Ginkgolide K delays the progression of osteoarthritis by regulating YAP to promote the formation of cartilage extracellular matrix.

Osteoarthritis (OA) is a degenerative disease characterized by cartilage wear and degradation. Ginkgolide K (GK) is a natural compound extracted from Ginkgo biloba leaves and possesses anti-inflammatory and anti-apoptotic effects. We found that the biological characteristics of GK were highly consistent with those of OA medications. This study aimed to determine and verify the therapeutic effect of GK on OA and mechanism of its therapeutic effect. For the in vivo experiment, OA rats were regularly injected in the articular cavity with GK, and the curative effects were observed after 4 and 8 weeks. For the in vitro experiment, we treated OA chondrocytes with different concentrations of GK and then detected the related indices of OA. Through the in vivo and in vitro experiments, we found that GK could promote the production of major components of the cartilage extracellular matrix. Transcriptome sequencing revealed that GK may activate hypoxia-inducible factor 1 alpha via the hypoxia signaling pathway, which, in turn, activates yes-associated protein and inhibits apoptosis of OA chondrocytes. GK has a therapeutic effect on OA and, therefore, has the potential to be developed into a new drug for OA treatment.

Effect of ginkgolide K on calcium channel activity in Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative dementia with the key pathological hallmark of amyloid deposits that may induce mitochondrial dysfunction. Ginkgolide K (GK) has been proven to have neuroprotective effects. The present study sought to explore the neuroprotective effect of GK through regulation of the expression of mitochondrial Ca2+ uniporter (MCU) in the pathology of AD. SH-SY5Y cells were cultured and the expression of MCU was enhanced by transfection of MCU recombinant vectors or knockdown by MCU small interfering RNA. The cells were treated with GK and amyloid ß (Aß). Thereafter, the effects of GK, MCU expression and Aß on viability and apoptosis of SH-SY5Y cells were examined via a WST-1 assay, flow cytometry and Caspase-3/8 activity assays, respectively. The effects of GK, MCU expression and Aß on the calcium levels in mitochondria were also examined. The regulatory effect of GK on MCU expression was examined by reverse transcription-quantitative PCR and western blot analysis. Furthermore, APP/PS1 mice received supplementation with GK and their cognitive ability was then examined through water maze tests, while the expression of MCU was examined using immunohistochemistry. The results indicated that enhancing the expression of MCU inhibited cell viability and promoted apoptosis. GK protected cells from amyloid-induced cytotoxicity by promoting cell viability and preventing cell apoptosis. The neuroprotective effect of GK was abolished when MCU expression was knocked down. GK decreased the expression of MCU in vitro and downregulation of MCU decreased the calcium level in mitochondria. Treatment with GK in APP/PS1 mice downregulated the expression of MCU in the brains and alleviated cognitive impairment. In conclusion, the present study demonstrated that the administration of GK protected neurons by preventing apoptosis. Furthermore, the neuroprotective effect of GK in neuronal cells was indicated to be related to the inhibition of MCU expression. Therefore, administration of GK may be a promising strategy for treating AD.

Ginkgolide K inhibited neurovascular unit injury against oxygen-glucose deprivation and reperfusion by regulating hypoxia-inducible factor-1α pathway / 中国药理学通报

Aim To investigate the protective effects of ginkgolide K (GK) on neurovascular unit injured by ischemic stroke and the potential mechanism associated with hypoxia-inducible factor-1α (HIF-1α) pathway. Methods The BV-2 cells and EA. hy926 cells suffered from oxygen-glucose deprivation and reperfusion (OGD/R) were applied to mimic the injury of neurovascular unit induced by cerebral ischemia in vitro. After 4 h OGD insult, BV-2 cells and EA. hy926 cells received reperfusion and treated with GK. The levels of inflammatory cytokines in the supernatant of BV-2 cells were detected, while the protective effects of GK on EA. hy926 cells were also evaluated after GK administration for 24 h. The p-Akt and p-Erk expressions were examined by Western blot after 1 h of GK treatment, while HIF-1α was detected after 6 h of GK treatment. In addition, PI3K inhibitor LY294002 was applied to further verify the potential mechanisms underlying the beneficial effects of GK. The expressions of p-Akt after 1 h of GK treatment, and the protein levels of HIF-1α pathway after 6 h of GK treatment were also analyzed by Western blot. Results GK significantly inhibited the levels of TNF-α, IL-6 and IL-1β in supernatant of BV-2 cells injured by OGD/R, through increasing p-Akt and decreasing p-Erk expressions, and then affecting HIF-1α pathway. In addition, LY294002 reduced the regulatory effect of GK. Furthermore, GK significantly improved viability and inhibited the release of LDH in supernatant of EA. hy926 cells suffered from OGD/R, and up-regulated the expressions of p-Akt, HIF-1α, HO-1 and VEGF, while cleaved caspase-3/9 was inhibited. Conclusions GK exerts multi-effects on reducing neurovascular unit injury induced by ischemic stroke, and the potential mechanism might be associated with the different regulatory effects of HIF-1α in different cells.

Ginkgolide K supports remyelination via induction of astrocytic IGF/PI3K/Nrf2 axis.

Although several therapies are approved, none promote re-myelination in multiple sclerosis (MS) patients, limiting their ability for sustained recovery. Thus, treatment development in MS has the opportunity to tackle the challenges, including experimental therapies targeting neuroprotection and re-myelination. Here, we provide a novel therapeutic target for Ginkgolide K (GK) that is now becoming a very critical natural compound to treat demyelination and neurodegeneration. GK improves behavioral dysfunction and demyelination in cuprizone (CPZ) model, followed by the migration and enrichment of astrocytes in the corpus callosum. Both in vitro and in vivo experiments demonstrates that GK triggers the upregulation of Nrf2/HO-1 in astrocytes and inhibition of p-NF-kB/p65, which is associated with the outcome of anti-inflammation and anti-oxidation by suppressing the production of IL-6 and TNFα as well as nitric oxide and iNOS in astrocytes. Further findings suggest that IGF/PI3K, but not BDNF, was induced in the corpus callosum after GK treatment, revealing that Nrf2 activation inhibited caspase-3 and apoptosis in O4+ oligodendrocytes possibly through IGF/PI3K signaling molecules. Since the current immunomodulatory therapies for MS have failed to prevent patients from entering the progressive phase of the disease, thus targeting Nrf2 in astrocytes with GK would be an ideal strategy for myelin protection and regeneration.

The therapeutic potential of ginkgolide K in experimental autoimmune encephalomyelitis via peripheral immunomodulation.

Multiple sclerosis is a T cell-mediated inflammatory, demyelinating disease of the central nervous system, accompanied by neuronal degeneration. Based on the anti-inflammatory effects of Ginkgolide K (GK), a platelet activating factor antagonist, we explored the possible application of GK in the treatment of MS. The results showed that GK effectively ameliorated the severity of experimental autoimmune encephalomyelitis. The intervention of GK inhibited the infiltration of inflammatory cells and demyelination in the spinal cord. At the same time, the expression of the inflammation-related molecules TLR4, NF-κB, and COX2 in the spinal cord was significantly lower in the GK-treated mice, indicating that GK intervention can inhibit the inflammatory microenvironment of the spinal cord in EAE mice. In mouse spleen lymphocytes, GK increased the proportion of regulatory T cells (Treg) and reduced the proportion of T helper 17 cells (Th17), modifying the imbalance between Th17/Treg cells. Additionally, GK shifted macrophage/microglia polarization from M1 to M2 cell type. Importantly, GK inhibited the expression of chemotactic molecules CCL-2, CCL-3 and CCL-5, thereby limiting the migration of inflammatory cells to the spinal cord. Our results provide the possibility that GK may be a promising naturally small molecule compound for the future treatment of MS.

Preparation and characterization of ginkgolide K-loaded PLGA-PEG nanoparticles and its neuroprotective activity in vitro / 中草药

Objective To prepare and characterize ginkgolide K-loaded mPEG-PLGA [poly (D,L-lactide-co-gly-colide)-block-poly (ethylene glycol)] polymer nanoparticles (GK-mPEG-PLGA-NPs) and to evaluate its neuroprotective effect on the H2O2-induced PC12 cells injury in vitro. Methods The PLGA-PEG-COOH polymer was selected as carrier and double emulsion solvent evaporation technique was employed to prepare the stealth nanoparticles. The encapsulation efficiency (EE) and drug load (DL) of GK-mPEG-PLGA-NPs were investigated by HPLC. The size distribution, zeta potential, and surface morphology of GK-mPEG-PLGA-NPs were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The in vitro release of GK-mPEG-PLGA-NPs was examined using phosphate buffer solution (pH 7.4) as the releasing medium for 24 h. The H2O2-induced PC12 cells injury models was established for the investigation of the protective effect of GK-mPEG-PLGA-NPs on nerve cells in vitro. Results EE and DL of GK-mPEG-PLGA-NPs was (83.40 ± 2.85)% and (3.26 ± 0.24) mg/g, respectively. The average diameter of GK-mPEG-PLGA-NPs was (93.19 ± 2.77) nm and zeta potential was (-11.93 ± 1.71) mV. The cumulative rate of drug release was (90.5 ± 4.0)% after 60 h in phosphate buffer solution. GK-mPEG-PLGA-NPs significantly inhibited the apoptosis of PC12 cells and the release of lactic dehydrogenase induced by H2O2. However, the protective action of GK-mPEG-PLGA-NPs on the H2O2-iduced PC12 cells injury was significantly weaker than that of GK. Conclusion Our results proved that GK-mPEG-PLGA-NPs had a sustained release behavior in vitro and the neuroprotective effect of GK-mPEG-PLGA-NPs on H2O2-induced PC12 cells, which indicates that GK-mPEG-PLGA-NPs has the prospect of application and deserves further research. Key words: ginkgolide K; mPEG-PLGA; in vitro release; in vitro neuroprotection; d

Protective effects of ginkgolide k on focal cerebral ischemia reperfusion injury in rats on the basis of amelioration of mitochondrial calcium uniporter / 中国药学杂志

OBJECTIVE: To explore whether the protective mechanism of ginkgolide K on cerebral focal ischemia reperfusion injury in rats induced by middle cerebral artery occlusion (MCAO) was associated with the amelioration of mitochondrial calcium uniporter ( MCU) or not. METHODS: Sprague Dawley (SD) rats were divided into 5 big groups randomly: sham operation group, MCAO group, GK added into RR group, GK group and GK added into SM group. The MCAO rat model were established after cerebral artery ischemia for 2 h and reperfusion for 22 h. Zea Longa 5 score system was used to evaluate neurological deficit score; Determination of brain water content and cerebral infarction areas were determined using gravimetric method and by triphenyltetrazolium chloride(TTC) staining method, respectively. In addition, malondialdehyde (MDA) and superoxide dismutase (SOD), nitric oxide synthase (NOS), nitric oxide (NO) were detected by Elisa. Additionally, mitochondrial[Ca2+] i concentration was estimated with the fluorescence spectrophotomete. The morphological change of the injured brains were observed by HE staining. The expression of caspase-3/8/9 protein were detected by Western blot. RESULTS: Compared with GK group, GK + RR group relieved obviously the neurological deficit score and reduced the cerebral infarction areas, brain water content, mitochondrial[Ca2+]i concentration and MDA, caspase-3/8/9 protein expression while enhance SOD activity. However, the effect of SM on the GK protective activity in MCAO rat injury was the opposite in comparison to GK + RR group. CONCLUSION: The stimulative effect of RR and the inhibitory effect of SM on the GK protection in MCAO rat had proves that the protective mechanism of GK on MCAO rat injury is associate with its down-regulation of the transport capacity of MCU, leading the attenuation of mitochondrial[Ca2+]i influx.

Ginkgolide K promotes angiogenesis in a middle cerebral artery occlusion mouse model via activating JAK2/STAT3 pathway.

Ginkgolide K (GK) is a new compound extracted from the leaves of Ginkgo biloba, which has been recognized to exert anti-oxidative stress and neuroprotective effect on ischemic stroke. While whether it plays an enhanced effect on angiogenesis during ischemic stroke remains unknown. The aim of this study was to investigate the effect of ginkgolide K on promoting angiogenesis as well as the protective mechanism after cerebral ischemia-reperfusion. Using the transient middle cerebral artery occlusion (tMCAO) mouse model, we found that GK (3.5, 7.0, 14.0 mg/kg, i.p., bid., 2 weeks) attenuated neurological impairments, and promoted angiogenesis of injured ipsilateral cortex and striatum after 14 days of cerebral ischemia-reperfusion in mice. Further, GK (3.5 mg/kg in vivo, 10 µM in vitro) significantly up-regulated the expressions of HIF-1α and VEGF in tMCAO mouse brains and in b End3 cells after OGD/R, and GK-induced upregulation of HIF-1α and VEGF in b End3 cells could be abolished by JAK2/STAT3 inhibitor AG490. Our results demonstrate that GK promotes angiogenesis after ischemia stroke through increasing the expression of HIF-1α/VEGF via JAK2/STAT3 pathway, which provide an insight into the novel clinical application of GK and its analogs in ischemic stroke therapy in future.

Ginkgolide K promotes astrocyte proliferation and migration after oxygen-glucose deprivation via inducing protective autophagy through the AMPK/mTOR/ULK1 signaling pathway.

Ischemic stroke is the leading cause of death around the world. Ginkgolide K (GK) has been used to treat ischemic stroke due to its neuroprotective potential. However, the molecular mechanism underlying the neuroprotective effect of GK in ischemic stroke is still almost blank. In this study, astrocytes were divided into four groups: control group, oxygen-glucose deprivation (OGD) group, OGD + GK group and OGD + GK + Compound C (CC) group. The viability and proliferation of astrocytes were examined by Cell Counting Kit-8 assay and 5-ethynyl-20-deoxyuridine (EdU) assay, respectively. Transwell migration and wound scratch assays were conducted to evaluate astrocyte migration. The protein expression in astrocytes were determined by western blot assay. We found that GK pretreatment promoted astrocyte proliferation and migration after OGD as shown by the increase in the viability of astrocytes, glial fibrillary acidic protein level, the number of EdU positive cells and migrated cells, and the migration distance. GK pretreatment induced autophagy after OGD, as indicated by upregulation of autophagy-related protein 7, Beclin-1 protein and increase of microtubule-associated protein 1 light chain 3 (LC3)-II/LC3-I, and downregulation of p62 protein. Moreover, GK pretreatment activated the AMP activated protein kinase (AMPK)/mammalian target of rapamycin (m-TOR)/ULK1 pathway in astrocytes following OGD. Notably, CC treatment blocked the promotory effect of GK on astrocyte proliferation and migration after OGD. Collectively, GK promoted astrocyte proliferation and migration after OGD via inducing protective autophagy through the AMPK/mTOR/ULK1 signaling pathway. Our findings suggested that GK might be a potential agent for cerebral ischemia/reperfusion injury.

[Effect of compatibility of ginkgolide A, ginkgolide B and ginkgolide K].

To investigate the best active compatibility of ginkgolide A, B and K (GA，GB，GK). The effects of GA, GB, GK alone, combinations of each two of them, and combinations of these three components on platelet-activating factor (PAF)-induced platelet aggregation activity and rat cerebral ischemia reperfusion model (tMCAO) were compared in this study. Different compatibilities of GA, GB and GK could significantly reduce the maximum aggregation rate of PAF-induced platelet aggregation, and the effect was most obvious in combination of the three. Different compatibilities of GA, GB and GK could alleviate the neural function, cerebral infarction volume and cerebral edema in the tMCAO model of rats to different degrees, and the effect of combinations of the three was stronger than those of combinations of two and single use. The combination of all of GA, GB and GK had the strongest effect on nerve injury caused by anti-platelet aggregation in tMCAO rats.

Ginkgolide K promotes the clearance of A53T mutation alpha-synuclein in SH-SY5Y cells.

Alpha-synuclein (α-syn) is associated to Parkinson's disease (PD). The aggregated form of α-syn has potential neurotoxicity. Thus, the clearance of α-syn aggregation is a plausible strategy to delay disease progression of PD. In our study, we found that the treatment of Ginkgolide B (GB) and Ginkgolide K (GK) reduced cell death, and enhanced cell proliferation in SH-SY5Y cells, which overexpressed A53T mutant α-syn. Surprisingly, GK, but not GB, promoted the clearance of A53T α-syn, which can be abolished by autophagy inhibitor 3-methyladenine, indicating that GK-induced autophagy intervened in the clearance of A53T α-syn. However, GK did not affect the NEDD4 that belongs to the ubiquitin ligase in the endosomal-lysosomal pathway. Furthermore, GK treatment inhibited the p-NF-kB/p65 and induced the PI3K, BDNF, and PSD-95. Taken together, GK increased the clearance of α-syn, reduced cell death, and triggered complex crosstalk between different signaling pathways. Although our results show a potentially new therapeutic candidate for PD, the details of this mechanism need to be further identified.

Effect of compatibility of ginkgolide A, ginkgolide B and ginkgolide K / 中国中药杂志

To investigate the best active compatibility of ginkgolide A, B and K (GA，GB，GK). The effects of GA, GB, GK alone, combinations of each two of them, and combinations of these three components on platelet-activating factor (PAF)-induced platelet aggregation activity and rat cerebral ischemia reperfusion model (tMCAO) were compared in this study. Different compatibilities of GA, GB and GK could significantly reduce the maximum aggregation rate of PAF-induced platelet aggregation, and the effect was most obvious in combination of the three. Different compatibilities of GA, GB and GK could alleviate the neural function, cerebral infarction volume and cerebral edema in the tMCAO model of rats to different degrees, and the effect of combinations of the three was stronger than those of combinations of two and single use. The combination of all of GA, GB and GK had the strongest effect on nerve injury caused by anti-platelet aggregation in tMCAO rats.

Ginkgolide K attenuates neuronal injury after ischemic stroke by inhibiting mitochondrial fission and GSK-3ß-dependent increases in mitochondrial membrane permeability.

Ginkgolide K (GK) belongs to the ginkgolide family of natural compounds found in Ginkgo biloba leaves, which have been used for centuries to treat cerebrovascular and cardiovascular diseases. We evaluated the protective effects of GK against neuronal apoptosis by assessing its ability to sustain mitochondrial integrity and function. Co-immunoprecipitation showed that Drp1 binding to GSK-3ß was increased after an oxygen-glucose deprivation/reperfusion (OGD/R) insult in cultured neuroblastoma cells. This induced Drp1 and GSK-3ß translocation to mitochondria and mitochondrial dysfunction, which was attenuated by GK. GK also reduced mitochondrial fission by increasing Drp1 phosphorylation at Ser637 and inhibiting mitochondrial Drp1 recruitment. In addition, GK exposure induced GSK-3ß phosphorylation at Ser9 and enhanced the interaction between adenine nucleotide translocator (ANT) and p-GSK-3ß. This interaction suppressed the interaction between ANT and cyclophilin D (CypD), which inhibited mitochondrial permeability transition pore (mPTP) opening. Similarly, suppression of mitochondrial fission by Mdivi-1 also inhibited GSK-3ß-induced mPTP opening. Treating mice with GK prevented GSK-3ß and Drp1 translocation to mitochondria and attenuated mitochondrial dysfunction after middle cerebral artery occlusion. We therefore propose that by inhibiting mitochondrial fission and attenuating mPTP opening, GK exerts neuroprotective effects that mitigate or prevent neuronal damage secondary to ischemic stroke.

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.

[Effects of ginkgolide A, B and K on platelet aggregation].

To investigate the effects of ginkgolide A (GA), ginkgolide B (GB) and ginkgolide K (GK) on platelet aggregation in rabbits, and compare the similarities and differences among these three components. The effects of different doses of ginkgolide A, B and K on platelet aggregation induced by platelet activating factor (PAF) were observed by using in vitro experiment. The results showed that three compounds could inhibit platelet aggregation induced by PAF in vitro, and the intensity was GK> GB> GA. It was further found that all of them can mobilize [Ca2+]i and enhance intracellular c-AMP level in a dose-dependent manner, which was consistent to the ability to antagonize PAF receptor. These findings indicated that GK was highly selective for PAF receptor, and may inhibit platelet aggregation by activating cAMP signaling pathway and inhibiting intracellular [Ca2+]i mobilization; GB and GA also had strong antagonism to PAF receptor, but the effect was weaker than that of GK.

[Effects of ginkgolide K on platelet aggregation activity and neuroprotection].

To investigate the antagonism effects of different concentrations of ginkgolide K(GK) on platelet activating factor (PAF)-induced platelet aggregation and neuroprotective effect on cells and animal models of ischemia-reperfusion injury. GK-containing serum in rabbit was prepared, and the effects of GK-containing serum on PAF-induced platelet aggregation was observed by platelet aggregation assay. The effect of different concentrations of GK on apoptosis of SH-SY5Y cells injured by oxygen-glucose deprivation/reoxygenation (OGD/R) was investigated by Hoechst 33342/PI double staining in OGD/R cell model. The focal cerebral ischemia-reperfusion model (I/R)was established in rats to detect the effects of GK on neurobehavioral scores and cerebral infarction volume. GK could inhibit PAF-induced platelet aggregation, reverse the apoptosis induced by OGD/R injury and improve the neurobehavioral score and cerebral infarction volume after cerebral ischemia-reperfusion injury in rats in a dose-dependent manner. GK can inhibit PAF-induced platelet aggregation and improve nerve injury after cerebral ischemia-reperfusion.

Effects of ginkgolide A, B and K on platelet aggregation / 中国中药杂志

To investigate the effects of ginkgolide A (GA), ginkgolide B (GB) and ginkgolide K (GK) on platelet aggregation in rabbits, and compare the similarities and differences among these three components. The effects of different doses of ginkgolide A, B and K on platelet aggregation induced by platelet activating factor (PAF) were observed by using in vitro experiment. The results showed that three compounds could inhibit platelet aggregation induced by PAF in vitro, and the intensity was GK> GB> GA. It was further found that all of them can mobilize [Ca2+]i and enhance intracellular c-AMP level in a dose-dependent manner, which was consistent to the ability to antagonize PAF receptor. These findings indicated that GK was highly selective for PAF receptor, and may inhibit platelet aggregation by activating cAMP signaling pathway and inhibiting intracellular [Ca2+]i mobilization; GB and GA also had strong antagonism to PAF receptor, but the effect was weaker than that of GK.

Effects of ginkgolide K on platelet aggregation activity and neuroprotection / 中国中药杂志

To investigate the antagonism effects of different concentrations of ginkgolide K(GK) on platelet activating factor (PAF)-induced platelet aggregation and neuroprotective effect on cells and animal models of ischemia-reperfusion injury. GK-containing serum in rabbit was prepared, and the effects of GK-containing serum on PAF-induced platelet aggregation was observed by platelet aggregation assay. The effect of different concentrations of GK on apoptosis of SH-SY5Y cells injured by oxygen-glucose deprivation/reoxygenation (OGD/R) was investigated by Hoechst 33342/PI double staining in OGD/R cell model. The focal cerebral ischemia-reperfusion model (I/R)was established in rats to detect the effects of GK on neurobehavioral scores and cerebral infarction volume. GK could inhibit PAF-induced platelet aggregation, reverse the apoptosis induced by OGD/R injury and improve the neurobehavioral score and cerebral infarction volume after cerebral ischemia-reperfusion injury in rats in a dose-dependent manner. GK can inhibit PAF-induced platelet aggregation and improve nerve injury after cerebral ischemia-reperfusion.

Pharmacokinetic studies of ginkgolide K in rat plasma and tissues after intravenous administration using ultra-high performance liquid chromatography-tandem mass spectrometry.

Ginkgolide K (GK), a derivative compound of ginkgolide B, has been recently isolated from the leaves of Ginkgo biloba. It is a powerful natural platelet activate factor (PAF) antagonist, and also has obvious protect effects for cerebral ischemia. However, no reports have been described for the pharmacokinetic study of GK. In this study, a simple, sensitive and reliable ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed for the determination of GK in rat plasma and tissues. Biological samples were pretreated by an efficient liquid-liquid extraction with ethyl acetate. The chromatographic separation was achieved on an Agilent ZORBAX SB-Aq column (4.6 mm × 50 mm, 1.8 µm) with a mobile phase of 0.5% aqueous formic acid (A)-menthol (B). Quantitation was carried out on a triple quadruple mass spectrometry using positive electrospray ionization in multiple reaction monitoring mode. Diazepam was used as internal standard (IS). The ion transitions monitored were set at m/z 407.10 → 389.20 and m/z 285.08 → 193.10 for GK and IS, respectively. The developed method was fully validated and successfully applied to the pharmacokinetics and tissue distribution study of GK after intravenous administration. The current results have indicated that pharmacokinetic parameters of GK vary in a dose-dependent manner with rapid elimination in 4h. The major distribution tissues of GK in rats were liver and kidney. This study would provide critical information to promote the future study of GK.