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.     

Variation of ginkgolides and bilobalide contents in leaves and cell cultures of<Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

Ginkgolides (GK) and bilobalide are valuable compounds that belong to the lactone terpene. The contents of these metabolites were determined by HPLC from female and male tree ofGinkgo biloba L. The productivity ofG. biloba cells was also compared with the corresponding individual trees. High variations in the ginkgolides and bilobalide were observed from different individuals, plant parts, and cultured cells. The ginkgolides and bilobalide contents were different depending on the plant parts. Callus was obtained from various plant tissues, and NAA was better at callogenesis than 2,4-D in both the female and male trees. The plants and their corresponding cells showed considerable variation in their ginkgolides and bilobalide concentrations. The ginkgolides and bilobalide contents were not correlated with the production between dominant trees and their corresponding cells. Light irradiation enhanced the production of GK-A and GK-B, however, the concentration of bilobalide decreased under dark conditions.     

Production of ginkgolides and bilobalide from optimized the<Emphasis Type="Italic">Ginkgo biloba</Emphasis> cell culture

The influence of various culture conditions on growth and ginkgolides (GKA and GKB), and bilobalide formation in callus and suspension cultures ofGinkgo biloba were investigated. Callus induced from the leaf petioles exhibited distinct morphological and physiological responses. The cell biomass and ginkgolides content varied among the cell lines brownish callus lines produced high levels of ginkgolides and bilobalide in spite of poor cell growth. Among the culture media used, MS medium showed significant effect on cell growth and ginkgolides production. Low concentration of sucrose (3%) improved cell growth, while higher sucrose levels (5 and 7%) improved ginkgolides production. Cultivation of callus cultures above 28°C dramatically reduced their growth rate; however the cell lines grown at 36°C showed increased levels of bilobalide content. A 2.5-L balloon type bubble bioreactor (BTBB) was successfully developed for the cell growth and ginkgolides production.     

Terpene trilactones from Ginkgo biloba are antagonists of cortical glycine and GABA(A) receptors

Glycine and gamma-aminobutyric acid, type A (GABA(A)) receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory synaptic transmission in the adult central nervous system. During development, the activation of these receptors leads to membrane depolarization. Ligands for the two receptors have important implications both in disease therapy and as pharmacological tools. Terpene trilactones (ginkgolides and bilobalide) are unique constituents of Ginkgo biloba extracts that have various effects on the central nervous system. We have investigated the relative potency of these compounds on glycine and GABA(A) receptors. We find that most of the ginkgolides are selective and potent antagonists of the glycine receptor. Bilobalide, the single major component in G. biloba extracts, also reduces glycine-induced currents, although to a lesser extent. Both ginkgolides and bilobalide inhibit GABA(A) receptors, with bilobalide demonstrating a more potent effect. Additionally, we provide evidence that open channels are required for glycine receptor inhibition by ginkgolides. Finally, we employ molecular modeling to elucidate the similarities and differences in the structure of the terpene trilactones to account for the pharmacological properties of these compounds and demonstrate a striking similarity between ginkgolides and picrotoxinin, a GABA(A) and recombinant glycine alpha-homomeric receptor antagonist.     

Pancreatic lipase inhibition activity of trilactone terpenes of Ginkgo biloba

The prevalence of obesity is increasing at an alarming rate, but, unfortunately, only a few drugs are currently available on the market. In the present study, the methanolic extract of Ginkgo biloba L. (Ginkgoaceae) was investigated as an inhibitor of pancreatic lipase (PL) in an attempt to explain its hypolipidaemic activity. In vitro assay of G. biloba leaves extract revealed a substantial PL inhibition activity (IC(50)?=?16.5 μg/mL). Further investigation was performed by employing theoretical docking simulations and experimental testing to uncover the active constituents responsible for G. biloba anti-lipase activity. Virtually, terpene trilactones, including ginkgolides and bilobalide, were found to fit within the binding pocket of PL via several attractive interactions with key amino acids. Experimentally, ginkgolides A, B, and bilobalide were found to inhibit PL significantly (IC(50)?=?22.9, 90.0, and 60.1 μg/mL, respectively). Our findings demonstrated that the hypolipidaemic effects of G. biloba extract can be attributed to the inhibition of PL by, at least in part, terpene trilactones. In conclusion, this work can be considered a new step towards the discovery of new natural safe hypolipidaemic PL inhibitors.     

Bilobalide and PC12 cells: A structure activity relationship study

Ginkgo biloba extracts have been postulated to beneficial for improving cognitive function and as such they have been used as a potential treatment of Alzheimer’s disease. The main active ingredients of the extract are terpene trilactones (TTLs), such as bilobalide (BB) and ginkgolides. Several structure–activity relationship (SAR) studies using ginkgolide scaffolds produced more biologically potent species by modification of the lactone moieties. However, modifications of BB scaffold have been limited, and no SAR studies on BB have been accomplished to date. Thus, the aim of this study was to elucidate how the modification of the lactone moieties of BB would affect their biological activities in a number of assays, including proliferating cell activity, neuroprotective effects against Aβ (1–40) peptides, and neurite outgrowth effects in PC12 neuronal cells. It appeared that the derivatives containing lactone groups showed similar biological activity to native BB, while those that possessed no lactone moieties exhibited lower neurite outgrowth effects. Thus, the results suggested that the lactone moieties of BB played an important role in exerting neurite outgrowth effects in PC12 cells.     

Ginkgolides and bilobalide: their physical, chromatographic and spectroscopic properties

Ginkgolides A, B, C, J, K, L and M and bilobalide are rare terpene trilactones that have been isolated from leaves and root bark of the Chinese tree Ginkgo biloba. The structures of the highly oxidized ginkgolides were independently elucidated in the 1960s by the groups of Nakanishi and Sakabe. Later these compounds were found to be potent and selective antagonists of platelet activating factor, which fact triggered much new research. During the past 40 years, much physical, chromatographic and spectroscopic data have been published on these compounds in various, sometimes inaccessible, sources. The published melting points, solubility in different solvents, ionization constants, chromatographic behaviour, specific optical rotations, UV, IR, MS and NMR data, and X-ray studies are summarized and, where necessary, discussed. The literature until April 2005 has been reviewed.     

Effect of supplementing terpenoid biosynthetic precursors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures

The effect of precursor feeding on the production of bilobalide and ginkgolides was studied with suspension cell cultures of Ginkgo biloba. The precursors greatly influenced the productivity of bilobalide and ginkgolides. Precursor supplementation increased the accumulation of both bilobalide and ginkgolides, and with positive effect on cell growth. The GA accumulation by cell cultures was influenced by precursors upstream in the metabolism, whereas the BB accumulation was under the influence of downstream precursors of the terpenoid biosynthetic pathway. Furthermore, precursor feeding modified the ratios of the BB, GA and GB in cells and cell cultures of G. biloba. The studies also aid in understanding effect of precursor feeding on the bilobalide and ginkgolides biosynthetic pathway.     

银杏叶萜内酯含量的变化及其与叶绿素荧光特性的关系

为探究银杏(Ginkgo biloba)叶萜类内酯含量和光合同化作用的关系,对其内酯含量和叶绿素荧光特性进行了研究。结果表明,不同采收时间银杏叶中白果内酯和银杏内酯含量有显著差异,总体上,5月份含量较低,此后逐渐升高,8月份达到高峰,然后快速下降,10月底最低;与此同时,银杏叶片的光合色素以及叶绿素荧光参数也呈现周期性变化。白果内酯以及萜内酯含量与叶绿素荧光参数Y(NPQ)之间呈极显著正相关关系,因此,可以通过银杏叶片的叶绿素荧光参数预测白果内酯和萜内酯含量。     

银杏药用物质的开发利用现状及其发展设想

本文概述了银杏叶的药理作用及其开发利用现状。目前,银杏药用物质的获取仍然是通过叶片提取这一途径,这同时存在地理环境、季节等诸多限制因素。而通过银杏组织、细胞培养获得其药用物质的技术研究刚刚起步,距离实际应用仍有相当的距离,但随着这项技术的不断发展,它不仅可用于生产银杏黄酮类和银杏内酯,而且有助于进一步研究银杏所含的特殊化学成分及其相关的代谢途径。     

不同银杏无性系叶药用成分差异及聚类分析

运用HPLC技术分析了54个银杏(Ginkgo biloba)无性系叶总黄酮和萜内酯及其组分含量的差异,并进行了聚类。结果表明,银杏无性系间叶中总黄酮、萜内酯及其组分含量存在遗传变异,且萜内酯及其组分含量的变异系数明显高于总黄酮及其组分。总黄酮含量较高的无性系有18、42、32和50号,其槲皮素、异鼠李素、山奈酚含量均较高。萜内酯含量较高的无性系为13、42、33、51和65号,其银杏内酯A(GA)、银杏内酯B(GB)、银杏内酯C(GC)及白果内酯(BB)含量均较高。通过对总黄酮-萜内酯进行联合复选,显示叶中总黄酮和萜内酯含量均较高的无性系为13、65、33、51、18、32和42号。这些无性系可通过嫁接、扦插直接在银杏采叶园进行推广种植,或作为叶用银杏新品种的育种材料。     

银杏悬浮培养细胞的生长、分化与萜内酯化合物的积累

研究了来源于银杏种子胚和幼苗茎的悬浮细胞的生长、分化和培养物中的白果内酯、银杏内酯A和B的含量变化。结果表明：在悬浮培养中,细胞聚集而成的细胞团大小、细胞中叶绿体的分化、外植体来源都影响培养物中的萜内酯的种类和含量,胚来源的悬浮细胞培养物中,银杏内酯B仅存在于直径<2mm的小细胞团悬浮培养中,且在<1 mm的细胞团中的含量最高,达0.437 mg /g(DW);而直径>3mm的细胞团悬浮培养物中只含有白果内酯和银杏内酯A。相同大小的悬浮细胞团中,胚来源的细胞中萜内酯含量高于茎来源的细胞。     

Isolation of ginkgolides A, B, C, J and bilobalide from G. biloba extracts

Ginkgolides A, B, C and J, together with bilobalide, are unique terpenoid components of the Ginkgo biloba tree. Due to similar chemical properties, their separation is quite tedious. We have developed an efficient and rapid protocol for separation of individual ginkgolides and bilobalide from G. biloba extracts. The procedure takes advantage of enhanced susceptibility of ginkgolides B and C to benzylation and the ease of separation of these products from ginkgolides A and J which do not react. The protocol is applicable to the previously reported enriched extracts prepared from G. biloba leaves. A single chromatographic step prior to benzylation provides bilobalide and mixture of ginkgolides A, B, C, and J. After benzylation, the individual ginkgolides are separated by chromatography.     

Ginkgolides and bilobalide protect BV2 microglia cells against OGD/reoxygenation injury by inhibiting TLR2/4 signaling pathways

Ginkgolide and bilobalide are major trilactone constituent of Ginkgo biloba leaves and have been shown to exert powerful neuroprotective properties. The aims of this study were to observe the inhibitory effects of ginkgolide and bilobalide on the activation of microglial cells induced by oxygen–glucose deprivation and reoxygenation (OGD/R) and the specific mechanisms by which these effects are mediated. For detecting whether ginkgolide and bilobalide increased cell viability in a dose-dependent manner, BV2 cells were subjected to oxygen–glucose deprivation for 4 h followed by 3 h reoxygenation with various concentrations of drugs (6.25, 12.5, 25, 50, and 100 μg/ml). The extent of apoptosis effect of OGD/R with or without ginkgolide and bilobalide treatment were also measured by Annexin V-FITC/PI staining. Similarly, the levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, and IL-10 were detected using a specific Bio-Plex Pro? Reagent Kit. The effects of ginkgolide and bilobalide on protein levels of TLR2/4, MyD88, p-TAK1, p-IKKβ, p-IkBα, NF-κB p65, Bcl-2, Bax, Bak, RIP3, cleaved-Caspase-3, cleaved PARP-1 and cellular localization of NF-κB p65 were evaluated by Western blot and double-labeled immunofluorescence staining, respectively. OGD/R significantly decreased the cell viability and increased the release of IL-1β, IL-6, IL-8, IL-10, TNF-α in BV2 microglia cells; these effects were suppressed by ginkgolide and bilobalide. Meanwhile, ginkgolide and bilobalide also attenuated the OGD/R-induced increases in TLR2, TLR4, MyD88, Bak, RIP3 levels and reversed cleaved caspase-3/caspase-3, Bax/Bcl-2 and cleaved PARP-1/PARP-1 ratio. Furthermore, ginkgolide and bilobalide also downregulated p-TAK1, p-IkBα, and p-IKKβ and inhibited the OGD/R-induced transfer of NF-κB p65 from cytoplasm to nucleus in BV2 microglia cells. The results showed that ginkgolide and bilobalide can inhibit OGD/R-induced production of inflammatory factors in BV2 microglia cells by regulating the TLRs/MyD88/NF-κB signaling pathways and attenuating inflammatory response. The possible mechanism of anti-inflammatory and neuroprotective effects of ginkgolides results from the synergistic reaction among each monomer constituents.     

Ginkgolides protect against amyloid-β1–42-mediated synapse damage in vitro

Background

The early stages of Alzheimer's disease (AD) are closely associated with the production of the Aβ_1–42 peptide, loss of synapses and gradual cognitive decline. Since some epidemiological studies showed that EGb 761, an extract from the leaves of the Ginkgo biloba tree, had a beneficial effect on mild forms of AD, the effects of some of the major components of the EGb 761 extract (ginkgolides A and B, myricetin and quercetin) on synapse damage in response to Aβ_1–42 were examined.

Results

The addition of Aβ_1–42 to cortical or hippocampal neurons reduced the amounts of cell associated synaptophysin, a pre-synaptic membrane protein that is essential for neurotransmission, indicating synapse damage. The effects of Aβ_1–42 on synapses were apparent at concentrations approximately 100 fold less than that required to kill neurons; the synaptophysin content of neuronal cultures was reduced by 50% by 50 nM Aβ_1–42. Pre-treatment of cortical or hippocampal neuronal cultures with ginkgolides A or B, but not with myrecitin or quercetin, protected against Aβ_1–42-induced loss of synaptophysin. This protective effect was achieved with nanomolar concentrations of ginkgolides. Previous studies indicated that the ginkgolides are platelet-activating factor (PAF) receptor antagonists and here we show that Aβ_1–42-induced loss of synaptophysin from neuronal cultures was also reduced by pre-treatment with other PAF antagonists (Hexa-PAF and CV6209). PAF, but not lyso-PAF, mimicked the effects Aβ_1–42 and caused a dose-dependent reduction in the synaptophysin content of neurons. This effect of PAF was greatly reduced by pre-treatment with ginkgolide B. In contrast, ginkgolide B did not affect the loss of synaptophysin in neurons incubated with prostaglandin E₂.

Conclusion

Pre-treatment with ginkgolides A or B protects neurons against Aβ_1–42-induced synapse damage. These ginkgolides also reduced the effects of PAF, but not those of prostaglandin E₂, on the synaptophysin content of neuronal cultures, results consistent with prior reports that ginkgolides act as PAF receptor antagonists. Such observations suggest that the ginkgolides are active components of Ginkgo biloba preparations and may protect against the synapse damage and the cognitive loss seen during the early stages of AD.     

Effects of extract of Ginkgo biloba leaves and its constituents on carcinogen-metabolizing enzyme activities and glutathione levels in mouse liver

The effects of a standardized extract of Ginkgo biloba L. leaves (EGb) and its terpene constituents, bilobalide and ginkgolides, on the activities of detoxification enzymes, i.e., glutathione S-transferases (GSTs) and DT-diaphorase, and glutathione contents, were investigated in the mouse liver. Oral treatment with EGb (100-1,000 mg/kg) and bilobalide (10-30 mg/kg) once a day for 4 days caused a dose-dependent elevation in GST activity. Ginkgolide A (30 mg/kg, for 4 days) also significantly elevated GST activity, whereas ginkgolide B and ginkgolide C at the same dose had no effects. EGb significantly increased the protein level of GST pi, and bilobalide significantly increased those of GST alpha and GST mu Moreover, EGb-treatment and bilobalide-treatment caused significant elevations in DT-diaphorase activity and in hepatic glutathione contents.     

Gingkolides and bilobalide: structure, pharmacology, and synthesis]

The review summarizes data on the structure, pharmacology properties and synthetic investigations of ginkgolides and bilobalide occurring in the ginkgo tree (Ginkgo biloba L.). Ginkgolide B is a powerful antagonist of platelet activating factor.     

EGb 761 (Ginkgo biloba) protects cochlear hair cells against ototoxicity induced by gentamicin via reducing reactive oxygen species and nitric oxide-related apoptosis

Gentamicin is an effective and powerful antibiotic. Extended use or excessive dosages of which can result in irreversible damage to the inner ear. The development of otoprotective strategies is a primary and urgent goal in research of gentamicin ototoxicity. Ginkgo biloba leaves and their extracts are among the most widely used herbal products and/or dietary supplements in the world. We investigated the protection of EGb 761 (a standardized preparation of EGb) on gentamicin ototoxicity and the involvement of reactive oxygen species (ROS) and nitric oxide (NO)-related mechanisms using in vitro organ cultures and an in vivo animal model. Gentamicin induced hair cell damage in cochlear cultures that could be prevented by EGb 761. EGb 761 also significantly reduced gentamicin-induced ROS and NO production. Furthermore, EGb 761 inhibited cellular apoptosis in cultured cochleae treated with gentamicin. In guinea pigs with gentamicin application onto the round window membrane, the mean auditory brain stem response threshold, ratio of cochlear hair cell damage and apoptosis were significantly elevated compared with those in the control group, and this could be prevented by oral administration of EGb 761. Individual EGb 761 components quercetin, bilobalide, ginkgolide A and ginkgolide B, but not kaempferol, significantly prevented gentamicin-induced hair cell damage. These results indicate that EGb 761 has a protective effect against gentamicin ototoxicity through a reduction in the formation of ROS and NO and subsequent inhibition of hair cell apoptosis in the cochlea.     

Effect of biotic elicitors on the accumulation of bilobalide and ginkgolides in Ginkgo biloba cell cultures

The effect of biotic elicitors on the production of bilobalide and ginkgolides in Ginkgo biloba cell suspension cultures was studied. The treatment of cell cultures with Candida albicans and Staphylococcus aureus as elicitors increased the amounts of bilobalide (BB), ginkgolide A (GA) and ginkgolide B (GB), with slight growth inhibition. The native bacterial elicitor was more effective for secondary metabolite accumulations both in cells and culture medium than autoclaved. However, exposure times of the cells to the elicitors strongly influenced the production of BB, GA and GB. This study suggests that biotic elicitors can regulate the production of BB, GA and GB either directly or indirectly. These results also describe the establishment of optimum conditions that determine the effects of biotic elicitors on secondary metabolism of bilobalides.