Components Of Ginkgo Biloba Extracts Research Articles

Amide Derivatives of Ginkgolide B and Their Inhibitory Effects on PAF-Induced Platelet Aggregation.

Ginkgolides are the most important components of Ginkgo biloba extracts, whose lactone can be hydrolyzed in the aqueous environment. Although the hydrolyzed products have complex structures and their functions are not well-understood, opening the lactone ring is an important strategy in producing novel derivatives of ginkgolide. The preparation of a single pure aminolyzed ginkgolide for the study of its bioactivity and understanding of the process of aminolysis are challenging. To obtain stable aminolyzed products, four amide derivatives (2–5) of ginkgolide B (GB, 1) were prepared via the ring-opening reaction of its lactone with propylamine. These products were purified and fully identified by high-resolution mass spectrometry (HRMS) and nuclear magnetic resonance (NMR) spectroscopy and were further evaluated for their ability to inhibit the PAF-induced platelet aggregation of rabbit platelets in vitro. Compound 2, which was obtained by selective aminolysis of the lactone ring C of GB, showed a much better inhibitory activity of platelet aggregation (IC50, 15 nM) than the parent compound GB (IC50, 442 nM). The other three products (3–5), which were obtained by the aminolysis of lactone rings C and F of GB, did not show platelet aggregation inhibitory activity. The results greatly extended our understanding of the chemistry of GB and provided important structural information for the exploration and development of new drugs based on ginkgolides in G. biloba.

Ginkgolide B ameliorates NLRP3 inflammasome activation after hypoxic-ischemic brain injury in the neonatal male rat

IntroductionPerinatal hypoxic-ischemic (HI) insult is an important cause of brain injury in neonates. The development of novel treatment strategies for neonates with HI brain injury is urgently needed. Ginkgolide B (GB) is a main component of Ginkgo biloba extracts with a long history of use in traditional Chinese medicine. However, it is unknown whether GB could play a protective role in hypoxic stress in immature animals. MethodsUsing neonatal hypoxic-ischemic (HI) brain injury model of rat pups, neurological score, infarct size, and brain edema were evaluated after HI injury. The activation of microglia and the production of IL-1β and IL-18 were detected by immunohistochemistry and ELISA, respectively. A priming signal (NF-κB P65) and an activation signal (Caspase-1) of NLRP3 inflammasome activation were detected by western blot analyses. ResultsGB administrated 30 min prior to ischemia induction can improve neurological disorder, reduce infarct volume and alleviate cerebral edema. Compared with the HI groups, GB inhibited the activation of microglia and decreased the production of IL-1β and IL-18 in neocortex. Furthermore, GB reduced NLRP3 expression mainly in microglia, and significantly inhibited the expression of Caspase-1 and the nuclear translocation of NF-κB P65, preventing NLRP3 inflammasome activation. ConclusionsGB ameliorates hypoxic-ischemic brain injury in the neonatal male rat via inhibiting NLRP3 inflammasome activation.

Combining Metabolic Profiling and Gene Expression Analysis to Reveal the Biosynthesis Site and Transport of Ginkgolides in Ginkgo biloba L.

The most unique components of Ginkgo biloba extracts are terpene trilactones (TTLs) including ginkgolides and bilobalide. Study of TTLs biosynthesis has been stagnant in recent years. Metabolic profiling of 40 compounds, including TTLs, flavonoids, and phenolic acids, were globally analyzed in leaf, fibrous root, main root, old stem and young stem extracts of G. biloba. Most of the flavonoids were mainly distributed in the leaf and old stem. Most of phenolic acids were generally distributed among various tissues. The total content of TTLs decreased in the order of the leaf, fibrous root, main root, old stem and young stem. The TTLs were further analyzed in different parts of the main root and old stem. The content of TTLs decreases in the order of the main root periderm, the main root cortex and phloem and the main root xylem. In old stems, the content of TTLs in the cortex and phloem was much higher than both the old stem periderm and xylem. The expression patterns of five key genes in the ginkgolide biosynthetic pathway were measured by real-time quantitative polymerase chain reaction (RT-Q-PCR). Combining metabolic profiling and RT-Q-PCR, the results showed that the fibrous root and main root periderm tissues were the important biosynthesis sites of ginkgolides. Based on the above results, a model of the ginkgolide biosynthesis site and transport pathway in G. biloba was proposed. In this putative model, ginkgolides are synthesized in the fibrous root and main root periderm, and these compounds are then transported through the old stem cortex and phloem to the leaves.

Ginkgolide B functions as a determinant constituent of Ginkgolides in alleviating lipopolysaccharide-induced lung injury.

Ginkgolides are the major bioactive components of Ginkgo biloba extracts, however, the exact constituents of Ginkgolides contributing to their pharmacological effects remain unknown. Herein, we have determined the anti-inflammatory effects of Ginkgolide B (GB) and Ginkgolides mixture (GM) at equivalent dosages against lipopolysaccharide (LPS)-induced inflammation. RAW 264.7 cell culture model and mouse model of LPS-induced lung injury were used to evaluate in vitro and in vivo effects of GB and GM, respectively. In RAW 264.7 cells, GB and GM at equivalent dosages exhibit an identical capacity to attenuate LPS-induced inducible nitric oxide synthase mRNA and protein expression and subsequent NO production. Likewise, GB and GM possess almost the same potency in attenuating LPS-induced expression and activation of nuclear factor kappa B (p65) and subsequent increases in tumor necrosis factor-α mRNA levels. In LPS-induced pulmonary injury, GB and GM at the equivalent dosages have equal efficiency in attenuating the accumulation of inflammatory cells, including neutrophils, lymphocytes, and macrophages, and in improving the histological damage of lungs. Moreover, GB and GM at equivalent dosages decrease the exudation of plasma protein to the same degree, whereas GM is superior to GB in alleviating myeloperoxidase activities. Finally, though GB and GM at equivalent dosages appear to reduce LPS-induced IL-1β mRNA and protein levels and IL-10 protein levels to the same degree, GM is more potent than GB to attenuate the IL-10 mRNA levels. Taken together, this study demonstrates that GB functions as the determinant constituent of Ginkgolides in alleviating LPS-induced lung injury.

PXR Mediated Protection against Liver Inflammation by Ginkgolide A in Tetrachloromethane Treated Mice.

The pregnane X receptor (PXR), a liver and intestine specific receptor,, has been reported to be related with the repression of inflammation as well as activation of cytochromosome P450 3A (CYP3A) expression. We examined the effect of PXR on tetrachloromethane (CCl4)-induced mouse liver inflammation in this work. Ginkgolide A, one main component of Ginkgo biloba extracts (GBE), activated PXR and enhanced PXR expression level, displayed both significant therapeutic effect and preventive effect against CCl4-induced mouse hepatitis. siRNA-mediated decrease of PXR expression significantly reduced the efficacy of Ginkgolide A in treating CCl4-induced inflammation in mice. Flavonoids, another important components of GBE, were shown anti-inflammatory effect in a different way from Ginkgolide A which might be independent on PXR because flavonoids significantly inhibited CYP3A11 activities in mice. The results indicated that anti-inflammatory effect of PXR might be mediated by enhancing transcription level of IκBα through binding of IκBα. Inhibition of NF-κB activity by NF-κB-specific suppressor IκBα is one of the potential mechanisms of Ginkgolide A against CCl4-induced liver inflammation.

Synthesis, in silico and in vivo blood brain barrier permeability of ginkgolide B cinnamate

Ginkgolide B, one of the important components of Ginkgo biloba extracts, has been revealed to exhibit great potential in therapy of cerebrovascular diseases. However the lack of permeability greatly limited it from further clinical application. Based on the prediction model for blood brain barrier (BBB) permeation, herein a potential brain-targeting analog ginkgolide B cinnamate (GBC) was successfully synthesized and characterized. After intravenous administration of GBC or GB, liquid chromatography tandem mass spectrometry (LC–MS/MS) was conducted to determine the analog in rat plasma and brain. The results showed that GBC had a significant increase in BBB permeability. A significant 1.61-times increase in half-life was observed for GBC and the drug targeting index (DTI) value was calculated to be 9.91. The experiment results matched well with the predicted one, which revealed that BBB permeability prediction model combined with in vivo study could be used as a quick, feasible and efficient tool for brain-targeting drug design.

Ginkgolide B protects against cisplatin-induced ototoxicity: enhancement of Akt-Nrf2-HO-1 signaling and reduction of NADPH oxidase.

Cisplatin is a widely used chemotherapeutic drug for the treatment of various cancers. However, the ototoxicity severely limited its maximum dose. The present study was designed to evaluate the effect of Ginkgolide B (GB), a major component of Ginkgo biloba extracts, on cisplatin-induced ototoxicity and to elucidate the molecular mechanism in vitro and in vivo. In HEI-OC1 auditory cells, GB concentration-dependently inhibited the reduction of cell viability and increase in apoptosis exerted by cisplatin. Cisplatin-activated mitochondrial apoptotic molecular events were significantly inhibited by GB. In addition, GB notably suppressed the increase in NOX2 and p47(phox) expression and the decrease in nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression in cisplatin-exposed cells. Inhibition of Nrf2 using SiRNA and blockage of HO-1 by zinc protoporphyrin IX (ZnPP) suppressed the protective effects of GB. Moreover, GB prevented cisplatin-induced reduction of Akt phosphorylation and LY294002, an inhibitor of PI3K/Akt signaling, blocked the anti-apoptotic effect of GB in cisplatin-treated cells. Furthermore, the protective effect of GB was tested in cisplatin-exposed rats. GB treatment markedly protected animals against cisplatin-induced hearing loss and vestibular dysfunction. Inhibition of Akt and HO-1 significantly suppressed the improvement in hearing loss and vestibular dysfunction in GB-treated rats. We demonstrate that GB decreases ROS generation through reducing NOX2 expression and enhancing activity through Akt-Nrf2-HO-1 pathway, resulting in inhibition of mitochondrial apoptosis and final reduction of cisplatin-induced ototoxicity in vitro and in vivo. Our findings have gained an insight into the mechanism of GB-exerted protective effect against cisplatin-induced ototoxicity.

Effects of Ginkgolide A on Okadaic Acid-induced Tau Hyperphosphorylation and the PI3K-Akt Signaling Pathway in N2a Cells

Alzheimer's disease is the most common form of dementia leading to the irreversible loss of neurons, and Tau hyperphosphorylation has an important role in the pathology of Alzheimer's disease. Ginkgolide A is one of the active components of Ginkgo biloba extracts which has been proven to have neuroprotective effects, but the effect of ginkgolide A on Tau hyperphosphorylation has not yet been reported. In this study, the effects of ginkgolide A on cell viability, Tau hyperphosphorylation, and the PI3K-Akt signaling pathway in N2a cell lines were explored, and methods such as the MTT assay, ELISA, and Western blots techniques were used. The results showed that ginkgolide A could increase cell viability and suppress the phosphorylation level of Tau in cell lysates, meanwhile, GSK3β was inhibited with phosphorylation at Ser9. Moreover, treatment of the cells with ginkgolide A promoted phosphorylation of PI3K and Akt, suggesting that the activation of the PI3K-Akt signaling pathway may be the mechanism for ginkgolide A to prevent the intracellular accumulation of p-Tau induced by okadaic acid and to protect the cells from Tau hyperphosphorylation-related toxicity.

Ginkgo May Sensitize Ovarian Cancer Cells to Cisplatin

Ginkgolide B (GB), the primary active component ofGinkgo bilobaextracts, may have antitumor properties. The objective of this study was to determine the effects and possible mechanisms of GB in ovarian cancer cells. In this study, human ovarian cancer cell lines (SKOV3 and CAOV3) were treated with different concentrations of GB alone or in combination with Cis-diaminodichloroplatinum (CDDP). An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine cell viability. The apoptosis rates of cells were measured by flow cytometric analysis. The expression of apoptosis-associated and proliferation-associated proteins was detected by Western blot. The cytotoxicity of GB was analyzed using a lactate dehydrogenase assay. Treatment with 100 µM GB for 3 days significantly inhibited SKOV3 and CAOV3 cell proliferation by 57.3% and 63.1% compared with control cells, respectively, as determined by MTT assay. Similarly, the apoptotic cell population was increased when treated with GB in a dose-dependent manner both in SKOV3 and CAOV3 cells. These effects were characterized by the upregulation of p21, p27, cleaved capase-3, and cleaved caspase-8 and downregulation of cyclin D1. In addition, a combined treatment of low concentrations of GB and CDDP showed an additive effect on the inhibition of SKOV3 cell proliferation. Furthermore, GB had significantly less cytotoxicity than CDDP in normal human ovarian surface epithelial cells. This study suggests that GB can be proposed as an effective antiproliferative and apoptosis-inducing agent with interesting translational application in ovarian cancers, used in addition to conventional chemotherapy.

1H‐NMR Analysis of Terpene Trilactones (TTLs) in Ginkgo biloba: Green Female Leaves Contain the Most TTLs

Terpene trilactones (TTLs), unique components of Ginkgo biloba extracts, are believed to play important roles in the biological activity of these materials. The investigation of seasonal and gender-related variations in the natural content of TTLs in the leaves is a challenging problem that must be addressed in order to establish more efficient extraction/isolation protocols for TTLs. The aims of this work were (i) to modify the extraction/isolation protocols of TTLs from G. biloba leaves by means of boiling and filtration procedures, and (ii) to investigate seasonal and gender-related variations in the TTL content of the leaves via (1) H-NMR analysis. When extracting TTLs from G. biloba leaves, procedures for boiling and filtration were improved in this work. Moreover, quantitative (1) H-NMR analysis using DMF as a reference was performed and correlated to the colour (green/yellow) and gender (male/female) variations in the natural compositions of TTLs in the leaves. Extraction procedures were modified to include boiling in ethyl acetate and filtration was achieved with celite. (1) H-NMR analysis of TTLs suggested that green female leaves contained the largest amount of TTLs, while no TTLs were present in yellow male leaves. The present results provide a method for quickly supplying laboratory-scale quantities of TTLs from natural sources to enable the study of their structure-activity relationships.

Ginkgolide B Induces Apoptosis via Activation of JNK and p21‐Activated Protein Kinase 2 in Mouse Embryonic Stem Cells

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signaling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards the proliferation and development of mouse embryonic stem cells and blastocysts in vitro, and causes developmental injury in vivo. However, the precise molecular mechanisms underlying its actions are currently unknown. Here, our study further revealed that GKB induced apoptotic biochemical changes, including activation of JNK, caspase-3, and p21-activated protein kinase 2 (PAK2), in ESC-B5 mouse embryonic stem cells. Treatment of ESC-B5 cells with a JNK-specific inhibitor (SP600125) reduced GKB-induced activation of both JNK and caspase-3, indicating that JNK activity is required for GKB-induced caspase activation. Experiments using caspase-3 inhibitors and antisense oligonucleotides against PAK2 showed that caspase-3 activation is required for PAK2 activation and both of these activations are required for GKB-induced apoptosis in ESC-B5 cells.

Injury effects of ginkgolide B on maturation of mouse oocytes, fertilization, and fetal development in vitro and in vivo

Ginkgolide B (GKB), the major active component of Ginkgo biloba extracts, exerts both stimulatory and inhibitory effects on apoptotic signaling. Previous studies by our group demonstrated that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell number, hinders early postimplantation blastocyst development, and increases early-stage blastocyst death. Here, we further investigate the effects of GKB on oocyte maturation, and subsequent pre- and postimplantation development in vitro and in vivo. In our experiments, GKB induced a significant reduction in the rate of oocyte maturation, fertilization, and in vitro embryonic development. Treatment of oocytes with 1-6 microM GKB during in vitro maturation (IVM) led to increased resorption of postimplantation embryos and decreased placental and fetal weights. Data obtained using an in vivo mouse model further disclosed that consumption of drinking water containing 3-6 microM GKB led to decreased oocyte maturation and in vitro fertilization, as well as early embryo developmental injury, specifically, inhibition of development to the blastocyst stage in vivo. To our knowledge, this is the first study to investigate the impact of GKB on maturation of mouse oocytes, fertilization, and sequential embryonic development.

The Signaling Cascades of Ginkgolide B-Induced Apoptosis in MCF-7 Breast Cancer Cells

Ginkgolide B, the major active component of Ginkgo biloba extracts, can bothstimulate and inhibit apoptotic signaling. Here, we demonstrate that ginkgolide B caninduce the production of reactive oxygen species in MCF-7 breast cancer cells, leading toan increase in the intracellular concentrations of cytoplasmic free Ca2+ and nitric oxide(NO), loss of mitochondrial membrane potential (MMP), activation of caspase-9 and -3,and increase the mRNA expression levels of p53 and p21, which are known to be involvedin apoptotic signaling. In addition, prevention of ROS generation by pretreatment withN-acetyl cysteine (NAC) could effectively block intracellular Ca2+ concentrationsincreases and apoptosis in ginkgolide B-treated MCF-7 cells. Moreover, pretreatment withnitric oxide (NO) scavengers could inhibit ginkgolide B-induced MMP change andsequent apoptotic processes. Overall, our results signify that both ROS and NO playedimportant roles in ginkgolide B-induced apoptosis of MCF-7 cells. Based on these studyresults, we propose a model for ginkgolide B-induced cell apoptosis signaling cascades inMCF-7 cells.

Effects of Ginkgolide B on 6-OHDA-induced apoptosis and calcium over load in cultured PC12

Ginkgolide B, one of the major components of Ginkgo biloba extracts, is a potent platelet-activating factor (PAF) receptor antagonist, which is also regarded as having neuroprotective effects on the CNS. The aim of this research is to observe the effects of Ginkgolide B on the PC12 apoptosis induced by 6-hydroxydopamine (6-OHDA) and to explore whether these effects are related to the changes of intracellular Ca2+ and Calbindin D28K mRNA in PC12 cells. In the present work, the damage of PC12 cells was induced by 100μM 6-OHDA. The cells survival rate was examined by MTT assays. The intracellular free calcium concentration in PC12 cells was measured by using the fluorescent Ca2+ indicator fluo-3/AM. Semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was employed to determine the expression of Calbindin D28K mRNA in PC12. The data show that the Ginkgolide B inhibited PC12 cells apoptosis induced by 6-OHDA in a dose-dependent manner, and decreased the activity of caspase-3. In addition, Ginkgolide B increased the expression of Calbindin D28K mRNA and inhibited 6-OHDA-induced elevation in the intracellular calcium concentration. Our results showed that the Ginkgolide B inhibited the apoptosis of PC12 induced by 6-OHDA, and the protective effects of Ginkgolide B on PC12 cells are mediated, at least in part, by up-regulating the Calbindin D28K mRNA and by decreasing the intracellular calcium concentration.

Dosage Effects of Ginkgolide B on Ethanol‐Induced Cell Death in Human Hepatoma G2 Cells

Ginkgolide B is a major active component of Ginkgo biloba extracts, which has been shown to confer anticancer effects by inducing apoptosis or inhibiting oxidative stress generation. Ethanol induces a wide range of cellular toxicities, many of which have been linked to free radical generation. To further elucidate the cellular effects of ginkgolide B, we examined the dose-response effect of ginkgolide B on ethanol-induced toxicity in human Hep G2 cells. TUNEL and MTT assays revealed that ethanol (50-400 mM) induced apoptotic cell death in human Hep G2 cells, and that this effect was inhibited by low (5-25 microM) doses of ginkgolide B, but enhanced by high (50-100 microM) doses of ginkgolide B. Additional experiments revealed that ethanol treatment directly increased intracellular oxidative stress; this effect was enhanced by high doses of ginkgolide B but decreased following treatment with low concentrations of ginkgolide B. The dose-response effects of ginkgolide B on reactive oxygen species (ROS) generation were directly correlated with cell apoptotic biochemical changes including c-Jun N-terminal kinase (JNK) activation, caspase-3 activation, and DNA fragmentation. These results indicate that treatment dosage may determine the effect of ginkgolide B on ethanol-induced ROS generation and cell apoptosis, and support the notion that an appropriate dosage of ginkgolide B may aid in decreasing the toxic effects of ethanol.

Ginkgolide B induces apoptosis and developmental injury in mouse embryonic stem cells and blastocysts

Ginkgolide B, the major active component of Ginkgo biloba extracts, can both stimulate and inhibit apoptotic signalling. We previously showed that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early post-implantation blastocyst development and increases early-stage blastocyst death. Here, we report more detailed examinations of the cytotoxic effects of ginkgolide B on mouse embryonic stem cells (ESCs) and blastocysts and their subsequent development in vitro and in vivo. Using cell culture assay model, we revealed in our results that ginkgolide B treatment of ESCs (ESC-B5) induced apoptosis via reactive oxygen species (ROS) generation, c-Jun N-terminal kinase (JNK) activation, loss of mitochondrial membrane potential (MMP) and the activation of caspase-3. Furthermore, an in vitro assay model showed that ginkgolide B treatment inhibited cell proliferation and growth in mouse blastocysts. Finally, an in vivo model showed that treatment with 10 microM ginkgolide B caused resorption of post-implantation blastocysts and fetal weight loss. Our results reveal for the first time that ginkgolide B retards the proliferation and development of mouse ESCs and blastocysts in vitro and causes developmental injury in vivo.

Ginkgolides induce apoptosis and decrease cell numbers in mouse blastocysts

In this report, we examine the cytotoxic effect of ginkgolides, the major components of Ginkgo biloba extracts, on the blastocyst stage of mouse embryos and on subsequent early postimplantation embryonic development in vitro. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed that blastocysts treated with 5 or 10 μM ginkgolide A or ginkgolide B showed increased apoptosis versus untreated controls. This could be correlated with the observation that ginkgolide-treated blastocysts showed a significant reduction in the average number of total cells in the blastocyst and trophectoderm/inner cell mass lineage versus controls. In addition, ginkgolide-pretreated blastocysts showed normal levels of implantation on culture dishes in vitro, but significantly fewer embryos reached the later stages of embryonic development in the treatment groups versus the controls, instead dying at relatively early stages of development. Our results collectively indicate that ginkgolide treatment of mouse blastocysts induces apoptosis, decreases cell numbers, retards early postimplantation blastocyst development, and increases early-stage blastocyst death. These novel findings provide important new insights into the effect of Ginkgo biloba extracts on mouse blastocysts.