不同成熟期栀子果实有效成分累积的研究

根据果实颜色可以将栀子成熟期划分为全青期、2/3青1/3黄期、半青半黄期、1/3青2/3黄期、全黄期、枯黄期.采用HPLC法测定栀子果实在不同成熟时期3种主要有效成分栀子苷、绿原酸、西红花苷-1的含量.结果表明:栀子苷、绿原酸、西红花苷-1含量均出现两个高峰期,峰形陡峭;高峰出现的时期不完全一致.栀子苷的含量在全青期和1/3青2/3黄期最高,而西红花苷-1的含量在全青期和全黄期最高.全青期为栀子苷和西红花苷-1的最佳采收期.根据果实颜色及时采收才能获得高含量的目标活性成分.     

基于亲和层析方法研究栀子苷的药物靶标

目的:运用亲和层析方法初步探索栀子苷的特异结合蛋白。方法:采用环氧氯丙烷(Epichlorohydrin,ECH)作为活化试剂对琼脂糖凝胶进行环氧基修饰,通过偶联栀子苷与环氧活化的琼脂糖凝胶(epoxy activated Sepharose CL-6B,EAS6B),制备以栀子苷为配基的亲和介质,以高效液相色谱法验证其偶联。利用栀子苷-EAS6B亲和介质,从小鼠脑组织总蛋白中筛选特异结合蛋白。结果:优化偶联条件后,得到最佳的活化反应条件:15%ECH、0.8 mol/L Na OH于37℃反应3 h,环氧基密度达到122μmol/m L,琼脂糖凝胶亲和介质的偶联量为19.53%,偶联率为17.08μmol/m L。从小鼠脑组织总蛋白中筛选得到3个栀子苷特异结合蛋白的条带,经质谱鉴定,发现主要是由热休克蛋白、脑酸溶性蛋白和谷氨酰胺合成酶等组成。结论:确立了琼脂糖凝胶环氧活化的最佳条件,制备了栀子苷-EAS6B亲和介质并应用其筛选出栀子苷的结合蛋白。     

采用β-葡萄糖苷酶两相催化法水解栀子苷的研究

本研究探索采用有机溶剂/水两相系统作为反应体系来进行栀子苷的酶解反应。以栀子苷和京尼平在水相溶剂中的分配系数作为考察指标,从4种有机溶剂/水两相系统中筛选适合采用两相催化反应体系水解栀子苷的两相溶剂系统。经筛选发现,正丁醇/水两相系统为最佳反应系统,其中栀子苷在水相中的分配比为74.2%,而京尼平仅为22.1%。以正丁醇/水两相系统作为的反应体系,采用β-葡萄糖苷酶催化栀子苷水解,酶解条件为反应温度为50℃,pH为5.0,转速为180 rpm,并采用HPLC-UV对反应过程进行检测。栀子苷转化率在反应10 h后达到86.6%。     

栀子果实成熟度对栀子苷含量积累的影响

目的:考察栀子果实成熟度与栀子苷含量积累的关系,为确定最适采收期奠定基础。方法:以不同成熟度的栀子果实为样品,采用高效液相色谱法测定栀子苷含量,并进行对比。结果:6个不同成熟度果实样品的栀子苷含量随成熟度提高而下降,果实全青渐变色时栀子苷含量最高(8.469%);在果实3/4红前含量下降缓慢(-3.71%),差异不明显;果实全红后含量下降显著(-12.18%～32.55%)。结论:药用栀子果实的采摘应以果实全青渐变到果实3/4红前及时采摘。     

不同采收期水栀子果实中西红花苷Ⅰ、西红花苷Ⅱ和栀子苷含量变化研究

建立水栀子药材中西红花苷Ⅰ、西红花苷Ⅱ超高效液相色谱法(UPLC)含量测定的方法,研究不同采收期的水栀子药材中西红花苷Ⅰ、西红花苷Ⅱ和栀子苷含量的变化趋势。西红花苷Ⅰ、西红花苷Ⅱ含量测定采用80%甲醇超声提取,通过UPLC-DAD法测定。色谱条件:Aglient Pro120 EC-C18(50 mm×4.6 mm,2.7μm)色谱柱,以水(A)-乙腈(B)为流动相梯度洗脱,0~5 min,5%~20%B;5~17 min,20%~27%B;柱温15℃,流速0.8m L/min,检测波长440 nm。栀子苷含测采用《中国药典》2015年版一部栀子项下的含量测定方法检测。通过实验,红花苷Ⅰ、西红花苷Ⅱ分别在49.5560~955.6000 ng、5.9045~1180.9000 ng线性范围内与各自峰面积呈良好的线性关系(r=0.9996、r=0.9998),平均回收率分别为98.17%、99.67%(n=9)。表明建立的西红花苷Ⅰ、西红花苷Ⅱ含量测定方法简便,稳定性、重复性、分离度均好;随着采收期的延长,样本中西红花苷Ⅰ、西红花苷Ⅱ的含量逐步增加,而栀子苷含量先降低后再逐步增加到一个平衡值。本研究为水栀子药材最佳采收期的确定提供了实验依据。     

栀子苷和牛血清白蛋白相互作用研究(英文)

在模拟生理条件下,利用紫外和荧光光谱法研究栀子苷和牛血清白蛋白相互作用。通过Stern-Volmer方程和Lineweaver-Burk考察栀子苷对牛血清白蛋白内源性荧光的猝灭机制,分别在298 K、310 K和322 K下利用结合常数和结合位点数计算反应体系的热力学参数。结果表明,当温度为298 K、302 K和322 K时,栀子苷对牛血清白蛋白的猝灭常数分别为4.632×104、3.515×104和3.575×104mol/L,结合常数KA分别为1.805×104、2.546×104和4.165×104,结合位点数分别为1.334、1.112和0.944,栀子苷对牛血清白蛋白的猝灭方式属于静态猝灭;热力学参数ΔG0,ΔH0,ΔS0,表明栀子苷与牛血清白蛋白结合作用力为静电引力,根据Frster非辐射共振能量转移理论,计算出栀子苷与牛血清白蛋白之间的结合距离为1.78 nm。     

矿质元素及pH值与栀子中栀子苷含量的相关性

为了探讨栀子苷含量与土壤及药材中的矿质元素的相关关系,用主成分分析方法对8个不同产地的栀子土壤中13种矿质元素及pH与药材中12种矿质元素进行了多因素分析.结果表明:Fe和Ni、Fe和Al、Mn和Co之间呈极显著正相关.影响栀子苷含量的Mg和pH贡献率分别为49.614%和20.826%,两者对栀子苷的变异贡献率最大.同时进行了栀子苷含量的变化与主成分的多元相关分析,证实了矿质元素与中药功效之间存在相关性.     

基于TLR3/TRIF通路探讨栀子苷抗流感病毒引起的病毒性肺炎的实验研究

中药在预防和治疗流感病毒方面具有独特的优势,栀子苷具有抗炎抗病毒作用,但栀子苷保护流感病毒引起的肺损伤的作用机制尚不明确。为了基于Toll样受体3(Toll-like receptor 3,TLR3)/β干扰素TIR结构域衔接蛋白(TIR-domain-containing adaptor inducing interferon-β,TRIF)信号传导途径探讨栀子苷对流感病毒诱导的肺损伤的调节作用,本研究按照随机数字表法将108只小鼠分为空白对照组、模型组、阳性对照组(50mg/kg利巴韦林)、低剂量栀子苷组(5mg/kg栀子苷)、中剂量栀子苷组(10mg/kg栀子苷)、高剂量栀子苷组(20mg/kg栀子苷)、TLR3激动剂组、TLR3激动剂+阳性药物组、TLR3激动剂+高剂量栀子苷组。采用流感病毒亚甲型鼠肺适应株A/FM/1/47(H1N1)建立流感病毒性肺炎小鼠模型,摘取肺组织,测定小鼠肺指数,HE染色观察肺组织病理学变化,酶联免疫法检测肺组织白介素-6(Interleukin-6,IL-6)、肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)和干扰素-β(Interferon-β,IFN-β)水平,qRT-PCR检测肺组织TLR3和TRIF mRNA表达,Western Blot检测肺组织TLR3、TRIF和p-NF-κB65蛋白表达。结果显示,与空白对照组相比,模型组小鼠肺指数、IL-6、TNF-α水平显著升高(P0.05),TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白水平均显著升高(P0.05)。与模型组比,中、高剂量栀子苷组和阳性对照组小鼠肺指数、IL-6和TNF-α水平均显著降低(P0.05),TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白水平均显著降低(P0.05)。与阳性对照组比,高剂量栀子苷组小鼠肺指数、IL-6、TNF-α、TLR3和TRIF的mRNA和蛋白、p-NF-κB65蛋白均无显著差异(P0.05)。与空白对照组相比,TLR3激动剂组小鼠肺指数显著升高(P0.05),与TLR3激动剂组相比,TLR3激动剂+高剂量栀子苷组小鼠肺指数显著降低(P0.05),TLR3激动剂+阳性药物组小鼠肺指数无显著差异(P0.05)。本研究揭示,栀子苷可能通过调节TLR3/TRIF通路介导的p-NF-κB65活性增强机体的抗病毒能力。     

高速逆流色谱分离制备栀子苷

以栀子苷粗提取物为原料,采用高速逆流色谱法分离栀子苷,溶剂系统为A:乙酸乙酯∶正丁醇∶水(2∶1.5∶3)和B∶正丁醇∶水(1∶1),上相为固定相,下相为流动相,流速为2.0 mL/min,转速为850 r/min,温度控制在25℃,纯度用HPLC测定.结果表明,利用溶剂系统A和B进行HSCCC制备栀子苷,使栀子苷含量从50.75％(HPLC)分别提高至86.6％和91.8％,回收率分别为81.36％和78.12％.     

UPLC-HDMS法测定大鼠灌胃栀子柏皮片后小檗碱与栀子苷的药代动力学

目的:通过UPLC-HDMS法测定小檗碱和栀子苷在大鼠灌胃栀子柏皮片后的药代动力学。方法:用固相萃取柱法处理血浆样本后,以正离子扫描方式,采用萃取离子监测(EIC)方式进行检测,采用Winnolin软件计算药物动力学参数。结果:小檗碱的达峰时间为1.89±0.56 h,其AUC值为793±87 ng/mL/h,药代参数显示该成分在体内浓度较低,消除较快;栀子苷的达峰时间为2.09±0.88 h,其AUC值为4687±345 ng/mL/h,药代参数显示该成分在体内表观分布容积较大,消除也较快。结论:利用高灵敏度的液质联用仪器在血中成功检测到了小檗碱及栀子苷,提示小檗碱及栀子苷为栀子柏皮片体内潜在药效作用物质。     

Geniposide inhibits glucolipotoxicity and cooperates with Txnip knockdown to potentiate cell adaption to endoplasmic reticulum stress in pancreatic beta cells

Thioredoxin‐interacting protein (Txnip), a negative regulator of thioredoxin, has become an attractive therapeutic target to alleviate metabolic diseases. Our previous data demonstrated that geniposide improved glucose‐stimulated insulin secretion by accelerating Txnip degradation and prevented the early‐stage apoptosis of pancreatic β cells induced by palmitate, but the underlying mechanisms are still unclear. The objective of this study is to identify the role of Txnip in geniposide preventing the apoptosis of pancreatic β cells induced by high glucose and palmitate (HG/PA). The results revealed that geniposide attenuated HG/PA‐induced cell apoptosis and the expression of Bax and caspase‐3, while increasing mitochondrial membrane potential and the anti‐apoptotic protein levels of heme‐oxygenase‐1 (HO‐1) and Bcl‐2 in INS‐1 rat pancreatic β cells. Knockdown of the Txnip gene raised the levels of anti‐apoptotic proteins HO‐1 and Bcl‐2 and geniposide potentiated the effect of Txnip when the INS‐1 cells were challenged by HG/PA. Furthermore, geniposide enhanced the adoptive unfolded protein response by increasing the phosphorylation of PERK/eIF2α and IRE1α in HG/PA‐treated INS‐1 cells. The results together suggest that geniposide might be useful to antagonize glucolipotoxicity and Txnip might be a pleiotropic cellular factor in pancreatic β cells.     

Geniposide Increases Unfolded Protein Response-Mediating HRD1 Expression to Accelerate APP Degradation in Primary Cortical Neurons

Altered proteostasis induced by amyloid peptide aggregation and hyperphosphorylation of tau protein, is a prominent feature of Alzheimer’s disease, which highlights the occurrence of endoplasmic reticulum stress and triggers the activation of the unfolded protein response (UPR), a signaling pathway that enforces adaptive programs to sustain proteostasis. In this study, we investigated the role of geniposide in the activation of UPR induced by high glucose in primary cortical neurons. We found that high glucose induced a significant activation of UPR, and geniposide enhanced the effect of high glucose on the phosphorylation of IRE1α, the most conserved UPR signaling branch. We observed that geniposide induced the expression of HRD1, an ubiquitin-ligase E3 in a time dependent manner, and amplified the expression of HRD1 induced by high glucose in primary cortical neurons. Suppression of IRE1α activity with STF-083010, an inhibitor of IRE1 phosphorylation, prevented the roles of geniposide on the expression of HRD1 and APP degradation in high glucose-treated cortical neurons. In addition, the results from RNA interfere on HRD1 revealed that HRD1 was involved in geniposide regulating APP degradation in cortical neurons. These data suggest that geniposide might be benefit to re-establish proteostasis by enhancing the UPR to decrease the load of APP in neurons challenged by high glucose.     

Preparative isolation and purification of geniposide from gardenia fruits by centrifugal partition chromatography

The iridoid glycoside, geniposide was purified by centrifugal partition chromatography (CPC) with a two-phase solvent system composed of ethyl acetate:isopropanol:water (3:2:5, v/v) from an 80% methanolic extract of fruits of Gardenia jasminoides. Preparative CPC yielded 56.2 mg of geniposide in a one-step separation of 500 mg of extract, with a purity of 95% as determined by HPLC. Isolated geniposide was identified from its 1H-NMR, 13C-NMR and MS spectra.     

人参抗疲劳作用研究进展

人参是古今著名的滋补强壮良药,药理研究、临床观察和流行病学调查,结果均证实人参具有多种生物活性。本文着重从人参抗中枢疲劳、抗自由基、调节糖代谢和乳酸代谢,以及对血红蛋白含量的影响等方面进行综述。     

Geniposide Protects Primary Cortical Neurons against Oligomeric Aβ1-42-Induced Neurotoxicity through a Mitochondrial Pathway

Mitochondrial dysfunction plays a key role in the progression of Alzheimer’s disease (AD). The accumulation of amyloid-beta peptide (Aβ) in the brains of AD patients is thought to be closely related to neuronal mitochondrial dysfunction and oxidative stress. Therefore, protecting mitochondria from Aβ-induced neurotoxicity is an effective strategy for AD therapeutics. In a previous study, we found that geniposide, a pharmacologically active compound purified from gardenia fruit, has protective effects on oxidative stress and mitochondrial dysfunction in AD transgenic mouse models. However, whether geniposide has a protective effect on Aβ-induced neuronal dysfunction remains unknown. In the present study, we demonstrate that geniposide protects cultured primary cortical neurons from Aβ-mediated mitochondrial dysfunction by recovering ATP generation, mitochondrial membrane potential (MMP), and cytochrome c oxidase (CcO) and caspase 3/9 activity; by reducing ROS production and cytochrome c leakage; as well as by inhibiting apoptosis. These findings suggest that geniposide may attenuate Aβ-induced neuronal injury by inhibiting mitochondrial dysfunction and oxidative stress.     

Metabolomics and Proteomics Annotate Therapeutic Properties of Geniposide: Targeting and Regulating Multiple Perturbed Pathways

Geniposide is an important constituent of Gardenia jasminoides Ellis, a famous Chinese medicinal plant, and has displayed bright prospects in prevention and therapy of hepatic injury (HI). Unfortunately, the working mechanisms of this compound are difficult to determine and thus remain unknown. To determine the mechanisms that underlie this compound, we conducted a systematic analysis of the therapeutic effects of geniposide using biochemistry, metabolomics and proteomics. Geniposide significantly intensified the therapeutic efficacy as indicated by our modern biochemical analysis. Metabolomics results indicate 9 ions in the positive mode as differentiating metabolites which were associated with perturbations in primary bile acid biosynthesis, butanoate metabolism, citrate cycle (TCA cycle), alanine, aspartate and glutamate metabolism. Of note, geniposide has potential pharmacological effect through regulating multiple perturbed pathways to normal state. In an attempt to address the benefits of geniposide based on the proteomics approaches, the protein-interacting networks were constructed to aid identifying the drug targets of geniposide. Six identified differential proteins appear to be involved in antioxidation and signal transduction, energy production, immunity, metabolism, chaperoning. These proteins were closely related in the protein-protein interaction network and the modulation of multiple vital physiological pathways. These data will help to understand the molecular therapeutic mechanisms of geniposide on hepatic damage rats. We also conclude that metabolomics and proteomics are powerful and versatile tools for both biomarker discovery and exploring the complex relationships between biological pathways and drug response, highlighting insights into drug discovery.     

Geniposide Regulates Glucose-Stimulated Insulin Secretion Possibly through Controlling Glucose Metabolism in INS-1 Cells

Glucose-stimulated insulin secretion (GSIS) is essential to the control of metabolic fuel homeostasis. The impairment of GSIS is a key element of β-cell failure and one of causes of type 2 diabetes mellitus (T2DM). Although the K_ATP channel-dependent mechanism of GSIS has been broadly accepted for several decades, it does not fully describe the effects of glucose on insulin secretion. Emerging evidence has suggested that other mechanisms are involved. The present study demonstrated that geniposide enhanced GSIS in response to the stimulation of low or moderately high concentrations of glucose, and promoted glucose uptake and intracellular ATP levels in INS-1 cells. However, in the presence of a high concentration of glucose, geniposide exerted a contrary role on both GSIS and glucose uptake and metabolism. Furthermore, geniposide improved the impairment of GSIS in INS-1 cells challenged with a high concentration of glucose. Further experiments showed that geniposide modulated pyruvate carboxylase expression and the production of intermediates of glucose metabolism. The data collectively suggest that geniposide has potential to prevent or improve the impairment of insulin secretion in β-cells challenged with high concentrations of glucose, likely through pyruvate carboxylase mediated glucose metabolism in β-cells.