【作者】 侯勇；

【导师】 董俊兴；

【作者基本信息】 安徽医科大学 ， 药学， 2019， 硕士

【摘要】 杨树花为杨属植物毛白杨（Populus tomentosa Carr.）、加拿大杨（Populus canadensis Moench.）或同属数种植物的雄花序,中国大部分地区均有分布。其作为我国一种传统中草药,被收录于2015版《中国兽药典》,一直作为治疗家禽痢疾等疾病的首选用药。国内外研究显示,其具有显著的抗菌、抗炎、抗氧化、抗病毒等药理活性,主要成分包括黄酮类和酚苷类化合物。为进一步丰富传统中草药杨树花的研究资料,本课题选取毛白杨的雄花序作为研究对象,对其所含化学成分进行系统的研究。同时对所得部分化合物进行了体外细胞毒、抗氧化及抗炎活性评价,发现两种化合物抗炎效果显著,为抗炎症疾病药物的研发提供候选。将毛白杨雄花序30kg以70%乙醇提取,萃取得石油醚部位约350g,乙酸乙酯部位约500g,正丁醇部位约400g。分别对乙酸乙酯及正丁醇部位进行系统分离纯化。综合利用硅胶柱色谱、Sephadex LH-20凝胶柱色谱、重结晶、制备型HPLC等技术方法,得到48个化合物。结合化合物的理化性质并利用HR-ESI-MS、¹H NMR、¹³C NMR、HSQC、HMBC等波谱技术鉴定了其中42个化合物的结构,分别为:金圣草黄素（1）,芹菜素（2）,木犀草素（3）,五桠果素（4）,山柰酚（5）,槲皮素（6）,杨梅素（7）,鼠李秦素（8）,乔松素（9）,柚皮素（10）,异野樱素（11）,圣草酚（12）,花旗松素（13）,二氢杨梅素（14）,芹菜素-7-O-β-D-葡萄糖苷（15）,木犀草素-7-O-β-D-葡萄糖苷（16）,香叶木素-7-O-β-D-葡萄糖苷（17）,山柰甲黄素-7-O-β-D-葡萄糖苷（18）,5-羟基二氢黄酮-7-O-β-D-葡萄糖苷（19）,山柰酚-3-O-β-D-葡萄糖（1→2）-[α-L-鼠李糖（1→4）]-β-D-葡萄糖苷（20）,芹菜素-7-O-β-D-（-6″-p-香豆酰基）-葡萄糖苷（21）、紫丁香苷（22）,水杨苷（23）,对羟基苯丙烯酸葡萄糖酯（24）,水杨苷-2′-苯甲酸酯（25）,苯甲醇-β-D-葡萄糖苷（26）,特里杨苷（27）,柳匍匐苷（28）,2-羟基环己基-6’-O-对香豆素-β-D-吡喃葡萄糖苷（29）,4′-羟基-1′-O-（β-D-葡萄糖基）-苯基-2-羟基苯甲酸酯（30）,熊果酸（31）,桦木酮酸（32）,胡萝卜苷（33）,1,3,6-三羟基-2-甲基蒽醌-3-O-α-鼠李糖-（1→2）-β-D-（6′-O-乙酰基）-葡萄糖苷（34）,1,3,6-三羟基-2-甲基蒽醌-3-O-α-鼠李糖-（1→2）-β-D-葡萄糖苷（35）,1,3-二羟基蒽醌（36）,邻苯二酚（37）,3,3′,4,4′-四羟基联苯（38）,香豆酸（39）,原儿茶酸（40）,咖啡酸（41）,1,3-二咖啡酰基-2-乙酰基甘油（42）。其中,化合物30为新化合物,化合物1,4,7,13¹5,17¹9,26,30³2,38为首次从杨属植物中分离得到,化合物1,4,7,8,10¹5,18,19,22,26,29³2,37,38,40,42为首次从该植物中分离得到。体外细胞毒活性评价结果显示黄酮类化合物对细胞生长抑制作用较大,其中以化合物3,6,9,11作用最为显著,其对各种细胞株IC₅₀值均小于15μg/mL。采用ABTS法和FRAP法综合评价部分化合物体外抗氧化活性,结果显示化合物6,7,14具有显著的体外抗氧化活性,化合物3,12,13,16,41,42活性较强,化合物1,2,4,5,17,28,30活性较弱。对部分化合物以LPS诱导小鼠巨噬细胞RAW 264.7细胞株NO释放量抑制程度筛选抗炎活性化合物,结果显示化合物27,42具有显著的NO释放抑制作用。细胞活力检测结果初步限定化合物27与42的中毒剂量。ELISA法测定结果显示二者均对TNF-α,IL-6,IL-1β三种因子释放具有明显抑制作用,且呈剂量依赖性。综上这些结果初步显示了化合物27,42具有明显的炎症调节作用,对其下一步活性机制的研究具有重要意义。更多还原

【Abstract】 The male inflorescence of Populus tomentosa Carr.,Populus canadensis Moench.or other plants of Populus are all referred as Flos populi.It is widely distributed in most parts of China.It is regarded as the first drug for treating diarrhea in poultry and included in Chinese Veterinary Pharmacopoeia（2015 Edition）.Previous research had shown that the main chemical constituents of Flos populi were flavonoids and phenolics.And it had been indicated possessing anti-bacterial,antioxidant,anti-inflammatory,antiviral and some other pharmacological activities notably.In order to enrich the research materials about it,we investigated the chemical constituents of the male inflorescence of Populus tomentosa Carr.systemically.Meanwhile,we evaluated the anti-tumor,antioxidant and anti-inflammatory activities of some compounds and found two known compounds might be potential candidates for the treatment of inflammatory diseases in this study.Dried male inflorescence of Populus tomentosa Carr.（30 kg）were extracted with 70%EtOH and partitioned obtain petroleum ether extract（350 g）,EtOAc extract（500 g）and n-butanol extract（400 g）.The EtOAc and n-butanol extract were separated and purified systematically.Using chromatographic methods such as silica gel,Sephadex LH-20,preparative HPLC and recrystallization repeatedly,forty-eight compounds were isolated and forty-two were identified based on the physiochemical properties and spectroscopic data.They were identified as chrysoeriol（1）,apigenin（2）,luteolin（3）,dillenetin（4）,kaempferol（5）,quercetin（6）,myricetin（7）,rhamnazin（8）,pinocembrin（9）,naringenin（10）,isosakuranetin（11）,eriodictyol（12）,taxifolin（13）,dihydromyricetin（14）,apigenin-7-O-β-D-glucopyranoside（15）,luteolin-7-O-β-D-glucopyranoside（19）,diosmetin-7-O-β-D-glucopyranoside（17）,kaempferide-7-O-β-D-glucopyranoside（18）,5-hydroxy-flavanone-7-O-β-D-glucopy-ranoside（19）,kaempferol-3-O-{[2-O-α-L-rhamnopy-ranosyl-4-O-β-D-glucopyranosyl]-β-D-glucopyranoside}（20）,apigenin-7-O-（-6″-O-p-coumaroyl）-β-D-glucopyranoside（21）,syringin（22）,salicin（23）,p-coumaric acid glucoside（24）,tremuloidine（25）,benzyl alcohol-O-β-D-glucopyranoside（26）,tremulacin（27）,salireposide（28）,2-hydroxycyclohexyl-6′-O-p-coumaroyl-β-D-glucopyranoside（29）,4′-hydroxy-1′-O-（β-D-glucoside）-benzyl-2-hydroxybenzoate（30）,ursolic acid（31）,betulonic acid（32）,daucosterol（33）,2-methyl-1,3,6-trihydroxy-9,10-anthraquinone-3-O-（6′-O-acetyl）-α-rhamnosyl（1→2）-β-D-glucoside（34）,2-methyl-1,3,6-trihydroxy-9,10-anthraquinone-3-O-α-rhamnosyl（1→2）-β-D-glucoside（35）,xanthopurpurin（36）,catechol（37）,3,3′,4,4′-tetrahydroxybiphenyl（38）,coumaric acid（39）,protocatechuic acid（40）,caffeic acid（41）,2-acetyl-1,3-dicaffeoylglycerol（42）.Compound 30 is a new compound.Compound 1,4,7,13-15,17-19,26,30-32,38 were isolated from the genus Populus for the first time.Compound1,4,7,8,10-15,18,19,22,26,29-32,37,38,40,42 were isolated from the plant for the first time.The cytotoxic effects in vitro of some compounds were evaluated and the flavonoid compounds had the most significant activity.Compound 3,6,9,11 exhibited significant cytotoxicities against most cell lines with IC₅₀ values of less than 15μg/mL.The antioxidant properties in vitro of some compounds were tested by the ABTS and FRAP assays.From the results,compounds 6,7 and 14 exhibited remarkable antioxidant activities.Compounds 3,12,13,16,41 and 42 exhibited obvious antioxidant activities.By contrast,compounds 1,2,4,5,17,28 and 30 were found to possess moderate activities.Moreover,we assessed the anti-inflammatory effects of other some compounds in terms of their abilities to inhibit the production of NO by LPS-stimulated the murine macrophage cell line RAW 264.7 with Griess method and found compounds 27 and 42could decrease NO level markedly.The cell viability assays had ensured the toxic doses of them.Additionally,treatment with compounds 27 and 42 attenuated the release of TNF-α,IL-6 and IL-1βin LPS-stimulated RAW 264.7 cells respectively in a concentration-dependent manner.The findings suggested that they had anti-inflammatory activities evidently and contributed to the potential underlying mechanisms of them further.更多还原