~毛黄堇Corydalis tomentella Franch.为罂粟科紫堇属植物，民间作为治疗肝炎和肝硬化的特效草药使用，疗效显著。但其化学成分尚不明确，药理作用缺乏基础数据，药材鉴定缺乏有效手段，严重制约其临床应用。本研究基于本草基因组学、植物化学及中药药理学的方法和技术，对毛黄堇药材鉴定、活性成分及肝保护作用开展研究。为药材质量控制及临床应用提供依据，对人工栽培、重要化合物生物合成具有指导意义。主要研究结果如下：
    1.采用紫堇属最大样本量开展毛黄堇及同属物种DNA条形码鉴定研究，结果显示DNA条形码能有效鉴定毛黄堇与同属物种。从5条（ITS, ITS2, matK, rbcL 和 psbA-trnH）核基因组及叶绿体基因组DNA条形码序列中，初步筛选出鉴定效率较高的3条（ITS, ITS2, matK)，基于毛黄堇及同属131个样本进一步评估鉴定效率最高的条形码及分析方法。结果显示，DNA条码对毛黄堇及其同属物种鉴定效率最高为69.6%。单序列中，ITS（65.2%）鉴定效率最高；单序列及复合序列中，ITS+matK (69.6%)鉴定效率最高；比较各分析方法，BLAST提供最高鉴定效率，DNA条形码能有效鉴定毛黄堇与同属物种。聚类分析显示，毛黄堇与石生黄堇亲缘关系最近。
    2.高通量测序紫堇属首个叶绿体基因组，有效鉴定毛黄堇与石生黄堇。Illumina HiSeq4000高通量测序平台测序毛黄堇及同属近缘种石生黄堇叶绿体基因组，组装获得190kb的毛黄堇叶绿体基因组和189kb的石生黄堇叶绿体基因组。总计注释107个叶绿体基因，包括73个蛋白质编码基因、25个tRNA和４个rRNA基因。毛黄堇叶绿体基因组具有较为特殊的结构，高度扩增的IR区和极度收缩的SSC区（9636bp），以及高比例的散在和串联重复序列。通过叶绿体基因组全局比对和提取70个共有蛋白编码基因序列建立系统进化树，能有效鉴定毛黄堇与近缘种石生黄堇。
    3.高通量测序毛黄堇核基因组，组装获得高质量基因组。PacBio Sequel三代测序平台7个SMRT cells共测序产生27.64 Gb (约107×)原始数据，过滤后N50 为9.63 kb。Survey分析显示，毛黄堇基因组约258.56Mb，具有非常低的杂合度(约0.3%)。最终组装生成252Mb的基因组，Contig N50为 2.36Mb，覆盖预测基因组97.3%，BUSCO评估显示毛黄堇基因组完整性95.70%。共注释到25,595个基因座位，编码36177个蛋白质，43.54% (110,107,533bp)的重复序列，BUSCO评估毛黄堇蛋白编码序列注释完整性84.8%。CAFÉ分析显示，毛黄堇扩张基因1374个，收缩基因2532个。根据time tree的标准生物钟以及系统发育树的进化节点计算分化时间，毛黄堇与博落回聚在一枝，其分化时间大约为36.48百万年前。13C稳定同位素标记酪氨酸培养毛黄堇植株，HPLC-Q/TOF MS检测代谢通路中化合物同位素标记。结果显示，关注通路中22个化合物中21个检测到被同位素标记，标记的形式与预测通路中化合物代谢规律一致，证实毛黄堇中脱氢卡维丁等生物碱合成上游途径与酪氨酸途径合成异喹啉生物碱一致。结合化合物结构、酶特性及同位素示踪，推测从已知途径终产物金黄紫堇碱起始，经过2步脱氢酶和2步甲基转移酶催化合成脱氢卡维丁。从毛黄堇基因组中，筛选到与脱氢卡维丁等生物碱合成相关22个关键酶同源基因，根、茎、叶、花不同部位转录组和生物碱含量分析显示，关键酶基因表达与生物碱含量具有一定相关性。
    4.从毛黄堇总生物碱中首次分离、鉴定6个生物碱成分。75%乙醇回流提取毛黄堇总生物碱，制备液相色谱仪分离、制备单体生物碱成分，UPLC-ESI-Q-TOF-MS液质联用仪，AM600型核磁共振仪鉴定化合物结构。共分离得到6个单体成分，鉴定为：脱氢卡维丁、脱氢阿卜卡维丁、脱氢异阿卜卡维丁、黄连碱、脱氢碎叶紫堇碱和甲基黄连碱，6个化合物均为首次从毛黄堇中分离。不同部位毛黄堇药材化学成分差异显著，其主要活性成分脱氢卡维丁在花和根中含量较高，茎、叶中偏低，黄连碱特异性分布在花中。野生和人工栽培毛黄堇化学成分差异不显著，表明人工栽培药材可以作为野生药材的替代品。不同采收期化学成分差异显著，3月采收毛黄堇主要化学成分含量最高。
    5.研究表明毛黄堇具有显著的酒精性急性肝损伤保护作用。采用酒精所致小鼠急性肝损伤及四氯化碳所致急性肝损伤模型对毛黄堇水提物肝保护活性进行评价。结果显示，毛黄堇水提物各剂量组(200 mg/kg、400 mg/kg、600 mg/kg)均能显著降低酒精所致急性肝损伤小鼠ALT和AST酶活性（P<0.01，P<0.05），减轻肝组织受损程度，对酒精性急性肝损伤具有保护作用。四氯化碳急性肝损伤结果显示，毛黄堇水提物在低剂量（200 mg/kg）时对四氯化碳所致急性肝损伤小鼠ALT活性有微弱的抑制作用。
    本研究以疗效确切的传统中草药毛黄堇为研究对象，为药材鉴定提供有效方法，揭示其药效物质基础，明确其主要药理活性，为毛黄堇药材地方和国家用药标准制定提供科学依据。
 

~corydalis tomentella franch., a species of the genus corydalis in papaveraceae，has been used as a folk herbal medicine for the treatment of hepatitis and cirrhosis. however, its chemical composition is still unclear, and pharmacological effect lacks basic data, which seriously restricts the clinical application. based on the methods and techniques of herbgenomics, phytochemistry and pharmacology, we studied the identification of medicinal materials，the active ingredients and main pharmacodynamics. this study provided the evidence for the clinical applications, it is of great significance for the artificial cultivation and the biosynthesis of important compounds.
1.the largest sample size of corydalis was used for identification of c. tomentella and its related species by dna barcoding. three regions (its, its2, matk) with high identification efficiency were screened out from five dna barcode (its, its2, matk, rbcl and psba-trnh) of nuclear genome and chloroplast genome. based on 131 samples of corydalis, different region and analysis methods were compared to screen the most suitable barcode and its analysis method. the result shows that, its (65.9%) provided the highest identification efficiency in single sequence, its+matk (69.6%) shows the highest identification efficiency in single sequence and its combination, and blast exhibited the highest identification efficiency in all analysis. dna barcoding can effectively identify c. tomentella and its related species.
2. high-throughput sequencing of the first chloroplast genome of corydalis for identification of c. tomentella and its related species. c. tomentella and c. saxicola were sequenced by illumina hiseq4000 high throughput sequencing platform. the 190 kb chloroplast genome of c. tomentella and 189 kb of c. saxicol were obtained. a total of 107 genes were annotated, including 73 protein-coding genes, 25 trnas and 4 rrna sequences. the chloroplast genome of c. tomentella exhibited the special structure, highly expanded ir region and extremely contracted ssc region (9636 bp), as well as high proportion of scattered and tandem repeats. the mltree was established by 70 common protein coding gene sequences. the result showed that c. tomentella was closely related to c. saxicola, but far from papaver somniferum l. and macleaya cordata (willd.)r.br., and c. tomentella and c. saxicola can be identified succesfully by chloroplast genome.
 3. high-throughput sequencing of the nuclear genome of c. tomentella and high-quality genome been obtained. we performed whole genome sequencing for c. tomentella and generated 27.64 gb (~107× coverage) raw data, filtered subreads 4,077,968, n50 9.63 kb. the genome were estimated as a extremely low level of heterozygosity (about 0.3%) and 258.56 mb of size. finally, 252mb genome was assembled, covering 97.30% of predicted genome size. the c. tomentella genome showed 95.70% consistent assessments using busco embryophyta subset. in total of 25,595 protein-coding gene models were annotated. the 84.8% annotation completeness with 15.2% missing buscos were assessed for c. tomentella protein sequences. café analysis showed that there were 1374 expansion genes and 2532 contraction genes in c. tomentella. according to the standard clock of time tree and the evolutionary node of phylogenetic tree, the differentiation time of c. tomentella and m. cordata was calculated as 36.48 million years ago. c. tomentella was cultured with 13c stable isotope labeled tyrosine. the result shows that 21 of the 22 compounds were identified by hplc-q/tof ms in the downstream metabolic pathway, which confirmed the tyrosine pathway was the possible pathway of alkaloid biosynthesis of c. tomentella. 22 key enzymes related to alkaloid biosynthesis such as dehydrocavidine were screened from the genome of c. tomentella, the analysis of tranome and alkaloid content of different parts(roots, stems, leaves and flowers) showed that the expression of key enzymes genes was significantly correlated with the content of alkaloid.
   4. isolation and identification of 6 alkaloids from total alkaloids in c. tomentella for the first time. the monomer alkaloids were separated and prepared by preparative liquid chromatography. the compounds were identified by uplc-esi-q-tof-ms and am600 nmr. 6 monomers were isolated and their structures were identified as: dehydrocavidine, dehydroapocavidine, dehydroisoapocavidine, coptisine, dehydrocheilanthifoline and worenine. the chemical constituents of c. tomentella from different parts were significantly different. dehydrocavidine is the main active ingredient of c. tomentella, the content was higher in flowers and roots than the content in stems and leaves, whereas another ingredient, coptisine, only distributed in flowers. there was no significant difference in chemical constituents between wild and cultivated c. tomentella, which indicated that cultivated c. tomentella could be used as a substitute for wild herbs. the chemical constituents of different harvesting stages were significant different, the highest content was found in march.
5. we proved that c. tomentella has significant protective effect on alcoholic acute liver injury and strong anti-hepatitis b virus activity. the models of alcohol-induced acute liver injury and carbon tetrachloride-induced acute liver injury in mice were used to evaluate the hepatoprotective activity of aqueous extract of c. tomentella. the result shows that the aqueous extract could significantly reduce the alt and ast activity in mice with acute alcoholic liver injury (p< 0.01, p < 0.05) at different doses (200 mg/kg, 400 mg/kg, 600 mg/kg), and the highest activity was found at low doses (p< 0.01), which was better than that of bicyclol positive control. the result of carbon tetrachloride-induced acute liver injury shows that aqueous extract of c. tomentella provided a weak inhibitory effect on alt activity in acute liver injury induced by carbon tetrachloride at low dose (200 mg/kg), but no inhibitory effect was observed at high dose.
    in this study, we performed detailed researches on a traditional chinese medicine with definite curative effect. our researches provided an effective method for identification of medicinal materials and revealed the substance basis of pharmacodynamic. moreover, which clarified the main pharmacological activities, and provided scientific instruction for formulating local and national drug standards.