Ginkgolide Research Articles

Ginkgo diterpene lactones inhibit cerebral ischemia/reperfusion induced inflammatory response in astrocytes via TLR4/NF-κB pathway in rats.

Ginkgo biloba L. (Ginkgoaceae) is a traditional Chinese medicine known to treating stroke and other cardio-cerebrovascular diseases for thousands of years in China. Ginkgo diterpene lactones (GDL) attracted much attention because of their neuroprotective properties. To uncover the effects of GDL, which consist of ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), on ischemic stroke, as well as the underlying molecular mechanisms. We used middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen-glucose deprivation/reoxygenation (OGD/R) models mimicking the process of ischemia/reperfusion in vivo and in vitro, respectively. Anticoagulant effects of GDL were investigated on platelet activating factor (PAF), arachidonic acid (AA) and adenosine diphosphate (ADP)-induced platelet aggregation both in vivo and in vitro. We also evaluated the effects of GDL on lipopolysaccharide (LPS)-induced inflammatory response in primary cultured rats' astrocytes. Infarct size, neurological deficit score, and brain edema were measured at 72 h after MCAO. Immunohistochemistry was utilized to analyze neurons necrosis and astrocytes activation. Expression of pro-inflammatory cytokines, including tumor necrotic factor-α (TNF-α) and interleukin-1β (IL-1β) were detected using enzyme-linked immunosorbent assay (ELISA) and real time PCR. The levels of toll-like receptor 4 (TLR4) and nuclear factor κB (NF-κB) were assessed by real time PCR or Western blot. Compared with MCAO/R rats, GDL significantly reduced infarct size and brain edema, improved neurological deficit score. Meanwhile, GDL suppressed platelet aggregation, astrocytes activation, pro-inflammatory cytokines releasing, TLR4 mRNA expression and transfer of NF-κB from cytoplasm to nucleus. Furthermore, GDL alleviated OGD/R injury and LPS-induced inflammatory response in primary astrocytes, characterized by promoting cell viability, decreasing lactate dehydrogenase (LDH) activity, and inhibiting IL-1β and TNF-α releasing. In summary, GDL attenuate cerebral ischemic injury, inhibit platelet aggregation and astrocytes activation. The anti-inflammatory activity might be associated with the downregulation of TLR4/NF-κB signal pathway. Our present findings provide an innovative insight into the novel treatment of GDL in ischemic stroke therapy.

Ginkgolide B for Myocardial Ischemia/Reperfusion Injury: A Preclinical Systematic Review and Meta-Analysis

Ginkgolide B (GB) is an extract of dried Ginkgo biloba leaves and possesses various pharmacological activities in the cardiovascular system. Herein, we aim to assess the available preclinical evidence and possible mechanisms of GB for myocardial ischemia/reperfusion injury. The study quality score was assessed using the CAMARADES 10-item checklist. Rev-Man 5.3 software was used for data analyses. Nineteen studies with total 437 animals were included for analysis. Meta-analyses indicated that GB interventions significantly reduce myocardial infarct size and cardiac markers when compared with control (P < 0.05). The possible mechanisms via which GB exerts cardioprotective effects are mainly associated with anti-oxidation, anti-inflammation, anti-apoptosis, and improvement of energy metabolism. Our study indicates that GB might be a promising cardioprotective agent for myocardial ischemia/reperfusion injury and may contribute to future clinical trial design.

Ginkgolide B and bilobalide promote the growth and increase β-catenin expression in hair follicle dermal papilla cells of American minks.

The regeneration of proliferation potential of dermal papilla (DP) cells contributes to the treatment of hair loss disorders. Ginkgolide B (GKB) and bilobalide (BB) are two functional components isolated from Ginkgo biloba that can promote hair growth. In the current study, the effect of GKB or BB on DP cell viability and the related signaling was assessed. Hair follicles were isolated from minks, and the growth of hair follicles was measured under the administration of GKB or BB. DP cells isolated from minks were also subjected to GKB or BB. The administration of GKB or BB induced the growth of hair follicles. The viability of DP cells was also increased by GKB or BB as illustrated by methyl thiazolyl tetrazolium and flow cytometry detection. Moreover, the secretion of VEGF was enhanced by GKB or BB. At molecular level, the activities of Akt, ERK1/2, and β-catenin were induced by GKB, whereas BB only increased the activities of Akt and β-catenin. In conclusion, although the two components influenced the β-catenin signaling activity in distinct mechanisms, they both increased the viability of DP cells and promoted the cycle of hair follicles.

Bone marrow mesenchymal stem cells intro-ventricular transplantation combined with intraperitoneal injection of ginkgolide K for focal cerebral ischemia/reperfusion injury in rats

Objective To investigate the neuroprotective effect of combined application of bone marrow mesenchymal stem cells (BMSCs) and Ginkgolide K (GK) intraperitoneal injection for rat focal cerebral ischemia. Methods In all rats, the middle cerebral artery occlusion (MCAO) was produced by occlusion of right internal carotid artery with a nylon monofilament. Fifty male Sprague-Dawley (SD) rats were divided into five groups by the random digits table, 10 rats in each group, including Sham group, saline solution intraperitoneal injection group, GK intraperitoneal injection group, BMSCs+ saline solution intraperitoneal group, and BMSCs+ GK intraperitoneal injection group. The neurological defective scores were assessed at 72, 120, 168 h after reperfusion respectively in each experiment and all the animals were then decapitated to determine the brain infarct volume and brain water content after 168 h. The expression of vascular endothelial growth factor (VEGF) was detected by Western blotting. Results The expression of VEGF was increased significantly after intraperitoneal injection of GK. Combined treatment of BMSCs and GK could significantly decrease the number of apoptosis cells [(18.8±3.6)/HP, F=167.531, P<0.01] and infarct volumes [(17.31±5.24)%, F=34.910, P<0.01], brain water content in ischemia hemisphere [(73.55±0.53)%, F=65.128, P<0.01]. The values of neurological defective scores in BMSCs+ GK injection group were significantly lower than the rest groups [120 h: (3.51±0.46), F=15.473, P<0.01; 168 h: (2.03±0.35), F=21.711, P<0.01]. Conclusion Combined treatment of BMSCs intro-ventricular transplantation and GK intraperitoneal injection produces a synergistic neuroprotective effect. Key words: Vascular endothelial growth factor; Bone marrow; Mesenchymal stem cells; Ginkgolide K

Ginkgo Flavonol Glycosides or Ginkgolides Tend to Differentially Protect Myocardial or Cerebral Ischemia-Reperfusion Injury via Regulation of TWEAK-Fn14 Signaling in Heart and Brain.

Shuxuening injection (SXNI), one of the pharmaceutical preparations of Ginkgo biloba extract, has significant effects on both ischemic stroke and heart diseases from bench to bedside. Its major active ingredients are ginkgo flavonol glycosides (GFGs) and ginkgolides (GGs). We have previously reported that SXNI as a whole protected ischemic brain and heart, but the active ingredients and their contribution to the therapeutic effects remain unclear. Therefore, we combined experimental and network analysis approach to further explore the specific effects and underlying mechanisms of GFGs and GGs of SXNI on ischemia–reperfusion injury in mouse brain and heart. In the myocardial ischemia–reperfusion injury (MIRI) model, pretreatment with GFGs at 2.5 ml/kg was superior to the same dose of GGs in improving cardiac function and coronary blood flow and reducing the levels of lactate dehydrogenase and aspartate aminotransferase in serum, with an effect similar to that achieved by SXNI. In contrast, pretreatment with GGs at 2.5 ml/kg reduced cerebral infarction area and cerebral edema similarly to that of SXNI but more significantly compared with GFGs in cerebral ischemia–reperfusion injury (CIRI) model. Network pharmacology analysis of GFGs and GGs revealed that tumor necrosis factor-related weak inducer of apoptosis (TWEAK)–fibroblast growth factor-inducible 14 (Fn14) signaling pathway as an important common mechanism but with differential targets in MIRI and CIRI. In addition, immunohistochemistry and enzyme linked immunosorbent assay (ELISA) assays were performed to evaluate the regulatory roles of GFGs and GGs on the common TWEAK–Fn14 signaling pathway to protect the heart and brain. Experimental results confirmed that TWEAK ligand and Fn14 receptor were downregulated by GFGs to mitigate MIRI in the heart while upregulated by GGs to improve CIRI in the brain. In conclusion, our study showed that GFGs and GGs of SXNI tend to differentially protect brain and heart from ischemia–reperfusion injuries at least in part by regulating a common TWEAK–Fn14 signaling pathway.

Ginkgolides-loaded soybean phospholipid-stabilized nanosuspension with improved storage stability and in vivo bioavailability

The purpose of this study was to investigate the effects of soybean phospholipid, as a steric stabilizer, on improving dissolution rate, storage stability and bioavailability of ginkgolides. The ginkgolides coarse powder, hydroxypropyl methylcellulose (HPMC), soybean phospholipid and sodium dodecyl sulfate (SDS) were mixed and wet-milled to prepare nanosuspension S1. Nanosuspension S2 was obtained by the same technique except adding the soybean phospholipid. Results of particle size showed that particle size (D50) of S1 significantly decreased from 44.25 μm to 0.373 μm. Results of differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD) and transmission electron microscope (TEM) showed that ginkgolides in nanosuspension still maintained its crystallinity, and the nanoparticles were all nearly circular and uniformly dispersed. Then, pellets F1 and F2 were prepared by layering S1 and S2 onto the microcrystalline cellulose (MCC) spheres, respectively. The dissolution rate of ginkgolide A (GA) and ginkgolide B (GB) in F1 was 98.3% and 97.7% in 30 min, respectively. It was much higher than F2 (89.0% and 86.5%) and coarse powder of ginkgolides (22.3% and 24.6%). According to the results of stability test, the storage stability of F1 was improved compared with F2. In addition, compared with coarse powder of ginkgolides, the relative bioavailability of GA and GB in F1 were up to (221.84 ± 106.67) % and (437.45 ± 336.43) %, respectively. The above results demonstrated that soybean phospholipid added to the nanosuspension played an important role in improving drug dissolution rate, storage stability and in vivo bioavailability: (1) The amphiphilic soybean phospholipid interacted with the drug, with the hydrophobic part adsorbed on the surface of the poorly soluble drug and the hydrophilic part exposed to the aqueous medium. This increases the wettability of the nanoparticles, which ensure a good redispersibility of the drug particles. (2) It could self-assemble to form an interfacial phospholipid film by surrounding the individual nanoparticles, which can produce enough steric hindrance to prevent nanoparticles from aggregation and ensure a rapid dissolution rate. (3) Soybean phospholipid and its hydrolysate formed strong micellar solubilizing vehicles with bile salts in vivo, stimulated the absorption process of ginkgolides. Thus, soybean phospholipid was a promising steric stabilizer in nanosuspension drug delivery system.

Ginkgolide B promotes osteoblast differentiation via activation of canonical Wnt signalling and alleviates osteoporosis through a bone anabolic way.

Osteoporosis has become a worldwide problem as the population ages. Although many advances have been made in the treatment of osteoporosis in the past few years, the outcome are sometimes disturbing because of the adverse effects of these treatments. Further studies are still needed to identify novel alternate agents to improve the therapeutic effect. Ginkgolide B (GB), a derivative of Ginkgo biloba leaves, has numerous pharmacological effects, including anticancer and anti‐inflammation activities. However, the effect of GB on the regulation of osteoblast activity and bone formation effect has not yet been investigated. In this study, we showed the in vitro and in vivo effects of GB on osteoblast differentiation and bone formation. We found that GB promotes osteoblast differentiation of Bone Mesenchymal Stem Cells (BMSCs) and MC3T3‐E1 cells in vitro in a Wnt/β‐catenin‐dependent manner. In an in vivo study, we constructed a cranial defect model in rats and treated with GB. Histomorphometric and histological analyses confirmed that the usage of GB significantly promotes bone formation. Further study on ovariectomy (OVX) rats demonstrated that GB is capable of alleviating ovariectomy‐induced bone loss by enhancing osteoblast activity. Our findings indicate that GB is a potential therapeutic agent of osteoporosis through an anabolic way in bone.

Effects of ginkgo diterpene lactone meglumine on learning and memory in old mice and its mechanisms

Objective To study the effects of diterpene ginkgolides meglumine injection (DGMI) on memory impairment, activation of microglia and astrocytes and inflammatory cytokines in aged mice. Methods Twenty aged mice (22 months old) were randomly divided into two groups: aged mouse group(n=10) and DGMI group(n=10). Another 10 mice (2 months old) were selected as young mouse control group. The mice in DGMI group were received 5 mg/kg DGMI per day by tail vain injection for 4 weeks.The mice in the other two groups were received the same amount normal saline for 4 weeks. The Morris water maze was used to evaluate the function of spacial learning and memory after administration of drugs. The expression of CD11b, GFAP, IL-1β, IL-6, TNF-α and NFκB in mice brain hippocampus were detected by Western blot. Results (1) The escape latency time of aged mouse group was significantly longer than that of young mouse control group from the 2nd day to the 7th day(P<0.01). The times of platform crossing, time and distance in target quadrant of aged mouse group were significantly shorter than those of young mouse group (all P<0.01). Compared with aged mouse group, DGMI significantly reduced the escape latency time of DGMI group (P<0.01). DGMI increased the times of platform crossing, time and distance in target quadrant of aged mouse group (P<0.01). (2) The expressions of CD11b, GFAP in young mouse control group, aged mouse group and DGMI group were as follows respectively: CD11b: (1.036±0.023), (1.757±0.046), (1.214±0.024); GFAP: (1.022±0.071), (1.344±0.021), (1.086±0.073). DGMI reduced the expression of CD11b and GFAP in hippocampus compared with aged mouse group (t=5.556, P<0.01; t=5.484, P<0.01). (3) The expressions of IL-1β, IL-6, TNF-α and NFκB in young mouse control group, aged mouse group and DGMI group were as follows respectively: IL-1β: (1.003±0.057), (2.062±0.105), (1.182±0.084); IL-6: (1.018±0.024), (1.583±0.052), (1.152±0.031); TNF-α: (1.021±0.054), (1.449±0.053), (1.211±0.036); p-NFκB: (1.052±0.034), (1.782±0.113), (1.158±0.066). DGMI reduced the expression of p-NFκB(t=6.547, P<0.01) and pro-inflammatory cytokines including IL-1β(t=8.513, P<0.01), IL-6(t=3.421, P<0.01) and TNF-α(t=5.562, P<0.01) in hippocampus compared with aged mouse group. Conclusion DGMI can improve the ability of learning and memory in aged mice. The mechanism may be related with inhibiting activity of microgliosis, astrocytosis, NFκB and neuroinflammaton. Key words: Diterpene ginkgolides meglumine injection; Aged mice; Learning and memory; Neuroinflammation

Enhancing the Astrocytic Clearance of Extracellular α-Synuclein Aggregates by Ginkgolides Attenuates Neural Cell Injury.

The accumulation of aggregated forms of the α-Synuclein (α-Syn) is associated with the pathogenesis of Parkinson's disease (PD), and the efficient clearance of aggregated α-Syn represents a potential approach in PD therapy. Astrocytes are the most numerous glia cells in the brain and play an essential role in supporting brain functions in PD state. In the present study, we demonstrated that cultured primary astrocytes engulfed and degraded extracellular aggregated recombinant human α-Syn. Meanwhile, we observed that the clearance of α-Syn by astrocytes was abolished by proteasome inhibitor MG132 and autophagy inhibitor 3-methyladenine (3MA). We further showed that intracellular α-Syn was reduced after ginkgolide B (GB) and bilobalide (BB) treatment, and the decrease was reversed by MG132 and 3MA. More importantly, GB and BB reduced indirect neurotoxicity to neurons induced by α-Syn-stimulated astrocytic conditioned medium. Together, we firstly find that astrocytes can engulf and degrade α-Syn aggregates via the proteasome and autophagy pathways, and further show that GB and BB enhance astrocytic clearance of α-Syn, which gives us an insight into the novel therapy for PD in future.

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.

Identification of eupatilin and ginkgolide B as p38 ligands from medicinal herbs by surface plasmon resonance biosensor-based active ingredients recognition system

Screening of bioactive ligands for a certain protein target from medicinal herbs is a highly important yet challenging task during drug discovery process. In this study, a surface plasmon resonance biosensor-based active ingredient recognition system (SPR-AIRS) was applied to screen p38 mitogen-activated protein kinase (p38) ligands from herbal extracts. After p38 protein was immobilized on a SPR chip and the suitability of SPR-AIRS was validated, thirty-four p38-related medicinal herbs were selected and pre-screened. Two medicinal herbs having high response signal with p38-immobilized chip, Folium Ginkgo and Herba Artemisiae Scopariae, were injected into SPR system for ligand fishing. Among them, two active compounds, eupatilin (EPT) and ginkgolide B (GKB), were identified as p38 ligands, and then the KD values of EPT and GKB were measured as 21.68 ± 2.21 and 44.71 ± 1.80 μM, respectively. They can inhibit p38 activities significantly and bind to the ATP binding site on p38. Furthermore, EPT and GKB can inhibit cell proliferation (IC50 = 30.31 ± 6.84 and 42.97 ± 0.83 μM), induce apoptosis and G2/M cell cycle arrest against K562 cell line. This is the first time that EPT and GKB are reported as effective p38 binding ligands. These results prove that SPR-AIRS could be an effective method to screen active compounds acting on a specific protein from complex systems.

Antioxidant effects of ginkgolides and bilobalide against cerebral ischemia injury by activating the Akt/Nrf2 pathway in vitro and in vivo.

Ginkgolide terpenoid lactones, including ginkgolides and bilobalide, are two crucial bioactive constituents of extract of Ginkgo biloba (EGb) which was used in the treatment of cardiovascular and cerebrovascular diseases. The aims of this study were to investigate the antioxidant effects and mechanism of ginkgolides (ginkgolide A (GA), ginkgolide B (GB), ginkgolide K (GK)) and bilobalide (BB) against oxidative stress induced by transient focal cerebral ischemia. In vitro, SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD) for 4h followed by reoxygenation with ginkgolides and BB treatments for 6h, and then cell viability, superoxide dismutase (SOD), and ROS were respectively detected using kit. Western blot was used to confirm the protein levels of hemeoxygenase-1 (HO-1), quinone oxidoreductase l (Nqo1), Akt, phosphorylated Akt (p-Akt), nuclear factor-E2-related factor2 (Nrf2), and phosphorylated Nrf2 (p-Nrf2). GB combined with different concentrations of LY294002 (PI3K inhibitor) were administrated to SH-SY5Y cells for 1h after OGD, and then p-Akt and p-Nrf2 levels were detected by western blot. In vivo, 2h of middle cerebral artery occlusion (MCAO) model was established, followed with reperfusion and GB treatments for 24 and 72h. The infarct volume ratios were confirmed by TTC staining. The protein levels of HO-1, Nqo1, SOD1, Akt, p-Akt, Nrf2, and p-Nrf2 were detected using western blot and immunohistochemistry (IHC). Experimental data in vitro confirm that GA, GB, GK, and BB resulted in significant decrease of ROS and increase of SOD activities and protein levels of HO-1 and Nqo1; however, GB group had a significant advantage in comparison with the GA and GK groups. Moreover, after ginkgolides and BB treatments, p-Akt and p-Nrf2 were significantly upregulated, which could be inhibited by LY294002 in a dose-dependent manner, meanwhile, GB exhibited more effective than GA and GK. In vivo, TTC staining indicated that the infarct volume ratios in MCAO rats were dramatically decreased by GB in a dose-dependent manner. Furthermore, GB significantly upregulated the protein levels of HO-1, Nqo1, SOD, p-Akt, p-Nrf2, and Nrf2. In conclusion, GA, GB, GK, and BB significantly inhibited oxidative stress damage caused by cerebral ischemia reperfusion. Compared with GA, GK, and BB, GB exerts the strongest antioxidant stress effects against ischemic stroke. Moreover, ginkgolides and BB upregulated the levels of antioxidant proteins through mediating the Akt/Nrf2 signaling pathway to protect neurons from oxidative stress injury.

Ginkgolide B ameliorates myocardial ischemia reperfusion injury in rats via inhibiting endoplasmic reticulum stress.

PurposeGinkgolide B (GB) is a terpene lactone component found in Ginkgo biloba, which has a protective role on ischemia reperfusion (I/R) injury. This study was aimed at exploring the protective mechanism of GB on the myocardial I/R.Patients and methodsMyocardial I/R model was established on Sprague Dawley rats. The levels of cardiac troponin I, cardiac troponin T, lactic dehydrogenase, and myoglobin were determined by a 200FR NEO automatic biochemical analyzer. Histological examination was performed through HE and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. The expression levels of p-PERK, p-IRE1α, ATF6, p-AKT, and mTOR were detected by Western blot.ResultsThe results exhibited that GB treatment suppressed the high levels of cardiac troponin I, cardiac troponin T, lactic dehydrogenase, and myoglobin and ameliorated the damaged and irregularly arranged myocardial cells induced by I/R injury significantly, indicating that GB could ameliorate myocardial I/R injury. Moreover, the high expression levels of endoplasmic reticulum (ER) stress key proteins caused by I/R injury were suppressed significantly by GB treatment, including p-PERK, p-IRE1α, and ATF6. GB treatment also decreased the number of apoptotic cells compared with I/R group. In addition, activation of ER stress by Tunicamycin treatment could counteract the protective effects of GB on I/R injury, suggesting that GB ameliorated myocardial I/R injury through inhibition of ER stress-induced apoptosis. Finally, the decreased p-AKT and p-mTOR expressions caused by I/R injury were upregulated by GB and inhibition of PI3K/AKT/mTOR pathway by LY294002 abolished the protective effects of GB on I/R injury, indicating that GB activated PI3K/AKT/mTOR pathway during I/R injury.ConclusionGB protected against myocardial I/R injury through inhibiting ER stress-induced apoptosis via PI3K/AKT/mTOR signaling pathway.

Oral administration of Ginkgolide B alleviates hypoxia-induced neuronal damage in rat hippocampus by inhibiting oxidative stress and apoptosis.

Objective(s):The aim of this study is to explore the potential neuroprotective effects of Ginkgolide B (GB), a main terpene lactone and active component in Ginkgo biloba, in hypoxia-induced neuronal damage, and to further investigate its possible mechanisms.Materials and Methods:54 Sprague-Dawley rats were randomly divided into three groups: the untreated control group (n=18); the hypoxia group (n=18; exposed to 6000 m simulated plateau altitude for six days); and the GB group (n=18; intragastric administration of 12 mg/kg GB three days prior to rapid adaption to 6000 m and on the first two days of hypoxia). After hypoxia exposure for six days, we dissected out the brain hippocampi and performed hematoxylin and eosin staining, Nissl staining, and TUNEL staining. Homogenates of the hippocampi were used to test the oxidative stress indices including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase. Bax and caspase-3 expression in the hippocampal tissue was measured using qRT-PCR.Results:Treatment with GB before exposure to hypoxia could protect neural cells and increase the number of Nissl bodies. TUNEL and qRT-PCR results demonstrated that GB treatment could decrease apoptotic cells in different areas of the hippocampus. Antioxidant defense systems such as SOD, GSH, and catalase were decreased (P<0.05), and the concentration of MDA was reduced significantly in the hippocampi of rats of the GB group (P<0.05).Conclusion:GB could alleviate hypoxia-induced neuronal damage in rat hippocampus by inhibiting oxidative stress and apoptosis.

Cardioprotection of Ginkgolide B on Myocardial Ischemia/Reperfusion-Induced Inflammatory Injury via Regulation of A20-NF-κB Pathway.

Inflammation urges most of the characteristics of plaques involved in the pathogenesis of myocardial ischemia/reperfusion injury (MI/RI). In addition, inflammatory signaling pathways not only mediate the properties of plaques that precipitate ischemia/reperfusion (I/R) but also influence the clinical consequences of the post-infarction remodeling and heart failure. Here, we studied whether Ginkgolide B (GB), an effective anti-inflammatory monomer, improved MI/RI via suppression of inflammation. Left anterior descending (LAD) coronary artery induced ischemia/reperfusion (I/R) of rats or A20 silencing mice, as well as hypoxia/reoxygenation (H/R) induced damages of primary cultured rat neonatal ventricular myocytes or A20 silencing ventricular myocytes, respectively, served as MI/RI model in vivo and in vitro to discuss the anti-I/R injury properties of GB. We found that GB significantly alleviated the symptoms of MI/RI evidently by reducing infarct size, preventing ultrastructural changes of myocardium, depressing Polymorphonuclears (PMNs) infiltration, lessening histopathological damage and suppressing the excessive inflammation. Further study demonstrated that GB remarkably inhibited NF-κB p65 subunit translocation, IκB-α phosphorylation, IKK-β activity, as well as the downstream inflammatory cytokines and proteins expressions via zinc finger protein A20. In conclusion, GB could alleviate MI/RI-induced inflammatory response through A20-NF-κB signal pathway, which may give us new insights into the preventive strategies for MI/RI disease.

Ginkgolide B enhances gemcitabine sensitivity in pancreatic cancer cell lines via inhibiting PAFR/NF-кB pathway

Gemcitabine resistance will occur by time after the initial response in pancreatic cancer. Ginkgolide B (GB), a major terpene lactone component of Ginkgo biloba leaves, is a highly selective and competitive inhibitor for platelet-activating factor (PAF) receptor. In the present study, we evaluated the effect of GB on gemcitabine sensitivity in pancreatic cancer cell lines in vitro and in vivo. Cell viability assay, flow cytometry, dual luciferase reporter assay and tumor xenograft model were used to evaluate cell proliferation, apoptosis, nuclear factor kappa b (NF-кB) activity in vitro and tumor growth in vivo. Western blot, immunohistochemistry (IHC) and immunofluorescence were used to shown different protein expression levels. We found the half maximal inhibitory concentration (IC50) of gemcitabine was significantly downregulated by GB in a dose-dependent manner. Furthermore, GB could suppress cell proliferation, increase cell apoptosis and repress tumor growth when combined with gemcitabine, but had no effect when treated alone. Gemcitabine could upregulate PAFR and phosphorylated NF-кB/p65 expression, and increase NF-кB activity, but this was largely suppressed in combination with GB. GB could suppress PAFR expression in a dose-dependent manner. Knockout of PAFR significantly decreased phosphorylated NF-кB/p65 expression, inhibited NF-кB activity, increased gemcitabine sensitivity and cell apoptosis. Besides, GB had no influence on gemcitabine IC50 in IκBα-SR stably expressed BxPC-3 and CAPAN1. Our results suggested that GB could enhance gemcitabine sensitivity in pancreatic cancer cell lines by suppressing PAFR/NF-кB pathway. Thus GB may have therapeutic potential when used in combination with gemcitabine in pancreatic cancer.

Ginkgolide B promotes neuronal differentiation through the Wnt/\u03b2-catenin pathway in neural stem cells of the postnatal mammalian subventricular zone

Chinese herbal medicines (CHMs) have been used to treat human diseases for thousands of years. Among them, Ginkgo biloba is reported to be beneficial to the nervous system and a potential treatment of neurological disorders. Since the presence of adult neural stem cells (NSCs) brings hope that the brain may heal itself, whether the effect of Ginkgo biloba is on NSCs remains elusive. In this study, we found that Ginkgo biloba extract (GBE) and one of its main ingredients, ginkgolide B (GB) promoted cell cycle exit and neuronal differentiation in NSCs derived from the postnatal subventricular zone (SVZ) of the mouse lateral ventricle. Furthermore, the administration of GB increased the nuclear level of β-catenin and activated the canonical Wnt pathway. Knockdown of β-catenin blocked the neurogenic effect of GB, suggesting that GB promotes neuronal differentiation through the Wnt/β-catenin pathway. Thus, our data provide a potential mechanism underlying the therapeutic effect of GBE or GB on brain injuries and neurodegenerative disorders.

Comparing the role of Ginkgolide B and Ginkgolide K on cultured astrocytes exposed to oxygen‑glucose deprivation.

Ginkgolide B (GB) and ginkgolide K (GK) are two main active monomers of ginkgolides that present a unique group of diterpenes found naturally in the leaves of the Ginkgo biloba tree. Astrocytes are the most abundant cell type within the central nervous system (CNS) and serve essential roles in maintaining healthy brain function. The present study compared the biological effects of GB and GK on astrocytes exposed to oxygen-glucose deprivation (OGD). The results demonstrated that GB and GK exhibit many different actions. The level of the platelet-activating factor (PAF) was elevated on astrocytes exposed to OGD, and inhibited by GB and GK treatment. Although GB and GK inhibited the expression of p-NF-κB/p65, GK exerted stronger anti-inflammatory and antioxidant effects on astrocytes exposed to OGD than GB by inhibiting interleukin (IL)-6 and tumor necrosis factor-α, and inducing IL-10 and the nuclear factor-erythroid 2-related factor 2/HO-1 signaling pathway. When compared with GB treatment, GK treatment maintained high levels of phosphoinositide 3-kinase/phosphorylated-protein kinase B expression, and induced a marked upregulation of Wnt family member 1 and brain derived neurotrophic factor, indicating that GK, as a natural plant compound, may have more attractive prospects for clinical application in the treatment of neurological disorders than GB.

Optimizing the Formulation for Ginkgolide B Solid Dispersion.

The main purpose of our study is to optimize the formulation for Ginkgolide B (GB) solid dispersion in order to improve its dissolution in water. For the preparation of GB solid dispersion, we use a solvent method. The optimized formulation consists of GB : PVPK30 = 1:10, PVPK30 as the carrier, and ethanol : dichloromethane = 1:1 as the solvent. They were treated ultrasonically for 10 min at 60°C. The results from scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), and particle size analysis show that the morphological appearance of GB changed significantly in solid dispersion. The original crystal form of GB no longer existed, but it was uniformly dispersed within PVPK30 in a non-crystalline form. It is probably because the -C=O in GB forms hydrogen bonds with the -OH of PVPK30 or urea; this produces a nice solid dispersion and significantly improves the dissolution of GB in water. When GB is in a solid dispersion system, GB’s dissolution in water could be enhanced from 30% to 80%. Furthermore, it may even be produced as a solid agent.

Ginkgolide B exerts anti-inflammatory and chondroprotective activity in LPS-induced chondrocytes.

Osteoarthritis (OA) is one of degenerative and chronic diseases of articular joints. Articular cartilage is an avascular tissue, and its primary cellular component is chondrocytes. The main characteristic of OA is non-classic inflammation and cartilage degeneration. Ginkgolide B (GB) is a component of Ginkgo biloba which has diverse bioactivities. The present study uses an in vitro experimental model to detect the underlying anti-inflammatory and chondroprotective effects of GB and provides a new way for future clinical therapy of OA. Rat chondrocytes were isolated, cultured and treated with 1 μg/mL lipopolysaccharide (LPS) and/or different concentrations of GB. Cell Counting Kit-8 (CCK-8) was used to test the cell viability of chondrocytes, and chondrocytes apoptosis was detected using a cell apoptosis kit. Collagen-II and aggrecan expression were detected by immunohistochemistry. Relative expression of genes was detected by real-time PCR and western blot. Ginkolide B did not inhibit chondrocyte proliferation, and ginkgolide B inhibited LPS induced matrixdegradation in chondrocytes. Ginkgolide B also reversed LPS-induced collagen-II and aggrecan decreased in chondrocytes via upregulated synthesis-related gene expression and downregulated matrix-degrading enzyme gene expression. Furthermore, we found that ginkgolide B significantly inhibited LPS-induced MAPK pathway activation. The results of our study suggest that ginkgolide B exerted anti-inflammatory and chondroprotective effects in LPS-induced chondrocytes, and might be an underlying therapy for OA afterwards.