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.

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。

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。

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。

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。

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。

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．

黄连不同部位水浸液自毒作用研究

目的:研究黄连不同部位水浸液对黄连生长的自毒作用。方法:采用生物测定方法,研究不同浓度的黄连根系分泌物、根茎浸提液、植株残体分解产物、根际土浸提液对黄连种子萌发、幼苗及成株生长的影响。结果:黄连不同部位水浸液对黄连生长有较强的抑制作用。结论:自毒作用是造成黄连连作障碍的重要原因之一,本研究对黄连的生产与管理具有重要意义。

外源5-氨基乙酰丙酸对盐胁迫下黄连幼苗光合参数及其叶绿素荧光特性的影响

该实验以2年生黄连幼苗为材料,在100mmol·L-1的NaCl模拟盐胁迫条件下,经不同浓度的外源5-氨基乙酰丙酸(ALA)处理后,测定黄连幼苗光合色素含量、叶绿素荧光参数及气体交换参数等光合生理指标的变化,探寻提高黄连幼苗在盐胁迫条件下抗性能力的途径。结果显示:(1)NaCl胁迫下黄连幼苗的光合生理受到显著抑制,在经过不同浓度的ALA处理后,显著提高了叶绿素a、叶绿素b、总叶绿素和类胡萝卜素等光合色素的含量。(2)盐胁迫下,黄连植株的的净光合速率(Pn)、气孔导度(Gs)及叶片蒸腾速率(Tr)均发生下降,并且随着胁迫时间和胁迫浓度的增加下降幅度逐渐增大,胞间CO2浓度(Ci)则呈上升趋势,说明盐胁迫下黄连净光合速率降低的主要影响因素是非气孔因素。(3)用ALA处理后,黄连的Pn、Gs及Tr均有不同程度的提高,Ci也有不同程度的降低,并且不同的浓度梯度存在着显著的效果差异。(4)ALA处理还提高了最大荧光(Fm,1.234)、最大光化学效率(Fv/Fm,0.849)、PSⅡ有效光化学效率(Fv′/Fm′,0.685)、PSⅡ实际光化学效率(ΦPSⅡ,0.545)和光化学淬灭系数(qP,0.872)的水平,有效降低了初始荧光(F0,0.211)和非光化学淬灭系数(NPQ,0.251)的水平。研究表明,外源ALA通过提高黄连幼苗叶片的光合色素含量,减少过剩激发能的耗散,提高光合电子传递效率,有效缓解了盐胁迫对黄连叶片PSⅡ的伤害,提高了植株的抗盐能力。

黄连酒炙过程中5种生物碱的变化研究

目的探究黄连炮制过程中5种生物碱的量变和质变。方法采用高效液相色谱法测定不同炮制方法、不同炮制温度对黄连酒炙品中生物碱含量的变化。结果酒炙黄连随着加热温度的增加,小檗碱、巴马汀、药根碱、黄连碱、表小檗碱在160℃时含量最高,此后含量逐渐降低。表小檗碱和黄连碱分别在170、240℃时含量降至最低以致高效液相色谱仪无法检出,在170℃时开始出现新的化学成分小檗红碱。结论黄连酒炙过程中5种生物碱发生了量变与质变,黄酒闷润与加热是关键的步骤,可以促进黄连生物碱的含量增加或转化为新的化学物质。辅料黄酒不能被乙醇替代。

加速溶剂萃取法快速提取黄连中的生物碱

探讨了快速溶剂萃取法(ASE)提取黄连中生物碱的可行性,并比较了该方法与回流提取法和超声提取法的优越性。以黄连中盐酸小檗碱的提取率为指标,以高效液相色谱法(HPLC)为检测方法,用正交实验对快速溶剂萃取法从黄连中提取盐酸小檗碱的工作条件进行优化。最佳仪器参数:提取溶剂为80%乙醇+0.5%HCl,提取温度为130℃,静态提取时间为10 min,提取次数为1次。快速溶剂萃取法可作为黄连中生物碱分析测定的前处理方法。

黄连的毛细管电泳特征指纹图谱研究

目的 建立黄连的高效毛细管电泳指纹图谱。方法 采用毛细管电泳考察了不同批药材季铵碱的保留时间和峰面积的相关性 ,并以小檗碱作为参照物计算了各色谱峰的相对保留时间和峰面积比值。结果 不同批号的黄连样品色谱组分的保留时间和峰面积的相关系数均大于 0 .9990。结论 建立的黄连高效毛细管电泳指纹图谱稳定、可靠 ,可作为黄连的特征指纹图谱。

黄连的研究进展

概述了黄连栽培的发展应用现状 ,化学、药理、临床及栽培生产的研究进展 ,提出了黄连栽培生产及新药开发的发展方向。

不同用量的吴茱萸炮制黄连后成分的比较

目的 考察不同用量的吴茱萸炮制黄连对小檗碱的影响以及萸黄连中吸收吴茱萸成分的情况。方法 以小檗碱、吴茱萸碱、吴茱萸次碱为指标 ,采用高效液相色谱法测定黄连 ,吴茱萸 ,10 %,2 0 %,30 %,40 %,5 0 %萸黄连中的成分含量。结果 黄连用吴茱萸炮制后小檗碱的含量减少 ,2 0 %,30 %萸黄连中小檗碱的含量较高 ;萸黄连中确实吸收了吴茱萸中的成分。结论 吴茱萸炮制黄连的用量以 2 0 %为宜。

应用M-H琼脂进行黄连对252株临床菌株的体外抗菌活性研究

目的 评价黄连对 2 5 2株临床菌株的体外抗菌活性。方法 采用新方法 (M- H琼脂代替营养琼脂 )对黄连进行金黄色葡萄球菌、表皮葡萄球菌和肠球菌等体外抗菌活性检测。结果 黄连对金黄色葡萄球菌、表皮葡萄球菌和肠球菌的 MIC5 0 分别为 0 .16 1,0 .16 1和 0 .32 2 m g/ m L。结论 黄连对金黄色葡萄球菌。