Effect of Methyl Jasmonate on the Terpene Trilactones,Flavonoids, and Phenolic Acids in Ginkgo biloba L. Leaves: Relevance to Leaf Senescence

The present study compared the effects of natural senescence and methyl jasmonate (JA-Me) treatment on the levels of terpene trilactones (TTLs; ginkgolides and bilobalide), phenolic acids, and flavonoids in the primary organs of Ginkgo biloba leaves, leaf blades, and petioles. Levels of the major TTLs, ginkgolides B and C, were significantly higher in the leaf blades of naturally senesced yellow leaves harvested on 20 October compared with green leaves harvested on 9 September. In petioles, a similar effect was found, although the levels of these compounds were almost half as high. These facts indicate the importance of the senescence process on TTL accumulation. Some flavonoids and phenolic acids also showed changes in content related to maturation or senescence. Generally, the application of JA-Me slightly but substantially increased the levels of TTLs in leaf blades irrespective of the difference in its application side on the leaves. Of the flavonoids analyzed, levels of quercetin, rutin, quercetin-4-glucoside, apigenin, and luteolin were dependent on the JA-Me application site, whereas levels of (+) catechin and (−) epicatechin were not. Application of JA-Me increased ferulic acid and p-coumaric acid esters in the petiole but decreased the levels of these compounds in the leaf blade. The content of p-coumaric acid glycosides and caffeic acid esters was only slightly modified by JA-Me. In general, JA-Me application affected leaf senescence by modifying the accumulation of ginkogolides, flavonoids, and phenolic acids. These effects were also found to be different in leaf blades and petioles. Based on JA-Me- and aging-related metabolic changes in endogenous levels of the secondary metabolites in G. biloba leaves, we discussed the results of study in the context of basic research and possible practical application.     

Quantum chemical investigation on solvation of Ginkgolides

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Quantitative analysis of bilobalide and ginkgolides from Ginkgo biloba leaves and Ginkgo products using (1)H-NMR

1H-NMR spectrometry was applied to the quantitative analysis of the bilobalide, ginkgolides A, B, and C in Ginkgo biloba leaves and six kinds of commercial Ginkgo products without any chromatographic purification. The experiment was performed by the analysis of each singlet H-12, which were well separated in the range of delta 6.0-7.0 in the (1)H-NMR spectrum. However, the H-12 protons of bilobalide and ginkgolides may have overlapped with H-6 or H-8 protons of the Ginkgo flavonoids. Therefore, the optimum (1)H-NMR solvent for the analysis of the compound was selected through the evaluation of solvent effects on the resolution of these signals from the compounds. Acetone-d(6)-benzene-d(6) (50 : 50) was found to be the best one among the solvents evaluated. The quantity of the compounds was calculated by the relative ratio of the intensity of each compound to the known amount of internal standard (25 microgram), phloroglucinol. This method allows rapid and simple quantitation of underivatized bilobalide and ginkgolides in 5 min without any pre-purification steps.     

Simultaneous determination of ginkgo flavonoids and terpenoids in plasma: Ammonium formate in LC mobile phase enhancing electrospray ionization efficiency and capacity

Extracts from Ginkgo biloba leaves confer their therapeutic effects through the synergistic actions of flavonoid and terpenoid components. We herein describe the development of an LC-MS/MS-based method for simultaneous determination of flavonoids (quercetin, kaempferol, and isorhamnetin) and terpenoids (bilobalide, ginkgolides A, B, C, and J) in acid-hydrolyzed plasma by circumventing cross-interference between the flavonoids and terpenoids identified. Notably, inclusion of ammonium formate (0.2 mM) in the mobile phase generated beneficial LC-electrolyte effects, including increased ESI efficiency and capacity, with the result that the newly developed procedure exhibits the highest analytical performance reported to date for ginkgo-associated studies. The method yields high sensitivity, negligible matrix interference and cross-interference, wide linear dynamic ranges, high sample throughput, and quite small initial sample size. The assay utility to dog pharmacokinetic measurements of commercial ginkgo products yielded the most comprehensive data on systemic exposure to the ginkgo compounds to date. The newly developed multi-analyte procedure should be widely useful.     

Identification and the retention index of bilobalide and ginkgolides using capillary gas chromatography

Ginkgo biloba L. is known to contain the unique terpene trilactone compounds bilobalide and ginkgolides. Capillary gas chromatography is used for the quantitative identification of bilobalide and the main ginkgolides (ginkgolide A, B, and C). The retention indices of these compounds are also determined. Retention indices of bilobalide and ginkgolide A, B, and C substitute the use of their standards at their routine identification. Our procedure does not require temperature-programmed operation.     

The pharmacokinetics study of ginkgolide A,B and the effect of food on bioavailability after oral administration of ginkgolide extracts in beagle dogs

Ginkgolides are the primarily active components in Ginkgo products that are popular worldwide. However, few studies have evaluated the bioavailability of ginkgolides and the effects of food on it after oral administration of ginkgolides. In this article, pharmacokinetics and absolute bioavailability of the primary components in ginkgolide extracts were evaluated in beagle dogs. For the first time, we showed that the fed dogs had significantly increased area under the concentration–time curve and peak concentration relative to the fasted dogs based on the data from both the prototype form and total lactones of ginkgolide A (GA) and ginkgolide B (GB). In terms of the free form of the prototype ginkgolides, the absolute bioavailabilities of GA and GB were 34.8 and 5.2% in the fasted dogs, respectively, which significantly increased to an average of 78.6 and 17.0%, respectively, in the fed dogs. In terms of acidified total lactones, the absolute bioavailabilities of GA and GB were 7.5 and 14.5% in the fed dogs, and the percentages declined to 4.1 and 3.7% in the fasted dogs, respectively. It was suggested that administration of ginkgolides after meals could promote the in vivo exposure and the bioavailability of GA and GB, and hence potentially enhance therapeutic outcomes.     

Liquid chromatography/electrospray tandem mass spectrometry of terpenoid lactones in Ginkgo biloba

Ginkgo biloba (ginkgo) is one of most frequently used botanical dietary supplements. The bioactive constituents include the terpenoid lactones consisting of bilobalide and the ginkgolides A, B, C and J. A new assay based on high-performance liquid chromatography/electrospray tandem mass spectrometry (LC/MS/MS) was developed for the measurement of the terpenoid lactones in ginkgo products such as leaf powder and extracts. Initially, the MS/MS fragmentation pathways of ginkgolides were investigated to identify abundant fragment ions that might be useful for the sensitive and selective detection of ginkgolides and bilobalide during LC/MS/MS. Then, sample preparation and clean-up procedures were streamlined to maximize throughput by taking advantage of the selectivity of LC/MS/MS detection. Analyte recoveries exceeded 90%, the intra-assay and inter-assay relative standard deviations were <5%, the relative error was <8% and the limits of detection and quantification were 3.6-120 and 11-350 fmol, depending on the analyte that was injected on to the LC column. Therefore, this LC/MS/MS assay facilitated the rapid quantitative analysis of ginkgolides A, B, C and J and bilobalide in ginkgo dietary supplements with excellent recovery, reproducibity, accuracy and sensitivity.     

Advances in Supercritical Carbon Dioxide Extraction of Bioactive Substances from Different Parts of Ginkgo biloba L.

Ginkgo biloba L. has always been a popular area of research due to its various active ingredients and pharmacological effects. Ginkgo biloba is rich in ginkgo flavonoids, ginkgolides, and ginkgolic acid, with anti-inflammation, antioxidation, neuroprotection, anti-platelet agglutination, hypolipidemic effect, anti-cancer, and anti-radiation properties. There are many methods to extract and separate the active components of ginkgo. Among them, supercritical carbon dioxide fluid extraction (SFE-CO₂) is known for its green, clean, and environment-friendly properties. In this paper, the pharmacological activities, the active components, and structures of different parts of ginkgo, the extraction methods of its effective ingredients, and the application of the SFE-CO₂ method for the extraction and separation of active ingredients in Ginkgo biloba from leaves, seeds, pollen, and roots were reviewed, in order to make best use of ginkgo resources, and provide support and references for the development of SFE-CO₂ of active components from Ginkgo biloba.     

Liquid chromatography-electrospray mass spectrometric studies of ginkgolides and bilobalide using simultaneous monitoring of proton,ammonium and sodium adducts

A liquid chromatographic-electrospray mass spectrometric method was developed for the determination of ginkgolides and bilobalide and was applied to the analysis of commercial products of Ginkgo biloba leaf extracts. Adducts of these compounds with ammonium, proton and sodium were detected and their relative abundance depended on the electrospray fragmentor voltage. The relative standard deviation (RSD) was improved from > 17% to < 6%, when three adduct ions of (M + H)+, (M + NH4)+ and (M + Na)+ were used for quantification compared with single ion monitoring. The characteristic mass spectra of bilobalide were different from those of ginkgolides; in particular, dimers of (2M + Na)+ were observed for bilobalide only. Analysis of 26 commercial ginkgo products revealed large variations in the composition and concentrations of ginkgolides and bilobalide in herbal products.     

Ginkgolides and glycine receptors: a structure-activity relationship study

Ginkgolides from the Ginkgo biloba tree are diterpenes with a cage structure consisting of six five-membered rings and a unique tBu group. They exert a variety of biological properties. In addition to being antagonists of the platelet activating factor receptor (PAFR), it has recently been shown that native ginkgolides are potent and selective antagonists of the inhibitory glycine receptor. Forty new ginkgolide derivatives have been prepared in good to high yields on milligram scales and investigated for their antagonistic properties at homomeric alpha 1 glycine receptors, thus providing the first structure-activity relationship study of ginkgolides at glycine receptors. A high-throughput screening assay showed that native ginkgolide C was the most potent ligand, and that manipulation of any of the hydroxyl groups led to loss of activity at alpha 1 glycine receptors.     

Lactone-free ginkgolides via regioselective DIBAL-H reduction

The lactone rings of ginkgolide A are converted into corresponding tetrahydrofuran moieties via DIBAL-H reduction followed by deoxygenation of the formed lactols with Et3SiH-BF3.Et2O to produce a series of lactone-free ginkgolides.     

Recent advances in the total synthesis of pharmaceutically relevant diterpenes

A detailed overview is presented of recent total synthesis approaches to four different diterpenoids--the ginkgolides, tonantzitlolone, ingenol and eleutherobin. Due to their structural complexity, all pose major challenges for total synthesis, guaranteeing diverse strategic tours de force and a rich synthetic chemistry. Additionally, all four diterpenoids are of major relevance because of their biological (target) or pharmaceutical properties.     

Development and validation of a gas chromatographic-mass spectrometric method for simultaneous identification and quantification of marker compounds including bilobalide,ginkgolides and flavonoids in Ginkgo biloba L. extract and pharmaceutical preparations

A gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the simultaneous determination of seven major chemical markers (bilobalide, ginkgolides A, B, C, kaempferol, quercetin and isorhamnetin) in phytopharmaceuticals of Ginkgo biloba L. The intra-day relative standard deviations (RSD) and inter-day RSD's were based on the analysis of the standardized Ginkgo biloba L. extract on the same day and on the following 3 consecutive days. The intra-day RSD's ranged from 1.21% (bilobalide) to 6.20% (kaempferol). The inter-day RSD's ranged from 2.10% (bilobalide) to 10.42% (isorhamnetin). Mean recoveries ranged from a low of 63.0 +/- 5.3% (isorhamnetin) to a maximum of 103.5 +/- 6.0% (ginkgolide A). Calibration curves were linear in ranges between 2.73 and 36.36 microg/ml for the markers. Limits of detection ranged from a low of 0.5 microg/ml (bilobalide) to a high of 2.5 microg/ml (quercetin). The limits of quantitation were a low of 1.1 microg/ml (gingkolides A, B, C) to a high of 7.5 microg/ml (kaempferol). The method was applied to a standard extract (>6% total terpenoids and >24% total flavonoids) and six ginkgo capsule phytopharmaceuticals.     

Simultaneous determination of ginkgolides A,B, C and bilobalide by LC‐MS/MS and its application to a pharmacokinetic study in rats

The study of pharmacokinetics of Ginkgo biloba extracts in Traditional Chinese Medicine was relatively recent. In this study, a simple, quick and sensitive LC‐MS/MS analytical method was developed for the determination of ginkgolides A, B, C and bilobalide in rat plasma. The analytes were completely separated from the endogenous compounds on an Agilent Zorbax Eclipse plus C₁₈ column (50 mm × 3.0 mm, 1.8 µm) using an isocratic elution. The single‐run analysis time was as short as 5.0 min. Sample preparation for protein removal was accomplished used a simple methanol precipitation method, after SPE showing a simultaneous extraction and cleanup of extracts allowing for a direct analysis. Extraction recoveries in rat plasma for ginkgolides A, B, C and bilobalide ranged from 75.6% to 89.0%. The calibration curves were determined over the ranges 0.5–20,000 ng/mL for ginkgolides A, B, C and bilobalide respectively. The lower limits of quantification (LLOQ) of the analytes were 0.5 ng/mL. Inter‐day and intra‐day precision and accuracy were below 15% and between 85 and 115%, respectively. Finally, the developed method was successfully applied to a pharmacokinetic study following oral administration of the Ginkgo biloba extracts to the male ICR rats. Copyright © 2015 John Wiley & Sons, Ltd.     

LC-ESI-MS Determination of Bilobalide and Ginkgolides in Canine Plasma

A sensitive and selective method using liquid chromatography with electrospray ionization mass spectrometric detection was developed for the quantification of bilobalide and ginkgolides in canine plasma. The analytes were extracted with diethyl ether-dichloromethane-isopropanol (6:3:1, v/v) after spiking the samples with daidzein (internal standard). The lower limit of quantification (LLOQ) of the method was 2.5 μg L⁻¹ for ginkgolide B and 10.0 μg L⁻¹ for bilabolide, ginkgolide A and ginkgolide C. The accuracy of the method was within 15% of the actual values over a wide range of plasma concentrations. The intra-day and inter-day precision was better than 15% (R.S.D.). Finally, the LC-ESI-MS method was successfully applied to study the pharmacokinetics of ginkgolides and bilabolide after administration of Ginkgo biloba extracts to dogs.     

Liquid chromatography/atmospheric pressure chemical ionization mass spectrometry of terpene lactones in plasma of volunteers dosed with Ginkgo biloba L. extracts

Liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-ITMS) was applied to evaluate the levels of ginkgolides A and B and bilobalide in plasma of volunteers after administration of Ginkgo biloba extracts in free (Ginkgoselect) or phospholipid complex (Ginkgoselect Phytosome) forms, providing 9.6 mg of total terpene lactones. The maximum plasma concentrations, C(max), of total ginkgolides A, B and bilobalide were 85.0 and 181.8 microg/mL for Ginkgoselect and Ginkgoselect Phytosome, respectively. The C(max) values were reached at 120 min for the free form and at 180--240 min for the phospholipid complex form. In both cases, the mean elimination half-life of each terpene lactone was in the range 120--180 min. Due to its sensitivity (about 1 ng/mL) and specificity, LC/APCI-ITMS proved to be a very powerful tool for pharmacokinetic studies of these phytochemicals.     

An efficient etherification of Ginkgol biloba extracts with fewer side effects in a micro-flow system

In this study, etherification of ginkgolide B and dimethylaminoethyl chloride hydrochloride was investigated as a model reaction in a micro-flow system (MFS), providing the resulting ethers in high yield with fewer side effects. Meanwhile, this novel process in MFS worked well for other ginkgolides from Ginkgol biloba and halides, giving moderate yields.     

Ginkgolides: selective acetylations,translactonization, and biological evaluation

Protocols for selective acetylation of the hydroxyl groups of ginkgolide C have been developed. These acetylations have given rise to various ginkgolide C acetates and iso-ginkgolide C acetates, the latter having a rearranged skeleton resulting from translactonization. These acetyl derivatives, as well as ginkgolides A and B acetates have been investigated for their ability to bind to a cloned platelet-activating factor (PAF) receptor.     

Preparation and Application of Molecularly Imprinted Polymers for Flavonoids: Review and Perspective

The separation and detection of flavonoids from various natural products have attracted increasing attention in the field of natural product research and development. Depending on the high specificity of molecularly imprinted polymers (MIPs), MIPs are proposed as efficient adsorbents for the selective extraction and separation of flavonoids from complex samples. At present, a comprehensive review article to summarize the separation and purification of flavonoids using molecular imprinting, and the employment of MIP-based sensors for the detection of flavonoids is still lacking. Here, we reviewed the general preparation methods of MIPs towards flavonoids, including bulk polymerization, precipitation polymerization, surface imprinting and emulsion polymerization. Additionally, a variety of applications of MIPs towards flavonoids are summarized, such as the different forms of MIP-based solid phase extraction (SPE) for the separation of flavonoids, and the MIP-based sensors for the detection of flavonoids. Finally, we discussed the advantages and disadvantages of the current synthetic methods for preparing MIPs of flavonoids and prospected the approaches for detecting flavonoids in the future. The purpose of this review is to provide helpful suggestions for the novel preparation methods of MIPs for the extraction of flavonoids and emerging applications of MIPs for the detection of flavonoids from natural products and biological samples.     

Metabolic Engineering of Microbial Cell Factories for Biosynthesis of Flavonoids: A Review

Flavonoids belong to a class of plant secondary metabolites that have a polyphenol structure. Flavonoids show extensive biological activity, such as antioxidative, anti-inflammatory, anti-mutagenic, anti-cancer, and antibacterial properties, so they are widely used in the food, pharmaceutical, and nutraceutical industries. However, traditional sources of flavonoids are no longer sufficient to meet current demands. In recent years, with the clarification of the biosynthetic pathway of flavonoids and the development of synthetic biology, it has become possible to use synthetic metabolic engineering methods with microorganisms as hosts to produce flavonoids. This article mainly reviews the biosynthetic pathways of flavonoids and the development of microbial expression systems for the production of flavonoids in order to provide a useful reference for further research on synthetic metabolic engineering of flavonoids. Meanwhile, the application of co-culture systems in the biosynthesis of flavonoids is emphasized in this review.