Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researc...Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B exhibited tissue-specific expression in plants.This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.展开更多
Stephania Tetrandrae Radix is one of the common traditional Chinese medicines,which has bitter and pungent taste as well as cold properties. It can subside edema,get rid of rheumatism and relieve pain. Therefore,it is...Stephania Tetrandrae Radix is one of the common traditional Chinese medicines,which has bitter and pungent taste as well as cold properties. It can subside edema,get rid of rheumatism and relieve pain. Therefore,it is mainly used for the treatment of rheumatism arthralgia,edema,dysuria,athlete's foot,swollen wet sores and other diseases in traditional Chinese medicine( TCM). Stephania Tetrandrae Radix is mainly composed of dual-benzyl isoquinoline alkaloids,including tetrandrine,fangchinoline and so on. Modern pharmacology research shows that Stephania Tetrandrae Radix and its main components have a wide range of pharmacological activity in the anti-inflammatory,anti-pathogenic microorganisms,anti-tumor,anti-hypertension,anti-arrhythmia,anti-myocardial ischemia,anti-fibrosis,anti-silicosis,inhibiting scar and other aspects,with broad application prospect. Stephania Tetrandrae Radix is often applied with compatibility of other Chinese medicines in clinically,and has achieved obvious effects in the treatment of rheumatoid arthritis,cardiovascular disease,cancer,hypertension,liver ascites and other diseases. There are some representative prescriptions,such as Fangji Fuling decoction,Fangji Huangqi decoction,Jijiao Lihuang pill,Xuanbi decoction,compound Hanfangji granule and so on. In this paper,the pharmacological effects and clinical applications of Stephania Tetrandrae Radix in the past ten years are reviewed,providing the reference for its further development and application.展开更多
A new artifact bisbenzylisoquinoline,2,2′-N,N-dichloromethyltetrandrine(1),has been ob- tained from the root of Stephania tetrandra and its structure has been advanced on the basis of spectroscopic and chemical evide...A new artifact bisbenzylisoquinoline,2,2′-N,N-dichloromethyltetrandrine(1),has been ob- tained from the root of Stephania tetrandra and its structure has been advanced on the basis of spectroscopic and chemical evidence.展开更多
Stephania tetrandra is a medicinal plant that contains four main types of benzylisoquinoline alkaloids(BIAs):mono-BIAs,bis-BIAs,aporphine-type BIAs(APT-BIA),and tetrahydroprotoberberines(THBs).S.tetrandra is particula...Stephania tetrandra is a medicinal plant that contains four main types of benzylisoquinoline alkaloids(BIAs):mono-BIAs,bis-BIAs,aporphine-type BIAs(APT-BIA),and tetrahydroprotoberberines(THBs).S.tetrandra is particularly rich in the bis-BIAs,tetrandrine and fangchinoline,which are critical quality markers and major components of the traditional Chinese medicine“Fangji”.However,changes in their BIA profiles and associated metabolic regulatory networks over time remain unclear.In this study,metabolomics and transcriptomics were integrated to investigate the dynamic accumulation patterns and underlying mechanisms of these alkaloids across three developmental stages:Autumnal Equinox(AuEq),Winter Solstice(WinSo),and Vernal Equinox(VeEq).Quantification revealed that both alkaloids reached their highest concentrations at VeEq,with tetran-drine levels more than doubling from WinSo.Untargeted metabolomics identified 61 differentially accumulated BIAs,displaying stage-specific accumulation patterns.Hierarchical clustering grouped these BIAs into four structural categories,predominantly enriched at VeEq.Transcriptome profiling showed significant upregulation of genes involved in isoquinoline alkaloid biosynthesis during VeEq,especially key enzymes such as TyrAT,TYDC,4HPPDC,and NCS.Co-expression and protein interaction network analyses further identified abscisic acid(ABA)signaling pathways as major hormonal regulators.Notably,ABA levels and exogenous ABA application confirmed its pivotal role in promoting bis-BIA biosynthesis,acting in concert with reactive oxygen species(ROS)to fine-tune alkaloid production.Collectively,this study establishes a hormone-ROS-BIA regulatory framework that explains seasonal fluctuations in alkaloid content and provides new entry points for improving the medicinal quality and cultivation strategies of S.tetrandra.展开更多
Objective:The objective of the study was to develop a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of tetrandrine,fangchinoline,and cyclanoline in ...Objective:The objective of the study was to develop a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of tetrandrine,fangchinoline,and cyclanoline in rat plasma and to investigate their pharmacokinetics after oral administration of Stephaniae Tetrandrae Radix extracts.Methods:Sample pretreatment involved methanol pretreatment and liquid–liquid extraction of ethyl acetate from plasma with methanol.Tramadol was used as the internal standard.The analysis was performed using an high strength silica T3 column(100 mm×2.1 mm,1.8μm)and a gradient elution method consisting of mobile phase solution A(0.1%formic acid in water)and B(acetonitrile)at a flow rate of 0.4 mL/min.The detection was performed using a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode and using an electrospray ionization source in the positive ionization mode.Results:High efficiency was achieved with an analysis time of 4 min/sample.The calibration curve linear in the concentration range of 1250 ng/ml(R^(2)≥0.9900)and the lower limit of quantification is 1 ng/ml.The intraday and interday precision(relative standard deviation)values were lower than 9.4.Accuracy(relative error)was within 10.3%at all three quality control levels.Conclusions:This method was successfully applied in pharmacokinetics of tetrandrine,fangchinoline,and cyclanoline in rats after oral administration of Stephaniae Tetrandrae Radix extracts.The maximum plasma concentration(C_(max))of tetrandrine,fangchinoline,and cyclanoline was 124.71±16.08,84.56±3.28,and 57.61±6.26 ng/mL,respectively.The time to reach C_(max)was 10.39±3.04 for tetrandrine,10.17±3.04 for fangchinoline,and 6.40±3.16 for cyclanoline.The pharmacokinetic results might help further guide the clinical application of Stephaniae Tetrandrae Radix.展开更多
目的基于网络药理学分析方法探讨防己-红参抗结直肠癌的有效成分和潜在分子机制。方法通过中药系统药理学数据库和分析平台(Chinese Medicine System Pharmacology Database,TCMSP)筛选防己和红参的有效活性成分及对应靶点,与结直肠癌...目的基于网络药理学分析方法探讨防己-红参抗结直肠癌的有效成分和潜在分子机制。方法通过中药系统药理学数据库和分析平台(Chinese Medicine System Pharmacology Database,TCMSP)筛选防己和红参的有效活性成分及对应靶点,与结直肠癌相关靶点进行交集,获得药物抗结直肠癌的潜在靶点,构建药物-靶点网络关系图,筛选关键成分。通过String数据库构建蛋白质-蛋白质相互作用(PPI)网络图筛选核心靶点,其次在GEPIA和HPA数据库验证核心靶点表达情况。利用WebGestalt和Metascape数据库对潜在靶点进行基因本体论(GO)和京都基因与基因组大百科全书数据库(KEGG)富集分析。以P<0.05为差异有统计学意义。结果通过TCMSP平台筛选获得防己有效成分10种,红参有效成分19种,利用维恩图交集获得药物抗结直肠癌潜在靶点71个,对药物-靶点网络关系图进一步计算分析获得防己-红参抗结直肠癌5种关键成分,包括β-谷甾醇(beta-Sitosterol)、千金藤碱[(+)-Stepharine]、蝙蝠葛林碱(Menisperine)、木兰花碱(Magnoflorine)、人参皂苷Rh2(ginsenoside Rh2),通过PPI网络图筛选获得6个核心靶点,分别是肿瘤坏死因子(TNF)、热休克蛋白90ɑB1(HSP90AB1)、前列腺素内过氧化物合酶(PTGS2)、蛋白激酶A催化亚基(PRKACA)、多巴胺受体D2(DRD2)和5-羟色胺转运蛋白(SLC6A4)。GO富集分析显示,防己-红参抗结直肠癌主要参与细胞增殖、代谢进展、抗氧化活性等生物功能过程(均P<0.01);根据KEGG信号通路富集分析,防己-红参涉及结直肠癌、非小细胞肺癌、前列腺癌、动脉粥样硬化等疾病通路(均P<0.01),信号通路主要富集于cAMP、cGMP-PKG、PI3K/AKT(均P<0.01)。结论通过网络药理学分析方法从分子水平阐明防己-红参抗结直肠癌的有效成分和潜在作用机制,防己-红参可能通过多种成分、多个靶点、多条途径协同发挥抗癌效果.展开更多
目的:结合粉防己碱在生药及水提物中含量的测定,从效、毒两个维度来阐释粉防己与其主要生物碱粉防己碱的作用差异及两者之间的关系。方法:HPLC测定粉防己水提物中粉防己碱的含量的基础上,确定粉防己碱给药剂量,分别采用热板法、小鼠耳...目的:结合粉防己碱在生药及水提物中含量的测定,从效、毒两个维度来阐释粉防己与其主要生物碱粉防己碱的作用差异及两者之间的关系。方法:HPLC测定粉防己水提物中粉防己碱的含量的基础上,确定粉防己碱给药剂量,分别采用热板法、小鼠耳肿胀法对粉防己与粉防己碱的镇痛、抗炎作用进行比较研究;同时,选取给药12,24 d以及停药12 d 3个不同时间点实验动物总蛋白(TP),白蛋白(ALB),丙氨酸氨基转氨酶(ALT),天冬氨酸氨基转氨酶(AST),总胆红素(TBIL)作为观测指标,对粉防己与粉防己碱的肝脏毒性进行探查和比较。结果:1防己药材中粉防己碱的含量8.99 mg·g-1,粉防己水提液中粉防己碱的转移率为30.03%;2粉防己与粉防己碱均有明显的镇痛作用,但从短期镇痛效果而言,以粉防己碱为佳,从长期镇痛效果而言,以粉防己水提物为佳。粉防己碱的抗炎效果相对优于粉防己水提物的抗炎效果,但两者相比无显著性差异。3粉防己水提物和粉防己碱给药均可以造成一定程度的肝细胞受损,但两者的作用机制不同,且粉防己水提物造成的肝脏损伤停药后具有可逆性,而粉防己碱造成的肝脏损伤在停药后则继续发展。结论:粉防己碱作为粉防己的主要组分,并不能够完全表征粉防己的药效及毒性,同样的,粉防己中的其他组分也对粉防己碱的毒性作用没有明显的监制作用。粉防己碱与粉防己的效、毒作用表现及机制并不相同。展开更多
文摘Benzylisoquinoline alkaloids(BIAs)are a structurally diverse group of plant metabolites renowned for their pharmacological properties.However,sustainable sources for these compounds remain limited.Consequently,researchers are focusing on elucidating BIA biosynthetic pathways and genes to explore alternative sources using synthetic biology approaches.CYP80B,a family of cytochrome P450(CYP450)enzymes,plays a crucial role in BIA biosynthesis.Previously reported CYP80Bs are known to catalyze the 3′-hydroxylation of(S)-Nmethylcoclaurine,with the N-methyl group essential for catalytic activity.In this study,we successfully cloned a full-length CYP80B gene(St CYP80B)from Stephania tetrandra(S.tetrandra)and identified its function using a yeast heterologous expression system.Both in vivo yeast feeding and in vitro enzyme analysis demonstrated that St CYP80B could catalyze Nmethylcoclaurine and coclaurine into their respective 3'-hydroxylated products.Notably,St CYP80B exhibited an expanded substrate selectivity compared to previously reported wildtype CYP80Bs,as it did not require an N-methyl group for hydroxylase activity.Furthermore,St CYP80B displayed a clear preference for the(S)-configuration.Co-expression of St CYP80B with the CYP450 reductases(CPRs,StCPR1,and StCPR2),also cloned from S.tetrandra,significantly enhanced the catalytic activity towards(S)-coclaurine.Site-directed mutagenesis of St CYP80B revealed that the residue H205 is crucial for coclaurine catalysis.Additionally,St CYP80B exhibited tissue-specific expression in plants.This study provides new genetic resources for the biosynthesis of BIAs and further elucidates their synthetic pathway in natural plant systems.
基金Supported by the National Natural Science Fund of China(81360648)
文摘Stephania Tetrandrae Radix is one of the common traditional Chinese medicines,which has bitter and pungent taste as well as cold properties. It can subside edema,get rid of rheumatism and relieve pain. Therefore,it is mainly used for the treatment of rheumatism arthralgia,edema,dysuria,athlete's foot,swollen wet sores and other diseases in traditional Chinese medicine( TCM). Stephania Tetrandrae Radix is mainly composed of dual-benzyl isoquinoline alkaloids,including tetrandrine,fangchinoline and so on. Modern pharmacology research shows that Stephania Tetrandrae Radix and its main components have a wide range of pharmacological activity in the anti-inflammatory,anti-pathogenic microorganisms,anti-tumor,anti-hypertension,anti-arrhythmia,anti-myocardial ischemia,anti-fibrosis,anti-silicosis,inhibiting scar and other aspects,with broad application prospect. Stephania Tetrandrae Radix is often applied with compatibility of other Chinese medicines in clinically,and has achieved obvious effects in the treatment of rheumatoid arthritis,cardiovascular disease,cancer,hypertension,liver ascites and other diseases. There are some representative prescriptions,such as Fangji Fuling decoction,Fangji Huangqi decoction,Jijiao Lihuang pill,Xuanbi decoction,compound Hanfangji granule and so on. In this paper,the pharmacological effects and clinical applications of Stephania Tetrandrae Radix in the past ten years are reviewed,providing the reference for its further development and application.
文摘A new artifact bisbenzylisoquinoline,2,2′-N,N-dichloromethyltetrandrine(1),has been ob- tained from the root of Stephania tetrandra and its structure has been advanced on the basis of spectroscopic and chemical evidence.
基金supported by Zhejiang Provincial Natural Science Foundation of China under Grant No.LY24B020001Zhejiang Province Science and Technology Program under Grant No.2022C03142.
文摘Stephania tetrandra is a medicinal plant that contains four main types of benzylisoquinoline alkaloids(BIAs):mono-BIAs,bis-BIAs,aporphine-type BIAs(APT-BIA),and tetrahydroprotoberberines(THBs).S.tetrandra is particularly rich in the bis-BIAs,tetrandrine and fangchinoline,which are critical quality markers and major components of the traditional Chinese medicine“Fangji”.However,changes in their BIA profiles and associated metabolic regulatory networks over time remain unclear.In this study,metabolomics and transcriptomics were integrated to investigate the dynamic accumulation patterns and underlying mechanisms of these alkaloids across three developmental stages:Autumnal Equinox(AuEq),Winter Solstice(WinSo),and Vernal Equinox(VeEq).Quantification revealed that both alkaloids reached their highest concentrations at VeEq,with tetran-drine levels more than doubling from WinSo.Untargeted metabolomics identified 61 differentially accumulated BIAs,displaying stage-specific accumulation patterns.Hierarchical clustering grouped these BIAs into four structural categories,predominantly enriched at VeEq.Transcriptome profiling showed significant upregulation of genes involved in isoquinoline alkaloid biosynthesis during VeEq,especially key enzymes such as TyrAT,TYDC,4HPPDC,and NCS.Co-expression and protein interaction network analyses further identified abscisic acid(ABA)signaling pathways as major hormonal regulators.Notably,ABA levels and exogenous ABA application confirmed its pivotal role in promoting bis-BIA biosynthesis,acting in concert with reactive oxygen species(ROS)to fine-tune alkaloid production.Collectively,this study establishes a hormone-ROS-BIA regulatory framework that explains seasonal fluctuations in alkaloid content and provides new entry points for improving the medicinal quality and cultivation strategies of S.tetrandra.
基金financially supported by National Natural Science Foundation of China/81973439Heilongjiang University of Chinese Medicine Research Fund/201504+4 种基金National Natural Science Foundation of China/81803686Research Fund of Heilongjiang University of Traditional Chinese Medicine/201504Heilongjiang Postdoctoral Research Start-up Funding Project/LBH-Q16214Heilongjiang Science Foundation Project/H2018056Heilongjiang University of Traditional Medicine Talents Support Plan/2018RCD03。
文摘Objective:The objective of the study was to develop a rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometric method for the determination of tetrandrine,fangchinoline,and cyclanoline in rat plasma and to investigate their pharmacokinetics after oral administration of Stephaniae Tetrandrae Radix extracts.Methods:Sample pretreatment involved methanol pretreatment and liquid–liquid extraction of ethyl acetate from plasma with methanol.Tramadol was used as the internal standard.The analysis was performed using an high strength silica T3 column(100 mm×2.1 mm,1.8μm)and a gradient elution method consisting of mobile phase solution A(0.1%formic acid in water)and B(acetonitrile)at a flow rate of 0.4 mL/min.The detection was performed using a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode and using an electrospray ionization source in the positive ionization mode.Results:High efficiency was achieved with an analysis time of 4 min/sample.The calibration curve linear in the concentration range of 1250 ng/ml(R^(2)≥0.9900)and the lower limit of quantification is 1 ng/ml.The intraday and interday precision(relative standard deviation)values were lower than 9.4.Accuracy(relative error)was within 10.3%at all three quality control levels.Conclusions:This method was successfully applied in pharmacokinetics of tetrandrine,fangchinoline,and cyclanoline in rats after oral administration of Stephaniae Tetrandrae Radix extracts.The maximum plasma concentration(C_(max))of tetrandrine,fangchinoline,and cyclanoline was 124.71±16.08,84.56±3.28,and 57.61±6.26 ng/mL,respectively.The time to reach C_(max)was 10.39±3.04 for tetrandrine,10.17±3.04 for fangchinoline,and 6.40±3.16 for cyclanoline.The pharmacokinetic results might help further guide the clinical application of Stephaniae Tetrandrae Radix.
文摘目的基于网络药理学分析方法探讨防己-红参抗结直肠癌的有效成分和潜在分子机制。方法通过中药系统药理学数据库和分析平台(Chinese Medicine System Pharmacology Database,TCMSP)筛选防己和红参的有效活性成分及对应靶点,与结直肠癌相关靶点进行交集,获得药物抗结直肠癌的潜在靶点,构建药物-靶点网络关系图,筛选关键成分。通过String数据库构建蛋白质-蛋白质相互作用(PPI)网络图筛选核心靶点,其次在GEPIA和HPA数据库验证核心靶点表达情况。利用WebGestalt和Metascape数据库对潜在靶点进行基因本体论(GO)和京都基因与基因组大百科全书数据库(KEGG)富集分析。以P<0.05为差异有统计学意义。结果通过TCMSP平台筛选获得防己有效成分10种,红参有效成分19种,利用维恩图交集获得药物抗结直肠癌潜在靶点71个,对药物-靶点网络关系图进一步计算分析获得防己-红参抗结直肠癌5种关键成分,包括β-谷甾醇(beta-Sitosterol)、千金藤碱[(+)-Stepharine]、蝙蝠葛林碱(Menisperine)、木兰花碱(Magnoflorine)、人参皂苷Rh2(ginsenoside Rh2),通过PPI网络图筛选获得6个核心靶点,分别是肿瘤坏死因子(TNF)、热休克蛋白90ɑB1(HSP90AB1)、前列腺素内过氧化物合酶(PTGS2)、蛋白激酶A催化亚基(PRKACA)、多巴胺受体D2(DRD2)和5-羟色胺转运蛋白(SLC6A4)。GO富集分析显示,防己-红参抗结直肠癌主要参与细胞增殖、代谢进展、抗氧化活性等生物功能过程(均P<0.01);根据KEGG信号通路富集分析,防己-红参涉及结直肠癌、非小细胞肺癌、前列腺癌、动脉粥样硬化等疾病通路(均P<0.01),信号通路主要富集于cAMP、cGMP-PKG、PI3K/AKT(均P<0.01)。结论通过网络药理学分析方法从分子水平阐明防己-红参抗结直肠癌的有效成分和潜在作用机制,防己-红参可能通过多种成分、多个靶点、多条途径协同发挥抗癌效果.
文摘目的对防己科千金藤属植物粉防己(Stephania tetrandraS.Moore)的生物碱化学成分进行研究。方法采用反复硅胶柱色谱法、Sephadex LH-20柱色谱法等进行分离纯化,并通过光谱分析鉴定其化学结构。结果分离得到7个生物碱类化合物,分别鉴定为粉防己碱(tetrandrine,1)、防己诺林碱(fangchinoline,2)、2′-N-chloromethyltetrandrine(3)、氧化防己碱(oxofangchirine,4)、粉防己碱D盐酸盐(fenfangjine D hydrochloride,5)、荷苞牡丹碱((+)-dicentrine,6)、tazopsine(7)。结论化合物7为首次从该属植物中分离得到,化合物6为首次从该植物中分离得到。
文摘目的:结合粉防己碱在生药及水提物中含量的测定,从效、毒两个维度来阐释粉防己与其主要生物碱粉防己碱的作用差异及两者之间的关系。方法:HPLC测定粉防己水提物中粉防己碱的含量的基础上,确定粉防己碱给药剂量,分别采用热板法、小鼠耳肿胀法对粉防己与粉防己碱的镇痛、抗炎作用进行比较研究;同时,选取给药12,24 d以及停药12 d 3个不同时间点实验动物总蛋白(TP),白蛋白(ALB),丙氨酸氨基转氨酶(ALT),天冬氨酸氨基转氨酶(AST),总胆红素(TBIL)作为观测指标,对粉防己与粉防己碱的肝脏毒性进行探查和比较。结果:1防己药材中粉防己碱的含量8.99 mg·g-1,粉防己水提液中粉防己碱的转移率为30.03%;2粉防己与粉防己碱均有明显的镇痛作用,但从短期镇痛效果而言,以粉防己碱为佳,从长期镇痛效果而言,以粉防己水提物为佳。粉防己碱的抗炎效果相对优于粉防己水提物的抗炎效果,但两者相比无显著性差异。3粉防己水提物和粉防己碱给药均可以造成一定程度的肝细胞受损,但两者的作用机制不同,且粉防己水提物造成的肝脏损伤停药后具有可逆性,而粉防己碱造成的肝脏损伤在停药后则继续发展。结论:粉防己碱作为粉防己的主要组分,并不能够完全表征粉防己的药效及毒性,同样的,粉防己中的其他组分也对粉防己碱的毒性作用没有明显的监制作用。粉防己碱与粉防己的效、毒作用表现及机制并不相同。
