【作者】 陈勇；

【导师】 李祥；

【作者基本信息】 南京中医药大学 ， 中药学， 2013， 博士

【摘要】 番荔枝内酯类化合物(Annonaceous acetogenins, ACGs)是从番荔枝科植物中分离得到的一类有很强抗肿瘤活性的天然产物,是目前国际天然产物化学家研究的热点之一。该类化合物抗肿瘤的构效关系尚不十分清楚,无毒性-活性相关性、体内的药代动力学研究。目的：从番荔枝子中分离番荔枝内酯类化合物,研究其抗肿瘤作用的构效关系、体内药代动力学过程、毒性-活性相关性及分子作用机制。方法：1、用95%乙醇提取及液液萃取法富集番荔枝子中番荔枝内酯部位；采用柱层析、制备液相等技术对该部位进行分离纯化；采用质谱、各种色谱光谱技术对分离得到的单体化合物进行理化性质测定及结构鉴定,并运用Mosher法对分离得到的新化合物绝对构型进行测定；对洗脱得到的脂肪油部位进行甲酯化后处理后,采用GC分析与混合对照品对照,分析其主要成分及相对含量。2、对分离得到的番荔枝内酯类化合物进行结构分类,采用MTT法测试该类化合物对不同种人肿瘤细胞增殖的抑制作用,并结合化合物的结构类型分析其抗肿瘤作用的构效关系。3、采用健康鼠进行不同结构类型番荔枝内酯类化合物的毒性试验,以及采用实体瘤小鼠模型,观察不同结构类型番荔枝内酯类化合物的抗肿瘤药效,分析体内毒性-药效相关性。4、静脉给予大鼠番荔枝内酯单体三周,通过对大鼠脏器光镜及电镜检查,细胞中线粒体复合物Ⅰ活力、钙离子和ROS荧光强度、ATP含量、Bax、Bcl-2及case-3蛋白表达、线粒体DNA损伤、线粒体ND1、ND2基因及蛋白表达等指标检测来分析番荔枝内酯对大鼠的毒性及分子机制。5、采用液-液萃取法对番荔枝内酯类化合物血浆样品进行处理,并采用HPLC-MS技术分析静脉给予番荔枝内酯单体bullatacin在大鼠体内的药代动力学过程。结果：1、采用IR、UV、MS、NMR等技术鉴定了从番荔枝子中分离的30个化合物,其中22个为番荔枝内酯类化合物(12个新化合物),采用Mosher法鉴定了新化合物的立体构型。GC分析脂肪油主要成分为脂肪酸(80.9%),其中不饱和脂肪酸的含量达到51.1%。2、分离得到的番荔枝内酯类成分分为三类：单四氢呋喃型、邻双氢呋喃型和间双四氢呋喃型。MTT实验结果显示不同结构类型番荔枝内酯类化合物均有很强的肿瘤细胞抑制活性。构效关系分析表明了在单四氢呋喃型和邻双四氢呋喃型番荔枝内酯类化合物中,四氢呋喃环离末端内酯环越近活性越好；在邻双四氢呋喃型和间双四氢呋喃型番荔枝内酯类化合物中,三羟基取代的总碳数为37,分子量为622的活性最强；在邻双四氢呋喃型和间双四氢呋喃型番荔枝内酯类化合物中,4-OH取代位置与活性强弱并无一定的关系。3、三种不同结构类型的番荔枝内酯小鼠体内毒性-药效相关性研究表明邻双四氢呋喃型番荔枝内酯在小鼠体内有较好的抗肿瘤活性,但也表现出比单四氢呋喃和间双四氢呋喃型番荔枝内酯更高的毒性。4、静脉给予大鼠番荔枝内酯单体bullatacin后,300μg/kg剂量组大鼠血液生化指标ALT、AST、BUN和CR活性与空白对照组比较均上升；300μg/kg剂量组大鼠肝及肾组织光镜及电镜下检测发现组织细胞及线粒体出现病变；与空白对照组比较,各组大鼠各脏器线粒体复合物Ⅰ活性均有所下降,呈一定的剂量依赖性,肝和肾组织细胞内钙离子和ROS浓度比对照组升高；300μg/kg剂量组大鼠心、肝和肾脏器组织中的ATP含量与空白对照组相比降低；300μg/kg剂量组大鼠肝和肾脏器组织中的Bax蛋白表达、Bax/Bcl-2及case-3蛋白表达升高；各组大鼠肝和肾组织线粒体DNA提取物PCR扩增产物样本进行琼脂糖凝胶电泳后可见4.7Kb片断,均扩增出大小为0.86Kb条带,肾组织样本还扩增出另一个大小约1.5Kb的条带；与空白对照组比较,各组大鼠肝肾组织中ND1、ND2基因表达均有升高趋势,肝组织中ND1、ND2蛋白表达降低,肾组织中ND1、ND2蛋白表达升高。5、运用液-液萃取建立了Bullatacin血清样品的制备方法,HPLC-MS技术建立的血清样品含量测定方法的特异性、精密度、回收率和稳定性都达到要求,并应用于研究静脉给予番荔枝内酯在大鼠体内的药代动力学过程。结论：从番荔枝子中分离得到22个番荔枝内酯类成分,结构分为三类：单四氢呋喃型、邻双氢呋喃型和间双四氢呋喃型,其中12个为新化合物。四氢呋喃环及其相邻的取代羟基位置、取代羟基个数及羟基位置不同对其体外抗肿瘤活性有一定的影响。与其它两类番荔枝内酯类化合物相比,邻双四氢呋喃番荔枝内酯在小鼠体内有较好的抗肿瘤活性,但也表现出较高的毒性。300μg/kg剂量bullatacin连续给予大鼠三周对大鼠表现出一定的毒性,与空白对照组比较,bullatacin可引起大鼠血液生化指标上升、肝和肾脏器病理学改变,作用分子机制为其可引大鼠组织细胞中线粒体复合物Ⅰ活力降低及ATP含量下降、钙离子和ROS浓度上升、Bax蛋白表达、Bax/Bcl-2及case-3蛋白表达升高有关,其还可引起大鼠肝肾组织线粒体DNA损伤,对线粒体ND1及ND2基因和蛋白表达有一定的影响。液-液萃取和HPLC-MS技术可用于Bullatacin血清样品的制备及其在大鼠体内的药代动力学研究血样样品的含量测定。更多还原

【Abstract】 ACGs (Annonaceous acetogenins) isolated from the plants in Annonaceae have strong anti-tumor activity and are the hot interest of the international natural products chemists. However, The antitumor structure-activity relationship of these compounds is obscure, and the in vivo pharmacokinetics and toxicity-activity relationship study have not yet been reported.Objective:To isolate ACGs from Annona squamosa seeds and to investigate the structure-activity relationship of their anti-tumor effects, the in vivo pharmacokinetics, toxicity-activity relationship, and the molecular mechanism of action.Methods:1. Enrich the ACGs parts with95%ethanol extraction and liquid-liquid extraction. This part was isolated and purified by column chromatography and preparative liquid technologies. Using mass spectrometry, a variety of chromatographic spectroscopy was performed to determinate the physical and chemical properties and structures of the isolated compounds. The absolute configuration of isolated new compounds was also determined by the Mosher method. After methyl esterification of fatty oils part, the GC was used to analyze the composition and content with mixed reference substance.2. The isolated ACGs were structurally classified. MTT assay was used to test the inhibitory effect of these compounds against different kinds of human tumor cells. The structure-activity relationship of the anti-tumor effects based on the structures of the compound type was analyzed.3.Healthy mice and solid tumor mouse model were used to observe the toxicity and anti-tumor activity of different structure types of ACGs, respectively, for analysis of the toxicity-activity relationship in vivo.4. Rats were given bullatacin three weeks by tail vein intravenous. The heart, liver and kidney tissues of rat were observed using light microscopy and electron microscopy to analyze the toxicity of bullatacin in rats. The mitochondrial complex I activity, calcium and ROS fluorescence intensity, ATP content, Bax, Bcl-2and case-3expression, mitochondrial DNA damage, mitochondrial ND1and ND2gene and protein expression were detected to analyze the molecular mechanisms of toxic effects of bullatacin in rats.5. Process the plasma samples of bullatacin by liquid-liquid extraction, and analyze the pharmacokinetics of bullatacin in rats by tail vein intravenous using HPLC-MS.Results:1. Thirty compounds were isolated from seeds of Annona squamosa. The structures of22ACGs were identified using IR, UV, MS and NMR. Twelve were new compounds and their absolute configuration was determined using Mosher method. The main components of the fatty oil were fatty acids (80.9%), of which the contents of unsaturated fatty acids reached51.1%.2. Isolated ACGs were structurally divided into three types:mono-THF (tetrahydrofuran), adjacent bis-THF and nonadjacent bis-THF ACGs. MTT assay results showed that different structure types of ACGs have a strong inhibitory activity against tumor cell growth. Structure-activity relationship analysis showed that among mono-THF and adjacent bis-THF ACGs, compounds with less carbon numbers between THF ring and lactone ring exhibited better activity. ACGs bearing three substituted hydroxyl (total carbon number is37, molecular weight is622) exhibited strongest activity among adjacent bis-THF and nonadjacent bis-THF ACGs. There is no definite relationship between the position of substituted4-OH and the activity of adjacent bis-THF and nonadjacent bis-THF ACGs.3. Toxicity-activity relationship of three types of ACGs in vivo showed that adjacent bis-THF ACG had higher anti-tumor activity and toxicity than mono-THF and nonadjacent bis-THF ACGs.4. Rats were treated with bullatacin by tail vein intravenous. Results showed that ALT, AST, BUN and CR activity of300μg/kg dose group was increased compared with control group. Liver and kidney tissues of300μg/kg dose group presented lesions through light microscope and electron microscope detection. Compared with control group, liver mitochondrial complex I activity of all bullatacin treated groups were decreased in a dose-dependent manner. Besides, calcium, ROS concentrations of heart, liver and kidney tissues and Bax and case-3expression and Bax/Bcl-2ratio in liver and kidney tissues were increased. ATP contents of heart, liver and kidney tissue of300μg/kg dose treated rat were decreased compared with control group. Mitochondrial DNA PCR amplification of liver and kidney tissues was subjected to agarose gel electrophoresis and amplified a4.7Kb and0.86Kb stripe, another size of approximately1.5Kb bands amplified in rat kidney tissue samples of bullatacin treated group. Compared with control group, ND1and ND2gene expression in rat liver and kidney in bullatacin treated group were increased. ND1and ND2protein expression in liver tissue was decreased, and ND1, ND2protein expression in renal tissue increased.5. Liquid-liquid extraction and determination method by HPLC-MS technique of serum sample with bullatacin treatment were established. The stability, extraction recovery and matrix effect involved in the method were also validated. This method was applied to measure the plasma bullatacin concentrations after a single tail vein intravenous.Conclusions:22ACGs (12new ACGs) including mono-tetrahydrofuran, adjacent bis-and nonadjacent bis-tetrahydrofuran ACGs were isolated from Annona squamosa seeds. The position of tetrahydrofuran ring and its adjacent substituted hydroxy, the number and position of substituted hydroxyl have a certain impact of anti-tumor activity of ACGs in vitro. Compared with mono-THF and nonadjacent bis-THF ACGs, adjacent bis-tetrahydrofuran ACG has better anti-tumor activity in mice, but exhibit higher toxicity.300μg/kg dose of bullatacin for three weeks in rats showed some toxicity effects. Compared with control group, bullatcin caused blood biochemical parameters increased and liver and kidney tissues pathological changes. Bullatacin led to decrease in mitochondrial complex I activity and ATP content, and increase in calcium, ROS concentration, Bax and case-3expression and Bax/Bcl-2ratio, and mitochondrial DNA of rat liver and kidney damage with a certain impact of mitochondrial ND1and ND2gene protein expression in liver and kidney tissues of rats. Liquid-liquid extraction and HPLC-MS techniques can be used to prepare serum samples with bullatacin and determine its blood samples content in rats.更多还原