The resin of Ferula sinkiangensis has been traditionally utilized for treating gastrointestinal disorders,inflammation,tumors,various cancers,and alopecia areata.The primary bioactive constituents,sesquiterpene coumarins,have demonstrated notable therapeutic potential against neuroinflammation.In this study,a structure-guided fractionation method was used to isolate nine novel sesquiterpene coumarins from the resin of F.sinkiangensis.These compounds were characterized and structurally elucidated using comprehensive physicochemical and spectroscopic techniques,including calculated electronic circular dichroism(ECD).Anti-neuroinflammatory assays revealed that compounds 2,3,and 6 significantly inhibited nitric oxide(NO)production in lipopolysaccharide(LPS)-stimulated BV2 microglial cells,with IC_(50)values ranging from 1.63 to 12.25μmol·L^(−1).
DANG WenGUO TingtingZHOU DiMENG QingqiFANG MingxiaCHEN GangLIN BinHOU YueLI Ning
Pyran-and furanocoumarins are key representatives of tetrahydropyrans and tetrahydrofurans,respectively,exhibiting diverse physiological and medical bioactivities.However,the biosynthetic mechanisms for their core structures remain poorly understood.Here we combined multiomics analyses of biosynthetic enzymes in Peucedanum praeruptorum and in vitro functional verification and identified two types of key enzymes critical for pyran and furan ring biosynthesis in plants.These included three distinct P.praeruptorum prenyltransferases(PpPT1e3)responsible for the prenylation of the simple coumarin skeleton 7 into linear or angular precursors,and two novel CYP450 cyclases(PpDC and PpOC)crucial for the cyclization of the linear/angular precursors into either tetrahydropyran or tetrahydrofuran scaffolds.Biochemical analyses of cyclases indicated that acid/base-assisted epoxide ring opening contributed to the enzyme-catalyzed tetrahydropyran and tetrahydrofuran ring refactoring.The possible acid/base-assisted catalytic mechanisms of the identified cyclases were theoretically investigated and assessed using site-specific mutagenesis.We identified two possible acidic amino acids Glu303 in PpDC and Asp301 in PpOC as vital in the catalytic process.This study provides new enzymatic tools in the epoxide formation/epoxide-opening mediated cascade reaction and exemplifies how plants become chemically diverse in terms of enzyme function and catalytic process.
佛手作为一种药食两用的柑桔类水果,香豆素类化合物是其关键的生物活性成分之一。由于香豆素类化合物结构相似,并且有同分异构体,目前还没有快速准确的方法针对香豆素类化合物进行同时定量。该研究建立了一种超高效液相色谱三重四极杆质谱法(ultra-performance liquid chromatography tandem triple quaternary mass spectrometry,UPLC-QqQ-MS/MS),在14 min内定性并定量4个不同产地佛手柑中的41种香豆素类化合物,包括16种简单香豆素、23种呋喃香豆素和2种其他香豆素,其中有8组同分异构体。该方法灵敏度高、准确度高、精确度高,可用于佛手实际样品中香豆素的筛选和测定,这是迄今为止筛查并定量香豆素最全面和快速的方法。柠檬内酯是佛手中含量最高的香豆素,浙江佛手中检出的香豆素类化合物含量和种类均最多,并且有4种香豆素在佛手中首次检出。
Coumarins,derived from the phenylpropanoid pathway,represent one of the primary metabolites found in angiosperms.The alignment of the tetrahydropyran(THP)and tetrahydrofuran(THF)rings with the lactone structure results in the formation of at least four types of complex coumarins.However,the mechanisms underlying the structural diversity of coumarin remain poorly understood.Here,we report the chromosome-level genome assembly of Notopterygium incisum,spanning 1.64 Gb,with a contig N50 value of 22.7 Mb and 60,021 annotated protein-coding genes.Additionally,we identified the key enzymes responsible for shaping the structural diversity of coumarins,including two p-coumaroyl CoA 2′-hydroxylases crucial for simple coumarins basic skeleton architecture,two UbiA prenyltransferases responsible for angular or linear coumarins biosynthesis,and five CYP736 cyclases involved in THP and THF ring formation.Notably,two bifunctional enzymes capable of catalyzing both demethylsuberosin and osthenol were identified for the first time.Evolutionary analysis implies that tandem and ectopic duplications of the CYP736 subfamily,specifically arising in the Apiaceae,contributed to the structural diversity of coumarins in N.incisum.Conclusively,this study proposes a parallel evolution scenario for the complex coumarin biosynthetic pathway among different angiosperms and provides essential synthetic biology elements for the heterologous industrial production of coumarins.
