【作者】 许睿；

【导师】 高幼衡；

【作者基本信息】 广州中医药大学 ， 中药学， 2009， 博士

【摘要】 目的:冬青科冬青属植物救必应（Ilex rotunda Thunb.）始载于《岭南采药录》,现收载于《广东省中药材标准》,为岭南地区常用中草药,民间使用追溯久远。药材性苦、凉,有清热解毒、凉血止血、行气止痛等功效,用于外感表证,癍痧发热,咽喉肿痛,乳娥,腕腹胀痛,腹泻,痢疾,急性胃肠炎,风湿骨痛;外用治跌打损伤,痈疖疮疡,外伤出血,烧烫伤等。现已运用该药材开发出多种中成药,市场销路很好,占有率高,创造了很好的经济效益,该药材原植物在广东各地区分布广泛,来源十分丰富,极具开发应用价值。《中华人民共和国药典》（1977年版一部）曾收载该药材,但因其化学成分研究部分相对空白,有效成分不明确并且缺少薄层鉴别和含量测定等项,无法对该药材的质量进行有效全面的控制,因而在《中国药典》后续版本中未将其收录在内。现阶段该药材的制备工艺主要以水提醇沉,提取物干燥、粉碎直接入药的方法,由于该药材的主要有效成分不清楚,使得对于以该药材为主要组方的中成药质量难以得到很好的控制,鉴于该药材在临床上其明确的药理功效,为了更好的控制该药材的质量,掌握该药材中所含的化学成分组成,提高该药材的质量标准,在此基础上对药材进行二次开发有着十分重要的意义。其次,通过对该药材的化学成分研究,寻找其主要有效成分或以其主要有效成分结构母核为基础的药物先导化合物,并在此前提下再开发出以其主要成分为组方、功效更明显、服用更方便的新成方制剂或新剂型奠定基础。因此,以救必应药材相关的化学成分及药理学研究有其重要的研究意义。同时为该药材再次收录《中国药典》提供充分的实验依据。方法:化学成分研究,取救必应药材粗粉按95%热乙醇提取,减压回收溶剂,得提取物干膏,取提取物利用改良的索氏回流提取器（Soxhlet’s apparatus）将所得拌样流浸膏,按溶剂极性分为石油醚、氯仿、乙酸乙酯、丙酮、甲醇五个部位。所得不同极性部位,利用硅胶、Sephadex LH-20、反向ODS硅胶等分离技术进行分离纯化,得到单体化合物。综合运用紫外光谱（UV）、红外光谱（IR）、质谱（MS）及核磁共振光谱(¹H-NMR、¹³C-NMR、DEPT、¹H-¹H-COSY、HSQC、HMBC)等现代光谱和波谱学技术鉴定所得单体化合物的化学结构。体外单体抗肿瘤活性研究,运用MTT法分别取对数生长期的细胞株,胰酶消化,计数,将细胞悬液分别稀释成每毫升5×10⁵个细胞、10×10⁵个细胞,加入96孔板中,根据不同细胞株特性每孔加入3000～8000个细胞,200μl每孔。实验组加入分离得到的单体化合物,对照组加入浓度为0.1%的DMSO,每组设4个平行孔,置于5%CO₂、37℃培养箱中培养72h,中止前4h加入5g·L^-1MTT液10μl,继续培养4h,弃去培养液加入0.1ml DMSO,振荡30min,待结晶溶解并完全显色后在酶联检测仪上检测570nm波长下每孔的OD值,按公式:生长抑制率（%）=（1-用药组平均OD值/对照组平均OD值）×100%,求出生长抑制率。结果用BLISS法软件程序处理,得到半数抑制率IC₅₀。结果:从救必应中共分离得到22个化合物,通过理化性质和光谱解析鉴定了其中20个化合物的结构。其中,三萜及其苷类化合物8个,分别为齐墩果酸（Comp.19）,铁冬青酸（Comp.10）,19α,24-dihydroxyurs-12-en-3-one-28-oic acid（Comp.17）,3β,19α-dihydroxyurs-12-en-24,28-dioic acid（Comp.11）,Peduncloside（Comp.12）,木栓酮（Comp.1）,3-羟基齐墩果烷（Comp.2）,28-羟基-木栓酮（Comp.3）;二萜类2个,分别为阿贝苦酮（Comp.4）,sugereoside（Comp.6）;酚性化合物4个,分别为二丁香苷醚（Comp.5,disyringin ether）,紫丁香苷（Comp.8）,芥子醛葡萄糖苷（Comp.9）,救必应醇（Comp.16,rotundaol）;甾体类化合物2个,分别为β-谷甾醇（Comp.21）;β-胡萝卜苷（Comp.22）;其他类化合物4个,分别为硬脂酸（Comp.20）、十九烷酸（Comp.18）、葡萄糖（Comp.7）、肌醇（Comp.13）。其中新化合物两个,分别为Comp.5和Comp.16,命名为二丁香苷醚（disyringin ether）和救必应醇（rotundaol）。选取从救必应中分离得到的12个单体化合物,11种受试肿瘤细胞株:CNE1人鼻咽癌细胞株;CNE2人鼻咽癌细胞株;MDA-MB-435人乳腺癌细胞株;SW620人结肠癌细胞株;LoVo人结肠癌细胞株;Bel-7402人肺癌细胞株;A549人肺癌细胞株;HeLa人宫颈癌细胞株;Hep3B人肝癌细胞株;HepG2人肝癌细胞株;Glc-82人肺癌细胞株。结果显示三萜类化合物rotundic acid对CNE1,CNE2,HeLa,SW620,Hep3B,A549,MDA-MB-435肿瘤细胞株有体外抑制活性,半数抑制浓度(IC₅₀)分别为16.48μmol·L^-1,18.59μmol·L^-1,15.70μmol·L^-1,18.24μmol·L^-1,29.80μmol·L^-1,70.20μmol·L^-1,11.21μmol·L^-1;3β,19α-dihydroxyurs-12-en-24,28-dioic acid对SW620,Hep3B,LoVo,MDA-MB-435肿瘤细胞株有体外抑制活性,半数抑制浓度(IC₅₀)分别为20.07μmol·L^-1,7.37μmol·L^-1,32.22μmol·L^-1,27.79μmol·L^-1。结论:从救必应中共分离得到22个化合物,通过理化性质和光谱解析鉴定了其中20个化合物的结构。其中三萜及其苷类化合物8个,四环二萜类化合物2个,酚苷类化合物4个,甾体类化合物2个,脂肪酸类化合物2个,其他类2个。二丁香苷醚（disyringin ether）和救必应醇（rotundaol）为新化合物,木栓烷类型三萜类化合物为首次从该属植物中分到,二萜类化合物为首次从该种分到。对从救必应中分离得到的单体化合物进行体外抗肿瘤活性筛选,发现其中三萜类化合物rotundic acid和3β,19α-dihydroxyurs-12-en-24,28-dioic acid分别对多种人肿瘤细胞有明显的体外抑制作用,这两个三萜类化合物的体外抗肿瘤活性为首次报道。更多还原

【Abstract】 Objective:Ilex rotunda Thunb.（Aquifoliaceae） was early recorded in "Lin Nan Cai Yao Lu". Now,This medical plant is recorded in "Guangdong Chinese Medicine criterion",used as traditional Chinese medicine in the southern region of the People’s Republic of China, also named "Jiu-bi-ying".The property and flavor of Ilex rotunda is cold and bitter.Its efficiency include heat-clearing and detoxicating,cooling blood,stop bleeding, promoting qi circulation to relieve pain,et al.A literature search revealed that the clinical internal used for treatment of cold fever,sunstroke,tonsillitis,swell throat, abdominal distension,diarrhea,dysentery,acute gastroenteritis and rheumatism,at the same time,external used for treatment of fracture,pyocutaneous disease,bleeding wound,scald and burn.Now,a lot of patent medicine in china use this plant as the main ingredient.The patent medicine including "Jiubiying" have good perspective marketing. The original plant of "Jiubiying" is distributed widespread in Guangdong province, China.Because of these reasons,the intensive research and development of this plant is value and meaningful.Pharmacopoeia of the People’s Republic of China,edition 1977 （Pharmacopoeia of P.R.C） was recorded this plant as Chinese medicine,but the Pharmacopoeia of P.R.C hadnot been recorded this plant again in the following edition. The main reason is this plant was lacked of TLC identification and assaying,and the chemical composition or active component were unclear.Therefore,for fully quality control and develop a new patent medicine,the systematically phytochemical investigation and pharmacological research are very necessary and significant.Methods:Phytochemical research:The dried root bark of the plant（28.8kg） was extracted with hot 95%ethanol and concentrated under reduced pressure to give a deep brown syrup（12.3kg）.The syrup（3.0kg） was partitioned successively as five parts by Soxhlet’s apparatus,these parts were concentrated under reduced pressure to give brown syrup respectively.Petroleum ether part（15g）,chloroform part（92g）,ethyl acetate part （910g）,acetone part（950g） and methanol part（157g）,which was directly treated by silica gel column chromatography and rechromatographed over Sephadex LH20 or separated on ODS.The structures of these compounds were elucidated on the basis of spectroscopic analysis,including UV、IR、MS、¹H-NMR、¹³C-NMR、DEPT、¹H-¹H-COSY、HSQC and HMBC,et al.Antitumor research:Some bioactivce screening of the part chemical constituents isolated from Ilex rotunda had been done.In vitro experiments,MTT assay was used to determine the cell growth inhibitory rate of the several kind of cancer cell line before and after the compouds treatment,which isolated from the Ilex rotunda.The results were processed by BLISS computer programme to obtain the IC₅₀ of each cell line in every compounds.Results:Studied on the chemical constituents of Ilex rotunda:22 compounds had been isolated from the bark of Ilex rotunda.On the basis of their physiochemical and spectroscopic evidence,their chemical structures were elucidated as follows.Eight triterpenoids:friedelin（Comp.1）,3β-OH-oleanane（Comp.2）,28-OH-friedelin（Comp.3）, rotundic acid（Comp.10）,3β,19α-dihydroxyurs-12-en-24,28-dioic acid（Comp.11）, peduncloside（Comp.12）,19α,24-dihydroxyurs-12-en-3-one-28-oic acid（Comp.17）, oleanolic acid（Comp.19）.Four compounds based on phenolic glycoside:disyringin ether（Comp.5）,rotundaol（Comp.16）,syringin（Comp.8）,sinapaldehyde glucoside（Comp.9）.Two compounds based on diterpene:abbeokutone（Comp.4）, sugereoside（Comp.6）.Two steroides:β-sitosterol（Comp.21）,β-daucosterol（Comp.22）. Four other compounds:glucose（Comp.7）,inositol（Comp.13）,nonadecylic acid（Comp.18）,stearic acid（Comp.20）.Among these compounds,two are new compounds:Both are phenolic glycoside,Comp.5 and Comp.16,named disyringin ether and rotundaol.Studied on the antitumor activities screening of 12 compouds isolated from Ilex rotunda on 11 kinds of tumor cells line in vitro:two triterpenoid compouds had showed the positive results.There are rotundic acid（Comp.10）against the CNE1,CNE2,HeLa, SW620,Hep3B,A549,MDA-MB-435 tumor cells line and 3β,19α-dihydroxyurs-12-en-24,28-dioic acid（Comp.11）against SW620,Hep3B,LoVo, MDA-MB-435,Bel-7402 tumor cells line.Conclusion:Advanced research on the chemical constituents and the vitro antitumor activities of Ilex rotunda.22 compouds were isolated from the bark of Ilex rotunda.The structures of 8 triterpenoids,4 phenolic glycosides,2 diterpene compouds,2 sterides and 4 other compounds were established on the basis of physicochemical evidence and spectral characteristics.Both the friedelane-type triterpenoid discovered from genus Ilex and the two diterpenes isolated from Ilex rotunda were the first time.The screening of antitumor effect of 12 compouds on 11 tumor cell lines shows two triterpenoids,rotundic acid and 3β,19α-dihydroxyurs-12-en-24,28-dioic acid, have significant antitumor activities.The antitumor effect of two triterpenoids are first reported in this dissertation更多还原