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

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

脑淋巴阻滞后SAH大鼠脑组织含水率和血管内皮细胞损害及银杏内酯的影响

目的探讨脑淋巴引流阻滞（CLB）及银杏内酯对大鼠蛛网膜下腔出血（SAH）脑组织含水率与血管内皮超微结构的影响。方法选雄性成年Wistar大鼠，采用枕大池内新鲜自体动脉血二次注入法建立大鼠SAH模型，用颈淋巴管结扎和颈淋巴结摘除法制作大鼠cLB模型。将动物分为：正常对照组，SAH组，SAH＋CLB组，SAH＋CLB＋生理盐水组（NS），SAH＋CLB＋20mg／kg银杏内酯组（Gl20组）和SAH＋CLB＋80mg／kg银杏内酯组（G180组）6组。干-湿重比较法测定脑组织含水率，透射电镜观察毛细血管内皮超微结构。结果脑组织含水率SAH组较正常对照组有所增加，SAH＋CLB组较SAH组增加明显（P〈0．01），SAH＋CLB＋G1组与SAH＋CLB组比较均明显降低（P〈0．01）。SAH＋CLB＋G180组较SAH＋CLB＋G120组有所降低（P〈0．05），对照组以外各组神经元及毛细血管内皮超微结构均有不同程度的损伤，SAH＋CLB＋G180组损伤明显减轻。结论CLB可增加SAH脑水肿，并能加重脑内血管内皮的损伤。银杏内酯可降低脑淋巴阻滞后SAH大鼠的脑组织含水率，对血管内皮细胞具有保护作用。     

Optimization of extraction technique and validation of developed RP-HPLC-ELSD method for determination of terpene trilactones in Ginkgo biloba leaves

Terpene trilactones are potent and selective antagonists of platelet activating factor. The present study deals with standardization of efficient extraction method and validation of newly developed simple, sensitive and rapid reversed phase high performance liquid chromatographic method with evaporative light scattering detection (RP-HPLC-ELSD) method for the quantitative determination of ginkgolide A (GA), ginkgolide B (GB), ginkgolide C (GC), ginkgolide J (GJ) and bilobalide (BB) within 8 min in Ginkgo biloba leaf extract. The analysis was conducted on a Zorbax RP-C₁₈ column with gradient elution of methanol–water–tetrahydrofuran. The method was validated for accuracy, precession, limit of detection and quantification. The drift tube temperature of evaporative light scattering detector was set to 90 °C and nitrogen flow rate was 1.5 standard liter/min (SLM).     

银杏内酯、刺五加皂甙、人参皂甙对氯化钴诱导化学缺氧的PC12细胞的保护作用

目的：研究银杏内酯、刺五加皂甙、人参皂甙对氯化钴诱导化学缺氧的PC12细胞的保护作用。方法：采用四甲基偶氮唑(MTT)比色分析法观察银杏内酯(37．5μg／ml)、刺五加皂甙(50μg／ml)、人参皂甙(60μg／ml)对氯化钴诱导化学缺氧的PC12细胞活性的影响。结果：银杏内酯、刺五加皂甙、人参皂甙能显著提高氯化钴诱导缺氧的PC12细胞的活性。结论：银杏内酯、刺五加皂甙、人参皂甙对氯化钴诱导缺氧损伤的PC12细胞有明显的保护作用。     

银杏内酯对拟老年痴呆模型大鼠学习记忆能力的影响

目的:探讨银杏内酯对拟老年痴呆大鼠学习记忆能力影响的可能机制。方法:大鼠海马微量注射冈田酸(OA),银杏内酯灌胃。水迷宫实验检测大鼠学习记忆能力;银染观察大鼠脑内神经原纤维缠结(NFT);免疫组化观察大鼠脑内bax、bcl-2的表达。结果:与对照组比较,模型组大鼠学习记忆能力下降,NFT表达增多明显,bcl-2免疫阳性神经元减少明显,bax免疫阳性神经元增多明显;与模型组相比,银杏内酯组大鼠学习记忆能力明显提高,NFT表达减少明显,bcl-2免疫阳性神经元增多明显,bax免疫阳性神经元减少明显。结论:银杏内酯提高拟老年痴呆大鼠的学习记忆能力可能与其抑制OA对tau蛋白的磷酸化,减少神经细胞的凋亡,从而对神经元产生保护作用有关。     

银杏内酯类化合物的来源、结构修饰与新剂型研究进展

银杏内酯是从银杏叶、根中分离得到一类萜内酯类化合物,作为强效血小板活化因子(Platelet activating factor,PAF)拮抗剂以及非特异性甘氨酸受体(Glycine receptor,GlyR)和γ-氨基丁酸(γ-Amino-Bu-Tyric acid,GABA)受体拮抗剂,目前广泛用于心脑血管疾病。自发现银杏叶提取物中的内酯成分的PAF拮抗活性后,众多科学家对此类化合物的构效关系、结构修饰、药理药效方面开展了大量研究。作为天然药物银杏内酯有着疗效好、毒副作用小的优势,具有良好的发展趋势,银杏内酯、银杏内酯衍生物及其制剂的研发将会带来更好的经济效益和社会效益,造福人类。本文对银杏内酯的研究及最新进展进行综述,并回顾其开发历程及相关研究,以期为天然药物的开发带来新的思考。      

银杏内酯预处理对拟AD大鼠海马CA1区神经元NFT和ChAT的影响

目的研究银杏内酯（ginkgolides）预处理对拟阿尔茨海默病（Alzheimer’s Disease，AD）模型大鼠中海马CA1区神经原纤维缠结（neurobfillary tangles，NFT）和胆碱乙酰基转移酶（choline acetyl transferase，CHAT）的影响。方法通过在大鼠海马CA1区微量注射冈田酸（Okadaic acid，OA）（0．4mmoL／L，0．5μL／次，隔天1次，共7次）建立拟AD大鼠模型；银杏内酯预处理组于模型制作前2周灌胃（50mg／kg，1次／d），然后通过Morris水迷宫观察大鼠行为学变化，改进的Bielschowsky染色观察海马CA1区NFT的变化，免疫组化法观察海马CA1区ChAT的表达。结果拟AD模型组大鼠出现认知、学习记忆能力减退，海马CA1区出现较多NFT，而ChAT表达减少；银杏内酯预处理组大鼠明显改善上述情况。结论OA的神经毒性可以诱导大鼠学习记忆能力的降低，并且导致胆碱能神经元损伤，功能低下；而银杏内酯预处理组可以显著改善模型大鼠的学习记忆能力，抑制NFT的形成，提高ChAT的活性。     

DGMI alleviates OGD/R-induced cell injury by regulating inflammatory and apoptosis signaling pathways

Diterpene ginkgolides meglumine injection (DGMI), a kind of Ginkgo biloba special extract injection, is now used for the treatment of ischemic stroke in convalescence. In the present study, we aimed to confirm whether DGMI could suppress inflammatoryresponses and apoptosis and explore the potential mechanisms underlying these effects. Cell viability and lactate dehydrogenase (LDH) release were measured by MTS and LDH assays after the cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R). The extent of anti-apoptotic effect of DGMI was detected by flow cytometry using Annexin V-FITC/PI double staining assay kit. Pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IL-10, were quantified by a specific Bio-Plex Pro^TM Reagent Kit. Additionally, activities of TLR2/4, NF-κB p65, MAPK pathway and apoptosis-related proteins as well as cellular localization of NF-κB p65 were determined by Western blotting analysis and immunofluorescence staining, respectively. DGMI at 50 μg/mL significantly increased the cell viability and decreased the secretion of IL-1β, IL-6, IL-10 and TNF-α in OGD/R-induced BV2 microglia cells. These effects were also confirmed by LDH assay and Annexin V-FITC/PI staining. Meanwhile, DGMI not only inhibited the protein expressions of TLR2, TLR4, MyD88, p-TAK1, p-IkBα, p-IKKβ and Bak, but also decreased the cleaved caspase-3/caspase-3, Bax/Bcl-2 and cleaved PARP-1/PARP-1 ratio in OGD/R-induced BV2 microglia cells. Furthermore, OGD/R-enhanced p-JNK1/2 and p-p38 MAPK expressions and nuclear translocation of NF-κB p65 were also partially inhibited by DGMI. The present study showed that inflammatoryresponses were triggered in BV2 microglia cells activated by OGD/R, leading tothe release of pro-inflammatory cytokines and apoptosis. DGMI suppressed the inflammatory response and apoptosis by regulating the TLR/MyD88/NF-κB signaling pathways and down-regulation of p-JNK1/2 and p-p38 MAPK activation.     

银杏苦内脂B对急性坏死性胰腺炎大鼠胰腺组织NF-kB p65、IkBαmRNA表达的影响

目的观察NF—kBp65、IkBαmRNA在急性坏死性胰腺炎（ANP）大鼠胰腺组织中的表达以及银杏苦内脂B（BN52021）对其表达的影响。方法180只Wistar大鼠随机分为对照组（NC组）、ANP模型组（ANP组）、BN52021治疗组（BN组），每组大鼠分别在术后1h、2h、3h、6h、12h和24h6个时间点处死，抽血测定血淀粉酶含量，取胰腺组织行病理学检查，RT—PCR法检测NF—kB p65、IkBαmRNA表达。结果血清淀粉酶和病理学改变符合ANP。BN52021能降低ANP大鼠的血清淀粉酶含量和改善胰腺的病理损害。ANP组和BN组NF—kB p65mRNA均呈双峰改变，第1峰在1h，第2峰分别在24h和12h，6h时降至最低。ANP组NF—kB p65mRNA表达在2h、3h、12h和24h较NC组显著增加（P〈0．05或0．001），仅在1h时较BN组有显著差异（P〈0．041），其他时间点无显著差异；但较NC组各时点显著升高（P〈0．05或0．001）。ANP组IkBαmRNA表达仅在24h点较NC组明显增加（P=0．000），BN组在1h、6h和12h较ANP组显著增加（P〈0．01），在1h、12h和24h也较NC组显著增加（P〈0．01）。结论NF—kBp65mRNA在ANP大鼠胰腺组织表达上调，IkBαmRNA仅在造模后24h时表达上调。BN52021可能通过上调IkBαmRNA的表达，破坏NF-kBp65mRNA和IkBαmRNA的平衡，从而发挥其治疗作用。