Effect of Ginkgolide in Ischemic Stroke patients with large Artery Atherosclerosis: Results from a randomized trial.

BACKGROUND: Dual antiplatelet therapy is considered beneficial in acute ischemic stroke (AIS) patients with intracranial artery stenosis (ICAS), with more bleeding events. Ginkgolide is shown to reduce platelet activation after infarction, which might be of benefit in AIS. We aimed to explore the effect of Ginkgolide in AIS patients with ICAS. METHODS: This was a randomized, double-blinded, placebo-controlled trial conducted at 61 centers in China. Within 72 h after onset, consecutive patients diagnosed as AIS with ICAS were randomized to either Ginkgolide or placebo treatment. The primary outcome was the composite of mortality and recurrent stroke (ischemic or hemorrhagic) during first 4 weeks in an intention-to-treat analysis. Secondary functional outcome was assessed by modified Rankin Scale and improvement of stroke severity was assessed by National Institution of Health Stroke Scale at day 28. Safety outcome was measured by the rate of severe adverse event (SAE). RESULTS: There were 936 patients randomized to either Ginkgolide or placebo treatment. Their average age was 64.2 ± 10.4 years old and 36.0% of the patients were female. The composite index event occurred in six patients in placebo group, and none occurred in Ginkgolide group (risk ratio 1.01; 95% CI 1.00-1.02). There were more patients who achieved favorable outcome in Ginkgolide group, compared with that of the placebo group (OR 2.16, 95%CI 1.37-3.41). SAE occurred in five (1.1%) patients in the Ginkgolide group and three (0.6%) in the placebo group (OR0.60, 95CI% 0.14-2.53). Intracranial hemorrhage occurred in 1/473 (0.2%) in the placebo group. CONCLUSIONS: Ginkgolide, working as PAF antagonist, may reduce recurrent stroke in AIS with ICAS patients within 72 hours after onset. It might be an optional treatment in moderate-to-severe AIS patients with ICAS. (http://www.chictr.org.cn Number as ChiCTR-IPR-17012310).

Diterpene Ginkgolides Meglumine Injection inhibits apoptosis induced by optic nerve crush injury via modulating MAPKs signaling pathways in retinal ganglion cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Diterpene Ginkgolides Meglumine Injection (DGMI) is made of extracts from Ginkgo biloba L, including Ginkgolides A, B, and K and some other contents, and has been widely used as the treatment of cerebral ischemic stroke in clinic. It can be learned from the "Compendium of Materia Medica" that Ginkgo possesses the effect of "dispersing toxin". The ancient Chinese phrase "dispersing toxin" is now explained as elimination of inflammation and oxidative state in human body. And it led to the original ideas for today's anti-oxidation studies of Ginkgo in apoptosis induced by optic nerve crush injury. AIM OF THE STUDY: To investigate the underlying molecular mechanism of the DGMI in retinal ganglion cells (RGCs) apoptosis. MATERIALS AND METHODS: TUNEL staining was used to observe the anti-apoptotic effects of DGMI on the adult rat optic nerve injury (ONC) model, and flow cytometry and hoechst 33,342 staining were used to observe the anti-apoptotic effects of DGMI on the oxygen glucose deprivation (OGD) induced RGC-5 cells injury model. The regulation of apoptosis and MAPKs pathways were investigated with Immunohistochemistry and Western blotting. RESULTS: This study demonstrated that DGMI is able to decrease the conduction time of F-VEP and ameliorate histological features induced by optic nerve crush injury in rats. Immunohistochemistry and TUNEL staining results indicated that DGMI can also inhibit cell apoptosis via modulating MAPKs signaling pathways. In addition, treatment with DGMI markedly improved the morphological structures and decreased the apoptotic index in RGC-5 cells. Mechanistically, DGMI could significantly inhibit cell apoptosis by inhibiting p38, JNK and Erk1/2 activation. CONCLUSION: The study shows that DGMI and ginkgolides inhibit RGCs apoptosis by impeding the activation of MAPKs signaling pathways in vivo and in vitro. Therefore, the present study provided scientific evidence for the underlying mechanism of DGMI and ginkgolides on optic nerve crush injury.

Polymeric Nanoparticles-Based Brain Delivery with Improved Therapeutic Efficacy of Ginkgolide B in Parkinson's Disease.

PURPOSE: Ginkgolide B (GB) is a terpene lactone derivative of Ginkgo biloba that is believed to function in a neuroprotective manner ideal for treating Parkinson's disease (PD). Despite its promising therapeutic properties, GB has poor bioavailability following oral administration and cannot readily achieve sufficient exposure in treated patients, limiting its clinical application for the treatment of PD. In an effort to improve its efficacy, we utilized poly(ethylene glycol)-co-poly(ε-caprolactone) (PEG-PCL) nanoparticles as a means of encapsulating GB (GB-NPs). These NPs facilitated the sustained release of GB into the blood, thereby improving its ability to accumulate in the brain and to treat PD. METHODS AND RESULTS: Using Madin-Darby canine kidney (MDCK) cells, we were able to confirm that these NPs could be taken into cells via multiple nonspecific mechanisms including micropinocytosis, clathrin-dependent endocytosis, and lipid raft/caveolae-mediated endocytosis. Once internalized, these NPs tended to accumulate in the endoplasmic reticulum and lysosomes. In zebrafish, we determined that these NPs were readily able to undergo transport across the chorion, gastrointestinal, blood-brain, and blood-retinal barriers. In a 1-methyl-4-phenylpyridinium ion (MPP+)-induced neuronal damage model system, we confirmed the neuroprotective potential of these NPs. Following oral administration to rats, GB-NPs exhibited more desirable pharmacokinetics than did free GB, achieving higher GB concentrations in both the brain and the blood. Using a murine PD model, we demonstrated that these GB-NPs achieved superior therapeutic efficacy and reduced toxicity relative to free GB. CONCLUSION: In conclusion, these results indicate that NPs encapsulation of GB can significantly improve its oral bioavailability, cerebral accumulation, and bioactivity via mediating its sustained release in vivo.

Preparation of Ginkgolide Solid Dispersions with Low-Molecular-Weight Chitosan and Assessment of their Protective Effect on Isoproterenol- Induced Myocardial Injury.

BACKGROUND: Ginkgolides are widely used in cardio-protective therapy; however, poor bioavailability currently limits their application. OBJECTIVE: The purpose of this study was to demonstrate whether solid dispersions prepared with Low- Molecular-Weight Chitosan (LMWC) could improve the protective effect of ginkgolides on Myocardial Injury (MI). METHODS: Ginkgolide Solid Dispersions (GKSD) were prepared with LMWC. Their properties were then characterized using differential scanning calorimetry, X-ray diffraction, scanning electron microscopy and Fourier transform infrared spectroscopy. In vivo pharmacokinetic studies were performed in rats, and the protective effect of GKSD on MI was investigated by western blotting and immunohistochemical analyses. RESULTS: Drug dissolution testing showed that GDSD were released at a significantly higher rate than ginkgolides, dissolved by alternative methods, suggesting that LMWC facilitates the release of ginkgolides. Differential scanning calorimetry, X-ray diffraction, scanning electron microscopy, and Fourier transform infrared spectroscopy all showed that GKSD was amorphous. In-vivo testing revealed larger AUC0-t, higher Cmax, and shorter Tmax for GKSD compared to that in original ginkgolides. Myocardial injury was induced in rats with isoproterenol to test the protective effect of GKSD. GKSD alleviated MI and reduced myocardial fibrosis, as observed by Hematoxylin and Eosin staining. Compared with the crude drug group, the secretion of malonyl dialdehyde and nitric oxide and expression of NOX-2 and NOX-4 were lower. The activities of the cardiac marker enzymes SOD, CAT, GPX, GPX-1, and GSH were higher in GKSD-administered rats, indicating a beneficial effect of GKSD in eliminating free radicals during myocardial injury. Additionally, western blotting and immunohistochemical analysis showed that GKSD markedly reduced the expression of signaling proteins RHOA, ROCK1, ROCK2, and RAC1. CONCLUSION: Solid dispersions prepared with low molecular weight chitosan improved the oral bioavailability of ginkgolide and enhanced its protective effect on myocardial injury.

Diterpene Ginkgolides Exert an Antidepressant Effect Through the NT3-TrkA and Ras-MAPK Pathways.

BACKGROUND: Depression is a highly prevalent mental illness that severely impacts the quality of life of affected individuals. Our recent studies demonstrated that diterpene ginkgolides (DG) have antidepressant effects in mice. However, the underlying molecular mechanisms remained much unclear. METHODS: In this study, we assessed the antidepressant effects of chronic DG therapy in rats by evaluating depression-related behaviors, we also examined potential side effects using biochemical indicators. Furthermore, we performed an in-depth molecular network analysis of gene-protein-metabolite interactions on the basis of metabolomics. RESULTS: Chronic DG treatment significantly ameliorated the depressive-like behavioral phenotype. Furthermore, the neurotrophin signaling-related NT3-TrkA and Ras-MAPK pathways may play an important role in the antidepressant effect of DG in the hippocampus. CONCLUSION: These findings provide novel insight into the mechanisms underlying the antidepressant action of DG, and should help advance the development of new therapeutic strategies for depression.

Intraperitoneal injection of ginkgolide B, a major active compound of Ginkgo biloba, dose-dependently increases the amount of wake and decreases non-rapid eye movement sleep in C57BL/6 mice.

The terpene lactones of Ginkgo biloba extract, namely ginkgolides (A, B, and C) and bilobalide, possess antioxidant, anti-inflammatory, and neuroprotective effects. They are widely prescribed for the treatment of cerebral dysfunctions and neurological impairments. In addition, they demonstrate antagonistic action at the gamma-aminobutyric acid type A and glycine receptors, which are members of the ligand-gated ion channel superfamily. In the present study, the effects of ginkgolides (A, B, and C) and bilobalide on sleep in C57BL/6 mice were investigated. Ginkgolide B was found to dose-dependently increase the amount of wake and decrease that of non-rapid eye movement sleep without changes in the electroencephalography power density of each sleep/wake stage, core body temperature and locomotor activity for the first 6 h after intraperitoneal injection. Of note, the amount of wake after injection of 5 mg/kg of ginkgolide B showed a significant increase (14.9 %) compared with that of vehicle (P = 0.005). In contrast, there were no significant differences in the amount of sleep, core body temperature, and locomotor activity in the mice injected with ginkgolide A and C. Bilobalide briefly induced a decrease in locomotor activity but did not exert significant effects on the amounts of sleep and wake. The modes of action of the wake-enhancing effects of ginkgolide B are unknown. However, it may act through the antagonism of gamma-aminobutyric acid type A and glycine receptors because it is established that these inhibitory amino acids mediate sleep and sleep-related physiology. It is of interest to further evaluate the stimulant and awaking actions of ginkgolide B on the central nervous system in clinical and basic research studies.

Highly stabilized nanocrystals delivering Ginkgolide B in protecting against the Parkinson's disease.

As a major cause of neurodegeneration in the elderly, Parkinson's disease (PD) has attracted intense research attention. PD results from a decline in the numbers of dopaminergic neurons. Due to low levels of plasma exposure and the drug efflux properties of neuronal cells, orally delivering anti-PD drugs is challenging. Nanocrystals (NCs) can increase dissolution velocities and saturation solubility, improving oral bioavailability and brain uptake. In this study, Ginkgolide B (GB), a potent anti-Parkinsonism compound, was selected to verify the utility of NCs to effectively accumulate GB in both the blood and brain. Highly stabilized GB-NCs had small sizes, high rates of dissolution, enhanced cellular uptake and permeability. The GB-NCs could protect neurons against cytotoxicity induced by MPP+, and showed no toxicity in zebrafish. Fluorescent imaging in zebrafish indicated high levels of the NCs in both the gut and brain. When orally administrated to rats, the GB-NCs showed higher drug plasma levels and neuronal drug distributions when compared to control groups. Importantly, in MPTP-induced PD model, GB-NCs treatment resulted in improved behavior, reduced dopamine deficiency, and elevated dopamine metabolite levels. In summary, these highlight the fabrication of GB-NCs as effective drug carriers for the neuronal delivery of anti-PD therapies.

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 ±â€¯106.67) % and (437.45 ±â€¯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 ameliorates myocardial ischemia reperfusion injury in rats via inhibiting endoplasmic reticulum stress.

PURPOSE: Ginkgolide 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 METHODS: Myocardial 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. RESULTS: The 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. CONCLUSION: GB protected against myocardial I/R injury through inhibiting ER stress-induced apoptosis via PI3K/AKT/mTOR signaling pathway.

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 A Prevents the Amyloid-ß-Induced Depolarization of Cortical Neurons.

Utilizing the N-methyl-d-aspartate (NMDA) receptor antagonist as a strategy, memantine is the only agent available for clinically treating mild to severe Alzheimer's disease (AD). Our aim was to develop novel similar herb-based drugs. Using a screening platform, ginkgolide A (GA), a pure compound extracted from Ginkgo biloba, was found to attenuate amyloid ß (Aß)-induced abnormal depolarization in mouse primary cortical neurons. Using receptor agonists, it was determined that GA inhibits both NMDA receptors and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Furthermore, the Aß-induced increase in c-Jun N-terminal kinase phosphorylation in neurons was prevented by GA. Body weight, glutamate oxaloacetate transaminase, glutamic-pyruvic transaminase, liver histology, and kidney histology were similar when the wild-type/AD animal model mice with and without GA treatment were compared. This pure compound improves the memory of wild-type mice. Our findings indicate that GA has great potential clinically for the treatment of AD because it might target NMDA receptors just like memantine.

Comparing the role of Ginkgolide B and Ginkgolide K on cultured astrocytes exposed to oxygen­glucose 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 oxygen­glucose deprivation (OGD). The results demonstrated that GB and GK exhibit many different actions. The level of the platelet­activating factor (PAF) was elevated on astrocytes exposed to OGD, and inhibited by GB and GK treatment. Although GB and GK inhibited the expression of p­NF­κB/p65, GK exerted stronger anti­inflammatory and antioxidant effects on astrocytes exposed to OGD than GB by inhibiting interleukin (IL)­6 and tumor necrosis factor­α, and inducing IL­10 and the nuclear factor­erythroid 2­related factor 2/HO­1 signaling pathway. When compared with GB treatment, GK treatment maintained high levels of phosphoinositide 3­kinase/phosphorylated­protein 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.

Diterpene ginkgolides meglumine injection protects against paraquat-induced lung injury and pulmonary fibrosis in rats.

In this study, we aimed to investigate the effects of diterpene ginkgolides meglumine injection (DGMI) on paraquat (PQ)-induced lung injury and pulmonary fibrosis in rats. Male SD rats were challenged by PQ (20?mg/kg, i.p.) with or without either DGMI (1.25, 2.5, 5?mg/kg, i.p.) or Edaravone (EDA, 6?mg/kg, i.p.) posttreatment 2?h after PQ administration. Lung tissues were removed for biochemical analyses and pathological examinations on day 1, day 3, day 7, day 14 and day 21. Results showed that the administration of DGMI significantly increased the survival of PQ-challenged rats. At the same time, DGMI reversed the increase of Malondialdehyde (MDA) level and the decrease of Super Oxide Dismutase (SOD) level in lung tissues. Moreover, lung to body weight ratio, Interleukin-1beta (IL-1?), Interleukin-6 (IL-6) and Tumor necrosis factor-alpha (TNF-?) levels in lung tissues were reduced compared with the model group. H&E and Masson staining revealed that DGMI (5?mg/kg) alleviated histological injury and pulmonary fibrosis, and EDA (6?mg/kg) exerted approximate effects. Immunohistochemistry staining presented that the benefit effects of DGMI were associated with its ability to activate Akt-Nrf-2 pathway. In conclusion, these results suggest that DGMI possesses potential role in future therapies for PQ-induced lung injury and pulmonary fibrosis.

Enhancing amplification of late-outgrowth endothelial cells by bilobalide.

Transfusion of autologous late-outgrowth endothelial cells (OECs) is a promising treatment for restenosis after revascularization. Preparing cells by in vitro amplification is a key step to implement the therapy. This study aimed to demonstrate that bilobalide, a terpenoid, enhances the OEC amplification. Human-, rabbit- and rat OECs and a mouse femoral artery injury model were used. Expanding OECs used endothelial growth medium-2 as the standard culture medium while exploring the mechanisms used endothelial basal medium-2. Proliferation assay used MTT method and BrdU method. Migration assay used the modified Boyden chamber. Intracellular nitric oxide, superoxide anion, hydroxyl radical/peroxynitrite and H2 O2 were quantified with DAF-FM DA, dihydroethidium, hydroxyphenyl fluorescein and a H2 O2 assay kit, respectively. Activated ERK1/2 and eNOS were tested with the Western blot. Bilobalide concentration-dependently enhanced OEC number increase in vitro. Transfusion of bilobalide-based human OECs into femoral injured athymia nude mouse reduced the intimal hyperplasia. Bilobalide promoted OEC proliferation and migration and increased the intracellular nitric oxide level. L-NAME, a NOS inhibitor, inhibits but not abolishes OEC proliferation, migration and ERK1/2 activation. Bilobalide concentration-dependently enhanced the eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation in activated OECs. Bilobalide alleviates the increase in hydroxyl radical/peroxynitrite, superoxide anion and H2 O2 in proliferating OECs. In conclusion, nitric oxide plays a partial role in OEC proliferation and migration; bilobalide increases OEC nitric oxide production and decreases nitric oxide depletion, promoting the OEC number increase; Bilobalide-based OECs are active in vivo. The findings may simplify the preparation of OECs, facilitating the implementation of the autologous-OECs-transfusion therapy.

Formulation Screening and Freeze-Drying Process Optimization of Ginkgolide B Lyophilized Powder for Injection.

The purpose of this study was to prepare ginkgolide B (GB) lyophilized powder for injection with excellent appearance and stable quality through a formulation screening and by optimizing the freeze-drying process. Cremophor EL as a solubilizer, PEG 400 as a latent solvent, and mannitol as an excipient were mixed to increase the solubility of GB in water to more than 18 times (about from 2.5 × 10-4 mol/L (0.106 mg/mL) to 1.914 mg/mL). Formulation screening was conducted by orthogonal design where the content of GB in the solution before lyophilization (using external standard method of HPLC) and reconstitution time after lyophilization were the two evaluation indexes. The optimized formulations were GB in an amount of 2 mg/mL, Cremophor EL in an amount of 16% (v/v), PEG 400 in an amount of 9% (v/v), mannitol in an amount of 8% (w/v), and the solution pH of 6.5. Through four single-factor experiments (GB adding order, preparation temperature of GB solution, adding amount, and adsorption time of activated carbon), the preparation process of GB solution was confirmed. The glass transition temperature of maximally GB freeze-concentrated solution was - 17.6°C through the electric resistance method. GB lyophilized powder began to collapse at - 14.0°C, and the fully collapsed temperature was - 13.0°C, which were determined by freeze-drying microscope. When the collapse temperature was determined, the primary drying temperature was obtained. Thereby, the freeze-drying curve of GB lyophilized powder was initially identified. The freeze-drying process was optimized by orthogonal design, the qualified product appearance and residual moisture content were the two evaluation indexes. The optimized process parameters and process were (1) shelf temperature, decreased from room temperature to - 45.0°C, at 0.5°C/min in 2 h; (2) shelf temperature increased from - 45.0 to - 25.0°C, at 0.1°C/min, maintained for 3 h, and the chamber pressure was held at 10 Pa; (3) shelf temperature was increased from - 25.0 to - 15.0°C at 0.1 °C/min, maintained for 4 h, and the chamber pressure was held at 10 Pa; and (4) shelf temperature was increased from - 15.0 to 20.0°C at 1.0 °C/min, maintained for 4 h, and the chamber pressure was raised up to 80 Pa. In these lyophilization process conditions, the products complied with relevant provisions of the lyophilized powders for injection. Meanwhile, the reproducibility was satisfactory. Post-freezing annealing had no significantly beneficial effects on shortening the freeze-drying cycle and improving the quality of GB lyophilized powder.

Pharmacokinetics of the prototype and hydrolyzed carboxylic forms of ginkgolides A, B, and K administered as a ginkgo diterpene lactones meglumine injection in beagle dogs.

Ginkgo diterpene lactones meglumine injection (GDLI) is a commercially available product used for neuroprotection. However, the pharmacokinetic properties of the prototypes and hydrolyzed carboxylic forms of the primary components in GDLI, i.e., ginkgolide A (GA), ginkgolide B (GB), and ginkgolide K (GK), have never been fully evaluated in beagle dogs. In this work, a simple, sensitive, and reliable method based on ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS) was developed, and the prototypes and total amounts of GA, GB, and GK were determined in beagle dog plasma. The plasma concentrations of the hydrolyzed carboxylic forms were calculated by subtracting the prototype concentrations from the total lactone concentrations. For the first time, the pharmacokinetics of GA, GB, and GK were fully assessed in three forms, i.e., the prototypes, the hydrolyzed carboxylic forms, and the total amounts, after intravenous administration of GDLI in beagle dogs. It was shown that ginkgolides primarily existed in the hydrolyzed form in plasma, and the ratio of hydrolysates to prototype forms of GA and GB decreased gradually to a homeostatic ratio. All of the three forms of the three ginkgolides showed linear exposure of AUC to the dosages. GA, GB, and GK showed a constant half-life approximately 2.7, 3.4, and 1.2 h, respectively, which were consistent for the forms at three dose levels (0.3, 1.0, and 3.0 mg·kg-1) and after a consecutive injection of GDLI for 7 days (1.0 mg·kg-1).

Ginkgolide A ameliorates non-alcoholic fatty liver diseases on high fat diet mice.

Non-alcoholic fatty liver disease (NAFLD) is one of the most common diseases worldwide and has continuously increased. NAFLD refers to a spectrum of diseases ranging from fatty liver to steatohepatitis, cirrhosis, and even to hepatocyte carcinoma. Excessive fatty acid enters the cell and the mitochondria undergo stress and unremoved ROS can trigger a form of cell apoptosis known as 'lipoapoptosis'. NASH arises from damaged liver hepatocytes due to lipotoxicity. NASH not only involves lipid accumulation and apoptosis but also inflammation. Ginkgo biloba has been tested clinical trials as a traditional medicine for asthma, bronchitis and cardiovascular disease. The effects of Ginkgolide A (GA), derived from the ginkgo biloba leaf, are still unknown in NAFLD. To determine the protective effects of GA in NAFLD, we examined the fatty liver disease condition in the non-esterified fatty acid (NEFA)-induced HepG2 cell line and in a high fat diet mouse model. The findings of this study suggest that GA is non-toxic at high concentrations in hepatocytes. Moreover, GA was found to inhibit cellular lipogenesis and lipid accumulation by causing mitochondrial oxidative stress. GA showed hepatoprotective efficacy by inducing cellular lipoapoptosis and by inhibiting cellular inflammation. The results demonstrated that GA may be feasible as a therapeutic agent for NAFLD patients.

Pharmacokinetics of ginkgolides A, B and K after single and multiple intravenous infusions and their interactions with midazolam in healthy Chinese male subjects.

PURPOSE: Ginkgo terpene lactones meglumine injection (GMI) is a novel preparation of traditional Chinese medicine that contains ginkgolides A, B and K (GA, GB, GK, respectively) as its primary components. In this study we evaluated the safety, tolerability and pharmacokinetics of these three ginkgolides after single and multiple intravenous infusions of GMI. We also investigated the effect of GMI on cytochrome P450 3A4 (CYP3A4) in healthy Chinese volunteers. METHODS: In this open-label, placebo-controlled study 15 subjects were randomly assigned to receive GMI or matched placebo (4:1 ratio). All subjects first received midazolam (MDZ) on day 1, followed by a 6-day washout. On Day 8, the subjects were started on once-daily dosing of either GMI or placebo for 14 days. Lastly, on Day 22 the subjects were given second dose of MDZ + GMI or MDZ + placebo. Plasma concentrations of ginkgolides, MDZ and its metabolite 1-hydroxy midazolam were quantified. RESULTS: The steady-state conditions of GA, GB and GK were achieved after 6 days of daily dosing. Following a single dose of GMI (Day 8) the area under the concentration-timecurve from zero to 24 h after administration (AUC0-24h) of GA, GB and GK (arithmetic ± standard deviation) was 4.10 ± 1.06, 4.61 ± 1.31 and 0.127 ± 0.102 h µg/mL, respectively; the corresponding values following multiple doses of GMI (Day 19) were 3.94 ± 1.16, 5.00 ± 1.55 and 0.118 ± 0.096 h µg/mL, respectively. The mean accumulation ratios were 0.95, 1.08 and 0.89 for GA, GB and GK, respectively. Additionally, the geometric mean [peak concentration (Cmax) and AUC0-24h] ratios of MDZ and 1-hydroxy midazolam were all within the specified acceptance ranges in the MDZ + placebo treatment and MDZ + GMI treatment. CONCLUSIONS: Our results show that GMI was well tolerated during the entire study. There was no systemic accumulation and no significant effects on the pharmacokinetics of MDZ in healthy Chinese male subjects after repeated dosing of GMI.

Protective effects of bilobalide against ethanol-induced gastric ulcer in vivo/vitro.

Bilobalide (BI) has been widely known as a unique constituent extracted from Ginkgo biloba. The aim of the current study was to reveal the potential efficacy as well as the underlying mechanism of the action of BI on ethanol-induced lesion in gastric mucosa in vivo/vitro. Ethanol (0.2ml/kg) was applied to induce gastric ulcer mice model. Our results indicated that treatment with BI markedly decreased the levels of interleukin-6 (IL-6), IL-1ß and tumor necrosis factor-α (TNF-α) in vivo. Additionally, BI intervation exhibited elevated myeloperoxidase (MPO) level in stomach, increased superoxide dismutase (SOD) activity and decreased malonaldehyde (MDA) content in serum and stomach when compared with those of the model group. It could be also observed that inhibited MAPK/NF-κB pathway expressions occurred after BI treatment both in vivo and in vitro. Taken together, BI exerted a gastro-protective effect against gastric ulceration, which was presumed to be associated with MAPK/NF-κB pathway.

Bilobalide alleviates depression-like behavior and cognitive deficit induced by chronic unpredictable mild stress in mice.

Bilobalide (BB), a unique constituent of Ginkgo biloba, has powerful neuroprotection and stress-alleviating properties. However, whether BB exerts a positive effect on depression and cognitive deficit induced by chronic stress is not known. The present study was designed to investigate the influence of BB on depression and cognitive impairments induced by chronic unpredictable mild stress (CUMS) in mice. During daily exposure to stressors for 5 consecutive weeks, mice were administered BB at the doses of 0, 3, or 6 mg/kg/day intraperitoneally. We replicated the finding that CUMS induced depression-like behavior and cognitive deficits as the CUMS+vehicle (VEH) group showed a significant increase in immobility in the tail suspension test, a decrease in the discrimination index of the novel object recognition task, and increased latency to platform and decreased number of platform crossings in the Morris water maze compared with the control+VEH group. Chronic administration of BB effectively reversed these alterations. In addition, the CUMS+VEH group showed significantly higher levels of baseline serum corticosterone than those of the control+VEH group and BB dose-dependently inhibited this effect. Our results suggest that BB may be useful for inhibition of depression-like behavior and cognitive deficits, and this protective effect was possibly exerted partly through an action on the hypothalamic-pituitary-adrenal axis.