Des(rhamnosyl)verbascoside抗HBV作用的定量蛋白质组学研究

为探究des(rhamnosyl)verbascoside体外抗乙型肝炎病毒(HBV)的活性作用和机制,本研究以des(rhamnosyl)verbascoside为实验药物对HepG2.2.15细胞进行干预,实验分为药物干预组和对照组,采用串联质谱标签(Tandem Mass Tag,TMT)蛋白质组学方法对提取的总蛋白进行分析。结果表明,共筛选得到300个差异表达蛋白,其中有109个上调蛋白,191个下调蛋白。基因本体论(Gene Ontology,GO)分析结果显示,差异蛋白主要参与DNA复制(DNA replication)、鞘糖脂代谢(Glycosphingolipid metabolic process)、细胞增殖(Cell proliferation)、寡糖分解代谢(Oligosaccharide catabolic process)等生物学过程,以及DNA聚合酶活性(DNA polymerase activity)、丝氨酸型羧肽酶活性(Serine type carboxypeptidase activity)、DNA引物酶活性(DNA primase activity)等分子功能。京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)分析结果显示,差异蛋白主要参与细胞代谢(Metabolism)、遗传信息传导(Genetic information processing)、生物系统通路(Organismal systems)等相关信号通路。亚细胞定位分析表明,差异蛋白大多定位在细胞质和细胞核。本研究共筛选出13个与抗HBV密切相关的蛋白。通过定量蛋白组学初步揭示des(rhamnosyl)verbascoside可能通过增加HGF、SORT1、MAN2B1,减少PRIM1、PRIM2、POLA1、POLD3、POLD2、POLD1、POLE、ERCC2、LAMC1、SDC1等蛋白表达来起到体外抗HBV的作用。

Neuroprotective effects of acteoside in a glaucoma mouse model by targeting Serta domain-containing protein 4

AIM:To explore the therapeutic effect and main molecular mechanisms of acteoside in a glaucoma model in DBA/2J mice.METHODS:Proteomics was used to compare the differentially expressed proteins of C57 and DBA/2J mice.After acteoside administration in DBA/2J mice,anterior segment observation,intraocular pressure(IOP)monitoring,electrophysiology examination,and hematoxylin and eosin staining were used to analyze any potential effects.Immunohistochemistry(IHC)assays were used to verify the proteomics results.Furthermore,retinal ganglion cell 5(RGC5)cell proliferation was assessed with cell counting kit-8(CCK-8)assays.Serta domain-containing protein 4(Sertad4)mRNA and protein expression levels were measured by qRT-PCR and Western blot analysis,respectively.RESULTS:Proteomics analysis suggested that Sertad4 was the most significantly differentially expressed protein.Compared with the saline group,the acteoside treatment group showed decreased IOP,improved N1-P1 wave amplitudes,thicker retina,and larger numbers of cells in the ganglion cell layer(GCL).The IHC results showed that Sertad4 expression levels in DBA/2J mice treated with acteoside were significantly lower than in the saline group.Acteoside treatment could improve RGC5 cell survival and reduce the Sertad4 mRNA and protein expression levels after glutamate injury.CONCLUSION:Sertad4 is differentially expressed in DBA/2J mice.Acteoside can protect RGCs from damage,possibly through the downregulation of Sertad4,and has a potential use in glaucoma treatment.

Verbascoside exerts an anti-atherosclerotic effect by regulating liver glycerophospholipid metabolism

Verbascoside,abundant in olive mill wastewater,is a phenylethanolic glycoside with a wide range of pharmacological activities.Atherosclerosis(AS)is a common metabolic disease and abnormal lipid metabolism in liver is inseparable from its formation and development.In this study,the anti-atherosclerotic effect of verbascoside was evaluated by establishing an atherosclerosis model based on western diet feeding of apolipoprotein E-defi cient mice for 16 weeks.After 12 weeks of administration during the feeding period,the levels of total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)in the plasma of mice were signifi cantly decreased,the formation of arterial plaques was delayed,and the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and lactate dehydrogenase(LDH)in plasma were alleviated,showing the hepatoprotective effect.In addition,based on untargeted lipidomic analysis,verbascoside stabilized glycerophospholipid metabolism,modulated lipid metabolism disorders and reduced lipid deposition in the liver to achieve the therapeutic effi cacy against atherosclerosis by regulating cardiolipin(CL),ether-linked phosphatidylcholine(ether-PC),lysophophatidylcholine(LPC),phosphatidylcholine(PC),oxidized phosphatidylcholine(OxPC),oxidized phosphatidylethanolamine(OxPE),triacylglycerol(TG),sphingomyelin(SM)back to normal levels.

Mechanism of action of verbascoside in mice with Parkinson's disease based on molecular docking,molecular dynamics and in vivo experiments

Objective:To explore the improving effect of verbascoside on Parkinson's disease(PD)model mice and to clarify the possible mechanism.Methods:60 C57BL/6 male mice were randomly divided into normal group,model group,low,middle and high dose groups(30,60 and 120 mg/kg,respectively).After a week of adaptive culture,except for the blank group,the subacute model of PD was established by intraperitoneal injection of 1-methyl-4-phenyl-1(MPTP)in each group for 5 consecutive days.After a week of adaptive culture,except for the blank group,the subacute model of PD was established by intraperitoneal injection of 1-methyl-4-phenyl-1(MPTP)in each group for 5 consecutive days.The establishment of the model,the drug group was intragastrically administered continuously for 7 d,and the mice in the normal group were injected with the same amount of saline.The behavioral changes of mice were observed by open field test,pole climbing test and grip test;the morphology,apoptosis and Nissl bodies of substantia nigra neurons were observed by HE staining and Nissl staining;the ultrastructural changes of neurons were observed by transmission electron microscope;the positive expressions of tyrosine hydroxylase(TH),α-synuclein(α-Syn)and toll-like receptor 4(TLR4)were detected by immunohistochemistry.The protein levels of TH,α-Syn,TLR4,NF-κB and P-NF-κB in substantia nigra of mice were detected by Western blot,and the levels of inflammatory factors IL-1β,IL-6 and tumor necrosis factor-α(TNF-α)in serum were detected by Elisa.Molecular docking was used to verify the binding ability of verbascoside to TLR4/NF-κB signal pathway and related factors,and molecular dynamics was used to verify the binding mode and stability of verbascoside with compounds.Results:compared with the normal group,the total walking distance,average speed,free activity time,pole climbing time and forelimb grip in the model group were significantly longer than those in the normal group(P<0.05).The ultrastructural changes of neurons,nuclear lysis,deformation and mitochondrial damage were observed under transmission electron microscope,and the number and morphological changes of HE staining substantia nigra neurons were decreased(P<0.05).Nissl staining showed that the number of Nissl positive cells decreased(P<0.05),the positive expression of TH decreased and the positive expression of α-syn and TLR4 increased(P<0.05).Western blotting showed that the expression of TH protein decreased,the expression of α-syn,TLR4,NF-κB p65 protein increased(P<0.05),and the expression of IL-1β,IL-6 and TNF-α in serum increased significantly(P<0.05).Compared with the model group,the activity ability of mice in the treatment group was significantly improved(P<0.05),the morphology of neurons gradually recovered and the number of neurons increased(P<0.05).The number of Nissl positive cells increased(P<0.05).The expression of TH protein increased,while the expression of α-syn,TLR4,NF-κBp65 protein decreased(P<0.05),and the expression of IL-1β,IL-6,TNF-α in serum decreased significantly(P<0.05).The results of molecular docking and molecular dynamics simulation show that the combination of mulberry glycoside with the compound is good and the property is stable.Conclusion:verbascoside has protective effect on PD mice,and its mechanism may be related to TLR4/NF-κB pathway,and then regulate neuroimmunity.

玄参中苯丙素苷Acteoside对小鼠高尿酸血症的影响

目的探讨玄参中苯丙素苷Acteoside对小鼠高尿酸血症的影响及可能的作用机理。方法从玄参根中分离提取苯丙素苷成分Acteoside,采用次黄嘌呤造成小鼠高尿酸血症,观察Acteoside对小鼠高尿酸血症的影响,并进一步从体外观察Acteoside对黄嘌呤氧化酶的抑制作用。结果Acteoside能显著降低高尿酸血症小鼠体内的尿酸水平,体外实验显示其对黄嘌呤氧化酶有明显的抑制作用,IC50为12.25μgml-1。结论玄参中Acteoside在小鼠高尿酸血症中的降尿酸作用可能与其抑制黄嘌呤氧化酶作用有关。

Isolation and Purification of Plantamajoside and Acteoside from Plant Extract of Plantago asiatica L. by High Performance Centrifugal Partition Chromatography

Two phenylethanoid glycosides（PhGs）, plantamajoside and acteoside, were isolated and purified from the aerial parts ofPlantago asiatica for the first time by high performance centrifugal partition chromatography（HPCPC） with ethyl acetate, n-butanol, ethanol and water（0.5：0.5：0.1：1, volume ratio） as solvent system. A total of 45.6 mg of plantamajoside and 293.8 mg of acteoside were purified from 1341 mg of the n-butanol extract of P. asiatica, with a purity of 〉93.3% as determined by HPLC. The HPCPC fractions were analyzed by HPLC-DAD and the structures were identified by their retention time, UV, electrospray ionization multi stage tandem mass spectrometry（ESI-MSn） in the negative ion mode, and confirmed by NMR experiments. The characteristic fragment ions of ESI-MS of the two PhGs isolated from Plantago asiatica were discussed, which are specific and useful for the identification of the structures of PhGs.

Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra

Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson＇s disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson＇s disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson＇s disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson＇s disease were established and verbascoside（60 mg/kg） was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase m RNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were significantly higher than the Parkinson＇s model group. These findings suggest that the mechanism by which verbascoside treats Parkinson＇s disease is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra.

苯丙素苷Acteoside,Isoacteoside和Ligupurpuroside J的全合成

发展了一条合成苯丙素苷类化合物的通用路线,并依据此路线完成了苯丙素苷毛蕊花糖苷(Acteoside)和异毛蕊花糖苷(Isoacteoside)的全合成及紫茎女贞苷J(Ligupurpuroside J)的首次全合成.其中的关键步骤是应用金(Ⅰ)催化的鼠李糖邻炔基苯甲酸酯给体与多羟基裸露的2-苯乙基葡萄糖苷进行区域选择性糖苷化反应,成功构建天然苯丙素苷中常见的α-(1→3)糖苷键.该合成路线减少了保护基的使用,简洁高效.

Verbascoside—A Review of Its Antitumor Activities

Cancer is a set of diseases including abnormal growth of cells that can spread to another tissue. Verbascoside (or acteoside) is a naturally occurring, water-soluble secondary metabolite with significant biological properties, which is distributed widely in plant kingdom. Verbascoside is pharmacologically active compounds with many recent evidences that support its biological activities and safety. This review focuses on the recent studies that concerned with the antitumor activities of verbascoside alone and as a synergistic agent as well as nanoproduct. It also shows the latest advances in its antitumor effects, cytotoxic selectivity and its efficiencies in treating cancer, in vitro and/or vivo.

毛蕊花糖苷对腺嘌呤所致不育症大鼠的影响

目的 探讨毛蕊花糖苷对腺嘌呤诱导的不育症大鼠的作用及其机制。方法 大鼠随机分为空白组、模型组、阳性对照组(100 mg/kg复方玄驹胶囊)和毛蕊花糖苷低、高剂量组(50、100 mg/kg)。除空白组外,其余各组每天灌胃150 mg/kg腺嘌呤,连续14 d,建立不育症大鼠模型。造模结束后,各组灌胃给予相应剂量药物,连续给药28 d。观察各组大鼠一般活动情况,计算脏器(肝脏、肾脏、睾丸、附睾)指数,于显微镜下观察精子情况,HE染色观察大鼠肝脏、肾脏及睾丸组织病理形态,并进行精子发生评估(Johnsen评分法),ELISA法检测血清T、LH、FSH、GnRH水平,RT-qPCR和Western blot法检测大鼠睾丸组织mTOR、LC3B、ULK1 mRNA和蛋白表达。结果 与空白组比较,模型组大鼠肾脏指数升高(P<0.05),精子计数、精子活动率及精子指数降低(P<0.05),血清T、GnRH水平降低(P<0.05),LH、FSH水平升高(P<0.05),肾脏和睾丸组织病理损伤明显,睾丸组织mTOR mRNA和蛋白表达升高(P<0.05),LC3B、ULK1 mRNA和蛋白表达降低(P<0.05),p-mTOR蛋白表达降低(P<0.05);与模型组比较,毛蕊花糖苷高剂量组肾脏指数降低(P<0.05),精子计数、精子活动率升高(P<0.05),血清T水平升高(P<0.05),LH、FSH水平降低(P<0.05),肾脏和睾丸组织病理损伤有不同程度改善,睾丸组织mTOR mRNA和蛋白表达降低(P<0.05),LC3B、ULK1 mRNA和蛋白表达升高(P<0.05),p-mTOR蛋白表达升高(P<0.05),且毛蕊花糖苷高剂量作用与阳性对照药复方玄驹胶囊相当。结论 毛蕊花糖苷能有效提高不育症大鼠的精子质量,改善性激素紊乱、生殖功能及肾脏、睾丸组织病理性损伤,其作用机制可能与增强正性调控自噬,调节下丘脑-垂体-睾丸轴紊乱有关。

UPLC-MS/MS法测定毛蕊花糖苷干预不育症模型大鼠的睾酮水平

目的建立超高效液相色谱-串联质谱法(ultra high performance liquid chromatography tandem mass spectrometry,UPLCMS/MS)检测毛蕊花糖苷干预不育症模型大鼠血浆睾酮的含量,测定不育症模型大鼠血浆24 h睾酮含量,并借助药动学理论定量评价干预效果。方法大鼠含药血浆以睾酮-C3为内标,用液液萃取法,以12000 r·min^(-1)离心10 min,取上清液,5μL进样分析。以Acquity UPLCⅢBEH C_(18)(50 mm×2.1 mm,1.7μm)为色谱柱;以甲醇为流动相A、5 mL·L^(-1)甲酸溶液为流动相B进行梯度洗脱,洗脱程序:流速为0.2 mL∙min^(-1);柱温为35℃;样品室温度为4℃;运行时间为10 min;质量检测器使用ESI离子源,正模式检测,睾酮及内标睾酮-C3用于定量的母→子离子对分别为m/z 289.2→97.0、m/z 291.4→101.1。借助药动学软件分析,获取空白组和毛蕊花糖苷干预组大鼠体内24 h睾酮曲线下面积(area under the curve,AUC)数据。结果睾酮质量浓度在1~1000 ng·mL^(-1)范围内呈良好的线性关系。该方法日内、日间精密度RSD值均<10.84%,日内、日间准确度RE均<10.03%,样品提取回收率为95.82%~99.17%,置于进样器于4℃冰箱及长期冷冻稳定性RSD值均<10.63%。测定结果显示,毛蕊花糖苷干预组药峰浓度(peak concentration,C_(max))为7.92 ng·mL^(-1),空白组C_(max)为10.26 ng·mL^(-1)。与空白组比较,毛蕊花糖苷干预组大鼠体内睾酮24 h总AUC相对平稳。结论建立了UPLC-MS/MS测定不育症模型大鼠血浆睾酮含量的方法。从PK/PD角度比较分析推测出,给予毛蕊花糖苷干预后,不育症模型大鼠的24 h总体睾酮可基本恢复正常水平,从而发挥改善大鼠雄性生殖功能的作用。

毛蕊花糖苷对中枢神经系统的作用及其机制研究进展

毛蕊花糖苷广泛分布于兔耳草、地黄、车前子等多种植物中。其对中枢神经系统的作用尤其显著,特别在治疗神经退行性疾病有优良的药用价值,其机制主要通过抑制胱天蛋白酶3(Caspase-3)来减少神经元凋亡、促进神经干细胞增殖、减少自由基堆积、抑制氧化应激和炎症反应来保护神经细胞起到缓解帕金森病症状的作用;通过阻止β-淀粉样蛋白(Amyloid β-protein, Aβ)沉积、抑制凋亡和增强胆碱能活动来改善阿尔茨海默病认知功能障碍;通过减少脊髓神经元细胞损伤来治疗多发性硬化;此外,毛蕊花糖苷还通过增加脑内单胺类神经递质(Monoamine oxidase, MAO)含量起到抗抑郁作用;通过抗炎、减轻氧化应激、抑制神经元凋亡来减少神经元损伤来减轻脑缺血再灌注的损伤;通过减轻氧化应激来起到抗惊厥的作用;还能通过增加脑内神经营养因子(Neurotrophin, NT)、海马突触结合蛋白-1(Synaptotagmin-1, Syt1)和原肌球蛋白受体激酶A(Tropomycin receptor kinase A, TrkA)的表达,增加突触可塑性起到改善记忆的作用。通过查阅近年来相关文献,对毛蕊花糖苷在中枢神经系统方面的作用进行综述,希望能为毛蕊花糖苷的进一步开发研究提供理论依据。

毛蕊花糖苷抑制Erastin诱导的多巴胺能神经细胞系MN9D细胞铁死亡

背景:近年越来越多的研究证实多巴胺能神经元细胞铁死亡参与了帕金森病的发病,毛蕊花糖苷目前被证实具有抗氧化、抗炎和神经保护作用。目的:探讨毛蕊花糖苷对Erastin诱导的MN9D细胞铁死亡的保护效果及作用机制。方法:以MN9D细胞为研究对象,分为对照组、模型组(20μmol/L Erastin组)、Erastin+1μg/mL毛蕊花糖苷组、Erastin+5μg/mL毛蕊花糖苷组、Erastin+10μg/mL毛蕊花糖苷组。MN9D细胞在CO_(2)恒温培养箱中培养24 h,然后用不同质量浓度毛蕊花糖苷预处理8 h,再加入20μmol/L Erastin诱导24 h后,采用ELISA法检测还原型谷胱甘肽、超氧化物歧化酶、总铁离子、丙二醛水平,免疫组织化学法检测酪氨酸羟化酶的表达,Western blot法检测酪氨酸羟化酶、核因子红细胞-2相关因子2、血红素加氧酶1、谷胱甘肽过氧化物酶4蛋白表达。结果与结论:(1)与对照组相比,模型组还原型谷胱甘肽、超氧化物歧化酶水平明显减少(P<0.05),丙二醛和总铁离子水平明显增加(P<0.05);与模型组相比,毛蕊花糖苷1,5,10μg/mL组还原型谷胱甘肽、超氧化物歧化酶水平明显增加(P<0.05),丙二醛和总铁离子水平明减少(P<0.05);(2)与对照组相比,模型组酪氨酸羟化酶阳性细胞面积明显减少(P<0.05);与模型组相比,毛蕊花糖苷1,5,10μg/mL组酪氨酸羟化酶阳性细胞面积明显增加(P<0.05);(3)与对照组相比,模型组酪氨酸羟化酶、核因子红细胞-2相关因子2、血红素加氧酶1、谷胱甘肽过氧化物酶4的蛋白表达明显减少(P<0.05);与模型组相比,毛蕊花糖苷1,5,10μg/mL组酪氨酸羟化酶、核因子红细胞-2相关因子2、血红素加氧酶1、谷胱甘肽过氧化物酶4的蛋白表达明显增加(P<0.05)。结果提示:毛蕊花糖苷对Erastin诱导的MN9D细胞铁死亡具有明显的抑制作用,其机制可能通过作用于核因子红细胞-2相关因子2/血红素加氧酶1/谷胱甘肽过氧化物酶4通路实现的。

基于LC-MS法研究百合地黄汤单煎及共煎液中化学成分变化

目的测定仲景古方百合地黄汤水煎液成分,并对鲜百合和鲜地黄的水煎液所含成分进行对比,为优化百合地黄汤提取工艺提供初步依据。方法以液相色谱-质谱联用(LC-MS)技术为基础,定性分析百合地黄汤水煎液所含化学成分并进行归属及聚类分析,同时对百合水煎液、地黄汁所含化学成分进行比对。结果百合水煎液、鲜地黄汁中分别检测到35种、36种化学成分;古法煎煮后,在百合地黄汤水煎液中发现69种化学成分,其中有机酸类16种、糖类及苷类共6种、氨基酸及多肽类共8种、生物碱类3种;其中有8种物质为原百合水煎液与地黄汁共有,较两种原煎液新产生36种物质,且抗抑郁有效成分毛蕊花糖苷仅存在于共煎液中。结论古法百合地黄汤共煎液中抗抑郁有效成分比单煎液丰富,有助于抗抑郁有效成分的提取,LC-MS技术适用于复方提取液的含量测定,通过此研究,构建了基于质谱技术的中药复方化学物质组成表征的指纹图谱。

毛蕊花糖苷对冠心病大鼠炎症及脂代谢的影响

目的 探讨毛蕊花糖苷(verbascoside, Verb)对冠心病(CHD)大鼠炎症及脂代谢的影响。方法 将SD大鼠随机分为对照组、模型组、毛蕊花糖苷低、中、高剂量组,每组12只。对照组大鼠正常饲料喂养,其他组大鼠高脂饮食联合腹腔注射垂体后叶素诱导冠心病模型。建模后,对照组和模型组大鼠均每天灌胃2 mL生理盐水,低、中、高剂量组大鼠分别每日灌胃2 mL剂量为20,40,80 mg/kg的毛蕊花糖苷,共灌胃4周。末次给药后24 h,分别检测各组大鼠的心功能指标[射血分数(EF)和短轴缩短率(FS)]、血清心肌损伤标志物[肌酸激酶同工酶MB(CK-MB)和乳酸脱氢酶(LDH)]、血清脂代谢指标[总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)和游离脂肪酸(FFA)]和血清炎症指标[肿瘤坏死因子-α(TNF-α)、细胞间黏附分子-1(ICAM-1)和单核细胞趋化因子-1(MCP-1)]。通过苏木素伊红(HE)染色观察心肌形态。通过qRT-PCR检测心肌组织中AMP激活的蛋白激酶α(AMPKα)、过氧化物酶体增殖物激活受体α和γ(PPAR-α和PPAR-γ)和PPAR-γ激活分子1α(PGC-1α)的mRNA水平。通过Western blot检测心肌组织中核因子-κB(NF-κB) p65和AMPKα的磷酸化水平。结果 与对照组比较,模型组大鼠的EF和FS降低(P<0.05),血清中CK-MB、LDH、TC、TG、LDL-C、FFA、TNF-α、ICAM-1和MCP-1水平升高(P<0.05),血清HDL-C水平降低(P<0.05),心肌组织中NF-κB p65的磷酸化水平升高(P<0.05),心肌组织中AMPKα、PPAR-α、PPAR-γ和PGC-1α mRNA水平和AMPKα的磷酸化水平降低(P<0.05)。与模型组比较,低、中和高剂量组大鼠的EF和FS升高(P<0.05),血清中CK-MB、LDH、TC、TG、LDL-C、FFA、TNF-α、ICAM-1和MCP-1水平降低(P<0.05),血清HDL-C水平升高(P<0.05),心肌组织中NF-κB p65的磷酸化水平降低(P<0.05),心肌组织中AMPKα、PPAR-α、PPAR-γ和PGC-1α mRNA水平和AMPKα的磷酸化水平升高(P<0.05)。与低剂量组比较,中和高剂量组大鼠的EF和FS升高(P<0.05),血清中CK-MB、LDH、TC、TG、LDL-C、FFA、TNF-α、ICAM-1和MCP-1水平降低(P<0.05),血清HDL-C水平升高(P<0.05),心肌组织中NF-κB p65的磷酸化水平降低(P<0.05),心肌组织中AMPKα、PPAR-α、PPAR-γ和PGC-1α mRNA水平和AMPKα的磷酸化水平升高(P<0.05)。结论 毛蕊花糖苷有效改善了冠心病大鼠的心功能,减轻了心肌损伤,其机制可能与抑制NF-κB介导的炎症反应和激活AMPK介导的脂代谢基因转录和表达有关。

不同产地石吊兰药材UPLC指纹图谱及化学计量学分析

目的:建立不同产地石吊兰药材UPLC指纹图谱,结合相似度评价、聚类分析、主成分分析、正交偏最小二乘回归分析等化学计量手段评价不同产地石吊兰药材品质,为其质量控制研究提供方法参考和基础数据。方法:通过UPLC梯度洗脱建立65批次石吊兰药材的指纹图谱,采用中药色谱指纹图谱相似度评价系统(2012A版)对石吊兰UPLC指纹图谱进行相似度评价,并采用聚类分析、主成分分析以及正交偏最小二乘判别分析对石吊兰品质进行综合评价。结果:标定了15个共有峰,并指认了10个共有峰;65批样品与对照指纹图谱的相似度在0.316~0.954之间;聚类分析将65批样品分为两大类;主成分分析筛选出累积方差达79.202%贡献率的5个主成分,并通过各主成分载荷得到每个批次的综合得分,以反映综合质量;正交偏最小二乘判别分析筛选出黔产、滇产、桂产石吊兰的差异性成分为苯乙醇苷类和黄酮类成分。结论:建立的石吊兰UPLC指纹图谱结合化学计量学评价方法,简便可行,可为综合评价不同产地石吊兰药材的质量提供参考。

地黄中毛蕊花糖苷对OxLDL诱导的人脐静脉内皮细胞凋亡与自噬的影响

目的:研究从地黄中提取的毛蕊花糖苷对氧化低密度脂蛋白(OxLDL)诱导下人脐静脉内皮细胞(HUVEC)的保护作用,并进一步探究其对细胞炎症、凋亡与自噬的影响。方法:采用OxLDL(150μg/mL)处理HUVEC细胞建立内皮细胞损伤模型,实验设正常对照组、模型组及毛蕊花糖苷50、100μg/mL组。采用MTT法检测细胞相对存活率;亚硝酸盐显色法及ELISA法检测细胞上清液中NO、eNOS、IL-1β、IL-6、TNF-α含量;Annexin-V-PE/7AAD双染流式技术检测细胞凋亡;Flow Sight成像细胞仪检测自噬蛋白LC3A/B表达及自噬情况;Western Blot法检测细胞凋亡与自噬相关蛋白Bax、Bcl-2、LC3B的表达。结果:与模型组比较,毛蕊花糖苷可提高HUVEC细胞活力,降低炎症因子IL-1β、IL-6、TNF-α分泌及细胞凋亡率,促进细胞分泌NO与eNOS,升高LC3BⅡ/Ⅰ比值及自噬蛋白LC3A/B在细胞中标记的自噬百分率,降低凋亡相关蛋白Bax/Bcl-2比值(P<0.05或P<0.01)。结论:毛蕊花糖苷可抑制炎症因子,减少细胞凋亡,增加细胞保护性自噬,改善内皮功能障碍。

Complete biosynthesis of the phenylethanoid glycoside verbascoside

Verbascoside,which was first discovered in 1963,is a well-known phenylethanoid glycoside(PhG)that exhibits antioxidant,anti-inflammatory,antimicrobial,and neuroprotective activities and contributes to the therapeutic effects of many medicinal plants.However,the biosynthetic pathway of verbascoside remains to be fully elucidated.Here,we report the identification of two missing enzymes in the verbascoside biosynthesis pathway by transcriptome mining and in vitro enzymatic assays.Specifically,a BAHD acyltransferase(hydroxycinnamoyl-CoA:salidroside hydroxycinnamoyltransferase[SHCT])was shown to catalyze the regioselective acylation of salidroside to form osmanthuside A,and a CYP98 hydroxylase(osmanthuside B 3,30-hydroxylase[OBH])was shown to catalyze meta-hydroxylations of the p-coumaroyl and tyrosol moieties of osmanthuside B to complete the biosynthesis of verbascoside.Because SHCTs and OBHs are found in many Lamiales species that produce verbascoside,this pathway may be general.The findings from the study provide novel insights into the formation of caffeoyl and hydroxytyrosol moieties in natural product biosynthetic pathways.In addition,with the newly acquired enzymes,we achieved heterologous production of osmanthuside B,verbascoside,and ligupurpuroside B in Escherichia coli;this work lays a foundation for sustainable production of verbascoside and other PhGs in micro-organisms.

Plackett-Burman结合Box-Behnken响应面法优化赪桐中类叶升麻苷纯化工艺及其稳定性分析

为优化赪桐中类叶升麻苷的纯化工艺,并分析其稳定性,试验对8种不同型号的大孔树脂进行吸附-解吸能力比较,筛选出最优的大孔树脂型号;采用Plackett-Burman结合Box-Behnken响应面法,考察上样液浓度、上样液流速、洗脱液浓度等因素水平,优化赪桐中类叶升麻苷的纯化工艺。通过高效液相测定纯化物在不同温度、光照、pH、抗氧化剂等环境条件下类叶升麻苷的含量。结果表明:赪桐中类叶升麻苷最佳纯化工艺条件为ADS-7型号大孔吸附树脂、上样液浓度9.0 mg/mL、上样液流速3.0 m L/min、洗脱液浓度58%,验证试验中的类叶升麻苷回收率可达到76.04%,与所建模型预测接近;稳定性试验表明,温度越高、光照时间越久、pH越高、类叶升麻苷的稳定性越差。同时,添加抗氧化剂可以明显增强其稳定性。故纯化后的类叶升麻苷应在低温、避光、酸性环境中贮藏和使用,且可通过添加不同种抗氧化剂延长其使用期限。采用Plackett-Burman结合Box-Behnken响应面法优化赪桐中类叶升麻苷纯化工艺稳定可行,可为后续抗奶牛乳房炎中草药型饲料添加剂产品的研发及产品稳定性使用提供理论依据。