川牛膝(Cyathula officinalis Kuan.)RAPD和AFLP标记的多态性聚类分析

[目的]用RAPD和AFLP技术对采自主产区不同地域的川牛膝Cyathula officinalis Kuan.、头花杯苋Cyathula capitata(Wall) Moq及它们的杂交品种杂交牛膝Hybrid accsssion进行多态性和聚类分析研究。[方法]用酚-氯仿法提取的基因组DNA,经RAPD及AFLP程序扩增后分别用琼脂糖凝胶和PAGE电泳检测,使用NTSYSpc-2.10s软件用UPGMA方法对检测结果进行聚类。[结果]RAPD和AFLP技术在分析遗传多样性和品种鉴定方面各有优缺点;采自同一地方的川牛膝具有最近的亲缘关系,头花杯苋、杂交牛膝与其他采自各地的川牛膝品种之间的遗传差异较大。[结论]在川牛膝中,DNA的差异与地域性有较大关系,说明药材确有地道性的特征,从而在DNA水平上证明了药用植物上的地道性观点。

A new heterocyclic compound from Cyathula officinalis Kuan

A new heterocyclic compound,named 5,5 -diisobutoxy-2,2 -bifuran(1),and four known compounds(2-5) were isolated from the roots of Cyathula officinalis Kuan.Their structures were elucidated by spectroscopic methods.Among these compounds,5,5 - dibutoxy-2,2 -bifuran(2) was isolated for the first time from this plant.

Cyathula prostrata:A potent source of anticancer agent against daltons ascites in Swiss albino mice

Objective:To evaluate the anticancer activity of the methanolic extract of Cyathula prostrata in Ehrlich ascites carcinoma(EAC)-bearing mice with methotrexate as positive control in the advanced stage of tumorigenesis.Methods:EAC was induced in swiss albino mice by injecting10~6 cell/mL of tumor cell suspension intraperitoneal.The methanolic extract of Cyathula prostrata effect on the tumor cell viability,DNA fragmentation and MTT assay were carried out.Results:Methanolic extract attenuated percentage increased in the cell survival lime when compared to control group.However,the effect was less than that of methotrexat.Methotrexat and the extracts reversed the tumor-induced alterations in UNA fragmentation and MTT assay.Conclusions:The present study suggests that the methanol extract of Cyathula prostrata has significant anticancer activity and that is comparable to that of methotrexate.

Extraction Process and Antioxidant Activity of Polysaccharides from Cyathula Capitata( Wall. ) Moq.

[Objectives]To optimize the ultrasonic-assisted extraction process of polysaccharides from Cyathula Capitata( Wall.) Moq. and evaluate its antioxidant capacity in vitro. [Methods] The single factor orthogonal design was applied to explore the effects of ultrasonic time,solid-to-liquid ratio,extraction time and ethanol precipitation concentration on the extraction rate of crude polysaccharides from C. Capitata,to obtain the optimal extraction process; DPPH and ABTS^+ free radical scavenging ability was detected to evaluate its in vitro antioxidant activity.[Results]The optimal extraction process of C. Capitata crude polysaccharide was as follows: solid-to-liquid ratio was 40∶ 1,the extraction temperature was 50℃,the ultrasonic time was 80 min. Under such conditions,the extraction rate of C. Capitata crude polysaccharide was 10. 51%,and the purity was 56. 9%; C. Capitata crude polysaccharide has good scavenging ability to DPPH and ABTS^+ free radicals,and its IC_(50) values are 0. 468 and 0. 231 mg/mL,respectively. [Conclusions]The optimized process is simple and feasible,and the C. Capitata polysaccharide has good antioxidant activity in vitro. This experiment provides a basis for the further development and utilization of the C. Capitata polysaccharide.

Ethanol extract of Cyathulae Radix inhibits osteoclast differentiation and bone loss

Cyathulae Radix,a traditional Chinese medicine and a common vegetable,boasts a history spanning millennia.It enhances bone density,boosts metabolism,and effectively alleviates osteoporosis-induced pain.Despite its historical use,the molecular mechanisms behind Cyathulae Radix’s impact on osteoporosis remain unexplored.In this study,we investigated the effects and mechanisms of Cyathulae Radix ethanol extract(CEE)in inhibiting osteoporosis and osteoclastogenesis.Eight-week-old female mice underwent ovariectomy and were treated with CEE for eight weeks.Micro-computed tomography(micro-CT)assessed histomorphometric parameters,bone tissue staining observed distal femur histomorphology,and three-point bending tests evaluated tibia mechanical properties.Enzyme-linked immunosorbent assay(ELISA)measured serum estradiol(E2),receptor activator for nuclear factor B ligand(RANKL),and osteoprotegerin(OPG)levels.Osteoclastogenesis-related markers were analyzed via Western blotting(WB)and quantitative real-time polymerase chain reaction(qRT-PCR).Additionally,CEE effects on RANKL-induced osteoclast formation and bone resorption were investigated in vitro using tartrate-resistant acid phosphatase(TRAP)staining,qRT-PCR,and WB assay.Compared with the ovariectomy(OVX)group,CEE treatment enhanced trabecular bone density,maximal load-bearing capacity,and various histomorphometric parameters.Serum E2 and OPG levels significantly increased,while Receptor activator of nuclear factor-κB(RANK)decreased in the CEE group.CEE downregulated matrix metallopeptidase 9(MMP-9),Cathepsin K(CTSK),and TRAP gene and protein expression.In bone marrow macrophages(BMMs),CEE reduced mature osteoclasts,bone resorption pit areas,and MMP-9,CTSK,and TRAP expression during osteoclast differentiation.Compared with DMSO treatment,CEE markedly inhibited RANK,TNF receptor associated factor 6(TRAF6),Proto-oncogene c-Fos(c-Fos),Nuclear factor of activated T-cells cytoplasmic 1(NFATc1)expressions,and Extracellular regulated protein kinases(ERK),c-Jun N-terminal kinase(JNK),NF-kappa B-p65(p65)phosphorylation in osteoclasts.In conclusion,CEE significantly inhibits OVX-induced osteoporosis and RANKL-induced osteoclastogenesis,potentially through modulating the Estrogen Receptor(ER)/RANK/NFATc1 signaling pathway.

基于UPLC-Triple-TOF-MS技术初步分析雅安产道地中药材川牛膝中生物碱成分

采用乙醇提取、成盐、二氯甲烷萃取等方法初步富集川牛膝总生物碱,确定最优提取分离工艺;通过超高液相色谱串联三重四级杆飞行时间质谱法,采用Waters XSelect CSH-C_(18)色谱柱(2.1 mm×150 mm,1.7μm),梯度洗脱,流速为0.3 mL/min,测试波长254 nm,柱温为50℃,进样量为3μL,采用电喷雾离子源正离子模式,扫描范围为m/z 100~1500,并采用全扫描模式对样品数据进行收集,根据高分辨质谱结合二级质谱所得信息对川牛膝中生物碱类化学成分进行快速鉴定,通过对比分子离子峰和碎片离子的质荷比、Scifinder和Reaxy数据库及相关文献,推测出两种生物碱成分。研究建立的生物碱类化学成分提取与分析方法,为提高雅安产道地中药材川牛膝的质量控制水平及后续阐明药效物质基础提供了实验依据。

播种深度与土壤水分含量对川牛膝出苗率及幼苗生长的影响

分析不同播种深度与土壤水分含量对川牛膝(Cyathula officinalis Kuan)种子出苗率及幼苗生长的影响,探寻适宜的播种深度与苗床土壤水分含量。通过盆栽试验,设置不同的播种深度与土壤水分含量处理,通过称重进行补水,测定发芽势、出苗率及幼苗生长与生理指标。结果表明,播种深度与土壤水分含量对种子出苗率及幼苗生长有显著影响。播种深度为0.31~0.61 cm时,种子发芽势与出苗率较高;播种深度为0.61~0.92 cm时,幼苗叶片叶绿素含量高,抗逆性较好;土壤水分含量为60.71%时,种子发芽势与出苗率较高;土壤水分含量为53.35%~60.71%时,幼苗长势较旺;土壤水分含量为38.62%~53.35%时,幼苗抗逆性较好;土壤水分含量为45.98%~53.35%时,幼苗叶片叶绿素含量较高。川牛膝种子适宜播种深度为0.61 cm,播种后土壤水分含量保持在60.71%左右,可以获得较高出苗率,出苗较整齐;出苗后,土壤水分含量保持在53.35%左右,幼苗长势旺盛,叶片叶绿素含量高,抗逆性好。

基于网络药理学探讨川牛膝治疗高血压的作用机制

目的:利用网络药理学分析川牛膝治疗高血压的作用机制。方法:应用中药系统药理学数据库及分析平台(TCMSP)筛选出川牛膝的活性成分及其作用靶点,采用OMIM数据库,Drugbank数据库,TTD数据库,CTD数据库和DISGENT数据库检索与高血压相关的靶点基因。将川牛膝及高血压的共同靶点导入STRING数据库进行蛋白质-蛋白质相互作用分析(PPI);利用METASCAPE数据库进行核心靶点GO功能富集和KEGG通路富集分析。结果:川牛膝主要化学成分为槲皮素、杯苋甾酮、红苋甾酮、β-谷甾醇、齐墩果酸等;共筛选出关键共同靶点170个,包括AKT1、IL6、VEGFA、TP53、CASP3、TNF、JUN、EGF、MAPK1、EGFR、PTGS2、STAT3等;GO富集分析得到生物学进程主要涉及炎症反应、对伤害的反应、对脂多糖的反应、活性氧代谢过程、凋亡信号通路等;KEGG富集信号通路主要包含糖尿病并发症AGE-RAGE信号路径、肿瘤的途径、乙型肝炎、TNF信号通路、肿瘤中的蛋白质多糖等。结论:川牛膝治疗高血压存在多通路、多靶点的可能,揭示了川牛膝治疗高血压的潜在分子机制。

Structural Features of Fructans from the Root of Cyathula officinalis Kuan

Three fructans (CoPS1, CoPS2 and CoPS3) vrere isolated from the root of Cyathula officinalis Kuan, a traditional Chinese medicine. The structures of the fructans were determined by methylation, reductive-cleavage method combined with GC-MS analysis, and 13C NMR spectroscopy. These results show that the fructans (CoPS1, CoPS2 and CoPS3) are graminan type fructans, and comprised of (2→1)- and (2→6)-linked β-D-fructofuranosyl backbone residues containing high branches.

Triterpenoid saponins from the roots of Cyathula officinalis and their inhibitory effects on nitric oxide production

The present study was designed to investigate the chemical constituents of the roots of Cyathula officinalis. Compounds were isolated by silica gel, Sephadex LH-20, ODS column chromatography, and preparative HPLC. Their structures were determined on the basis of 1D and 2D NMR techniques, mass spectrometry, and chemical methods. One new oleanane-type triterpenoid saponin, 28-O-[α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl-(1→3)-β-D-glucopyranosyl] hederagenin(1), was isolated from the roots of Cyathula officinalis. The anti-inflammatory activities of the isolates were evaluated for their inhibitory effects against LPS-induced nitric oxide(NO) production in RAW 264.7 macrophages cells. Compounds 2, 4, and 6 exhibited moderate anti-inflammatory activities.

芪龙活络颗粒质量标准研究

目的建立芪龙活络颗粒的质量标准。方法采用薄层色谱法对制剂中黄芪、川牛膝、鸡血藤药材进行定性鉴别;采用高效液相色谱法测定丹酚酸B的含量,色谱柱为Eclipse XDB-C_(18)柱(150 mm×4.6 mm,5µm),流动相为乙腈-0.1%磷酸溶液(23∶77,V/V),流速为1.0 mL/min,检测波长为286 nm,柱温为30℃,进样量为10µL。结果薄层色谱斑点清晰,且阴性对照无干扰。丹酚酸B的进样量在0.0624~1.248µg范围内与峰面积线性关系良好(r=1.000,n=5);精密度、稳定性、重复性试验结果的RSD均小于2.00%(n=6);平均回收率为107.61%,RSD为4.28%(n=6)。5批样品中丹酚酸B的含量为1.09~1.32 mg/g。结论该方法专属性好、结果准确,可用于芪龙活络颗粒的质量控制。暂订芪龙活络颗粒中丹酚酸B的含量应不低于1.0 mg/g。

基于网络药理学和分子对接探讨川牛膝治疗继发性闭经的作用机制

目的:基于网络药理学和分子对接技术探讨川牛膝治疗继发性闭经(secondary amenorrhea, SA)的作用机制。方法:从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)检索川牛膝的活性成分及作用靶点,从GeneCards数据库获取SA的疾病靶点,采用R软件得到两者的交集靶点。借助STRING数据库构建蛋白质-蛋白质相互作用(protein-protein interaction, PPI)网络,通过Cytoscape 3.8.2软件进行聚类分析和拓扑分析从而筛选核心靶点。应用R软件进行基因本体(gene ontology, GO)和京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)富集分析(P<0.05)。最后,采用Autodock Vina软件进行分子对接验证。结果:川牛膝活性成分4个,即槲皮素、β-谷甾醇、甜菜素、红苋甾酮;川牛膝作用靶点178个,SA相关靶点1 366个,两者的交集靶点56个;基于Network Analyzer插件的拓扑分析筛选得到9个核心靶点,包括丝氨酸/苏氨酸蛋白激酶(serine/threorine protein kinase, AKT1)、丝裂原活化蛋白激酶1(mitogen activated protein kinase 1,MAPK1)、雌激素受体1(estrogen receptor 1,ESR1)等。GO功能分析主要涉及对类固醇激素的反应、活性氧代谢过程、上皮细胞增殖等;KEGG通路135条,主要包括HIF-1信号通路、内分泌抵抗、雌激素信号通路等。分子对接显示,活性成分(槲皮素、β-谷甾醇)与核心靶点(AKT1、MAPK1)具有较好的结合活性。结论:川牛膝治疗SA具有多成分、多靶点、多途径的特点,其可能通过槲皮素、β-谷甾醇等成分作用于AKT1、MAPK1、ESR1等靶点,调控雌激素信号通路、内分泌抵抗等通路。

Inokosterone Is a Potential Drug Target of Estrogen Receptor 1 in Rheumatoid Arthritis Patients:Analysis from Active Ingredient of Cyathula Officinalis

Objective:To elucidate the active compounds and the molecular mechanism of Cyathula Off/c/na//s as a drug treatment for rheumatoid arthritis(RA).Methods:The target genes of active ingredients from Cyathula Officinalis were obtained from bioinformatics analysis tool for the molecular mechanism of traditional Chinese medicine.The protein-protein interaction between the target genes were analyzed using STRING and Genemania.The transcriptome of RA patients compared to healthy people(GSE121894)were analyzed using R program package Limma.The relative expression of the target genes was obtained from the RNA-seq datasets.The molecular docking analyses were processed based on the molecular model of estrogen receptor 1(ESR1)binding with estradiol(PDB ID:1A52).The binding details were analyzed by SYBYL.Results:Inokosterone,ecdysterone,and cyaterone were the 3 active ingredients from Cyathula Officinalis that bind to target genes.Of all the significantly changed genes from RA patients,ESR1,ADORA1,and ANXA1 were significantly increased in mRNA samples of RA patients.Conclusion:ESR1,the transcription factor that binds inokosterone in the molecular binding analysis,is the target protein of Cyathula Officinalis.

川牛膝对淡水石斑鱼部分血清抗氧化指标的影响

试验旨在研究川牛膝对淡水石斑鱼部分抗氧化指标的影响。选取初重为(50.20±2.30) g的淡水石斑鱼450尾,随机分为5组,每组3个重复,每个重复30尾。在各组基础饲料中分别添加0、0.5%、1.0%、1.5%和2.0%的川牛膝粉。试验期28 d。结果显示,试验第21 d时,2.0%川牛膝添加组淡水石斑鱼血清谷胱甘肽过氧化物酶(GSH-Px)活性显著高于试验第14和第28 d时(P<0.05),且显著高于0.5%川牛膝添加组、1.0%川牛膝添加组和1.5%川牛膝添加组(P<0.05)。试验第14 d时,1.5%川牛膝添加组淡水石斑鱼血清超氧化物歧化酶(SOD)活性显著高于其他时间点(P<0.05)。试验第21 d时,1.5%川牛膝添加组淡水石斑鱼血清过氧化氢酶(CAT)活性显著高于试验第7和第14 d (P<0.05)。试验第28 d时,0.5%川牛膝添加组和1.0%川牛膝添加组淡水石斑鱼血清丙二醛(MDA)含量显著高于对照组(P<0.05)。试验第28 d时,1.0%川牛膝添加组淡水石斑鱼血清总抗氧化能力(T-AOC)显著高于对照组、0.5%川牛膝添加组和2.0%川牛膝添加组(P<0.05)。研究表明,川牛膝对淡水石斑鱼的抗氧化功能具有促进作用,但各抗氧化指标对应的最优川牛膝添加量不同,在养殖过程中可预先添加0.5%川牛膝粉,再根据实际情况适量调整川牛膝粉的添加量,以达到最优的养殖效果。

麻牛膝TONSOKU基因克隆、生信分析及其抑菌作用

目的克隆编码麻牛膝(Cyathula capitata(Wall.)Moq.)CcTSK蛋白的基因全长,通过基因重组技术制备表达质粒,对表达产物进行生物学预测和抑菌分析。方法以麻牛膝嫩叶为材料,完成CcTSK基因cDNA的克隆。采用生信软件分析该基因及编码蛋白的生物学特征。构建CcTSK基因原核表达载体,并转入大肠埃希菌BL21(DE3)感受态中,IPTG诱导表达,考察CcTSK-GST融合蛋白的表达菌株对低浓度抗生素的敏感性、自身生长变化及在较高浓度抗生素下的存活率。结果CcTSK基因的gDNA为12613 bp,CDS为3855 bp,共编码1284个氨基酸。CcTSK蛋白为亲水性不稳定蛋白,没有跨膜结构域,属于没有信号识别功能的非分泌蛋白,亚细胞定位预测该蛋白可能位于细胞核,并且具有核定位信号,含有TPR重复序列与LRR重复序列结构域,二级结构主要有α-螺旋(占53.89%)、无规卷曲(占32.79%)、延伸链(占8.88%)和β-转角(占4.44%)。经IPTG诱导后,CcTSK-GST融合蛋白的表达抑制大肠埃希菌生长。低浓度抗生素测试结果显示,CcTSK-GST表达菌株对氯霉素(chloramphenicol,Cam)最敏感;高浓度抗生素处理下,CcTSK-GST的表达使得抑菌现象显著增强。结论成功获得编码CcTSK蛋白的基因全长及原核表达质粒,并对其生物学特征进行了分析比较,该蛋白与拟南芥AtTSK蛋白有较高相似性。此外,CcTSK-GST融合蛋白的表达对大肠埃希菌增殖具有抑制作用,并且显著增强抗生素所产生的抑菌现象。

QuEChERS结合在线GPC-GC-MS/MS法测定川牛膝中24种农药残留

目的:建立QuEChERS结合在线凝胶渗透色谱-气相色谱-串联质谱法(GPC-GC-MS/MS法)测定川牛膝中24种农药残留的方法。方法:粉碎混匀后的川牛膝样品经乙腈提取,采用QuEChERS净化处理,以在线凝胶渗透色谱气质联用法进行分析,用农药残留的特征碎片离子建立气质联用检测条件,采用内标法定量。结果:在0.0~0.5μg/mL质量浓度范围内,线性关系良好,24种农药残留检测限范围为0.001~0.008 mg/kg,定量限为0.003~0.020 mg/kg,回收率为68.4%~111.9%,相对标准偏差为0.7%~8.8%。结论:该方法操作简便、批量处理时间短、定性定量准确、灵敏度高,适合道地药材川牛膝中农药残留的检测。

LC-MS/MS测定川牛膝中57种农药残留及风险评估

目的:采用液相色谱串联质谱法(LC-MS/MS)建立川牛膝中57种农药的测定方法,并采用此方法对348批川牛膝样品进行农药筛查。方法:样品经乙腈提取,分散固相萃取QuEChERS净化,采用LC-MS/MS进行测定,多反应监测模式采集数据,基质匹配外标法定量,采用点评估法对检出结果进行风险评估分析。结果:57种农药线性关系良好,r均大于0.996,57种农药的平均加样回收率为62.5%~113.3%,RSD%均小于10%。348批川牛膝中检出20种农药,其中有4种禁用农药(均未超出限量规定)、6种植物生长调节剂、10种常用农药;风险评估结果显示,川牛膝中农药残留风险较低,植物生长调节剂使用较普遍。结论:该方法简便、灵敏、准确,适用于川牛膝中多农药残留的快速筛查与检测,可为川牛膝的农药风险评估和规范化种植提供数据参考。

川牛膝薄层色谱鉴别方法的优化研究

目的:建立中药材川牛膝薄层色谱鉴别的新方法,为川牛膝的鉴别提供更优方案。方法:对川牛膝及其常见混伪品麻牛膝、杂牛膝、牛膝的薄层色谱条件进行考察,筛选并确定最优条件。结果:从供试品溶液制备、展开剂组成等方面进行了优化,并分别考察了影响薄层色谱效果的温度、湿度、稳定性、专属性等多个因素,建立了一种薄层特征明显、可有效地区别川牛膝及其混伪品的薄层色谱鉴别方法。结论:该方法操作简便,成本低,定性特征明显,重复性好,适用于川牛膝药材的薄层色谱鉴别,且比现有的药典方法效果更佳。

多重位点特异性PCR法同时鉴别牛膝和川牛膝配方颗粒

目的建立一种同时鉴别牛膝和川牛膝药材及配方颗粒的多重位点特异性PCR鉴别方法。方法通过比较牛膝属相近物种核基因ITS序列,设计牛膝和川牛膝的特异性引物,经过优化PCR反应体系及PCR反应条件,建立了牛膝和川牛膝配方颗粒的多重位点特异性PCR鉴别方法。结果在退火温度为58℃、循环次数为31时,牛膝和川牛膝配方颗粒分别可扩增出187 bp和164 bp的特异性条带。该方法对牛膝和川牛膝药材的掺伪检出限分别为1%、5%,对两者配方颗粒的掺伪检出限分别为10%、50%。结论所建立的多重位点特异性PCR鉴别可有效鉴别牛膝和川牛膝配方颗粒及其掺伪体系。

川牛膝药材、酒制饮片、标准汤剂与配方颗粒的UPLC特征图谱相关性研究

目的:采用UPLC法研究川牛膝药材、酒制饮片、标准汤剂与配方颗粒四者之间特征图谱的相关性,并考察整个过程成分传递性与差异性。方法:采用ACQUITY BEH C_(18)色谱柱(内径2.1 mm,柱长150 mm,粒径1.7μm),以乙腈为流动相A,0.2%磷酸溶液为流动相B,进行梯度洗脱,柱温30℃,流速为0.3 mL/min,检测波长为266 nm,进样体积1μL。结果:以5-羟甲基糠醛、杯苋甾酮等为主要参照,建立了15批川牛膝药材、酒制饮片、标准汤剂及3批配方颗粒的UPLC特征图谱。15批川牛膝药材中均呈现出7个共有的特征峰,炮制后的15批酒川牛膝饮片、标准汤剂及3批配方颗粒中均呈现8个共有特征峰,通过UPLC-ESI-MS分析指认出2个成分:5-羟甲基糠醛、杯苋甾酮。结论:建立了酒川牛膝药材、酒制饮片、标准汤剂与配方颗粒的UPLC特征图谱,确认川牛膝药材炮制为酒川牛膝饮片后新增5-羟甲基糠醛成分,除5-羟基糠醛外,川牛膝药材、酒制饮片、标准汤剂与配方颗粒的主要化学成分基本相同;UPLC特征图谱相关性良好,可以准确反映川牛膝药材至酒川牛膝配方颗粒的量值传递信息。所建立质量评价模式能够反映酒川牛膝4种形态多成分的整体面貌,为酒川牛膝配方颗粒质量控制及质量标准建立提供参考。