Integrated network pharmacology analysis and in vitro cell experiments to reveal the mechanisms of Ligusticum chuanxiong Hort.in the treatment of non-alcoholic fatty liver disease

Background:Non-alcoholic fatty liver disease(NAFLD)is a liver disease of unknown etiology.A traditional Chinese medicine Ligusticum chuanxiong Hort.(CX),it has been used about 2,000 years.Until now,the mechanism of action of CX on NAFLD remains unclear.Method:We first tested the toxicity of CX to AML12 cells with CCK-8.In vitro cell models of NAFLD were made using free fatty acid,and used Oil Red O staining tested lipid droplets.Then the active compounds of CX were collected from TCMSP and literatures,and SwissTargetPrediction,Search Tool for Interacting Chemicals,Encyclopedia of Traditional Chinese Medicin,Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine database were used to predict the targets of the compounds.DRUGBANK,Online Mendelian Inheritance in Man,Therapeutic Target Database,DisGeNET and GeneCards database were used to predict the targets of NAFLD.Use Venn diagram to obtained the intersection targets by,and analyzed the protein-protein intersection network.Use Kyoto Encyclopedia of Genes and Genomes and Gene Ontology to forecast the function of intersection genes.Molecular docking was used to evaluate the interaction between hub gene and active ingredients.Finally,use western blotting to determine the effects of CX on PPARA,PPARG,IL1B and TNF proteins.Result:CX can reduce the production of AML12 cell lipid droplets.A total of 15 chemical components were identified from CX.Folic acid,chrysophanol and sitosterol were the main components of CX against NAFLD.ALB,TNF,PPARG and PPARA proteins were the main targets of CX in the treatment of NAFLD.PPAR signaling pathway and fatty acid degradation were closely related to anti-NAFLD.Molecular docking results shows that folic acid was the main active ingredient of CX for NAFLD treatment,and TNF is the main potential target.The cellular NAFLD model showed that CX up-regulated the expression of PPARA and PPARG protein and down-regulated inflammatory factor IL-1B and TNF expression.Conclusion:Our study suggests that CX has a therapeutic effect on NAFLDA,which may be related to the PPAR pathway and the reduction of inflammatory cytokines.

Law of Occurrence and the Damage of Main Diseases and Pests in Ligusticum chuanxiong Hort Field

[Objective]The aim was to provide scientific basis for the prevention and treatment of the main diseases and pests. [Method]The systematic investigation was carried out in the process of seedling and field cultivation of Ligusticum chuanxiong. [Result]The main diseases were root rot,Powdery mildew and Spot blight,while the main pests were Epinotia leucantha Meyrick,Prodenia litura Fabr.,Scarabaeoidea,Tetranychus cinnabarinus Boisduval and Delia platura Meigen. [Conclusion]The law of occurrence of main diseases and pests were confirmed.

A rapid HPLC method for determination of coniferyl ferulate in Angelica sinensis and Ligusticum chuanxiong

Aim A reliable and rapid HPLC method was developed for quantitative determination of coniferyl femlate, an ester of ferulic acid, with multiple pharmacological activities in Angelica sinensis and Ligusticum chuanxiong, two commonly used Chinese medicines. Methods The determination was achieved by using a Zorbax ODS C18 analytical column （250 mm×4.6 mm ID, 5 μm） at isocratic elution of 1% aqueous acetic acid and acetonitrile （1：1） with diode-array detection （318 nm）. The calibration curve of coniferyl femlate showed good linearity （r^2 = 0.9995） within the test range. Results The developed method showed good precision with intra- and inter-day variations of 0.22% - 1.16% and 0.86% - 2.62% between the levels of 0.380 - 0.038 mg·mL^-1, respectively. The repeatability represented as RSD of coniferyl femlate was less than 2.7% for three levels （0.2 - 1.0 g of Angelica sinensis）, and the recovery was 105.3% with RSD of 3.2%. Conclusion The validated method was successfully applied to quantify coniferyl femlate in 12 samples of Danggui and Chuanxiong.

Pathogen Identification of the Anthracnose of Ligusticum chuanxiong Hort. and Inhibitory Effect of Four Fungicides on the Pathogen

[Objective] The research aimed to isolate and identify the pathogen of anthracnose of Ligusticum chuanxiong Hort,and make the antifungal test on four kinds of fungicides for selecting the optimum fungicides.[Method] The pathogenic fungi was separated according to Koch＇s rule and the shape characteristics were identified. The antifungal test of fungicides was made by using plate mycelium growth inhibition method.[Result] The pathogenic fungi was separated from leaves of Ligusticum chuanxiong Hort. and morphological characters were consistent with that of Colletotrichum gloeosporioides. In addition to chloroisobromine cyanuric acid,the other three fungicides could inhibit the mycelium growth. The growth inhibition rates of 1 000 times Metalaxyl＋mancozeb,zineb,Thiophanate-methyl for the mycelium were 26.8%,22.1%,59.8% respectively after 7 days.[Conclusion] The anthracnose was caused by Colletotrichum gloeosporioides. Ligusticum chuanxiong Hort. was a new record host plant of Colletotrichum gloeosporioides and the best inhibiting fungicide was Thiophanate methyl.

Optimization of ISSR-PCR Reaction Conditions for Genomic DNA of Ligusticum chuanxiong hort.

[Objective] To investigate the impacts of ISSR-PCR amplification factors, for the establishment and optimization of ISSR-PCR reaction system for Ligusticum chuanxiong hort. [Method] Using genomic DNA of Chuanxiong leaf extracted via an improved CTAB method as template, single factor analysis was performed to investigate the impacts of DNA template concentration, Mg2＋ concentration, dNTPs concentration, primer concentration, Taq DNA polymerase concentration on ISSR-PCR amplification and to optimize this system for Ligusticum chuanxiong hort. [Result] The ISSR-PCR amplification（25 μl） suitable for Ligusticum chuanxiong hort. was determined to be composed of 2.5 μl of 10×reaction buffer, 2.1 mmol/L MgCl2, 300 μmol/L dNTPs, 0.4 μmol/L primer, 1.0 U Taq DNA polymerase and 20-40 ng genomic DNA. [Conclusion] Our study laid basis for analyzing the genetic diversity of Ligusticum chuanxiong hort. resources distributed in 17 different areas of China.

辽藁本(Ligusticum jeholense)幼苗初生维管系统的发育

应用整体透明和石蜡连续切片等方法,对辽藁本幼苗初生维管系统的发育进行了观察.结果表明:该幼苗的轴向器官中,以子叶节区下部的初生维管系统先建立,向下发育形成了下胚轴和根的维管系统;再向上通过子叶节区中、上部的分生组织性组织与第1片真叶的叶迹相连;上胚轴-苗的维管系统向下发育与子叶迹相连,至此构成了该幼苗完整、连续的初生维管系统.此外,对幼苗侧生器官子叶片的三出-叉状脉的形成进行了观察,认为该叶脉序属于原始脉序类型.

川芎(Ligusticum chuanxiong Hort)种植地的土壤动物群落特征

采用大型手捡、Tullgren干漏斗和Baermann湿漏斗法对川芎种植基地土壤动物群落进行调查.结果显示,试验所采集土壤动物平均密度为3.13×104只/m2,其中,川芎样地土壤动物平均密度为1.35×104只/m2,11个类群;菜地土壤动物平均密度为4.91×104只/m2,15个类群.同功能种团分析表明,菜地的杂食性和腐食性土壤动物密度显著高于川芎地(P<0.05),植食性和捕食性显著低于川芎样地(P<0.05).菜地的DG和C指数高于川芎样地,而J和H'指数低于川芎样地.表明川芎种植对土壤动物群落分布与多样性特征产生一定影响.

Measurement and Correlation of the Solubility of Ligusticum Chuanxiong oil in Supercritical CO_2

Extraction of the Ligusticum Chuanxiong oil with supercritical CO2 （SC-CO2） was investigated at the temperatures ranging from 55℃ to 70℃ and pressure from 25 MPa to 35 MPa. The mass of Ligusticum Chuanxiong oil extracted increased with pressure at constant temperature. The initial slope of the extraction was considered as the solubility of oil in SC-CO2. Chrastil equation was used to correlate the solubility data of Ligusticum Chuanxiong oil. An improved Chrastil equation was also presented and was employed to correlate the solubility data, The correlation results show that the values of the average absolute relative deviation are 5.94% and 3.33% respectively, indicating the improved version has better correlation accuracy than that of Chrastil equation.

Comparative Morphology of the Leaf Epidermis in <i>Ligusticum</i>(Apiaceae) from China

To investigate the taxonomic value of leaf epidermal characters in developing a classification for Ligusticum (Apiaceae), thirty-eight species (including a cultivar) of the genus were examined using both light and scanning electron microscopy. Our results show that the infrageneric classification of Ligusticum based on the characters of involucel bracteoles, mericarps, and pollen grains was not supported as few epidermal characters were relatively constant at section level. Evidence from leaf epidermis, similar to that from molecular and morphological data, supports the separation of Tilingia (i.e. L. ajanense and L. tachiroei) from Ligusticum. Ligusticopsis was found to be indistinguishable from Ligusticum in epidermal characters. Furthermore, results from epidermal analysis, together with that from cytological evidence, fruit morphology as well as molecular phylogenetics suggest that L. delavayi should be transferred out of Ligusticum.

Study on the mechanism of “Ligusticum chuanxiong Hort.-Salvia miltiorrhiza” couplet medicine for coronary heart disease based on network pharmacology

Objective:To study the active components and gene targets of“Ligusticum chuanxiong Hort.-Salvia miltiorrhiza”couplet medicine for the treatment of coronary heart disease(CHD)based on network pharmacology,and to explore its mechanism.Methods:Based on oral bioavailability(OB)>30%and drug-like(DL)>0.18,the active components of“Ligusticum chuanxiong Hort.-Salvia miltiorrhiza”for CHD were screened and the targets of treating CHD were predicted by using TCMSP and GeneCards database.The active component-CHD target network was established by Cytoscape 3.7.2 software.The protein-protein interaction(PPI)network was constructed by utilizing String database.Finally,GO enrichment analysis and KEGG pathway enrichment analysis were performed by using Bioconductor and R language.Results:The study predicted 72 active components in total,including 7 Ligusticum chuanxiong Hort.and 65 Salvia miltiorrhiza,such asβ-sitosterol,tanshinone.Totally 96 target genen of active components were obtained,including PTGS1,NCOA2,NOS2,etc.Results of GO enrichment analysis showed 142 biological processes,related to adrenergic receptor activity,G protein-coupled amine receptor activity,etc.KEGG pathway enrichment analysis showed 131 pathways,including PI3K-Akt signaling pathway,IL-17 signaling pathway,HIF-1 signaling pathway,etc.Conclusion:“Ligusticum chuanxiong Hort.-Salvia miltiorrhiza”couplet medicine exerts therapeutic effects on CHD from multiple targets as PTGS1,PTGS2 and adrenergic receptor activity and PI3K-Akt signaling pathway.The study can provide reference for further researches on its mechanism and the pharmacological effects of Shenzhi Tongxin Capsule.

Effect of Ligusticum Chuanxiong on Hypoxic Pulmonary Hypertension in Rats and Its Mechanism

Objective: To explore the mechanism of Chuanxiong in alleviating hypoxic pulmonary hypertension in rats by inhibiting pulmonary vascular remodeling. Methods: Thirty healthy and clean male SD rats weighing (180 - 220) g were randomly divided into three groups (n = 10): normoxia group (n), hypoxia group (H) and Chuanxiong group (L). Group N was fed in normoxic environment, and the other two groups were fed in hypoxic (9% 11% O2) environment for 4 weeks, 8 h/D, 6 days a week. Rats in group L were gavaged with Ligusticum chuanxiong solution diluted with normal saline at the concentration of 300 mg/kg, and rats in group H were gavaged with equal volume of normal saline. After 4 weeks, the mean pulmonary artery pressure was measured. After pulmonary perfusion, the right ventricular free wall and left ventricle plus ventricular septum were taken to measure the right ventricular hypertrophy index. The changes of pulmonary morphology and ultrastructure were observed under light microscope. Results: Compared with group n, the average pulmonary artery pressure and right ventricular hypertrophy index in the other two groups increased, and the thickening of pulmonary vascular wall was obvious under microscope (P Conclusion: Ligusticum chuanxiong can relieve pulmonary artery pressure in rats by inhibiting pulmonary vascular remodeling.

外源Cd对川芎苓种萌发及幼苗生长的影响

本研究旨在探究不同浓度镉(Cd)对川芎苓种萌发和幼苗生长的影响。采用以石英砂为基质的水培试验,施加不同浓度CdCl 2·2.5 H 2O(0、0.1、0.25、0.5、1、2 mmol/L)溶液处理川芎苓种,研究Cd胁迫对川芎苓种萌发、幼苗生长、生理代谢及Cd含量的影响。结果显示,随着Cd浓度的增加,株高抑制率、根长抑制率上升;根、叶及节盘Cd含量显著升高;株高、根长、叶鲜重、根鲜重下降;抗氧化酶(过氧化氢酶、过氧化物酶、超氧化物歧化酶)活性、非酶系统物质(可溶性蛋白、谷胱甘肽、脯氨酸)含量以及丙二醛含量先升高后降低。研究表明,0.1~2.0 mmol/L Cd对川芎苓种萌发和早期幼苗生长均有明显的毒害作用,随着Cd胁迫浓度的升高毒害作用增强,其中川芎幼苗根对Cd胁迫最敏感。Cd胁迫下川芎幼苗生理生化指标整体上呈现低促高抑的Hormesis效应。川芎幼苗通过提高抗氧化酶活性和非酶系统物质积累,减少膜系统损伤、活性氧蓄积,缓解Cd毒害作用,提高耐受性。

茶芎化学成分及药理活性研究进展

茶芎Ligusticum sinense Oliv.Cv.Chaxiong为江西省特产中药材之一,主要含有苯酞类等化学成分,具有活血行气、祛风止痛等功效。同时,茶芎的有效成分在抗惊厥、抗癫痫、抗炎镇痛、保护心脑血管及改善学习记忆能力等方面具有较好的药理活性。通过查阅国内外文献,对近30年来报道的关于茶芎化学成分和药理活性方面的内容进行系统综述,为将来更好地开发和利用茶芎提供参考。

基于多元统计学分析^(60)Co-γ射线辐照对川芎挥发性成分的影响

川芎挥发性成分是其主要活性成分之一,也是评价川芎药用品质的重要指标。为探究^(60)Co-γ射线辐照对川芎挥发性成分的影响,本研究以不同吸收剂量处理川芎样品,采用电子鼻结合顶空固相微萃取-气相色谱-质谱技术(HS-SPME-GC-MS),通过主成分分析、线性判别分析、正交偏最小二乘法和聚类分析的多元统计学方法分析数据,以期了解川芎辐照前后挥发性成分的变化。电子鼻与GC-MS结果都表明:川芎气味主要由烷烃类、醇类、醛类、酮类贡献,10 kGy剂量以下的辐照处理对川芎挥发性气味成分影响不大;川芎经0 kGy(对照组)、3 kGy、7 kGy、10 kGy剂量辐照处理后,共鉴定出60种化合物,其中烃类23种,醇类12种,脂类10种,醛酮类4种,其他类11种;筛选到21种辐照前后的差异特征性指标物,它们在川芎挥发性成分中只占约12.92%,不是川芎的主要挥发性成分。经10 kGy剂量以下的辐照处理后,川芎中没有新生成的物质被鉴定出来。研究结果提示经10 kGy以下剂量处理川芎不会影响其主要挥发性成分。

辽藁本种质创新、栽培繁育及分子生物学研究进展

辽藁本作为我国大宗药材,随着市场需求量的增加,种植面积也在逐年扩大,但是辽藁本栽培种较少、栽培技术落后,分子生物学等方面研究不够深入,严重制约了相关产业的发展。文章综述了辽藁本在种质创新、栽培技术研究、种苗繁育、药用成分分析、分子生物学及微生物组学等方面的研究进展,展望了辽藁本未来研究应关注的方向,为辽藁本的进一步深入研究和产业发展提供科学依据。

基于全谱非靶向代谢组学技术的川芎不同部位代谢物深度解析

为了深度解析川芎中不同部位代谢物特征,本研究采用高效液相色谱法(HPLC)对川芎的根茎、茎、叶中的活性成分进行含量测定,并整合超高效液相色谱-质谱联用仪(UPLC-MS)和气相色谱-质谱联用仪(GC-MS)的全谱非靶向代谢组学技术,对川芎的根茎、茎和叶的挥发性成分、非挥发性成分进行全面的定性和定量分析。结果表明,川芎根茎中活性成分含量高于茎、叶。川芎的全谱非靶向代谢组学共检出2891个代谢物,总丰度为茎>叶>根茎,各部位化学成分种类相同、含量差异较大,包括氨基酸及其衍生物、萜类、酚酸类等32类化合物。其中LC-MS检出1726个代谢物,GC-MS检出1216个代谢物,两个平台共同检出51个代谢物。对川芎不同部位的差异代谢物进行分析,根茎与茎、根茎与叶、茎与叶中筛选到差异代谢物1683、2054和1844个,差异代谢物总丰度为根茎≈茎>叶。根茎中显著富集含氮化合物、酚类、其他类(糖类、内酯类),茎中的醇、胺类、醚类高度富集,叶中的萜类、酮类、黄酮类高度富集。川芎中大多数活性成分如川芎嗪、阿魏酸、藁本内酯等,呈现根茎>茎>叶的趋势,但茎和叶中也含有含量较高的阿魏酸、欧当归内酯A等重要活性成分,具有较高利用价值。通过KEGG富集分析结果推测差异代谢物可能与次生代谢产物的生物合成等途径相关。本研究深度解析了川芎不同部位的成分积累规律,为川芎资源的合理利用提供了科学依据。

基于正交设计优化新鲜川芎中挥发油提取工艺

为优化川芎挥发油提取工艺,建立以新鲜川芎为原料提取制备挥发油的新方法,采用正交试验设计对乙醇回流提取工艺条件进行优化,并进一步考察优选回收浓缩、油水分离等关键工艺参数。以藁本内酯和洋川芎内酯A作为指标成分,应用HPLC一测多评法进行含量测定。以提取量、油收率、油含量为评价指标,最终确定川芎挥发油提取制备新方法为:鲜川芎切片,加8倍量70%乙醇回流提取2次(以干药材计,第一次需测定鲜川芎水分,调节乙醇浓度为70%),每次1.5 h,滤液回收浓缩至2.0~3.0 g生药/mL,静置过夜,弃去水层,上层乳化层加热至60℃离心,分取油层,余下乳化层再加氯化钠至饱和并加热至60℃离心,合并油层,即得川芎挥发油。新方法所得川芎挥发油收率约为3%,藁本内酯和洋川芎内酯A含量达70%。该研究所建立的新方法生产设备要求低、操作简便易行,可实现川芎挥发油的高效制备,且含量高、品质优,为川芎挥发油的工业化生产提供了新思路和新方法。

基于网络药理学和分子对接探讨黄芪-川芎配伍治疗瘢痕的作用机制

基于网络药理学探讨黄芪-川芎配伍治疗瘢痕的作用机制。通过TCMSP数据库筛选出黄芪和川芎的有效成分及其靶点,同时使用OMIM、GeneCards和DrugBank数据库获取得到黄芪-川芎配伍治疗瘢痕的潜在交集靶点。采用Cytoscape3.7.0软件构建中药-有效成分-靶点-疾病网络,并通过STRING数据库构建PPI网络,利用微生信网站进行KEGG富集分析及GO功能分析可视化,再佐以分子对接验证。结果显示,共筛选得到川芎有效成分7个,对应靶点42个;黄芪有效成分20个,对应靶点462个;瘢痕靶点2216个。黄芪-川芎配伍治疗瘢痕的关键成分有槲皮素和山柰酚等;治疗瘢痕的关键靶点是TP53和白细胞介素6等。KEGG富集分析结果表明,黄芪-川芎主要与IL-17信号通路和TNF信号通路有关。分子对接表明,黄芪-川芎主要活性成分槲皮素、山柰酚和杨梅酮与关键靶点AKT1、IL6和TNF具有良好的稳定性。本文基于网络药理学和分子对接验证研究表明,黄芪和川芎可能通过多成分、多靶点和多通路来治疗瘢痕。

基于网络药理学与分子对接预测川芎-赤芍药对治疗动脉粥样硬化的作用机制

心血管疾病是全球范围内死亡率最高的一种疾病,其主要病理基础是动脉粥样硬化。为了探讨川芎-赤芍药对治疗动脉粥样硬化的潜在作用机制,利用网络药理学平台,挖掘了川芎-赤芍的有效成分及靶点并筛选核心成分;查找了动脉粥样硬化的靶点,然后与药物靶点取交集筛选候选靶点;构建候选靶点的蛋白互作网络得到核心靶点;对候选靶点进行GO功能富集分析与KEGG信号通路富集分析。最后将核心成分和核心靶点进行分子对接。结果从川芎-赤芍药对中筛选到包括核心成分β-谷甾醇、黄芩素、豆甾醇和杨梅酮在内的35个活性成分,获得川芎-赤芍药对治疗动脉粥样硬化的包括核心靶点AKT1、TNF、VEGFA、TP53在内的候选靶点58个,核心成分和核心靶点间均具有较好的结合能力,候选靶点主要参与了对氧水平下降的反应、对细菌来源分子的反应、对激素的反应等生物学过程,富集在癌症信号通路、脂质和动脉粥样硬化信号通路、化学致癌-受体激活通路、流体剪切力与动脉粥样硬化通路等相关通路上,以协同的形式通过调节细胞生长发育、降低血脂水平和控制促炎介质的分泌等对动脉粥样硬化发挥治疗作用,为川芎-赤芍药对的临床应用提供理论基础。

基于转录组和代谢组解析川芎对镉胁迫的响应机制

为了解不同水平镉(Cd)胁迫下川芎基因表达和代谢产物的变化规律,探究川芎对Cd胁迫的响应机制,利用盆栽试验,以不添加Cd溶液的清洁土壤为对照,设置1、3、6、10 mg·kg-1共4个Cd胁迫处理水平,采用转录组学测序(RNA sequencing,RNA-seq)和超高效液相色谱质谱联用(ultra performance liquid chromatography tandem mass spectrometry,UPLC-MS/MS)技术,筛选川芎抗Cd胁迫的关键基因与代谢通路。结果表明,共筛选到8569个差异表达基因,包括上调基因6859个,下调基因1710个。与对照相比,4个Cd处理组的共有差异基因仅1个,为CML19,可能是川芎抗Cd胁迫的关键基因。代谢组共标注和定量了1238种差异代谢物,在KEGG数据库中注释得到76条代谢通路,其中包括氨基酸代谢、氨酰基-tRNA的生物合成和脂肪酸的生物合成等。联合分析表明,当受到不同水平Cd胁迫时,川芎通过调节不同种类氨基酸来维持自身代谢平衡,在川芎抗Cd胁迫中发挥重要作用。研究结果为川芎抗Cd胁迫提供科学支撑与理论依据,为针对川芎资源的栽培育种及缓解Cd胁迫等相关研究提供基础。