The updated weeping forsythia genome reveals the genomic basis for the evolution and the forsythin and forsythoside A biosynthesis

Weeping forsythia (Forsythia suspensa,Oleaceae) is a deciduous broad-leaved tree species distributed in the warm temperate zone of China.However,the species still lacks a chromosome-level genome.In this study,the former draft genome (Accession No.WIPI00000000) of weeping forsythia was assembled into 14 chromosomes with a 712.9 Mb genome size.Weeping forsythia underwent a and b whole-genome duplication events.After the divergence between weeping forsythia and Olea europaea,1 453 gene families had a significant expansion,and 1 146 gene families had a significant contraction.The enrichment pathways and ontologies of expanded genes suggested that the tillering,photosynthesis and growth capacity of weeping forsythia were enhanced after the divergence of weeping forsythia and O.europaea.The contracted genes suggested that the resistance of weeping forsythia to cold and drought was weakened.The last glacial period led to a significant decline in the effective population size of weeping forsythia.Forty-six candidate genes were identified for the synthesis of the forsythin and forsythoside A by genomic and transcriptomic data.In this study,we improved the previous draft genome of weeping forsythia.Our genome will provide genomic resources for the subsequent evolution and breeding research of weeping forsythia.

Recent advances in the antiviral activity of Forsythiae Fructus

Forsythiae Fructus,a well-known traditional Chinese medicine preparation derived from the dried fruits of Forsythia suspensa(Thunb.)Vahl,has been historically utilized for its heat-clearing and detoxification properties.Forsythiae Fructus has been reported to exhibit antiviral activity such as against SARS-CoV-2 and influenza virus.This review highlights the recent updates on the effects and underlying action mechanisms of compounds in and active fractions from Forsythiae Fructus and Chinese formulae containing Forsythiae Fructus.

A new forsythenside from Forsythia suspensa

A new forsythenside, 4-hydroxy-4-[8-[[1-[（4-hydroxyphenyl）acetyl]-β-D-glucopyranosyl-6-]oxy]ethy1]-2,5-cyclohexadien-1- one, named forsythenside F, was isolasted from the plant of Forsythia suspensa （Thunb.） Vahl. The structure of the new compound was established on the basis of various spectroscopic analysis, including ^1H NMR, ^13C NMR, 2D NMR techniques （HMBC and HSQC）, and HR-ESI-MS.

Establishment and Verification of An Efficient Virus-induced Gene Silencing System in Forsythia

To understand the functional identification of large-scale genomic sequences in Forsythia,tobacco rattle virus(TRV)-mediated virus-induced gene silencing(VIGS),suitable for the plant,was explored in this study.The results showed that the TRV-mediated VIGS system could be successfully used in Forsythia for silencing the reporter gene FsPDS(Forsythia phytoene desaturase)using stem infiltration and leaf infiltrationmethods.All the treated plants were pruned below the injection site after 7–15 d infection;the FsPDS was silenced and typical photobleaching symptoms were observed in newly sprouted leaves at the whole-plant level.Meanwhile,this system has been successfully tested and verified through virus detection and qRT-PCR analysis.After the optimization,Forsythia magnesium chelatase subunit H(FsChlH)was silenced successfully in Forsythia using this system,resulting in yellow leaveswith decreased chlorophyll content.The system was stable,highly efficient and had greater rapidity and convenience,which made it suitable to study the function of genes related to physiological pathways such as growth and development,and metabolic regulation in Forsythia.

Forsythiae Fructus inhibits melanoma growth through activating MAPKs/Nrf2/HO-1 signaling and modulating glycerophospholipid metabolism

OBJECTIVE Forsythiae Fructus(Lianqiao)is a typical heat-clearing and detoxicating traditional Chinese medicine(TCM)herb,which has been traditionally used for treating cancer according to TCM theory.However,the underlying mechanism has not been fully explained.METHODS In this study,we investigated the antitumor effect of Forsythiae Fructus aqueous extract(FAE)on B16-F10 melanoma.RESULTS FAE strongly inhibited the tumor growth and metastasis formation in B16-F10 melanoma transplanted mice.The survival time of tumor-bearing mice was also significantly prolonged by FAE.The levels of ROS,MDA,TNF-αand IL-6 decreased,while GSH increased in the FAE treatment group,indicating FAE possesses strong anti-oxidative and anti-inflammatory activity.Western blotting analysis demonstrated that antioxidant proteins Nrf2 and HO-1,tumor suppressors P53 and p-PTEN,and the MAPK pathways in tumor tissues were upregulated by FAE treatment.Serum metabolomics analysis further uncovered that 17 metabolites mostly involving in glycerophospholipid metabolism were correlated with the antitumor effect of FAE.Notably,several lysophosphatidylcholines(LysoPCs)significantly decreased in tumor model group,while FAE treatment restored the changes of these phospholipids to about normal condition.LysoPC acyltransferase 1(LPCAT1)and autotaxin(ATX)highly expressed in melanoma and markedly downregulated by FAE were believed to be responsible for this modulation.CONCLUSION FAE exhibites strong antitumor activity against B16-F10 melanoma through activating MAPKs/Nrf2/HO-1 mediated anti-oxidation and anti-inflammation and modulating glycerophospholipid metabolism via downregulating LPCAT1 and ATX.Besides,it is suggested that serum LysoPCs could be potential biomarkers for the diagnosis and prognosis of melanoma.

Metabolomics Analysis of Metabolites in Forsythia suspense Fruit Using UPLC/ESI-Q TRAP-MS/MS

Forsythiae Fructus,the fruit of Forsythia suspense is a traditional Chinese hebal medicine that has the antiviral and antioxidant effects in China.Modern analytical chemistry studies showed that the extracts of Forsythiae Fructus contain many bioactive components,such as flavonoids,lignans,phenolic acids,and terpenoids,which can be used to anti-inflammatory and treat toxicity,tonsillitis,ulcers,pharyngitis and acute nephritis.In order to study the types and quantities of metabolites in Forsythiae Fructus,we isolated,identified and analysed metabolites between two varieties of Forsythiae Fructus using UPLC/ESI-Q TRAP-MS/MS.The results showed that a total of 407 metabolites were identified in Forsythiae Fructus using UPLC/ESI-Q TRAP-MS/MS,including 21 terpenoids,68 phenolic acids,63 flavonoids,43 amino acids and derivatives,22 alkaloids,55 lipids,24 lignans and coumarins,31 nucleotides and derivatives,29 organic acids,and 51 other metabolites.Among,lignans and coumarins,terpenoids,organic acids,lipids,and phenolic acids were rich in Forsythiae Fructus,which accounted for more than 60%of the total metabolite content.Differential metabolite analysis revealed that 80 metabolites differed significantly between the two types of Forsythiae Fructus.Our results greatly enrich the Forsythiae Fructus phytochemical composition database and provide valuable information for further study of the metabolites of Forsythiae Fructus.

Three New Compounds from Forsythia Suspensa (Thunb.) Vahl

Three new compounds, suspenolic acid (1), forsythenside A (2), and forsythenside B (3) have been isolated from the fruits of Forsythia suspensa. Their structures were elucidated by spectral methods including IR, H-1- and C-13-NMR, DEPT, H-1-H-1 COSY, H-1-C-13 COSY, NOE difference, HMBC, FABMS, CIMS, and chemical reactions.

Study on the mechanism of Fructus Forsythiae and Folium Nelumbinis on acute pharyngitis based on network pharmacology

Objective:To explore the mechanism of Lianqiao-Heye(Fructus Forsythiae and Folium Nelumbinis,FF-FN)drug pair in treating acute pharyngitis(AP)by network pharmacology.Methods:The chemical components and potential therapeutic targets of FF-FN drug pair were obtained from TCMSP firstly,and databases GeneCards,DrugBank,OMIM and PharmGKB were utilized to get the genes related to AP and the intersection of the results was obtained.Then Cytoscape software was used to construct drug-component-target network diagram to screen out the key compounds.Protein interaction network(PPI)was established using String database,and the core targets were screened by CytoNCA topology analysis.R language software was used for GO biological function analysis and KEGG pathway analysis.Finally,the docking verification of key compounds and core target molecules was carried out by AutoDock software.Results:A total of 38 active compounds and 917 potential therapeutic targets were obtained from FF-FN drug pair,while a total of 1534 targets were screened out for AP,among which,the number of intersection targets was 117.7 core targets were screened out from PPI core network,with JUN,TP53,CXCL8 and RELA included.A total of 2487 biological processes were involved in GO enrichment analysis,and 157 related pathways were screened out by KEGG.Based on results of molecular docking verification,the key compounds such as quercetin,luteolin and wogonin in FF-FN drug pair were proved capable of binding to the core targets and a good affinity was shown.Conclusion:FF-FN pair can intervene AP through multiple targets and multiple pathways,including PI3KAkts signaling pathway,AGE-RAGE signaling pathway and IL-17 signaling pathway.The combination of quercetin,luteolin,wogonin,kaempferol andβ-sitosterol with JUN,RELA,MAPK1,TNF and MYC can possibly be one of the mechanisms regarding to the therapeutic effect.

连翘(Forsythia suspensa(Thunb.)Vahl)的试管苗快速繁殖

【目的】连翘是中国常用中草药,也是庭院绿化的常用树种,需求量较大。建立连翘（Forsythia suspensa（Thunb.）Vahl）的试管苗快速繁殖体系,以期为连翘的大规模栽培育种奠定基础。【方法】以MS为基本培养基,添加不同浓度的6-BA与NAA诱导连翘幼茎外植体再生芽的发生;以1/2 MS为基本培养基,添加IAA、IBA或NAA等单一激素,诱导再生芽生根。【结果】结果表明,BA与NAA的比例对于新芽发生有重要影响,NAA的存在通常不同程度导致愈伤组织的发生,当NAA与6-BA比例达到2︰1时,茎段外植体完全脱分化形成愈伤组织。而不含有NAA,即仅含有6-BA单一激素情况下,更有利于芽的发生。对于生根诱导,低浓度IAA或NAA（0.1mg/L）均不能诱导连翘试管苗生根,高浓度（0.5mg/L）会导致茎基部先发生愈伤化,之后才有根从愈伤组织处长出。IBA在0.1~0.5mg/L范围内,均可以诱导90%以上的再生小芽生根。【结论】以连翘的幼茎为外植体诱导连翘的试管苗快速繁殖,MS＋6-BA 0.5 mg/L为最佳的芽诱导培养基;1/2 MS＋IBA 0.2mg/L为最佳的试管苗生根培养基,生根率达到90%以上。

Microwave assisted-semi bionic extraction of lignan compounds from Fructus Forsythiae by orthogonal design

Microwave assisted-semi bionic extraction (MASBE) process for lignans from Fructus Forsythiae was studied. The influences of solvent pH value, microwave power, dosage of solvent and irradiation time were investigated. Optimum extracting parameters were determined by orthogonal experiments as follows: pH value of solvent at first extraction is 5.5-6.0, that at the second extraction is 7-8; microwave power is 700 W; mass ratio of Fructus Forsythiae to water is 1-12; irradiation time is 10 min, and extracting times is two. Under these optimal conditions, the yield of lignans reaches 0.364%. Compared with the conventional extraction methods, the MASBE process has the advantages of high extraction rate, high extraction selectivity.

Pharmacokinetic interaction of Forsythia suspensa extract and azithromycin injection after single and co-intravenous administration in rats

Azithromycin and Chinese medicine forsythia are often used together to treat pediatric mycoplasma infections in China. We aimed to investigate the pharmacokinetic interaction of Forsythia suspensa extract and azithromycin after single and co-intravenous administration in rats. Male Sprague-Dawley rats received single(Forsythia suspensa extract or azithromycin) treatment or co-administration of Forsythia suspensa extract and azithromycin. Blood samples were collected at scheduled times, and drug concentrations were determined by HPLC-UV or HPLC-MS/MS methods. Both non-compartmental analyses and nonlinear mixed-effects modeling approaches were applied to fit pharmacokinetic data and evaluate the impact of co-administration. Pharmacokinetic analysis showed that the area under the curve of azithromycin and forsythiaside increased, and clearance decreased significantly(P < 0.05),after co-administration. The in vivo behavior of both azithromycin and forsythiaside could be appropriately described by the two-compartmental model. The final population pharmacokinetic model indicated that co-administration decreased the central volume of azithromycin and forsythiaside clearance significantly. Co-administration of Forsythia suspensa extract and azithromycin significantly decreased the clearance and increased exposure for both drugs. Pharmacokinetic data suggest that drug co-administration may increase efficiency.

连翘总黄酮提取工艺优化及抗炎活性研究

通过单因素试验和响应面试验设计,评价浸泡时间、料液比、乙醇浓度、提取温度和提取时间对连翘(Forsythia suspensa)总黄酮提取率的影响,建立数学模型,得出二次回归方程,预测连翘总黄酮提取的最佳工艺条件,并对新工艺提取的连翘总黄酮进行小鼠耳廓急性炎症肿胀抑制效果评价,测试连翘总黄酮提取物的抗炎活性。结果表明,最佳工艺条件为料液比1∶20、浸泡时间30 min、提取温度58℃、乙醇浓度60%、提取时间79 min。经验证,新工艺的平均提取率为14.54%。新工艺提取的连翘总黄酮提取液处理二甲苯急性致炎的小鼠耳廓的肿胀值为(15.61±2.73)mg,低于生理盐水处理后的肿胀值(17.21±2.65)mg,体外抗炎试验表明连翘总黄酮提取物可抑制小白鼠耳廓的炎症肿胀。引入浸泡环节的连翘总黄酮提取工艺具有提取率较高、稳定可靠的特点,新工艺提取的连翘总黄酮具有一定的抗炎效果。

不同产地连翘质量的研究现状分析

目的总结不同产地连翘质量控制的研究现状。方法通过收集连翘中主要化学成分的研究文献资料,对不同产地连翘中主要化学成分(总挥发油、连翘酯苷A、连翘酯苷B、连翘苷和微量元素钠、锰、锌、铁、镁、钾、钙、铬、锗、硒及重金属铅、镉、砷、汞、铜)的含量研究结果进行数据收集和比较。结果由统计分析结果可知,陕西、湖北、安徽的样本挥发油质量分数均值约为1.7%,为最高;河南、陕西和山西的连翘酯苷A含量排名靠前,分别为5.47%、4.90%、4.83%;山西连翘中的连翘酯苷B样本量最高且含量均值高于其他产地;山西连翘微量元素优势较大,山西运城、太原、临汾、晋中产地的连翘中的Mn、Fe、Mg、K、Ca、Cr、Ge、Se质量分数居于前列,Na、Zn分别是河南三门峡、河北邯郸产地较优;各产地连翘的重金属含量均未超标。结论山西、河南、陕西、河北作为连翘的主要产区,多种化学成分的质量分数普遍高于其他产区,品质更为优良。本文为开展不同产地连翘的共性和个性特征研究提供了数据支撑,为各产地连翘的质量控制提供了参考。

连翘叶茶对肝癌细胞增殖和迁移功能的影响及其作用机制

【目的】探究连翘叶茶粗提物及其主要成分连翘苷和连翘酯苷A对肝癌细胞LM3增殖和迁移功能的影响及其作用机制。【方法】利用水提法提取连翘叶绿茶和红茶,HPLC方法检测连翘绿茶和连翘红茶中有效成分连翘苷、连翘酯苷A的含量。采用CCK8实验,探究两种连翘叶茶粗提物、连翘苷和连翘酯苷A对肝癌细胞LM3增殖的影响;利用两种连翘叶茶粗提物、连翘苷和连翘酯苷A培养肝癌细胞LM3,通过细胞划痕实验,观察24、48、72、96 h细胞迁移情况,检测两种连翘叶茶粗提物、连翘苷和连翘酯苷A对肝癌细胞LM3迁移的影响;使用RT-PCR技术,检测增殖迁移相关基因Ki-67、Erk、PI3K和mTOR的表达,揭示连翘叶茶粗提物及其主要成分连翘苷和连翘酯苷A影响肝癌细胞增殖迁移功能的分子机制。【结果】连翘叶绿茶和红茶粗提物、连翘苷均显著抑制肝癌细胞LM3的增殖和迁移,连翘酯苷A显著抑制肝癌细胞LM3的迁移。其中,连翘绿茶粗提物在低、中和高浓度,培养时间24、48、72 h,均可明显抑制肝癌细胞增殖和迁移。连翘红茶粗提物随着浓度升高对LM3细胞增殖和迁移的抑制效果不断增强,在高浓度72 h抑制效果最佳。RT-PCR结果分析,其主要作用机制可能是通过降低Ki-67基因表达量来实现的。【结论】连翘叶绿茶和连翘叶红茶粗提物及连翘苷和连翘酯苷A均能够通过降低Ki-67表达抑制肝癌细胞LM3的增殖或迁移。

基于计算机视觉技术的连翘果实性状变化研究

目的:探究连翘果实发育与性状之间的关系,建立基于计算机视觉技术的连翘“辨状论质”方法。方法:采用Python库LabelMe对连翘显微横切图片进行标注,获取连翘果实显微横切各部位的面积占比。采用自制软件Lq_measure对连翘进行长度、宽度和单果面积的测量,同时称取连翘果皮(含隔膜)和种子的质量,并制作数据集。结果:连翘果实性状差异大,证实了计算机视觉技术筛选优质连翘具有工业化应用的前景,建立了一套以Lq_measure软件为基础的连翘性状研究实验操作流程,为连翘性状数据库的建立提供了基本方法,为基于连翘果型的中药“辨状论质”理论研究提供可靠技术支撑。结论:连翘种心性状可用于鉴别连翘的果实成熟度,连翘长宽比系数和连翘果皮重占比相关性为0.74,为以后的连翘质量控制提供了理论依据。

基于TLR4/MyD88/NF-κB信号通路探讨复方银翘合剂缓解哮喘小鼠炎症反应的作用研究

目的:研究复方银翘合剂对哮喘小鼠的作用,并探讨其可能的作用机制。方法:将72只小鼠随机分为6组,正常组、模型组、复方银翘合剂低、中、高剂量组和阳性药物组,每组12只。采用卵清蛋白诱导小鼠哮喘模型,观察小鼠一般症状并检测IgE浓度。收集小鼠肺泡灌洗液并染色计数,HE染色观察小鼠肺组织病理变化。ELISA检测小鼠肺泡灌洗液中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-4、IL-5、IL-13的水平。Western blot法检测肺组织中Toll样受体4(TLR4)、髓样分化因子88(MyD88)和核转录因子κB(NF-κB)蛋白表达水平。结果:与正常组比较,模型组小鼠肺泡灌洗液中总细胞数、嗜酸性粒细胞、淋巴细胞、中性粒细胞均显著增多(P<0.05),巨噬细胞显著减少(P<0.05),肺组织中支气管管壁增厚,炎性细胞浸润,TNF-α、IL-4、IL-5、IL-13的水平均显著提高(P<0.05),TLR4、MyD88和NF-κB蛋白表达水平均显著升高(P<0.05)。与模型组比较,复方银翘合剂中、高剂量组小鼠肺泡灌洗液中总细胞数、嗜酸性粒细胞、淋巴细胞、中性粒细胞均显著减少(P<0.05),巨噬细胞显著增多(P<0.05),TNF-α、IL-4、IL-5、IL-13的水平均显著降低(P<0.05),TLR4、MyD88和NF-κB蛋白表达水平均显著降低(P<0.05)。复方银翘合剂各剂量组比较结果显示,高剂量组的上述变化最明显。结论:复方银翘合剂可缓解哮喘小鼠症状,减轻肺组织病理变化和炎症水平,其作用机制可能与抑制TLR4/MyD88/NF-κB信号通路有关。

连翘种子品质检验及质量分级标准研究

目的建立连翘种子品质检验方法,制订连翘种子质量分级标准。方法通过测定不同产地连翘种子的生活力、净度、发芽率、含水量、千粒重、形态等指标,对16份来自全国连翘主产地的连翘种子进行品质检验,并且使用SPSS软件对所得数据进行K-均值聚类分析,初步制订连翘种子的质量分级标准。结果连翘一级种子黑褐色、圆润饱满,净度≥90%,千粒重≥3.2 g,含水量≤6.2%,生活力≥85%,发芽率≥85%;二级种子褐色、较饱满,净度≥75%,千粒重≥2.7 g,含水量≤5.5%,生活力≥65%,发芽率≥60%;三级种子黄棕色、瘦瘪,净度≥40%,千粒重≥2.2 g,含水量≤4.3%,生活力≥45%,发芽率≥35%。结论本研究制订了连翘种子分级标准,可为连翘种子的质量评价和人工栽培提供参考依据。

连翘配方颗粒与饮片的抗炎、抗肿瘤和抑菌效果的比较研究

目的采用斑马鱼体内模型实验和体外抗菌实验比较连翘配方颗粒和饮片的抗炎、抗肿瘤和抑菌的药效差异。方法将斑马鱼胚胎分为空白组、模型对照组、10μg/mL地塞米松处理组、连翘饮片处理组(剂量为400、600、800μg/mL)和连翘配方颗粒处理组(剂量为400、600、800μg/mL),分别构建硫酸铜、尾鳍横断和LPS显微注射斑马鱼炎症模型,采用中性粒细胞观察、病理组织切片、生存分析和实时定量荧光PCR实验评价连翘配方颗粒与饮片的抗炎药效差异,并从MAPK和NF-κB信号通路角度探讨其抗炎作用机理差异。将斑马鱼胚胎分为模型对照组、250 nmol/L索拉菲尼处理组、连翘饮片处理组(400、600、800μg/mL)和连翘配方颗粒处理组(400、600、800μg/mL),采用斑马鱼异种移植肿瘤细胞模型,通过荧光观察和病理组织切片评价连翘配方颗粒与饮片抗肿瘤的药效差异。将实验分为空白组、连翘饮片处理组(1.95、3.91、7.81、15.63、31.25、62.5、125、250、500 mg/mL)和连翘配方颗粒处理组(1.95、3.91、7.81、15.63、31.25、62.5、125、250、500 mg/mL),采用微量肉汤稀释法评价连翘配方颗粒与饮片的抑菌药效差异。结果与模型对照组比较,连翘配方颗粒及饮片均可抑制中性粒细胞聚集数量(P<0.001)和Tnfα、Il6、P38、Jnk、Erk和P65的mRNA表达(P<0.05或P<0.01或P<0.001),升高斑马鱼的存活率(P<0.01或P<0.001);减少人肺腺癌A549细胞、人结直肠癌HCT116细胞、人胰腺癌PANC-1细胞、人肝癌Hep3B细胞和人宫颈癌Hela细胞在卵黄囊内的聚集(P<0.05或P<0.01或P<0.001);并在15.63 mg/mL时起到抑菌作用。结论连翘配方颗粒及饮片均具有显著抗炎、抗肿瘤和抑菌作用,两者除在低剂量下抑制Il6、P65、Jnk的mRNA表达和HCT116细胞增殖有显著性差异外,其他作用均无明显差异。

基于选择清除分析挖掘连翘异型花柱候选基因

连翘(Forsythia suspensa (Thunb.) Vahl)为多年生木本植物,花柱二型具有自交不亲和性。挖掘连翘异型花柱发育候选基因,利于揭示连翘异型花柱的进化与发育机理。对40株长花柱型与短花柱型连翘植株进行全基因组重测序,检测样本基因组的SNP变异位点,开展选择清除分析。利用遗传分化系数Fst和核苷酸多态性πratio结合的方法筛选候选区域(前5%水平),对差异基因进行GO数据库和KEGG数据库功能注释分析,采用qRT-PCR对候选基因在花柱发育不同时期的表达量进行检测。选择清除分析显示受选择基因295个,主要在植物的生长发育以及代谢调控方面发挥重要作用。分析植物激素调节相关的部分通路,确定连翘异型花柱候选基因CYP734A1(EVM0010386)、BRI1(EVM0011829)、CYCD3(EVM0018316),基因表达结果显示CYP734A1基因在连翘短花柱发育的关键时期持续高表达,露冠期BRI1与CYCD3基因表达水平在短花柱型中显著高于长花柱型。本研究挖掘的候选基因在连翘长花柱与短花柱中存在差异表达,油菜素甾醇参与了连翘异型花柱的发育,是产生连翘花柱异型的原因之一。本研究为连翘异型花柱的进化以及遗传发育调控机制解析奠定基础并提供思路。