An Economical Method for Preparative Purification of Five Alkaloids from Coptis Chinensis Franch by High-Speed Counter-Current Chromatography Using Singled Prepared Solvent System by GC

Coptis chinensis Franch, a widely used Traditional Chinese Medicine, shows various kinds of bioactivity. The major active components of the herb are considered to be alkaloids. Thus, preparative separation of these alkaloids is critical important for further pharmacology and mechanism studies. In the paper, five alkaloids from C. chinensis were purified by HSCCC using the solvent system composed of chloro-form-metha- nol-water (2:1:1, v/v/v) single prepared. The content of each solvent in solvent system were determined by gas chromatography (GC), then according the ratios of solvents in each phase to prepare the mobile and stationary phase respectively. And a comparative study was carried out between together preparation and single preparation of the solvent system. The purities and recoveries of all the products were over 98.5% and 92%. However, 134 mL chloroform, 336 mL methanol and 452 mL water were saved when the two phase were singled by GC. Our research showed an economical method for separating alkaloids from C. chinensis by HSCCC using the solvent system single prepared by GC.

Modulation of gut microbiota by berberine and decocted Coptis chinensis Franch.in a high-fat diet-induced metabolic syndrome rat model

Objective:To investigate the effect of decocted Coptis chinensis Franch.(Huanglian Jianji,黄连煎剂,HLJJ) and berbenne on the gut microbiota of the rats with metabolic syndrome (MS) induced by high-fat diet (HFD).Methods:Sixty Sprague Dawley rats were randomly divided into normal diet (ND) group (n =15)and HFD group (n =45).After 8 weeks of feeding,the HFD group was further divided into three groups:a group provided with a combination of HLJJ and HFD for 4 weeks,a group provided with a combination of berberine and HFD for 4 weeks,and a group supplied only with HFD for 4 consecutive weeks.Epididymal fat weight,serum high-density lipoproteincholesterohC (HDL-C),lowdensity lipoproteincholesterol-C (LDL-C) and irisin levels were measured.In addition,the V3-V4 region of the cecum microbiota 16S rRNA gene amplicon was sequenced on the Illumina MiSeq platform.Results:MS was successfully induced in the rats with HFD for 8 weeks.After 12 weeks,serum irisin levels in the HFD + berberine group were significantly increased compared with those in the HFD group (P <.05).The high-quality tags were delineated into 1149 operational taxonomic units at a 97% similarity level.Furthermore,at the genus level,Akkermansia muciniphila (A.muciniphila),Bacteroides,and Ruminococcus were markedly increased,whereas Candidatus arthromitus,Prevotella,Phascolarctobacterium were in great decrease in the HLJJ group and the berberine group,compared with the HFD group.The abundance of A.muciniphila,Ruminococcus gnavus and Bacteroides was significantly higher in the berberine group,whereas,the abundance of Oscillospira was lower in the berberine group,compared with the HLJJ group.Conclusion:HLJJ and berberine changed the gut microbiota in rats with MS.The mechanism underlying the treatments of berberine and HLJJ on HFD-induced MS is partly different.

Network pharmacology and molecular dynamics study of the effect of the Astragalus-Coptis drug pair on diabetic kidney disease

BACKGROUND Diabetic kidney disease(DKD)is the primary cause of end-stage renal disease.The Astragalus-Coptis drug pair is frequently employed in the management of DKD.However,the precise molecular mechanism underlying its therapeutic effect remains elusive.AIM To investigate the synergistic effects of multiple active ingredients in the Astragalus-Coptis drug pair on DKD through multiple targets and pathways.METHODS The ingredients of the Astragalus-Coptis drug pair were collected and screened using the TCMSP database and the SwissADME platform.The targets were predicted using the SwissTargetPrediction database,while the DKD differential gene expression analysis was obtained from the Gene Expression Omnibus database.DKD targets were acquired from the GeneCards,Online Mendelian Inheritance in Man database,and DisGeNET databases,with common targets identified through the Venny platform.The protein-protein interaction network and the“disease-active ingredient-target”network of the common targets were constructed utilizing the STRING database and Cytoscape software,followed by the analysis of the interaction relationships and further screening of key targets and core active ingredients.Gene Ontology(GO)function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichments were performed using the DAVID database.The tissue and organ distributions of key targets were evaluated.PyMOL and AutoDock software validate the molecular docking between the core ingredients and key targets.Finally,molecular dynamics(MD)simulations were conducted to simulate the optimal complex formed by interactions between core ingredients and key target proteins.RESULTS A total of 27 active ingredients and 512 potential targets of the Astragalus-Coptis drug pair were identified.There were 273 common targets between DKD and the Astragalus-Coptis drug pair.Through protein-protein interaction network topology analysis,we identified 9 core active ingredients and 10 key targets.GO and KEGG pathway enrichment analyses revealed that Astragalus-Coptis drug pair treatment for DKD involves various biological processes,including protein phosphorylation,negative regulation of apoptosis,inflammatory response,and endoplasmic reticulum unfolded protein response.These pathways are mainly associated with the advanced glycation end products(AGE)-receptor for AGE products signaling pathway in diabetic complications,as well as the Lipid and atherosclerosis.Molecular docking and MD simulations demonstrated high affinity and stability between the core active ingredients and key targets.Notably,the quercetin-AKT serine/threonine kinase 1(AKT1)and quercetin-tumor necrosis factor(TNF)protein complexes exhibited exceptional stability.CONCLUSION This study demonstrated that DKD treatment with the Astragalus-Coptis drug pair involves multiple ingredients,targets,and signaling pathways.We propose a novel approach for investigating the molecular mechanism underlying the therapeutic effects of the Astragalus-Coptis drug pair on DKD.Furthermore,we suggest that quercetin is the most potent active ingredient and specifically targets AKT1 and TNF,providing a theoretical foundation for further exploration of pharmacologically active ingredients and elucidating their molecular mechanisms in DKD treatment.

黄连和三角叶黄连去甲乌药碱合成酶(NCS)基因克隆与原核表达分析

目的:探究黄连和三角叶黄连去甲乌药碱合成酶(NCS)基因的表达差异,为理解同属植物生物碱含量差异机制奠定基础。方法:采集相同生长年限的黄连和三角叶黄连,分析其叶片、根和根茎的生物碱含量和生物碱合成的关键基因表达量,对关键基因进行克隆并构建原核表达载体。结果:黄连和三角叶黄连各部位生物碱含量存在差异,且NCS基因与各生物碱含量关系最密切,生物信息学分析显示NCS基因在两种黄连中无明显差异,同时亲缘关系十分接近;成功构建NCS基因的原核表达载体。结论:黄连和三角叶黄连的NCS基因是生物碱合成的关键基因,其在根的表达差异可能是影响生物碱合成的重要因素。

不同育苗基质及激素处理对华重楼种子出苗的影响研究

针对重楼育苗常遇到的出苗时间长、出苗率低和种子播种后腐烂严重的问题,对最佳育苗方式、育苗基质及在较短时间内获得较多的重楼种苗等方面进行研究,旨在为重楼栽培和育苗方面提供技术参考。研究了不同厚度稻草覆盖、土壤与草炭不同配比、不同浓度KNO_(3)与质量浓度赤霉素(GA_(3))溶液浸种重楼种子对重楼种子出苗的影响。结果表明:在重楼种子播种前的土壤表面覆盖厚度为4 cm的稻草后播种重楼,再在重楼种子表面覆盖厚度为4 cm的稻草,重楼种子的出苗率最高,达90.8%,与其他覆盖厚度的差异具有高度统计学意义,出苗时间为播种后的第20月;土壤与草炭质量比为1∶3时,重楼种子的出苗率为73.0%,与其他质量比的出苗率的差异具有高度统计学意义(P<0.01),出苗时间为播种后的第20月;用19 mmol/L KNO_(3)、600 mg/L GA_(3)溶液浸种重楼种子,种子胚根长出率最高达56.67%,与排第2位溶液组合(KNO_(3)浓度为16 mmol/L、GA_(3)质量浓度为600 mg/L)胚根长出率的差异具有高度统计学意义(P<0.01),出苗时间为浸种处理后的第8月。3种育苗方式比较表明:稻草覆盖厚度为4 cm、土壤与草炭质量比为1∶3的育苗基质利于提高重楼种子的出苗率,KNO_(3)浓度为19 mmol/L、GA_(3)的质量浓度为600 mg/L的溶液浸种重楼种子,可大大缩短重楼种子的育苗时间,幼苗获得率约50%。

浇施磷酸二氢钾对华重楼幼苗生长与光合特性的影响

为了培育优质华重楼[Paris polyphlla Smith var.chinensis(Franch.)Hara]种苗,开展了二年生一叶期盆栽幼苗不同浓度磷酸二氢钾追肥研究,并测定其光合参数和生长量。结果表明,追施磷酸二氢钾后,华重楼幼苗的株高、地茎粗、光合色素及光合效率与对照相比均有显著提高。当磷酸二氢钾浓度为0~300 mg/L时,幼苗的株高、生根数量、茎鲜重、叶鲜重、叶绿素a、总叶绿素含量及类胡萝卜含量均随磷酸二氢钾浓度升高而增加,而地茎粗和叶绿素b含量则在磷酸二氢钾浓度为200 mg/L时达到最大值。当磷酸二氢钾浓度超过300 mg/L时,其相应的指标随着磷酸二氢钾浓度升高呈下降趋势。当磷酸二氢钾浓度达300 mg/L时,幼苗叶片净光合速率最大,胞间CO_(2)浓度最低。

十五种药用植物水提物对层出镰刀菌的抑制活性

开展植物提取物对层出镰刀菌(Fusarium proliferatum)的抑制活性研究,为其防治奠定理论基础,采用菌丝生长速率法测定药用植物水提物对层出镰刀菌的抑制及毒力作用。结果表明:黄连等15种药用植物提取物对层出镰刀菌菌丝生长有一定的抑制作用,当药用植物水提物浓度为20 mg干样/mL时,其抑菌率为0.24%~77.17%,其中以黄连的抑制效果最佳,黄连水提物对层出镰刀菌的毒力回归方程的EC50值为1.0630 mg干样/mL。黄连水提物在防治层出镰刀菌方面具有的良好应用潜力。

黄连重金属ATP酶基因CcHMA3的克隆和表达分析

目的:克隆黄连重金属ATP酶基因CcHMA3,并对其编码蛋白进行生物信息学以及差异表达分析。方法:根据黄连基因组和转录组数据,利用PCR扩增CcHMA3的全长cDNA并进行生物信息学分析。利用实时荧光定量PCR法(qRT-PCR)检测CcHMA3基因在黄连植株中的表达差异。结果:克隆获得CcHMA3基因,开放阅读框全长为3030 bp,编码1009个氨基酸;蛋白相对分子量为109.03 kD,理论等电点为6.7;蛋白序列中含有6个跨膜结构域和2个保守结构域,亚细胞定位预测表明CcHMA3位于质膜上,具有一定亲水性。qRT-PCR结果表明,CcHMA3基因在黄连不同组织中的相对表达量由高到低依次为须根、根茎、叶柄、叶片;当镉处理后,CcHMA3基因在须根中的表达量显著高于其他组织(P<0.05),且对不同镉胁迫处理时间存在明显不同的响应表达。结论:该研究为进一步深入研究CcHMA3基因在黄连重金属镉代谢过程中的调控机制,进而寻找可能的阻断方式奠定了实验基础。

Investigation of the differences between the “COLD” and “HOT” nature of Coptis chinensis Franch and its processed materials based on animal’s temperature tropism

The description and differentiation of the so-called "Cold" and "Hot" natures, the primary "Drug Naure" of Chinese medicine, is the focus of theoretical research. In this study, the divergency between the "Cold" and the "Hot" natures was investigated through examining the temperature tropism of mice affected by Coptis chinensis Franch and its processed materials by using a cold/hot plate differentiating technology. After exposure to C. chinensis Franch, the macroscopic behavioral index of the remaining rate (RR) on a warm pad (40℃ ) significantly increased (P<0.05), suggesting the enhancement of Hot tropism. The internal indexes of adenosine triphosphatase (ATPase) activity and oxygen consuming volume decreased significantly (P<0.05), suggesting the decapability of energy metabolism. This external behavior of Hot tropism might reflect the internal Cold nature of C. chinensis Franch. However, the processed materials of C. chinensis Franch exhibited a different Cold nature in temperature tropism compared with crude C. chinensis Franch (CC): the Cold nature of bile-processed C. chinensis Franch (BC) enhanced while the ginger-processed C. chinensis Franch (GC) changed inversely. The changing sequence was consistent with the theoretical prognostication. It is indicated that the external Cold & Hot natures of Chinese medicine may possibly reflect in an ethological way for the changes of animal’s temperature tropism which might be internally regulated by the body’s energy metabolism.

基于网络药理学探究黄连调控巨噬细胞干预动脉粥样硬化斑块稳定性的潜在机制

目的探究黄连调控巨噬细胞干预动脉粥样硬化斑块稳定性的潜在机制。方法对GEO数据库中的动脉斑块相关数据集进行差异分析得到巨噬细胞干预动脉粥样硬化斑块相关基因。从TCMSP数据库获取黄连的有效成分和潜在靶点。对两者取交集后找出发挥作用的化学成分和潜在靶点。对潜在靶点进行蛋白质-蛋白质相互作用(PPI)、基因本体(GO)、京都基因与基因组百科全书(KEGG)分析,探究黄连调控巨噬细胞干预动脉粥样硬化斑块稳定性的作用机制。结果稳定斑块组和破裂斑块组差异分析共找到892个差异基因。通过TCMSP共找出黄连中的11个化学成分,251个靶点。两者取交集后得到16个黄连调控巨噬细胞的潜在靶点。PPI结果显示,DPP4、TNFAIP6、IL6ST、POR、RUNX1T1、HMOX1、CAV1等16个交集基因之间有较强的相互作用关系,且DPP4、HMOX1、CAV1和VCAM1处于PPI网络的枢纽位置。GO结果表明,生物学过程(BP)与对脂多糖的反应、对细菌来源分子的反应、对T细胞激活的正向调节等有关。细胞组成(CC)与膜筏、膜微区、膜区等细胞器有关。分子功能(MF)参与肽酶活化剂活性、趋化因子活性等分子功能的调节。KEGG结果与流体剪切应力及动脉粥样硬化、NF-kappa B信号传导途径有关。结论黄连内槲皮素可能通过调节DPP4、HMOX1、CAV1等靶点影响斑块内巨噬细胞的信号传导途径,进而干预斑块稳定性。

基于网络药理学和分子对接探讨“寒凉”中药黄连、黄芩、黄柏治疗痴呆的作用机制

目的 基于网络药理学和分子对接探讨“寒凉”中药黄连、黄芩、黄柏(三黄)治疗痴呆的作用机制。方法 通过数据挖掘《宣明论方》,筛选出符合“寒凉”属性并且是治疗“痴呆症”的常见中药黄芩、黄连、黄柏。通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, TCMSP)数据库检索“三黄”的有效成分和作用靶点,在GeneCards、DrugBank等数据库检索痴呆症(dementia)靶点。利用Cytoscape3.7.2构建“药物-成分-靶点”网络,用VENNY2.1获得“三黄”和痴呆症的交集靶点后,构建蛋白质互作网络(protein-protein interaction networks, PPI),利用Metascape库对核心靶点进行GO、KEGG富集分析,并构建“三黄”药物-成分-靶点-通路-疾病网络,最后利用分子对接,以反向验证“三黄”活性成分与核心靶点之间的关系。结果 筛选出“三黄”治疗痴呆症的重要活性成分有槲皮素、β-谷甾醇、小檗碱、黄连碱等,关键核心靶点有TP53、AKT1、IL6、JUN、MAPK14、TNF等。KEGG通路分析有129条,主要有AGE-RAGE信号介导的糖尿病并发症通路、PI3K-Akt信号通路、钙信号通路等;GO功能中,分子功能117条,细胞组成61条,生物过程1 366条,主要涉及配体激活转录因子活性、DNA结合转录因子结合等分子功能,膜微区、突触后膜等细胞组成,活性氧代谢过程、对脂多糖的反应、炎症反应等生物过程。分子对接结果可以看出重要活性成分与核心靶点全部对接成功。结论 本研究结果表明,“三黄”特有的药性优势,可以通过多成分、多靶点、多通路的方式治疗痴呆症,为“三黄”治疗痴呆症临床应用和机制研究提供了依据。

川黄连黄酮类成分抗氧化活性研究

文章探究川黄连中黄酮类成分的抗氧化性能,采用正交实验设计法优化川黄连中黄酮类成分的提取工艺,并考查总黄酮提取物对1,1-二苯基-2-三硝基苯肼自由基(DPPH·)、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐阳离子自由基(ABTS+·)的清除率;提取川黄连中总黄酮的最佳工艺条件为:75%乙醇、料液比1∶15(g/mL)、回流提取时间1.5 h。在该工艺下,川黄连中黄酮的总含量为0.6768%。质量浓度分别为0.0036~0.02875 mg/m L、0.02875~0.2300 mg/mL,川黄连总黄酮对DPPH自由基和ABTS自由基的清除能力明显强于VC。川黄连总黄酮对两者均有较好的清除能力,且在一定浓度内体外抗氧化活性明显强于VC。从而得出川黄连黄酮类成分具有一定抗氧化能力的结论,说明生物碱之外的其他化学成分有较好的开发价值,本文为川黄连抗氧化活性的深入研究及开发利用提供了一定的参考。

华重楼愈伤组织培养及薯蓣皂苷含量测定

以华重楼[Paris polyphylla Smith var.chinensis(Franch.)Hara]植物多种器官为外植体,经不同消毒处理和培养基诱导培养后,观察其愈伤组织的诱导生长情况;并以TTC法检测诱导产生的愈伤组织中的薯蓣皂苷含量。结果表明,华重楼的隐芽、内层芽鞘、根茎、预处理后根茎和子房外植体经诱导培养后,均可不同程度地产生愈伤组织;其中以预处理后根茎外植体产生的愈伤组织生长速度最快,诱导率也最高,为33.5%。经检测结果显示,多次继代培养的华重楼愈伤组织中含有与华重楼对照药材和薯蓣皂苷对照品相同的薯蓣皂苷或其类似物。

核磁共振氢谱法鉴别黄连的研究

用 FT- NMR波谱仪测定了 6个不同产地黄连特征总提物的 1 HNMR图谱 ,并对特征信号进行了归属。从特征总提物中分得并鉴定了小檗碱和棕榈碱。分析结果指出 ,每种黄连的 1 HNMR图谱都显示出原小檗碱型生物碱的特征共振峰。

21个产地七叶一枝花中皂苷类成分的评价

目的对21个产地七叶一枝花Paris Polyphylla Smith var.chinensis(Franch.)Hara中的皂苷类成分进行评价。方法 HPLC法测定偏诺皂苷{偏诺皂苷-3-O-β-D-glu(1→3)[α-L-rha(1→2)]-β-D-glu(PGGR)、重楼皂苷Ⅵ、Ⅶ、H}和薯蓣皂苷(纤细薯蓣皂苷、重楼皂苷Ⅰ、Ⅱ、Ⅴ)的含有量,SPSS 22.0软件进行主成分分析和聚类分析。结果偏诺皂苷为七叶一枝花皂苷的主要种类。PGGR、重楼皂苷Ⅰ、Ⅴ、Ⅶ、H为该药材主要有效成分;7个产地的综合得分大于0,依次为巴中>青川>盐津>通江>南川>崇州>洪雅。21批样品分为4类,与聚类分析结果相似。结论四川巴中、青川、崇州、洪雅、通江,云南盐津,重庆南川适宜栽培七叶一枝花,可保证皂苷类成分的稳定性。

野生珍稀药用植物七叶一枝花的成分含量分析

利用分光光度计、高效液相色谱仪和等离子光谱仪对华中地区珍稀药用植物七叶一枝花[ParispolyphyllaSmithvar.chinensis(Franch.)Hara]根茎的化学成分薯蓣皂甙元、黄酮、19种游离氨基酸、微量元素进行了分析。结果显示:薯蓣皂甙元含量为0.62%,黄酮含量为0.0628%,总游离氨基酸含量为0.33%,微量元素中钙为0.77%,镁为0.20%,钾为0.98%,铁为0.20%,锌为44.8mg/kg,磷为746mg/kg。

干旱胁迫对黄连生理作用的影响

研究干旱胁迫对黄连MDA、Pro、Protein含量和SOD、POD、CAT活性的影响以及SNP(亚硝基铁氰化钠)对干旱胁迫的缓解作用,设置5个处理,分别记为S0,S1,S2,S3和S3S．结果表明:MDA含量随胁迫时间的延长和强度的增加呈上升趋势,S1,S2和S3与对照S0存在显著差异;Pro含量先升高后下降,S2与S0呈显著差异;SOD, POD活性下降,S3与S0呈显著差异;CAT活性先下降后升高;Protein含量下降．不同处理间CAT活性和Protein 含量无显著差异．SNP喷洒后,MDA和Pro的增加速率明显降低,SOD,POD和CAT下降的速率也有明显的降低,且对CAT活性的回升有促进作用．因此,0．1 mmol／L SNP能有效缓解干旱对黄连的伤害．

七叶一枝花中4种重楼皂苷同时测定与聚类分析

目的建立HPLC法同时测定七叶一枝花Paris polyphylla Smith.var chinensis(Franch.)Hara中重楼皂苷Ⅰ、Ⅱ、Ⅵ、Ⅶ的含有量,并进行聚类分析。方法该植物甲醇提取液的分析采用Waters XBridge C18色谱柱(4.6 mm×250 mm,5μm);流动相乙腈-水,梯度洗脱;体积流量1.0 m L/min;检测波长203 nm;柱温25℃;进样量20μL。考察样品农艺性状(株高、茎粗、叶长、叶宽、叶长宽比)。DPS7.05软件进行聚类分析。结果 7个产地(浙江、云南、江西、湖北、湖南、广西、四川)18批样品的农艺性状与重楼皂苷含有量有所差异,其中浙江、云南、广西产者含有量较高,江西、湖北产者较低。重楼总皂苷含有量与茎粗和叶长宽比呈显著正相关,与叶宽呈负相关,与经纬度相关性较低。各种质可划分为4个类群,其中类群Ⅰ农艺性状良好,重楼总皂苷含有量较高。结论以重楼总皂苷含有量为目标筛选七叶一枝花时,应优先考虑浙江泰顺、磐安、庆元、湖南永顺种源。

黄连ISSR反应条件优化的研究

以黄连(味连,Coptis ChinensisFranch.)基因组DNA为模板,通过单因子、双因子实验研究了ISSR反应体系中主要成分(Mg2+、dNTP、引物、模板、TaqDNA聚合酶)以及热循环参数(退火温度、循环数、变性时间、退火时间、延伸时间)对扩增结果的影响,并找出各自的最适条件,建立了适合黄连ISSR分析的反应体系和扩增程序,即在25μL反应体系中,内含1×PCR buffer、1.5mmol.L-1Mg2+、200μmol.L-1dNTP、0.3μmol.L-1引物、40ng模板、1UTaqDNA聚合酶。扩增程序为94℃预变性5min,然后进行35个循环:94℃变性30s,(据不同引物的退火温度)复性1min,72℃延伸1.5min,循环结束后72℃延伸7min,-4℃保存。这一优化系统的建立为今后利用ISSR标记技术进行黄连鉴定及种质遗传多样性分析提供了一个标准化程序。

七叶一枝花地上部分化学成分研究

目的:研究七叶一枝花地上部分的化学成分。方法:用95%乙醇提取七叶一枝花地上部分,通过硅胶柱层析、RP-18、Sephadex LH-20等色谱方法分离化合物,以波谱方法鉴定化合物的结构。结果:从七叶一枝花地上部分中分离得到10个化合物,分别鉴定为:β-谷甾醇(1)、麦角甾-7,22-二烯-3-酮(2)、β-蜕皮激素(3)、山柰酚(4)、胡萝卜苷(5)、木犀草素(6)、calonysterone(7)、木犀草苷(8)、槲皮素(9)、3β,5α,9α-三羟基-麦角甾-7,22-二烯-6-酮(10)。结论:其中,化合物2、6、10为首次从该属植物中分离得到。