A New Stigmasterol and a New Eremophilenolide from Ligularia dolichobotrys

A new stigmasterol 3, 7, 22-trihydoxystigmast-5-ene (1) and a new eremophilen- olide 8-methoxy-6-angeloyloxyeremophil-7(11)-en-8, 12-olide-14-oic acid (2) were isolated from Ligularia dolichobotrys Diels. Their structures were deduced on the basis of spectral data.

In vivo combination therapy of chloroquine and stigmasterol against Plasmodium berghei induced pathologies in mice

Objective:To investigate the effect of combination therapy of chloroquine and stigmasterol on Plasmodium berghei(thereafter referred to as P.berghei)malaria-induced organ pathologies.Methods:Thirty five mice weighing 20-30 g were placed into seven groups of five mice each and distributed as uninfected administered 100 mg/kg BW stigmasterol,uninfected administered only feed and water ad libitum,infected with P.berghei and-administered 50 mg/kg BW stigmasterol,100 mg/kg BW stigmasterol,100 mg/kg BW stigmasterol plus 5 mg/kg BW chloroquine,and 5 mg/kg BW chloroquine.The last group of mice served as P.berghei infected and not treated control.The levels of parasitemia,packed cell volume,and other biochemical parameters were measured.Results:Combination therapy of P.berghei infection with stigmasterol and chloroquine did not significantly(P>0.05)reduce parasitemia level while stigmasterol treatment alone significantly(P<0.05)reduced the parasitemia level.However,the P.berghei induced anemia was decreased significantly(P<0.05)upon treatment with a combination of chloroquine and stigmasterol as well as with stigmasterol alone.Furthermore,the combination of chloroquine and stigmasterol significantly(P<0.05)decreased the activities of serum alanine aminotransferase,aspartate aminotransferase,urea and spleen total proteins levels in P.berghei mice in comparison with the untreated group.Treatment of P.berghei infected mice with stigmasterol alone and in combination with chloroquine significantly(P<0.05)increased the level of serum creatinine while serum and spleen malondialdehyde levels were significantly(P<0.05)decreased.Levels of glutathione in spleen and kidney were insignificantly(P>0.05)altered upon treatment with both doses of stigmasterol as well as the combination therapy.Conclusions:This study concluded that the combination of stigmasterol and chloroquine could combat anemia and some organ pathologies associated with P.berghei infection.

In vivo combination therapy of chloroquine and stigmasterol against Plasmodium berghei induced pathologies in mice

Objective:To investigate the effect of combination therapy of chloroquine and stigmasterol on Plasmodium(P.)berghei malaria-induced organ pathologies.Methods:Totally 35 mice weighing 20-30g were placed into 7 groups of 5 mice each and distributed as uninfected administered 100 mg/kg BW stigmasterol,uninfected administered only feed and water ad libitum,infected with P.berghei and administered 50 mg/Kg BW stigmasterol,100 mg/kg BW stigmasterol,100 mg/kg BW stigmasterol plus 5 mg/kg BW chloroquine,and 5 mg/kg BW chloroquine.The last group of mice served as P.berghei infected and not treated control.The levels of parasitemia,packed cell volume,and other biochemical parameters were measured.Results:Combination therapy of P.berghei infection with stigmasterol and chloroquine did not significantly(P>0.05)reduce parasitemia level while stigmasterol treatment alone significantly(P<0.05)reduced the parasitemia level.However,the P.berghei induced anemia was decreased significantly(P<0.05)upon treatment with a combination of chloroquine and stigmasterol as well as with stigmasterol alone.Furthermore,the combination of chloroquine and stigmasterol significantly(P<0.05)decreased the activities of serum alanine aminotransferase,aspartate aminotransferase,urea and level of spleen total proteins in P.berghei infected mice in comparison with the untreated group.Treatment of P.berghei infected mice with stigmasterol alone and in combination with chloroquine significantly(P<0.05)increased the level of serum creatinine while serum and spleen malondialdehyde levels were significantly(P<0.05)decreased.Levels of glutathione in spleen and kidney were insignificantly(P>0.05)altered upon treatment with both doses of stigmasterol as well as the combination therapy.Conclusions:This study concluded that the combination of stigmasterol and chloroquine could combat anemia and some organ pathologies associated with P.berghei infection.

Stigmasterol protects human brain microvessel endothelial cells against ischemia-reperfusion injury through suppressing EPHA2 phosphorylation

Stigmasterol is a plant sterol with anti-apoptotic,anti-oxidative and anti-inflammatory effect through multiple mechanisms.In this study,we further assessed whether it exerts protective effect on human brain microvessel endothelial cells(HBMECs)against ischemia-reperfusion injury and explored the underlying mechanisms.HBMECs were used to establish an in vitro oxygen and glucose deprivation/reperfusion(OGD/R)model,while a middle cerebral artery occlusion(MCAO)model of rats were constructed.The interaction between stigmasterol and EPHA2 was detected by surface plasmon resonance(SPR)and cellular thermal shift assay(CETSA).The results showed that 10μmol·L−1 stigmasterol significantly protected cell viability,alleviated the loss of tight junction proteins and attenuated the blood-brain barrier(BBB)damage induced by OGD/R in the in vitro model.Subsequent molecular docking showed that stigmasterol might interact with EPHA2 at multiple sites,including T692,a critical gatekeep residue of this receptor.Exogenous ephrin-A1(an EPHA2 ligand)exacerbated OGD/R-induced EPHA2 phosphorylation at S897,facilitated ZO-1/claudin-5 loss,and promoted BBB leakage in vitro,which were significantly attenuated after stigmasterol treatment.The rat MCAO model confirmed these protective effects in vivo.In summary,these findings suggest that stigmasterol protects HBMECs against ischemia-reperfusion injury by maintaining cell viability,reducing the loss of tight junction proteins,and attenuating the BBB damage.These protective effects are at least meditated by its interaction with EPHA2 and inhibitory effect on EPHA2 phosphorylation.

A New Stigmasterol Ester from Aeschynomene indica

Objective To study the chemical constituents of Aeschynomene indica.Methods The constituents were isolated and purified by means of silica gel column chromatography and recrytallization,and the structures were elucidated by physicochemical properties and spectral analyses.Results Twelve compounds were obtained and elucidated as stigmasterol tritriacontanate(1),monotetracontane(2),taraxerol(3),stigmasterol(4),stearic acid(5),heptatria- contanoic acid(6),arachidic acid(7),ursolic acid acetate(8),quercetin(9),myricetin(10),myricetin-3-O-rhamnoside(11),and rutoside(12).Conclusion All the compounds are isolated from this plant for the first time and compound 1 is a new one.

豆甾醇治疗胶质母细胞瘤靶基因预测及相关预后模型的建立

目的 预测豆甾醇治疗胶质母细胞瘤(glioblastoma, GBM)的潜在靶基因并构建相关预后模型,以期揭示其抗胶质瘤的作用机制,并探讨这些靶基因在GBM患者预后中的作用。方法 通过数据库获得GBM差异表达基因及豆甾醇靶基因,使用韦恩图筛选豆甾醇治疗GBM的潜在靶基因并使用R语言进行富集分析。采用单因素COX回归分析和最小绝对收缩与选择算子(least absolute shrinkage and selection operator, LASSO)分析筛选与GBM患者预后相关的豆甾醇靶基因并构建相关的预后模型。采用逆转录实时荧光定量聚合酶链反应(real-time quantitative reverse transcription polymerase chain reaction, RT-qPCR)和Western blotting分析检测豆甾醇对相关靶基因表达的影响。结果 共获得31个豆甾醇治疗GBM的潜在靶基因。富集分析显示,这些靶基因与G蛋白偶联受体(G protein-coupled receptor, GPCR)信号通路的激活和脂质代谢的调控有关。回归分析筛选出2个与GBM预后相关的豆甾醇靶基因脂肪酸结合蛋白5(fatty acid binding protein 5,FABP5)和α-1B肾上腺素能受体(alpha-1B adrenergic receptor, ADRA1B),基于这2个基因构建的预后模型能够准确预测GBM患者的预后。豆甾醇能够抑制GBM细胞中这2种基因在转录及翻译水平的表达(FABP5:t=9.909,P=0.001;ADRA1B:t=3.319,P=0.029)。结论 豆甾醇的抗胶质瘤作用可能与GPCR信号通路及脂质代谢的调控有关。通过抑制FABP5及ADRA1B的表达,豆甾醇可能具有改善GBM患者预后的潜在作用。同时,以FABP5及ADRA1B的表达水平构建的预后模型在预测GBM患者预后及监测疗效方面可能能够发挥一定的作用。

基于网络药理学探讨补骨脂与儿童性早熟的关联性

【目的】基于网络药理学方法及分子对接技术,探讨补骨脂是否可能促进儿童性早熟及其潜在机制。【方法】通过BATMAN-TCM在线平台获取补骨脂主要活性成分及其作用靶点,从GeneCards数据库中获取性早熟相关疾病靶点,采用Cytoscape 3.7.1软件构建“活性成分-疾病靶点”可视化网络。基于STRING在线数据库进行蛋白质-蛋白质相互作用(PPI)的网络图构建。使用Metascape在线工具进行靶点基因的基因本体(GO)和京都基因与基因组百科全书(KEGG)通路的富集分析。从PubChem数据库中获取主要活性成分结构,从RCSB PDB数据库中获取核心靶点结构,导入Autodock进行分子对接,最后利用PyMOL软件绘制分子对接模拟图。【结果】得到12种补骨脂的活性成分,涉及274个靶点,其中11种活性成分和98个靶点与性早熟相关。主要活性化合物为豆甾醇、补骨脂酚、异补骨脂素、补骨脂查尔酮、补骨脂乙素、甲氧基补骨脂素,主要靶点有雌激素受体1(ESR1)、雌激素受体2(ESR2)、胰岛素样生长因子1(IGF1)、孕酮受体(PGR),主要涉及卵巢类固醇生成通路、Hippo信号通路。分子对接结果表明这些活性化合物与靶标具有良好的结合作用。【结论】补骨脂存在促进儿童性发育的可能性及具有潜在药理作用机制,分析结果可为儿童性早熟、青春期早发育等临床防治提供理论参考。

基于代谢组学解析抗感水稻差异代谢物β-谷甾醇对褐飞虱的杀虫活性

本研究利用代谢组学筛选抗性水稻中有活性的代谢物质,为开发控制褐飞虱Nilaparvata lugens的绿色植物源农药开拓思路。基于水稻品种对褐飞虱有抗性的差异,采用生长发育和存活率比较方法,验证了水稻品种Mudgo和TN1对褐飞虱的抗性差异;通过代谢组学分析抗褐飞虱水稻Mudgo和感褐飞虱水稻TN1代谢物的不同,筛选了品种间有显著差异的物质β-谷甾醇和无显著差异的物质豆甾醇作为褐飞虱生测物质,进行存活率和适合度功能验证,明确其对褐飞虱的功能效应。褐飞虱在Mugdo和TN1两种水稻品种上的生命参数间存在显著差异,在Mudgo水稻上发育历期延长,体重、体长和存活率降低。代谢组学分析共检测到69种差异代谢物,主要为脂质(36%)、碳水化合物(23%)、氨基酸(19%)、次生代谢物质(14%)、核酸(6%)及其他物质(1%)等6大类物质。褐飞虱人工饲料添加剂生测数据显示,褐飞虱取食20.0μg/mL和500.0μg/mL浓度β-谷甾醇,雌虫体重显著降低;取食500.0μg/mL浓度β-谷甾醇,雄虫体重显著降低。此外,取食不同浓度豆甾醇对褐飞虱体重均无显著性影响。β-谷甾醇对褐飞虱的存活率有显著降低作用,而豆甾醇只有在4.0μg/mL、100.0μg/mL和500.0μg/mL浓度下有降低作用。确认了水稻品种Mudgo对褐飞虱有显著的抗性,可显著降低存活率,抑制生长。代谢组学筛选到的69种差异代谢物可作为抗褐飞虱绿色植物源农药的备选库;其中,差异显著的代谢物质β-谷甾醇对褐飞虱体重和存活率有显著降低作用,可作为防治褐飞虱的植物源农药。

Semi-synthesis of Several Stigmasterol Saponins

Several stigamasterol saponins were concisely synthesized. Name!y, four monosaccharide （glucopyranose, galactopyranose, xylopyranose, 2-acetamido-2-deoxy-a-D-glucopyranose）, lactopyranose and chacotriose were coupled with 3-OH of stigmasterol. All the compounds were identified by NMR, IR and high resolusion MS.

植物甾醇中豆甾醇分离提纯的工艺研究

植物甾醇是一种存在于植物中的天然活性物质,被广泛应用于维生素D、多种激素和甾族化合物的合成。目前工业生产常以大豆油脂的脱臭馏出物为原料,通过甲酯化提取混合植物甾醇。已知豆甾醇在环己酮和正戊醇中的溶解度随温度降低而下降。采用溶剂结晶法,分离提纯植物甾醇中的豆甾醇单体。结果表明:采用料液比1∶3,加热至65℃溶解后进行控温降温养晶,经过3次结晶过程豆甾醇单体含量(本文中甾醇含量均为质量分数)可达到76.89%,收率46.37%。

Phytochemical investigation and simultaneous estimation of bioactive lupeol and stigmasterol in Abutilon indicum by validated HPTLC method

Objective:To perform a simultaneous quantitative estimation of two biologically active triterpenoid compounds lupeol and a steroid compound,stigmasterol,in Abutilon indicum(A.indicum)using high-performance thin-layer chromatography(HPTLC).Methods:TLC aluminum plates precoated with silica-gel 60 F254(20 cmí10 cm)were used with a mobile phase of toluene-methanol-formic acid(7.0:2.7:0.3,v/v/v)and densitometric determination of these compounds was carried out at 530 nm in reflectance/absorbance mode.Results:Compact bands for lupeol and stigmasterol were obtained at R_(f)0.52±0.02 and 0.28±0.05.The limit of detection(45 and 18 ng/band),limit of quantification(135 and 54 ng/band),recovery(98.2%-99.7%and 97.2%-99.6%)and precision(≤2.18 and 1.91)were satisfactory for lupeol and stigmasterol respectively.Linearity range for lupeol and stigmasterol were 100-1000(r^(2)=0.9994)and 50-500 ng/band(r^(2)=0.9941)and the contents were estimated as(0.59±0.10)%and(0.83±0.10)%w/w respectively.The total phenolic,flavonoid,proanthocyanidin,alkaloidal and saponin contents of methanolic extract of A.indicum were also measured in this work.According to International Conference on Harmonization(ICH)guidelines,the method was validated for linearity,precision,accuracy,and recovery,limit of detection,limit of quantification,specificity,and robustness.Conclusions:The HPTLC method was found to be reproducible,accurate,and precise and could detect these two compounds at nanogram level from the A.indicum.

六味地黄汤干预肾纤维化作用机制的网络药理学分析

目的基于网络药理学方法及体内外实验探讨六味地黄汤(LWDHD)干预肾纤维化的潜在作用机制。方法(1)通过TCMSP平台检索筛选LWDHD的活性成分及其作用靶点;通过GeneCards、OMIM、TTD数据库检索肾纤维化疾病相关靶点;利用R语言VennDiagram包对LWDHD活性成分靶点和肾纤维化疾病相关靶点取交集,得到共同(交集)靶点,即为LWDHD治疗肾纤维化的关键靶点;运用Cytoscape 3.9.1软件构建LWDHD治疗肾纤维化的“活性成分-关键靶点”网络,分析核心活性成分;通过STRING平台构建共同靶点蛋白互作(PPI)网络;分别通过DAVID数据库、R语言软件的Pathview包对共同靶点进行GO功能及KEGG通路富集分析;采用AutoDock Vina软件对核心活性成分与核心靶点进行分子对接。(2)体内实验:将24只雄性SD大鼠随机分为4组:假手术组、模型组、LWDHD组(6.75 g·kg^(-1))及依那普利组(10 mg·kg^(-1)),每组6只。采用单侧输尿管结扎术(UUO)建立肾纤维化大鼠模型。各组以相应药物灌胃给药(10 mL·kg^(-1)),每日1次,连续7 d。采用HE、MASSON染色法观察大鼠肾脏组织病理变化和胶原沉积情况。(3)体外实验:将体外培养的HK-2细胞分为正常对照组(10%空白血清)、模型组(10%空白血清+200μmol·L^(-1)CoCl_(2))、LWDHD组(10%含药血清+200μmol·L^(-1)CoCl_(2));采用200μmol·L^(-1)CoCl_(2)模拟肾内缺氧环境,同时加入空白或含药血清,培养24 h后收集细胞;采用Western Blot法检测HK-2细胞HIF-1α蛋白表达水平。结果(1)共得到LWDHD活性成分40个,活性成分作用靶点194个,肾纤维化疾病相关靶点5135个,共同(交集)靶点即LWDHD治疗肾纤维化的关键靶点159个。筛选核心活性成分主要有槲皮素、豆甾醇、山柰酚、β-谷甾醇、薯蓣皂苷元等,核心靶点有热休克蛋白90α家族A类成员1(HSP90AA1)、c-Jun氨基末端激酶(JUN)、蛋白激酶B(AKT1)及缺氧诱导因子1α(HIF-1α)等。GO功能主要与细胞因子介导信号通路、基因表达正向调控、凋亡过程负调控、缺氧反应等生物过程相关;KEGG关键通路有动脉粥样硬化、癌症途径、病毒感染、晚期糖基化终末产物-糖基化终末产物受体(AGE-RAGE)通路、肿瘤坏死因子(TNF)信号通路、白细胞介素17(IL-17)信号通路、HIF-1信号通路等。槲皮素与HSP90AA1、HIF-1α结合力中等,豆甾醇与HSP90AA1、JUNE、AKT1、HIF-1α结合力中等,薯蓣皂苷元与HIF-1α有较强的结合力。(2)体内实验显示,与假手术组比较,模型组大鼠出现肾间质炎性细胞浸润、空泡化、肾小管萎缩,部分肾小管代偿性扩张;肾组织纤维化明显,可见大量染成蓝色的胶原纤维,胶原容积分数显著升高(P<0.01)。与模型组相比,LWDHD组及依那普利组大鼠肾组织病变程度减轻,炎性细胞减少,肾小管结构损伤明显减轻;肾组织蓝色胶原纤维明显减少,纤维化程度明显减轻,胶原容积分数显著降低(P<0.01)。(3)体外实验显示,与正常对照组比较,模型组细胞HIF-1α蛋白表达水平显著升高(P<0.01);与模型组比较,LWDHD组细胞HIF-1α蛋白表达水平明显降低(P<0.05)。结论LWDHD治疗肾纤维化作用可能是通过槲皮素、豆甾醇、薯蓣皂苷元等多种活性成分,作用于HSP90AA1、JUNE、AKT1、HIF-1α等多靶点,通过AGE-RAGE信号通路、TNF信号通路、IL-17信号通路、HIF-1信号通路等多条信号通路来实现的。

基于网络药理学探讨豆甾醇抗胃癌的靶点与机制

豆甾醇是来源于食物中的一种植物不饱和甾醇,在胃癌防治中展现出良好的应用前景。基于网络药理学探讨豆甾醇抗胃癌的作用靶点及分子机制。借助PharmMapper数据库得到药物相关靶点,通过疾病数据库Genecard和OMIM(Online Mendelian Inheritance in Man)获得胃癌相关靶点;对潜在标靶进行GO(Gene Ontology)、KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析得到相关作用通路;随后,利用STRING数据库分析治疗靶点之间蛋白的相互作用,借助Cytoscape3.8.0中CytoHubba插件构建蛋白质相互作用网络以获得Hub基因,预测豆甾醇抗胃癌的作用靶点及机制。借助数据库得到豆甾醇潜在胃癌治疗靶点19个,涉及77个生物过程与10条信号通路;通过蛋白互作网络取排名前5的Hub基因,分别为TERT、MET、SRC、MDM2、HIF1A。结果显示,网络药理学可以准确预测豆甾醇抗胃癌的作用靶点并揭示其分子机制与PI3K(Phosphatidylinositide 3-kinases)/AKT(蛋白激酶B)和RAS(Rat Sarcoma)通路上TERT、MET、SRC、MDM2、HIF1A这些关键基因的表达有关。

甾醇分子差异对脂质体结构稳定性的影响

通过添加胆固醇、β-谷甾醇和豆甾醇,采用乙醇注入法结合动态高压微流射技术制备甾醇脂质体,研究不同甾醇对脂质体膜结构稳定性的影响。结果表明:β-谷甾醇和豆甾醇的加入,使甾醇脂质体粒径从(48.35±0.41)nm分别增至(106.27±0.90)nm和(107.27±0.59)nm,Zeta电位保持不变,均在-30 mV左右;电子显微镜观察发现,添加甾醇的脂质体形成了稳定的磷脂双分子层结构;甾醇可以使脂质体的盐稳定性和pH稳定性增强,但对温度稳定性无显著影响;β-谷甾醇和豆甾醇的加入使脂质体膜的流动性、疏水性和微极性都显著减小,这表明脂质体磷脂双分子膜的结构变得更加致密。

香青兰化学成分研究

目的:研究香青兰化学成分。方法:采用活性追踪分离的方法,对香青兰全草进行了系统萃取,反复柱层析。结果:从总浸膏中分离得到了1个化合物,命名为α,从石油醚层和氯仿层分别得到3个化合物,分别命名为S2、S3和L1。4个化合物鉴定为:化合物α为田蓟甘(tilianin),化合物L1为熊果酸(ursolie acid),化合物S2为豆甾醇(stigmasterol),化合物S3为乌发醇(uvqol)。结论:化合物S2、S3和化合物L1均为该植物首次获得。

香附化学成分研究

目的:研究莎草科植物香附的化学成分。方法:用色谱法分离化合物,根据理化性质和光谱数据鉴定结构。结果:分离鉴定了6个化合物:大黄素甲醚(1)、十六烷酸(2)、β-谷甾醇(3)、豆甾醇(4)、Catenarin(5)、胡萝卜苷(6)。结论:化合物1、4、5为首次从该植物中分离得到。

混合植物甾醇中豆甾醇和β-谷甾醇的高效液相色谱分析

应用高效液相色谱对豆甾醇、β_谷甾醇和重结晶法分离精制的混合甾醇进行了分析测定 ;实验采用的色谱柱为HypersilODS反相柱(4.6×150mm ,5μm) ,流动相为甲醇 ,紫外检测波长为210nm ,恒溶剂洗脱 ,混合植物甾醇在10min内得到了很好的分离 ;实验结果表明 ,采用液相色谱法能够快速准确地测定混合甾醇中豆甾醇和 β_谷甾醇的含量。

混合甾醇中β-谷甾醇和豆甾醇的紫外光谱法分析测定

用紫外光谱法测定了β-谷甾醇和豆甾醇与Deniges磺基醋酸汞络合物的紫外光谱,探讨了显色反应时间、显色剂用量及浓硫酸与冰醋酸配比对紫外吸收光谱的影响。结果表明在一定浓度范围内,甾醇络合物的吸光度A与其浓度成线性关系。并利用紫外光谱法测定了混合甾醇中β-谷甾醇和豆甾醇的含量,此法所得结果与用气相色谱法测试的结果相近。

马桑化学成分研究

以河南省西峡县采集的马桑为原料 ,经乙醇回流提取 ,通过硅胶柱、高效液相制备柱对氯仿段进行分离 ,得到9个化合物 ,根据理化性质和光谱数据鉴定其结构分别为豆甾醇、β-谷甾醇、高级脂肪酸及其酯 ,其中 ,8个化合物为首次从该植物中得到 .

显齿蛇葡萄化学成分的研究

目的:研究显齿蛇葡萄的化学成分。方法:用色谱法和光谱分析法分离和鉴定其化学成分。结果:分离并鉴定了7个化合物,分别为蛇葡萄素(Ⅰ),杨梅素(Ⅱ);杨梅甙(Ⅲ),没食子酸(Ⅳ),β-谷甾醇(Ⅴ),豆甾醇(Ⅵ),二氢槲皮素(Ⅶ)。结论:化合物Ⅳ、Ⅴ、Ⅵ、Ⅶ现为首次从该植物中得到,化合物Ⅶ为首次从蛇葡萄属植物中分得。