Comprehensive analysis of JAZ family members in Ginkgo biloba reveals the regulatory role of the GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis.

Ginkgo biloba L., an ancient relict plant known as a 'living fossil', has a high medicinal and nutritional value in its kernels and leaves. Ginkgolides are unique diterpene lactone compounds in G. biloba, with favorable therapeutic effects on cardiovascular and cerebrovascular diseases. Thus, it is essential to study the biosynthesis and regulatory mechanism of ginkgolide, which will contribute to quality improvement and medication requirements. In this study, the regulatory roles of the JAZ gene family and GbCOI1/GbJAZs/GbMYC2 module in ginkgolide biosynthesis were explored based on genome and methyl jasmonate-induced transcriptome. Firstly, 18 JAZ proteins were identified from G. biloba, and the gene characteristics and expansion patterns along with evolutionary relationships of these GbJAZs were analyzed systematically. Expression patterns analysis indicated that most GbJAZs expressed highly in the fibrous root and were induced significantly by methyl jasmonate. Mechanistically, yeast two-hybrid assays suggested that GbJAZ3/11 interacted with both GbMYC2 and GbCOI1, and several GbJAZ proteins could form homodimers or heterodimers between the GbJAZ family. Moreover, GbMYC2 is directly bound to the G-box element in the promoter of GbLPS, to regulate the biosynthesis of ginkgolide. Collectively, these results systematically characterized the JAZ gene family in G. biloba and demonstrated that the GbCOI1/GbJAZs/GbMYC2 module could regulate ginkgolides biosynthesis, which provides a novel insight for studying the mechanism of JA regulating ginkgolide biosynthesis.

Multiple Mechanistic Models Reveal the Neuroprotective Effects of Diterpene Ginkgolides against Astrocyte-Mediated Demyelination via the PAF-PAFR Pathway.

Currently, therapies for ischemic stroke are limited. Ginkgolides, unique Folium Ginkgo components, have potential benefits for ischemic stroke patients, but there is little evidence that ginkgolides improve neurological function in these patients. Clinical studies have confirmed the neurological improvement efficacy of diterpene ginkgolides meglumine injection (DGMI), an extract of Ginkgo biloba containing ginkgolides A (GA), B (GB), and K (GK), in ischemic stroke patients. In the present study, we performed transcriptome analyses using RNA-seq and explored the potential mechanism of ginkgolides in seven in vitro cell models that mimic pathological stroke processes. Transcriptome analyses revealed that the ginkgolides had potential antiplatelet properties and neuroprotective activities in the nervous system. Specifically, human umbilical vein endothelial cells (HUVEC-T1 cells) showed the strongest response to DGMI and U251 human glioma cells ranked next. The results of pathway enrichment analysis via gene set enrichment analysis (GSEA) showed that the neuroprotective activities of DGMI and its monomers in the U251 cell model were related to their regulation of the sphingolipid and neurotrophin signaling pathways. We next verified these in vitro findings in an in vivo cuprizone (CPZ, bis(cyclohexanone)oxaldihydrazone)-induced model. GB and GK protected against demyelination in the corpus callosum (CC) and promoted oligodendrocyte regeneration in CPZ-fed mice. Moreover, GB and GK antagonized platelet-activating factor (PAF) receptor (PAFR) expression in astrocytes, inhibited PAF-induced inflammatory responses, and promoted brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) secretion, supporting remyelination. These findings are critical for developing therapies that promote remyelination and prevent stroke progression.

Genome-wide transcriptome analysis reveals the regulatory network governing terpene trilactones biosynthesis in Ginkgo biloba.

Ginkgo biloba L. is currently the only remaining gymnosperm of the Ginkgoaceae Ginkgo genus, and its history can be traced back to the Carboniferous 200 million years ago. Terpene trilactones (TTLs) are one of the main active ingredients in G. biloba, including ginkgolides and bilobalide. They have a good curative effect on cardiovascular and cerebrovascular diseases because of their special antagonistic effect on platelet-activating factors. Therefore, it is necessary to deeply mine genes related to TTLs and to analyze their transcriptional regulation mechanism, which will hold vitally important scientific and practical significance for quality improvement and regulation of G. biloba. In this study, we performed RNA-Seq on the root, stem, immature leaf, mature leaf, microstrobilus, ovulate strobilus, immature fruit and mature fruit of G. biloba. The TTL regulatory network of G. biloba in different organs was revealed by different transcriptomic analysis strategies. Weighted gene co-expression network analysis (WGCNA) revealed that the five modules were closely correlated with organs. The 12 transcription factors, 5 structural genes and 24 Cytochrome P450 (CYP450) were identified as candidate regulators for TTL accumulation by WGCNA and cytoscape visualization. Finally, 6 APETALA2/ethylene response factors, 2 CYP450s and bHLH were inferred to regulate the metabolism of TTLs by correlation analysis. This study is the comprehensive in authenticating transcription factors, structural genes and CYP450 involved in TTL biosynthesis, thereby providing molecular evidence for revealing the comprehensive regulatory network involved in TTL metabolism in G. biloba.

A novel method for ginkgolide biosynthesis elucidation based on MeJA induction and differential metabolomics.

Ginkgolides from Ginkgo Biloba have significantly therapeutic effect to cardiovascular and cerebrovascular diseases. However, the biosynthetic pathway of ginkgolides has not been fully elucidated until now. As ginkgolides are synthesized in the ginkgo roots, the accumulation of ginkgolides intermediate metabolites varies greatly between roots and leaves. As Methyl jasmonate (MeJA) can effectively enhance the biosynthesis of ginkgolides, a novel method based on MeJA induction and differential metabolomics was used to screen the differentially intermediate metabolites among ginkgo leaves, roots and roots-MJ-3. Two differential intermediate metabolites (dehydroabietadienal and 1, 2, 3, 4, 4a, 9, 10, 10a-Octahydro-6-hydroxy-7-isopropyl-1, 4a-dimethyl-1-phenanthrenemethanol) were identified in ginkgo roots by UPLC-QTOF-MS. Then, a new ginkgolides biosynthetic pathway was proposed based on differential metabolomics. This study provides a novel method for the elucidation of nature product precursor and is helpful to promote the clarification of ginkgolides biosynthetic pathway.

The therapeutic effects of ginkgolides in Guillain-Barré syndrome and experimental autoimmune neuritis.

BACKGROUND: Guillain-Barré syndrome (GBS) is an acquired immune-mediated inflammatory peripheral neuropathy. The immune regulation of ginkgolides have been revealed in recent years. We herein investigate the potential therapeutic effects of ginkgolides both on GBS and its animal model, experimental autoimmune neuritis (EAN). METHODS: EAN in C57BL/6 mice induced by subcutaneous injection with peripheral nerve myelin P0 protein peptide 180-199 (P0 peptide) were treated with ginkgolides at three different doses. GBS patients were randomly divided into two groups, the experimental group and the control group. The experimental group were treated with ginkgolides as soon as diagnosed. RESULTS: Our data indicated that ginkgolides administration daily ameliorated the score of EAN and delayed the peak of disease in EAN mice. Ginkgolides also down-regulated the proportions of T helper (Th) 17 cells in EAN spleens. Furthermore, we also found that administration of ginkgolides significantly decreased the levels of interferon (IFN)-γ and interleukin-12 (IL)-12 in GBS patients. CONCLUSIONS: Our results suggested that ginkgolides ameliorated the clinical score of EAN through down-regulating the proportions of Th 17 cells. Ginkgolides also suppressed inflammation response by decreasing pro-inflammatory cytokines IFN-γ and IL-12, suggesting ginkgolides had potential therapeutic effects on GBS patients and EAN in the future.

Ginkgolide B­induced AMPK pathway activation protects astrocytes by regulating endoplasmic reticulum stress, oxidative stress and energy metabolism induced by Aß1­42.

Ginkgolide B (GB), the diterpenoid lactone compound isolated from the extracts of Ginkgo biloba leaves, significantly improves cognitive impairment, but its potential pharmacological effect on astrocytes induced by ß­amyloid (Aß)1­42 remains to be elucidated. The present study aimed to investigate the protective effect and mechanism of GB on astrocytes with Aß1­42­induced apoptosis in Alzheimer's disease (AD). Astrocytes obtained from Sprague Dawley rats were randomly divided into control, Aß, GB and GB + compound C groups. Cell viability and apoptosis were analyzed using Cell Counting Kit­8 and flow cytometry assays, respectively. Protein and mRNA expression levels were analyzed using western blotting and reverse transcription­quantitative PCR, respectively. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH­Px), reactive oxygen species (ROS) and ATP were determined using the corresponding commercial kits. The findings revealed that GB attenuated Aß1­42­induced apoptosis and the 5' adenosine monophosphate­ activated protein kinase (AMPK) inhibitor compound C reversed the protective effects of GB. In addition, GB reversed Aß1­42­induced oxidative damage and energy metabolism disorders, including decreases in the levels of SOD, GSH­Px and ATP and increased the levels of MDA and ROS in astrocytes, while compound C reversed the anti­oxidative effect and the involvement of GB in maintaining energy metabolism in astrocytes. Finally, GB decreased the expression levels of the endoplasmic reticulum stress (ERS) proteins and the apoptotic protein CHOP and increased both mRNA and protein expression of the components of the energy metabolism­related AMPK/peroxisome proliferator­activated receptor γ coactivator 1α/peroxisome proliferator­activated receptor α and anti­oxidation­related nuclear respiratory factor 2/heme oxygenase 1/NAD(P)H dehydrogenase (quinone 1) pathways and downregulated the expression of ß­secretase 1. However, compound C could antagonize these effects. In conclusion, the findings demonstrated that GB protected against Aß1­42­induced apoptosis by inhibiting ERS, oxidative stress, energy metabolism disorders and Aß1­42 production probably by activating AMPK signaling pathways. The findings provided an innovative insight into the treatment using GB as a therapeutic in Aß1­42­related AD.

XQ-1H promotes cerebral angiogenesis via activating PI3K/Akt/GSK3ß/ß-catenin/VEGF signal in mice exposed to cerebral ischemic injury.

Stroke still ranks as a most lethal disease worldwide. Angiogenesis during the chronic phase of ischemic stroke can alleviate ischemic injury and attenuate neurological deficit. XQ-1H is a new compound derived from the structure modification of ginkgolide B, which exerts anti-inflammation and neuroprotection against cerebral ischemic injury during the acute or subacute phase. However, whether XQ-1H facilitates angiogenesis and neural functional recovery during the chronic phase remains unclear. This research was designed to explore whether XQ-1H promotes angiogenesis after ischemic stroke and to preliminarily elucidate the mechanism. In vitro, XQ-1H was found to facilitate proliferation, migration and tube formation in bEnd.3 cells. In vivo, XQ-1H raised the CD31 positive microvessel number and increased focal cerebral blood flow in mice exposed to cerebral ischemic injury, and improved the neurological function. Mechanism studies revealed that XQ-1H exerted angiogenesis promoting effect via the PI3K/Akt/GSK3ß/ß-catenin/VEGF signal pathway, which was reversed by LY294002 (the specific inhibitor of PI3K/Akt). In conclusion, XQ-1H exerts angiogenetic effect both in vivo and in vitro, which is a potential agent against ischemic stroke during chronic phase.

Anti-neoplastic Effect of Ginkgolide C through Modulating c-Met Phosphorylation in Hepatocellular Carcinoma Cells.

Ginkgolide C (GGC) derived from Ginkgo biloba, has been reported to exhibit various biological functions. However, the anti-neoplastic effect of GGC and its mechanisms in liver cancer have not been studied previously. Hepatocyte growth factor (HGF)/c-mesenchymal-epithelial transition receptor (c-Met) pathway can regulate tumor growth and metastasis in hepatocellular carcinoma (HCC) cells. This study aimed to evaluate the anti-neoplastic effect of GGC against HCC cells and we observed that GGC inhibited HGF-induced c-Met and c-Met downstream oncogenic pathways, such as PI3K/Akt/mTOR and MEK/ERK. In addition, GGC also suppressed the proliferation of expression of diverse tumorigenic proteins (Bcl-2, Bcl-xL, Survivin, IAP-1, IAP-2, Cyclin D1, and COX-2) and induced apoptosis. Interestingly, the silencing of c-Met by small interfering RNA (siRNA) mitigated c-Met expression and enhanced GGC-induced apoptosis. Moreover, it was noted that GGC also significantly reduced the invasion and migration of HCC cells. Overall, the data clearly demonstrate that GGC exerts its anti-neoplastic activity through modulating c-Met phosphorylation and may be used as an effective therapy against HCC.

XQ-1H attenuates ischemic injury in PC12 cells via Wnt/ß-catenin signaling though inhibition of apoptosis and promotion of proliferation.

10-O-(N,N-dimethylaminoethyl)-ginkgolide B methanesulfonate (XQ-1H) is a new derivative of ginkgolide B and has previously been proven to exert neuroprotective effects on ischemic injury. However, it is not clear whether XQ-1H affects the cell survival and proliferation in oxygen-glucose deprivation/reoxygenation (OGD/R) damaged PC12 cells. Our results showed that OGD/R improved cell viability after 24 hr of posttreatment with XQ-1H (10 or 5 µM), inhibiting cell injury and apoptosis by upregulating the expression of brain-derived neurotrophic factor, nerve growth factor, and antiapoptotic B-cell lymphoma-extra large, while reducing proapoptotic cleaved caspase-3 protein. By introducing the Wnt/ß-catenin signaling inhibitor XAV-939 and 5-bromo-2'-deoxyuridine staining, it was proved that XQ-1H promoted the proliferation of PC12 cells in a Wnt-signal-dependent manner via inhibiting the activation of glycogen synthase kinase-3ß after phosphatidylinositol 3-kinase/protein kinase B signal activation, thereby activating Wnt1, ß-catenin, and the expression of downstream neurogenic differentiation 1 and cyclin D1, which was comparable to Wnt/ß-catenin signaling agonist 4,6-disubstituted pyrrolopyrimidine. We conclude that XQ-1H, after OGD/R damage to PC12 cells, may limit cell apoptosis in a Wnt/ß-catenin signal-dependent manner, promoting cell proliferation and survival.

A sensitive LC-MS/MS method to determine ginkgolide B in human plasma and urine: application in a pharmacokinetics and excretion study of healthy Chinese subjects.

1. Ginkgolide B (GB), the most active of the ginkgolides, has been developed as a new drug for the treatment of vascular insufficiency; however, the pharmacokinetics of GB remain unclear. Here, we investigated the pharmacokinetics and urine excretion properties of GB in healthy Chinese subjects administered single- and multiple-dose injectable GB based on a new LC-MS/MS method.2. GB pharmacokinetics were found to be dose-dependent from 20 to 60 mg. GB reached a steady state by day 6 with once-daily dosing at 40 mg. Systemic exposure to GB, as characterised by AUC0-∞, indicated accumulation following repeated once-daily dosing for seven consecutive days. The mean urinary cumulative excretion rate of GB in response to 20, 40, and 60 mg GB was 41.9 ± 18.5%, 32.9 ± 12.2%, and 43.9 ± 8.5%, respectively.3. Dose-proportional pharmacokinetics of GB were observed after intravenous administration in healthy subjects. A gradual reduction in the volume of distribution and slight change in mean resistance time led us to conjecture the limited accumulation of GB based on distribution equilibrium in vivo.4. This comprehensive study of the clinical pharmacokinetics of GB will provide useful information for its application and further development.

A loss-of-function mutation of an inhibitory zinc- and proton-binding site reduces channel blocker potency in the glycine receptor.

The zinc ion (Zn2+) and proton (H+) are critical regulators for the glycine receptor chloride channel in physiological and pathological conditions. Both ions bind to the H109 residue at the extracellular agonist binding domain. However, whether the H109 residue affects the conformation of the remote channel pore is not yet known. In this study, we focus on the loss-of-function mutation, H109A, and use the inhibitory potencies of six structurally-diverse channel pore blockers (niflumic acid, picrotoxin, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) with various molecular volumes to measure the H109A mutation's effect on channel pore conformation. We found that their inhibitory potencies were mostly reduced by the H109A mutation and the extents of reduction were positively correlated with the molecular volumes of the blockers. In addition, we also found that the H109A mutation slowed both the blocking and unblocking rates of the blockers. Taken together, we propose that the H109A mutation might "narrow" the channel pore, although other forms of conformational change cannot be excluded. This further provides an implication that the H109 residue might allosterically control the channel pore conformation, and that Zn2+ or H+ binding to this site might also alter the conformation of the channel pore.

Effects of ß-glucosidase and α-rhamnosidase on the Contents of Flavonoids, Ginkgolides, and Aroma Components in Ginkgo Tea Drink.

Ginkgo tea is a kind of health food produced from Ginkgo biloba leaves. The market of Ginkgo tea encountered many difficulties because of its bad palatability and vague function statement. In this study, two kinds of glycosidase were used to improve the flavor of Ginkgo tea, and three kinds of bioactivities were selected to investigate the health care function of the tea infusion. The aroma components extracted by headspace absorb (HSA) method during the making of Ginkgo tea were analyzed by GC-MS. The flavonoids and ginkgolides released into the tea infusion were studied by HPLC. A combination of ß-glucosidase (ß-G) and α-rhamnosidase (α-R) was applied during the making of the tea. The contents of characteristic aroma components and the release of total flavonoids and ginkgolides were increased significantly by adding ß-G and α-R. The composition of flavone glycosides was changed greatly. The free radical scavenging, inhibition of inflammatory cell activation, and tumor cytotoxicity activities of the tea were demonstrably improved. According to the release of active components, Ginkgo tea can be brewed repeatedly for at least three times. The enzymes used here show potential application prospects in the making of Ginkgo tea or tea drink to get higher contents of flavonoids, ginkgolides, and aroma components.

Insight into the metabolic mechanism of Diterpene Ginkgolides on antidepressant effects for attenuating behavioural deficits compared with venlafaxine.

Depression is a severe and chronic mental disorder, affecting about 322 million individuals worldwide. A recent study showed that diterpene ginkgolides (DG) have antidepressant-like effects on baseline behaviours in mice. Here, we examined the effects of DG and venlafaxine (VLX) in a chronic social defeat stress model of depression. Both DG and VLX attenuated stress-induced social deficits, despair behaviour and exploratory behaviour. To elucidate the metabolic changes underlying the antidepressive effects of DG and VLX, we investigated candidate functional pathways in the prefrontal cortex using a GC-MS-based metabolomics approach. Metabolic functions and pathways analysis revealed that DG and VLX affect protein biosynthesis and nucleotide metabolism to enhance cell proliferation, with DG having a weaker impact than VLX. Glutamate and aspartate metabolism played important roles in the antidepressant effects of DG and VLX. Tyrosine degradation and cell-to-cell signaling and interaction helped discriminate the two antidepressants. L-glutamic acid was negatively correlated, while hypoxanthine was positively correlated, with the social interaction ratio. Understanding the metabolic changes produced by DG and VLX should provide insight into the mechanisms of action of these drugs and aid in the development of novel therapies for depression.

Pharmacokinetics, Metabolism and Disposition of [14C]XQ-1H After Intravenous Administration to Male Rats.

OBJECTIVE: This study describes the in vivo pharmacokinetics and metabolism of [14C]labeled XQ-1H in male rats. METHODS: XQ-1H is a methanesulfonate of XQ, 10-O-(N,N-dimethylaminoethyl)-ginkgolide B, a derivative of ginkgolide B (GB) with enhanced water solubility. Since it is very difficult to synthesize radiolabeled GB, the results obtained in this study may provide helpful insight to further ADME investigation of GB and its analogue compounds. After an i.v. administration of [14C]XQ-1H to male rats, XQ (the freebase form of XQ-1H) was extensively hydrolyzed, moderately metabolized, and mainly excreted in feces (71.5% of the dose) via the biliary route. RESULTS: The main enzyme mediated metabolic pathways were mono- and di-demthylation. Using the radiolabel form of XQ-1H, the temporal binding of XQ to red blood cells was observed. CONCLUSION: Binding of XQ to RBCs may lower the blood's viscosity and thus provide symptomatic improvement of ischemic stroke patients.

Overview of Medicinally Important Diterpenoids Derived from Plastids.

BACKGROUND: FL 32608 Terpenoids are hydrocarbon compounds derived from common fivecarbon isoprene (C5H8) building blocks. They are formed through the condensation and subsequent modification of isoprene units in various ways including - among others - cyclization and/or oxygenation. Their synthesis is localized either to the chloroplast and/or to the cytoplasm/peroxisome/ endoplasmic reticulum and mitochondrion. Terpenoids represent a very large and diverse class of metabolites and play important roles in plant growth and development. In addition, they have been intensively used in human health care, disease treatment and in dietary supplements. Approximately 60% of natural products known so far are terpenoids. CONCLUSION: This review briefly summarizes the biosynthetic pathways of major plant terpenoids. Then, five well-known and medicinally important diterpenoids, including paclitaxel, tanshinone, ginkgolide, triptolide and oridonin are discussed in detail. Their structures, occurrence, extraction and identification methods, pharmacological properties and clinical uses are also reviewed. Finally, the prospects of using biotechnology to produce medicinally important terpenoids are also briefly discussed.

A Bilobalide-Producing Endophytic Fungus, Pestalotiopsis uvicola from Medicinal Plant Ginkgo biloba.

For screening bilobalide (BB)-producing endophytic fungi from medicinal plant Ginkgo biloba, a total of 57 fungal isolates were isolated from the internal stem, root, leaf, and bark of the plant G. biloba. Fermentation processes using BB-producing fungi other than G. biloba may become a novel way to produce BB, which is a terpene trilactones exhibiting neuroprotective effects. In this study, a BB-producing endophytic fungal strain GZUYX13 was isolated from the leaves of G. biloba grown in the campus of Guizhou University, Guiyang city, Guizhou province, China. The strain produced BB when grown in potato dextrose liquid medium. The amount of BB produced by this endophytic fungus was quantified to be 106 µg/L via high-performance liquid chromatography (HPLC), substantially lower than that produced by the host tissue. The fungal BB which was analyzed by thin layer chromatography (TLC) and HPLC was proven to be identical to authentic BB. The strain GZUYX13 was identified as Pestalotiopsis uvicola via morphology and ITS rDNA phylogeny. To the best of our knowledge, this is the first report concerning the isolation and identification of endophytic BB-producing Pestalotiopsis spp. from the host plant, which further proved that endophytic fungi have the potential to produce bioactive compounds.

GABAA receptor cysteinyl mutants and the ginkgo terpenoid lactones bilobalide and ginkgolides.

The terpenoid lactones from Ginkgo biloba, bilobalide and ginkgolides, have been shown to act as negative modulators at α1ß2γ2L GABAA receptors. They have structural features similar to those of the chloride channel blocker picrotoxinin. Unlike picrotoxinin, however they are not known to produce convulsant effects. Using two-electrode voltage clamp electrophysiology, this study compared the effect of mutation of 2', 6' and 15' pore facing M2 domain residues to cysteine on the action of picrotoxinin, bilobalide and ginkgolides at α1ß2γ2L GABAA receptors expressed in Xenopus oocytes. Picrotoxinin was affected by mutation differently from the ginkgo terpenoid lactones. Although some of these compounds were affected by the mutation at same position and/or subunit, the changes in their potency were found to be dissimilar. The results suggest that the intracellular pore binding site for picrotoxinin, bilobalide, ginkgolide A, ginkgolide B and ginkgolide C is comprised of 2'ß-6'ß6'γ, 2'α2'ß-6'α6'ß, 2'α2'ß2'γ-6'ß6'γ, 2'α, 2'ß2'γ-6'ß and 2'α2'ß, respectively. Unlike bilobalide and ginkgolides, the inhibitory action of picrotoxinin was not affected by mutations at 15' position. It is proposed that 15'α15'ß, 15'ß, 15'α15'ß and 15'α15'ß15'γ forms an extracellular pore binding site for bilobalide, ginkgolide A, ginkgolide B and ginkgolide C, respectively. The lack of convulsant effects of bilobalide, and ginkgolide A and B may be associated in part with their different binding locations within the chloride channel.

Characterizations of the hydrolyzed products of ginkgolide A and ginkgolide B by liquid chromatography coupled with mass spectrometry.

Ginkgolides are diterpenoid trilactones responsible for the neuromodulatory properties of Ginkgo biloba extracts. They are to be hydrolyzed in aqueous solutions as mixed carboxylate forms potentially including three monocarboxylates, three dicarboxylates and one tricarboxylate. Characterizations of the hydrolyzed products are challenging because there is no way to prepare them individually. In this work, the major hydrolyzed products of ginkgolide A (GA) and ginkgolide B (GB) including all three monocarboxyaltes have been produced in buffers and subjected to liquid chromatography coupled with triple quadrupole MS and LTQ Orbitrap MS analysis. With the comparative analysis of the trilactone of GA and GB, it was highlighted a unique charge-driven fragmentation pathway of twice neutral losses of CO on the lactone-C. The monocarboxylates were accordingly identified based on the construction of their fragmentation pathways cross-linked with those of the trilactone. In brief, the lactone-C hydrolyzed product is characteristic of the absence of product ions between [M-H](-) and [M-H-C2H2O3](-) (m/z 351 for GA and m/z 367 for GB). The featured fragmentation pathway of the lactone-F hydrolyzed product is the cleavage of ring-A, yielding a fragment (m/z 295 for GA and m/z 309 for GB) followed with twice (GA) or triple (GB) neutral losses of CO. The most characteristic fragment of the lactone-E hydrolyzed product is [M-H-H2O-CO2-2CO](-) (m/z 307 for GA and m/z 323 for GB) in contrast to the other two monocarboxylates. The knowledge gained in this work was of special uses to investigate the biological fates and the corresponding pharmacological mechanisms of ginkgolides.

Molecular cloning and characterization of GbMECT and GbMECP gene promoters from Ginkgo biloba.

Ginkgolides are key pharmaceutical components in Ginkgo biloba. Using the cDNA sequence of the MECP and MECT genes to design primers, we obtained the promoters of these genes from Ginkgo genomic DNA using the genome walking method. The two promoters were 744 and 982 bp in length, respectively. The cis-elements of the GbMECPs and GbMECT promoters were predicted and analyzed using the plant cis-acting regulatory element database. We found major cis-elements in the sequence of the GbMECT and GbMECPs promoters. The GbMECP promoter contains six TATA boxes and eight CAAT boxes. The GbMECT contains five TATA boxes and seven CAAT boxes. Furthermore, some cis-elements in the promoters of GbMECPs and GbMECT included hormone and light-regulated elements, UB-B-induced elements, and stress-related dehydration-responsive elements. Expression analysis results showed that the MECP gene is mainly involved in responses to CCC (cycocel) and UV-B, and that MECT is mainly involved in responses to wounding treatment. These results also showed that the expression model was consistent with the cis-elements present. During the annual growth cycle, the level of GbMECPs was significantly correlated with terpene lactones accumulation in leaves. A fitted quadratic curve showed the best model for correlating GbMECPs with terpene lactones in leaves. These results will help us to understand the transcriptional regulatory mechanisms involved in key gene expression and ginkgolide accumulation in G. biloba.

Transformation of multi-component ginkgolide into ginkgolide B by Coprinus comatus.

BACKGROUND: As the strongest antagonist of the platelet activating factor, ginkgolide B (GB) possesses anti-ischemic, anti-oxidant and anti-convulsant properties, and it is used for the treatment of thrombosis in clinical practice. Till now, GB is usually obtained from extraction of Ginkgo biloba leaves through column chromatography with an extremely low yield and high cost, which can not meet clinical requirement. Therefore, it is urgent to find a new method to prepare GB. RESULTS: In the current study, we studied the ability and mechanism to transform multi-component ginkgolide into GB by Coprinus comatus in order to enhance the GB yield. Except for ginkgolide A (GA) and GB, all the other ginkgolides in the extract were transformed by the strain. In the case of culture medium containing 20 g/L glucose, the transformation product was identified as 12% GA and 88% GB by high performance liquid chromatography-Mass spectrometry (HPLC-MS), two stage mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR). Partial GA was also transformed into GB according to the yield (76%) and the content of GA in the raw ginkgolide (28.5%). Glucose was the key factor to transform ginkgolides. When glucose concentration in medium was higher than 40 g/L, all ginkgolides were transformed into the GB. Proteomic analysis showed that C. comatus transformed ginkgolide into GB by producing 5 aldo/keto reductases and catalases, and enhancing the metabolism of glucose, including Embden-Meyerhof pathway (EMP), hexose monophophate pathway (HMP) and tricarboxylic acid (TCA). CONCLUSIONS: C. comatus could transform ginkgolides into GB when the medium contained 40 g/L glucose. When the strain transformed ginkgolides, the glucose metabolism was enhanced and the strain synthesized more aldo/keto reductases and catalases. Our current study laid the groundwork for industrial production of GB.