【作者】 王莉；

【导师】 康文艺； 张永洲；

【作者基本信息】 河南大学 ， 中药学硕士（专业学位）， 2023， 硕士

【摘要】 斑花败酱（Patrinia punctiflora Hsu et H.J.Wang）隶属于败酱科（Valerianaceae）败酱属,是一种多年生草本植物。集中分布于河南、陕西和江苏等地,属于药食两用资源。浙江昌化民间用于消炎解凝、排脓、利尿;天台山民间用全草治阑尾炎;广西民间用根浸酒内服治跌打,叶外敷洗疮毒;江西民间用嫩叶作蔬菜食用。文献调研发现,败酱属植物常被用于治疗糖尿病的配方中。同时课题组前期已从同属植物黄花败酱中发现系列具有降糖活性的化学成分,并且对斑花败酱（Patrinia punctiflora Hsu et H.J.Wang）提取物进行了Hep G2细胞胰岛素抵抗活性筛选,发现在同等浓度下,斑花败酱提取物促进Hep G2细胞糖吸收的效果比黄花败酱（Patrinia scabiosaefolia Fisch.ex Link.）更加显著。为了深入挖掘败酱属植物的降低血糖活性成分,本课题选择该属植物——斑花败酱进行化学成分和活性筛选以及机制探究,以期对败酱属植物的深层次开发和利用提供有效依据。采用乙醇浸提法得到斑花败酱全草浸提物,通过大孔树脂（D-101 Macroporous Res in）以及（Silica gel、ODS、Sephadex LH-20）等层析柱,结合（Flash chromatography、Semipre-HPLC）等分离技术,经过各种波谱分析（1D-NMR、2D-NMR、MS、IR、UV、ORD）方法对斑花败酱的化学成分进行分离纯化及结构鉴定,并根据标准品和文献资料对比共鉴定出36个化合物结构,主要为环烯醚萜类、木脂素类、芳香醛类以及其他类型化合物,分别为:（1S,3R,5S,7S,8S,9S）-1-methoxy-7-hydroxy-8-methyl-3,8-epoxy-Δ^4,11-di hyronepetane（1）,（1S,3R,5S,7R,8S,9S）-1-methoxy-7-hydroxy-8-methyl-3,8-epoxy-Δ^4,11-dihy ronepetane（2）,patriscabioin M（3）,patriscabioin P（4）,8,9-didehydro-7-hydroxydolicho dial（5）,patriscabioin Q（6）,jatamanin N（7）,patrirscabioin R（8）jatamanin Y（9）,pa trinoside H（10）,patrinoside-aglucone（11）,stenopterin A（12）,（+）-nortrachelogenin（13）,matairesionol（14）,异落叶松脂素（15）,（7S,8R）-dihydrodehydrodiconiferyl alcoho（16）,落叶松脂醇（17）,丁香脂素（18）,（+）-medioresinol（19）,8-hydroxy-7’-epipinoresinol（20）,pinoresinol（21）,pinoresinol-4-O-β-D-glucopyranoside（22）,illiciumlignan C（23）,matairesinol 4’-O-β-D-glucopyranoside（24）,山奈酚-3,7-二-O-α-L-鼠李糖（25）,芥子醛（26）,alismoxide（27）,松柏醛（28）,丁香醛（29）,香草醛（30）,1H-indole-3-carbaldehyd e（31）,咖啡酸乙酯（32）,benzyl-β-D-glucopyranoside（33）,methyl-β-D-glucopyranoside（34）,（3S,5R,6S,7E）3,5,6-trihydroxy-7-megastigmen-9-one（35）,（-）-loliolide（36）。其中化合物4、6～10为新环烯醚萜类化合物。应用葡萄糖胺在Hep G2细胞上建立胰岛素抵抗模型,以罗格列酮为阳性对照药,从促进葡萄糖吸收角度研究斑花败酱化学成分对肝胰岛素抵抗的改善作用及分子机制,探究斑花败酱中具有改善胰岛素抵抗活性的化合物。结果显示,化合物1、3、9、13、18、26～29、31～32可不同程度地促进IR-Hep G2细胞的葡萄糖摄取;EC50值分别为185.47、41.71、32.25、60.96、36.52、62.94、93.59和56.49μM。对比MTT和GOD-PO D实验,从最佳剂量以及新化合物多种角度出发,通过Western blotting技术,检测新化合物9对Hep G2细胞内与胰岛素抵抗有关的一些关键蛋白表达的影响。结果表明新化合物9可以增加PI-3K、p-AKT、GLUT4和p-GSK3β蛋白表达水平,并抑制糖异生关键酶PEPCK和G6Pase蛋白的表达。综上结果表明,化合物9通过激活PI-3K/AKT信号通路,促进磷酸化Akt激活下游信号分子葡萄糖转运蛋白4（GLUT4）转移至质膜,同时,促进糖原合成、抑制糖异生的发生,进而改善胰岛素抵抗,从而起到改善胰岛素抵抗的作用。更多还原

【Abstract】 Patrinia punctiflora belongs to the Patrinia genus in the Valerianaceae,mainly distributed in southern Henan,southwestern Shaanxi,Jiangsu and other places,and is a traditional dual-use resource for medicine and food.In the region of Zhejiang Changhua,P.punctiflora as folk medicine is used for antiphlogistic,anticoagulant,purulent and diuretic and in Tiantai Mountain,its whole herbs is used to treat appendicitis,in Guangxi,its root dip wine is used to treat falls,and leaf external application to wash sore poison.in the region of Jiangxi,its tender leaves are used as vegetables.According to literature research,Patrinia plants are often used in the formula for the treatment of diabetes.At the same time,the research group has previously discovered a series of chemical components with hypoglycemic activity from P.scabiosaefolia.,and screened for Hep G2 cell insulin resistance activity for P.punctiflora.It was found that at the same concentration,the effect of P.punctiflora extraction on promoting glucose absorption in Hep G2cells is more significant than P.scabiosaefolia.In order to deeply explore the hypoglycemic active ingredients of Patrinia plants,this study selected P.punctiflora from this genus for chemical composition and activity screening,as well as mechanism exploration,in order to provide effective basis for the deep development and utilization of Patrinia plants.The ethanol extraction method was used to obtain the whole herb extract of P.punctiflor a.D-101 Macroporous Resin,Silica gel,ODS and Sephadex LH-20 were selected as separation materials.In combination with Flash chromatography and Semipre-HPLC separation technologies,the chemical constituents were systematically studied by means of NMR,MS,IR,UV,ORD an d other spectral analysis methods（HSQC,HMBC,¹H-¹H COSY and NOESY）.In addition,a tot al of 36 compounds were isolated and identified by comparison between standard substances and literature,mainly lignans,iridoids,aromatic aldehydes and other compounds,as follows:（1S,3R,5S,7S,8S,9S）-1-methoxy-7-hydroxy-8-methyl-3,8-epoxy-Δ^4,11-dihyronepetane（1）,（1S,3R,5S,7R,8S,9S）-1-methoxy-7-hydroxy-8-methyl-3,8-epoxy-Δ^4,11-dihyronepetane（2）,patriscabioin M（3）,patriscabioin P（4）,8,9-didehydro-7-hydroxydolichodial（5）,patriscabioin Q（6）,jatamanin N（7）,patrirscabio in R（8）jatamanin Y（9）,patrinoside H（10）,patrinoside-aglucone（11）,stenopterin A（12）,（+）-n ortrachelogenin（13）,matairesionol（14）,（+）-isolariciresinol（15）,（7S,8R）-dihydrodehydrodiconiferyl alcoho（16）,（+）-lariciresinol（17）,（+）-syringaresinol（18）,（+）-medioresinol（19）,8-hydroxy-7’-epipi noresinol（20）,pinoresinol（21）,pinoresinol-4-O-β-D-glucopyranoside（22）,illiciumlignan C（23）,matairesinol 4’-O-β-D-glucopyranoside（24）,kaempferol-3,7-di-O-α-L-rahmnoside（25）,sinapic alde hyde（26）,alismoxide（27）,coniferaldehyde（28）,syringaldehyde（29）,vanillin（30）,1H-indole-3-c arbaldehyde（31）,ethyl caffeate（32）,benzyl-β-D-glucopyranoside（33）,methyl-β-D-glucopyranosid e（34）,（3S,5R,6S,7E）3,5,6-trihydroxy-7-megastigmen-9-one（35）,（-）-loliolide（36）.Compounds 4,6～10 are new iridoids.Insulin resistance model was established on Hep G2 cells with glucosamine,rosiglitazone was used as positive control drug,and the improvement effect and molecular mechanism of the chemical components of Patrinia punctiflora on hepatic insulin resistance were studied from the perspective of promoting glucose absorption,and the compounds of Patrinia punctiflora with the activity of improving insulin resistance were explored.The results showed that compounds 1,3,9,13,18,26～29,31～32 could promote the glucose uptake of IR-Hep G2 cells to different degrees.By comparing the MTT and GOD POD experiments,the effects of new compound 9 on the expression of some key proteins related to insulin resistance in Hep G2 cells were detected by Western blotting technique from the perspective of the optimal dose and multiple perspectives of the new compound.The results showed that compound 9 increased the protein expression levels of PI-3K,p-AKT,GLUT4 and p-GSK3β,and inhibited the expression of PEPCK and G6Pase proteins,which are key gluconeogenetic enzymes.These results suggest that compound 9 can improve insulin resistance by activating the PI-3K/AKT signaling pathway,promoting phosphorylated Akt to activate the downstream signaling molecule glucose transporter 4（GLUT4）to the plasma membrane,promoting glycogen synthesis,inhibiting gluconogenesis,and thereby improving insulin resistance.更多还原