepsy but also improved the learning and memory ability. Therefore, GTTL may be a potential drug candidate for the prevention and/or treatment of epilepsy.

Natural accumulation of terpene trilactones in Ginkgo biloba leaves: variations by gender, age and season

Ginkgo terpene trilactones (TTLs) are a group of biologically active compounds in Ginkgo leaf extracts. The contents of TTLs in Ginkgo leaves directly affects the qualities of the related phytopharmaceutical products. In this study, the natural occurrence of TTLs in Ginkgo leaves within one vegetative season was investigated. The leaves were collected from six sampled trees with different age and gender at monthly intervals from June to November in Madison, WI. The contents of TTLs were quantified by RP-HPLC/RI procedure, validated by LC/sonic spray ionization-mass spectrometer (SSI-MS) assay, and the data were analyzed statistically. The natural accumulation of total TTLs in Ginkgo leaves fluctuated significantly with seasons with a peak appearing in August. The total amounts of TTLs accumulated in leaves from younger trees were higher than those from older trees; total TTLs in female trees were higher than in male trees. This survey on the natural occurrence of TTLs was expected to ensure the quality of Ginkgo product from the stage of incoming material with rich and consistent bioactive value in terms of Ginkgo terpene trilactones.

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.

Qualitative and Quantitative Analyses of Ginkgo Terpene Trilactones by Liquid Chromatography/Sonic Spray Ionization Ion Trap Mass Spectrometry

A liquid chromatography/sonic spray ionization mass spectrometry method (LC/SSI-MS) was developed for qualitative and quantitative analyses of ginkgo terpene trilactones. Five ginkgo terpene trilactones were successfully protonated for qualitative and quantitative analyses under the study conditions. The typical ion adducts were identified as (M + H)+, (M + NH4)+, and (M + Na)+. The limits of detection were achieved between 2.5 and 10 ng with RSD of 0.173-4.82% and a linear range of 10-80 ng with R2 = 0.991-0.999. This method was used to identify and quantify ginkgo terpene trilactones in extractions of ginkgo biloba leaves obtained from three different extraction methods. This is the first completely validated LC/MS method for quantification of ginkgo terpene trilactones. The factors that contributed to reduce the errors of identification and quantification of ginkgo terpene trilactones are systematically reported, and the advantages and disadvantages of LC/MS method in quantitative analysis are also discussed.

Sample Preparation and Determination of Ginkgo Terpene Trilactones in Selected Beverage, Snack, and Dietary Supplement Products by Liquid Chromatography with Evaporative Light-Scattering Detection

Ginkgo biloba leaf extract has been widely used in dietary supplements and more recently in some foods and beverages. Sample preparation procedures for determination of ginkgo terpene trilactones (including bilobalide and ginkgolides A, B, C, and J) in various sample matrixes were developed in this study. Ginkgo leaves and capsules were extracted with 5% KH2PO4 aqueous solution under sonication. Tea bags were extracted with boiling water, whereas drink samples were taken directly from the bottles. After filtration and the addition of NaCl to approximately 30% (w/v), the terpene trilactones in aqueous solutions were quantitatively extracted with ethyl acetate-tetrahydrofuran (4 + 1, v/v). Puff samples (a cereal-based fried snack item) were first defatted by using hexane or by using supercritical fluid extraction and then extracting under sonication with methanol-acetic acid (99 + 1, v/v). After evaporation of the organic phase, the terpene trilactones were redissolved in methanol and determined on a C18 reversed-phase column by liquid chromatography (LC) with evaporative light-scattering detection. The method of standard additions and gas chromatography with flame ionization detection were used for method validation. For most samples, the relative standard deviation was <10%. The identities of target compounds in ginkgo leaves and drink samples were confirmed by LC/electrospray ionization-tandem mass spectrometry.

A method for extraction and quantification of Ginkgo terpene trilactones.

A method was developed for the extraction and quantification of pharmacologically active terpene trilactones (ginkgolides, bilobalide) from the tissues of Ginkgo biloba L. and pharmaceutical ginkgo products by RP-HPLC, based on the theory of terpene trilactones ionization. Four ginkgolides (GA, GB, GC, GJ) and bilobalide (BB) from both the ginkgo leaves and commercially available ginkgo extracts were quantitatively extracted by using this method. The recovery rate of the method was 97.5-100% with RSD of 1.2-2.8%. The detection limit was 0.05-0.1 microg, and the linear range was 0.1-12 microg. This detection limit represents a marked improvement over previously reported methods, suggesting the new method is a viable technique for routine analysis of ginkgo terpene trilactones in natural and commercial samples. The method reported by van Beek et al. in 1991 (van Beek, T. A.; Scheeren, H. A.; Rantio T.; Melger, W. C.; Lelyveld, G. P. J. Chromatogr. 1991, 543, 375-387.) was used as a reference method to monitor the accuracy of extraction and analysis in this study. SSI-MS technique was used to identify isolated target components. Carbohydrase treatment and solubility of terpene trilactones in various solvents were also discussed.

Liquid chromatography-atmospheric pressure chemical ionisation/mass spectrometry: a rapid and selective method for the quantitative determination of ginkgolides and bilobalide in ginkgo leaf extracts and phytopharmaceuticals.

In order to evaluate the composition of active constituents in phytopharmaceutical preparations, valid analytical methods are required. For the determination of the active terpene constituents of Ginkgo biloba (the ginkgolides and bilobalide), a liquid chromatography-mass spectrometry (LC-MS) method has been developed using atmospheric pressure chemical ionisation (APCI) in the negative ion mode. This detection mode was found to be much more sensitive and selective compared to UV; indeed the ginkgo terpene trilactones lack strong UV chromophores and flavonoids interfere with their UV detection. LC-APCI/MS detection allowed a considerable reduction in analysis time when compared to LC-UV, because LC resolution was only needed between the pair of isomers ginkgolide B and ginkgolide J. All compounds were selectively detected by single ion monitoring of their specific deprotonated molecules [M-H]-. The samples were directly injected without pre-purification, and a fast gradient was applied, reducing the total time of analysis to 14 min. With this method, the ginkgo terpene trilactones were detected on-line in the picogram range. Several commercial ginkgo preparations on the Swiss market were analysed, and the ginkgolide and bilobalide contents were evaluated using the method described.

Selective dissolution and one step separation of terpene trilactones in ginkgo leaf extracts for GC-FID determination

Under ultrasonication, the ginkgo terpene trilactones, ginkgolides and bilobalide, in ginkgo extracts can be selectively dissolved in 10% aqueous NaH 2PO 4 solution at a temperature of 50–60°C and separated from the solution by extraction with a mixture of ethyl acetate/tetrahydrofuran in a capped vial. After derivatization, these terpene trilactones can be quantified using GC-FID. This method has a detection limit of 10 ng, and the RSD was 6% ( n=5). Twelve commercial GBE products in powder, liquid, tablet and capsule forms were analyzed. The total time required for analyzing these samples from sample preparation to final data processing was less than 6 h, and the total organic solvent consumption was less than 40 ml. This procedure proves to be a simple, fast, safe, and effective method for all types of Ginkgo biloba extracts (GBE) including the “complex” or “advanced” formulas.

Preparative Isolation and Separation Procedure for Ginkgolides A, B, C, and J and Bilobalide

A simple preparative method for the isolation and purification of ginkgolides A, B, C, and J and bilobalide (ginkgo terpene trilactones) was developed. As starting material, a commercially available leaf extract from Ginkgo biloba containing ≥ 6% ginkgo terpene trilactones was used. After a partition step with EtOAc, the enriched intermediate extract was separated into the individual terpenes by medium-pressure liquid chromatography on silica impregnated with 6.5% NaOAc with a gradient from petroleum ether−EtOAc to EtOAc−MeOH. After recrystallization from H2O−MeOH, all five terpenes could be isolated in high purity. After a selective extraction with H2O, leaves could also be used as a starting material.

Quantitation of bilobalide and ginkgolides A, B, C and J by means of nuclear magnetic resonance spectroscopy

AbstractA quantitative 1H NMR procedure for the determination of bilobalide and ginkgolides A, B, C and J in Ginkgo biloba leaves and in phytopharmaceuticals without prior chromatographic separation of the mixture has been developed. The method is based on the comparison of the integral of each H‐12 proton of the five ginkgo terpene trilactones with that of the olefinic protons of the internal standard (maleic acid). These protons are all well separated at 200 MHz and occur in a less crowded region of the NMR spectrum (6.15‐6.50 ppm). The selectivity, reproducibility and sensitivity are comparable with high pressure liquid chromatography (HPLC) with refractive index (RI) detection. The minimum amount that can be quantified within 30 min at 200 MHz is approximately 0.1 mg for all four ginkgolides and bilobalide. Advantages, in comparison with HPLC‐RI, are that no reference substances are needed and that for the occasional analysis of a limited number of samples a very significant time‐gain can be achieved.