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.

Evaluation and Exploration of Citrus Germplasm Resources in the Origin of Shatangju(Citrus flamea Hort.ex Tseng Shiyueju)

[Objectives]To protect the local rare citrus germplasm resources and further develop and cultivate new citrus varieties.[Methods]The germplasm resources of Shatangju(Citrus flamea Hort.Ex Tseng Shiyueju)in Sihui City of Guangdong Province and other 8 citrus varieties from the same origin were evaluated.The appearance characteristics of tree shape,the length and thickness of shoot at each shoot stage,the appearance characteristics of leaves and the fruit quality of each variety were investigated.[Results]Through the comparison of various citrus varieties grafted with sour mandarin,the results showed that the overall advantages of the length and thickness of the shoots of Shatang tangerine in summer and autumn were more prominent,which was conducive to fruit setting.The sprouting time of the three shoots of each variety was different,and there were sporadic sprouting phenomena in different seasons,so the growth was different,and the length and thickness of the shoots were different in different seasons.There were significant differences in shoot length and diameter among different varieties in the same season.The coefficient of variation of Shatangju's leaves was low,and the traits were stable;leaf shape,leaf base shape,leaf margin and other indicators among the three shoots of the same variety and the differences among the same shoots of different varieties were compared.All varieties had strong flavor,separately sweet and sour,sweet and sweet taste.Shatangju has many segments,orange pericarp,rough texture,easy peeling,and high oblate fruit shape.The orange pericarp was favored by the participants,while the rough and thick peel was not favored by the participants.[Conclusions]Based on the improvement of local varieties,it is recommended to improve the smoothness of pericarp,reducing the thickness of pericarp and improving the flavor of fruit will be conducive to increasing sales.In the cultivation process of various quality resources,especially in the period of fruit growth and development,the plan of nutrition and fertilization should be formulated according to the rules of fruit growth and development,the rules of yield formation and the rules of quality composition,so as to establish the integrated nutrient management system of various varieties.

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.

Study on Flowering, Cultivation, and Reproduction of Agave americana Var. Marginata Hort. (Agavaceae)

Agave americana Var. Marginata Hort. （Agavaceae） is a perennial herb. This paper summarized its classification, distribution, morphological characteristics, habit, flowering and fruiting, cultivation and reproduction, as well as multiple uses in daily life, which provided references for the exploitation and utilization of Agave americana Vat. Marginata Hort. （Agavaceae）.

金边阔叶麦冬(Liriope platyphylla Wang et Tangvar.variegata Hort.)的组织培养

将金边阔叶麦冬 (LiriopeplatyphyllaWangetTangvar.variegataHort.)不同部位外植体块 ,接种于附加不同激素配比的MS培养基上 ,实验结果表明 ,取自根状茎芽点 ,培养基为MS +BA 1.5mg/L +NAA 0 .5mg/L的不定芽诱导率最高 ( 10 0 % ) ,长势较旺 ,不定芽和愈伤组织均较多 ,但极易造成性状分离。用MS +BA 3.0mg/L +NAA 0 .5mg/L液体培养可以直接“芽生芽” ,避免了脱分化过程 ,从而稳定保持嵌合性。不定芽每月可转接 1次 ,适宜的生根培养基为 1/2MS大量 +IBA 0 .2 5mg/L。试管苗在蛭石中炼苗后即可移栽。

生菜(Lactuca sativa var ro mana Hort.)下胚轴愈伤组织的诱导与植株再生

通过对生菜下胚轴的愈伤诱导和植株再生的研究 ,发现诱导愈伤组织时 ,MS培养基中加 0 .5mg/L 6 BA + 1mg/L 2 ,4 D + 0 .1mg/LNAA效果最好 ,其出愈率达 94 %;愈伤组织分化为芽时 ,在MS + 1.0mg/L 6 BA + 0 .0 5mg/L NAA中分化频率最高 ,达 10 0 %.再生苗可在 1/ 2MS+ 0 .5mg/L NAA中诱导生根成完整植株 .

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.

RAPD Analysis of Twelve General Species of Gladiolus hybridus Hort.

RAPD analysis was applied in studying the classification and genetic relationship of 12 cultivars of Gladiolus hybridus Hort. Twenty-six arbitrary primers screened from 80 primers were used for further PCR and diversity analysis. A total of 164 RAPD sites were detected with a mean of 6.31 fragments amplified for each primer. A total of 151 polymorphic DNA fragments were detected among all ,the 164 amplified fragments, which accounted for a high level of 92.8% of all and could be used for identification of different cultivars. The result revealed that the germplasm resource of Gladiolus hybridus Hort. cultivar had a narrow genetic base on molecular level.

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。

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。

比利时杜鹃花类黄酮3'-羟化酶(F3'H)基因克隆及功能分析

【目的】类黄酮3’-羟化酶(flavanone 3’-hydroxylase,F3’H)是植物花青素合成过程中的关键酶,探究比利时杜鹃花(Rhododendron hybridum Hort)F3’H基因的功能及表达特性。【方法】以比利时杜鹃花不同发育时期的花瓣及盛花期的根、茎、叶为实验材料,从比利时杜鹃花转录本数据库中筛选类黄酮生物合成通路关键酶类黄酮3’-羟化酶基因序列信息并进行生物信息学分析,利用反转录(RT-PCR)技术克隆RhF3’H基因;利用紫外分光光度计测定了比利时杜鹃花花瓣不同时期的花青素含量,利用RT-qPCR技术对不同发育时期花瓣和成熟期的不同组织进行RhF3’H基因表达量分析;采用Gateway技术构建过表达载体35S:RhF3’H-GFP重组载体进行亚细胞定位验证;构建p1302-RhF3’H过表达载体对比利时杜鹃花花瓣进行侵染。【结果】成功获得比利时杜鹃花RhF3’H基因长度为1557 bp,编码518个氨基酸,具有保守的F3’H结构域,属于P450超家族;系统进化树分析显示,比利时杜鹃花RhF3’H与龙眼和荔枝F3’H蛋白亲缘关系最近;RT-qPCR结果显示,RhF3’H在比利时杜鹃花不同花瓣时期和根、茎、叶组织中均有表达,在不同花瓣发育时期中,盛开期和衰败期中RhF3’H基因的表达量较高,与花青素含量结果相一致;亚细胞定位分析显示,RhF3’H主要存在于细胞膜上;成功构建P1302-RhF3’H瞬时过表达载体,相较于CK和p1302,p1302-RhF3’H在杜鹃花花瓣中显著高表达,其花青素含量也显著增加。【结论】RhF3’H基因在花瓣细胞膜中表达,表达模式与花青素积累趋势一致;过表达RhF3’H基因促进了花青素的合成。

环割对“Hort 16A”猕猴桃枝叶营养和果实品质的影响

【目的】为探明不同环割处理对猕猴桃植株养分状况和果实品质的影响,找出最适环割方法,为制定猕猴桃配套栽培技术提供理论依据.【方法】以6年生"Hort16A"为试验材料,研究了不同环割部位、环割时期及环割程度对猕猴桃果实品质和结果蔓中可溶性糖、可溶性淀粉、可溶性蛋白的含量及功能叶中叶绿素含量等生理指标的影响.[结果]①不同环割部位处理结果表明,初花期在主干嫁接口上方5~10 cm、双主蔓分支点上方2 cm或直径≥1.5 cm的结果母蔓分支点上方2 cm处环割一周均可显著提高果实纵经、横径和单果重,但主干环割还可显著提高结果蔓可溶性蛋白、可溶性糖含量;两主蔓环割可显著提高果实叶黄素、可溶性固形物、总糖和Vc含量;结果母蔓环割可显著提高叶片叶绿素a、b及总量.②不同环割时期处理结果表明,主干上初花期环割比花后10、20 d环割的增产作用更显著,但主干上花后20 d环割的果实叶黄素、可溶性固形物、总糖、总酸含量最高.③不同环割程度处理结果表明,初花期主干环割1周的单果重最大,环割2周的果实叶黄素含量最高,环割3周在降酸增糖并提高结果蔓可溶性蛋白、可溶性糖含量上效果最显著.④灰色关联度分析后的排名结果显示,初花期两主蔓环割1周和结果母蔓环割1周分别位于第1、2位,而初花期主干上环割1、2、3周分别位于第8、7、6位.【结论】初花期两主蔓环割1周对提高"Hort 16A"植株养分状况和果实品质效果最好.

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.

银边红雀珊瑚（Pedilanthus tithymaloides（L）Poir．var．cuculatus Hort．）快速繁殖研究

顶芽或腋芽在添加６－苄基氨基嘌呤（６－ＢＡ）３ｐｐｍ和萘乙酸（ＮＡＡ）０．５ｐｐｍ的ＭＳ培养基培养３０ｄ，长４～６ｃｍ，腋芽３～７个。此时，将它们分切成单芽切段，转到添加６－ＢＡ２ｐｐｍ的ＭＳ培养基，继代培养周期２１ｄ，繁殖系数５～８。芽长２～３ｃｍ时用添加吲哚丁酸（ＩＢＡ）１ｐｐｍ的ＭＳ培养基进行生根培养，培养２１ｄ，生根率８７％，移栽成活率９３％。

川芎苓种内生燕麦镰刀菌拮抗川芎根腐病的研究

根腐病是川芎栽培的首要病害,严重影响川芎药材生产。生物防治技术是防控药用植物病害的重要手段。本研究以川芎苓种自身携带的内生潜在生防菌燕麦镰刀菌Fusarium avenaceum对抗川芎根腐病主要致病菌茄腐镰刀菌Fusarium solani,并回接至川芎无菌组培苗进行菌植互作实验和防效验证。结果显示,F.avenaceum对F.solani菌丝生长平均抑制率达48.22%;二者均能在川芎植株体内成功定植;单独接种F.solani浸染率为46.93%,病情指数为66.70%,接种F.avenaceum可显著降低病情指数;组培苗先感染F.avenaceum比先感染F.solani发病率低,当两菌复合回接且F.avenaceum优先接种时促进川芎根茎显著增大。由此说明,川芎苓种内生菌F.avenaceum可以作为F.solani所致川芎根腐病的生防菌,且“防病”效应优于“治疗”效应。

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。

木兰皮川芎提取液脂质体凝胶面膜的研制

以木兰皮和川芎提取液为主要原料,研究制备了一款可以改善痤疮后遗留的色素沉积、毛孔粗大问题的脂质体面膜,采用DPPH法对木兰皮、川芎提取液进行体外抗氧化活性测定;采用单因素实验优化脂质体和面膜配方;采用人体斑贴试验来验证面膜的安全性,通过皮肤保湿率的测定以及智能皮肤分析仪的检测来验证脂质体面膜的保湿功效。测定结果显示,该面膜的毛孔、色素、油脂、敏感等皮肤指标均出现显著性改善,具有良好的保湿、改善色素沉积、毛孔粗大效果,因此川芎木兰皮脂质体面膜可作为一种良好的护肤类产品进行开发利用。

Hort16A猕猴桃叶片直接再生不定芽研究

通过外植体直接再生不定芽方式进行器官再生,不经过愈伤组织,能使无性系后代保持原品种的特性。以Hort16A猕猴桃叶片为外植体,以MS培养基为基本培养基,设计不同类型和浓度的植物生长调节剂组合对诱-导分化效果进行研究。结果表明,经过4~5周暗培养后外植体褐化程度最低;诱导不定芽分化的最适植物生长调节剂组合为1.5 mg/L 6-BA＋0.5 mg/L IBA,在该浓度下,不定芽分化率为100%,平均出芽个数为6.9。

Hort16A猕猴桃顶芽培养

以Hort16A猕猴桃(Actinidia chinensis Planch.)顶芽为外植体,研究了不同植物生长调节剂组合对丛生芽诱导、生根培养的影响。结果表明,在丛生芽诱导培养中,培养基中生长调节剂最适浓度组合为6-BA 1 mg/L与NAA 0.03 mg/L,丛生芽的诱导率到达92%,芽的长势较好。生根培养中,在1/2MS培养基中生长调节剂IBA最适浓度为0.5 mg/L,生根率到达了98%。