椿皮中二氯甲烷提取部位化学成分及其抗肿瘤活性以及分散片制备工艺与质量标准的研究

发布时间：2018-09-10 21:09

【摘要】：第一部分椿皮中二氯甲烷提取部位化学成分的研究臭椿(Ailanthus altissima Swingle)又名樗木,为苦木科(Simaroubaceae)臭椿属落叶乔木,因叶上油腺破裂后有奇臭而得名,为中国本土树种,在我国分布广泛,除黑龙江、吉林和海南省外,全国各地均有分布。于18世纪作为观赏植物传入北美和欧洲,到目前为止,臭椿已经在欧洲广泛分布,同时分布在美国各地,现在已成为美国七个主要树种之一。椿皮为臭椿的干燥树皮或根皮,为我国民间常用中草药,具有清热、燥湿、止血、消炎、杀虫等功效,可用于治疗子宫出血、赤白带下、久泻久痢、湿热泻痢、便血及崩漏疾病。近年来研究表明,椿皮的提取物中因具有抗阿米巴原虫活性,故可作为除虫剂使用,同时经研究证实,它还具有明显的抗癌作用,可用于治疗结肠癌、宫颈癌、直肠癌等癌症,还具有抗非洲淋巴细胞瘤病毒和抗结核菌的作用。椿皮主要化学成分有苦木苦味素、挥发性成分、萜类化合物、生物碱类等,其中最引人注目的化学成分就是苦木苦味素,而苦木苦味素类化合物多为四环二萜内酯及五环二萜内酯,是苦木科植物的特征性成分。臭椿属是苦木苦味素类化学成分比较集中的属级单位,而臭椿是臭椿属中研究比较多的一种药用植物,本研究对臭椿皮的化学成分中二氯甲烷提取部位进行了研究。目的：以椿皮为研究对象,利用各种分离手段对其化学成分尤其是苦木苦味素类化学成分进行系统研究,并采用波谱学及光谱学方法,包括：13C-NMR,1H-NMR, HMQC,COSY, HMBC, NOESY等方法对分离得到的单体化合物进行平面结构和立体结构的鉴定,为发现新的药物先导性化合物奠定基础。方法：取干燥的椿皮50 Kg,以95%乙醇热回流提取,浓缩后得到总提物。将总提物分散在饱和氯化钠溶液中,分别以石油醚、二氯甲烷、乙酸乙酯和正丁醇萃取,分别浓缩,得到各提取部位,其中二氯甲烷萃取部位260 g。采用硅胶层析、葡聚糖凝胶、制备薄层和制备高效液相色谱法对上述二氯甲烷萃取部位的化学成分进行分离,得到的单体化合物采用现代化的分析方法如13C-NMR,'H-NMR, HMQC, COSY, HMBC和NOESY鉴定其化学结果。结果：从椿皮中分离并鉴定了9个化合物,鉴定结果如下：vanillin (1), stigmast-4-en-3-one (2), isoailanthone (3), shinjudilactone (4), ailanthone (5), shinjulactone B (6),6a-tigloyloxychaparrin(7),2-dihydroailanthone (8), 12-dihydroisoailanthone (9).第二部分椿皮中二氯甲烷提取部位及化合物抗肿瘤活性的研究椿皮始载于我国第一部药典《唐本草》中,为我国传统中药材,古时所用椿皮系指香椿和臭椿的干皮或根皮,其中以臭椿皮为好。我国历代都有对椿皮药用的记载：《食疗本草》载其有治痢疾、杀蛔虫之效；《活性论》中记载臭椿皮能治疗赤白痢、肠滑、痔疾泻血不止；《妇人良方》中记载用,以臭椿根与汉椒焙烧,加连根葱同煎后熏洗,可治疗妇女产后肠脱不收,与蜂蜜水煎服可治疗痔疮,单用椿皮的水煎液可治疗疮癣等。现代药理学研究发现,椿皮除了具有上述传统功效外,还兼有抗肿瘤、抗结核菌、抗EB病毒、抗阿米巴、抗疟等作用,尤以抗肿瘤作用明显,其所含的化学成分苦木苦味素有治疗结肠癌、宫颈癌、直肠癌等作用,是目前药理学研究的热点之一。本研究将椿皮的抗肿瘤活性研究作为分离、纯化和精制椿皮中各个化合物的重要依据,以“重在应用,面向开发”为本研究的指导原则。目的：以臭椿皮为研究对象,利用现代化的肿瘤细胞活性筛选技术手段,对椿皮中的各个提取部位以及各分离、纯化和制备的化合物进行系统的抗肿瘤活性筛选,以各个组分的肿瘤细胞增值抑制率为指标进行活性分析。分别以ailanthone和2-dihydroailanthone为代表化合物进行抗肿瘤作用机制的研究。方法：1以MTT法检测不同提取部位及化合物的抗肿瘤活性。试验设空白对照组、DMSO溶剂对照组及待测药物组(提取部位的浓度分别为1μg/mL,10 μg/mL和100 μg/mL;化合物的浓度分别为0.01 μg/mL,0.1μg/mL,1 μg/mL,10 μg/mL和100μg/mL)。2 Ailanthone和2-dihydroailanthone对MCF-7人乳腺癌细胞和U251人神经胶质瘤细胞抗肿瘤作用机制的研究。以浓度不同浓度的ailanthone和2-dihydroailanthone分别对两种肿瘤细胞进行增殖抑制率检测、细胞凋亡检测、细胞周期检测以及RT-PCR检测Bax和Bcl-2 mRNAd的表达和Western blot法检测Bax及Bcl-2蛋白表达的研究。结果：1测定了不同提取部位及化合物抗肿瘤活性的筛选,实验结果显示,对于13个肿瘤细胞株而言,不用的提取部位及化合物具有不同的抗肿瘤活性,而化合物2和4在高、中、低三个浓度的抗肿瘤活性均比较低。对化合物1、3、5、和6的IC50值进行了测定。实验结果显示,以上4个化合物对不同的肿瘤细胞的IC50值是不相同的,在13种肿瘤细胞上进行的IC50值测定数据显示,化合物5、6对13个肿瘤细胞的IC50值小于阳性药顺铂(cisplatin),表明它们对13个肿瘤细胞的抑制作用优于阳性对照药顺铂；化合物3对U251、Hela、AsPC-1、HCT116、AGS、SMMC-7721、 U-2OS、CCRF-CEM细胞的IC50值均小于阳性对照药顺铂,表明化合物3对这9个肿瘤细胞的抑制作用优于阳性对照药顺铂。化合物1对U251、Hela、HCT116、AGS、SMMC-7721、U-2OS、CCRF-CEM细胞的IC50值均小于阳性对照药顺铂,表明化合物1对这7个肿瘤细胞的抑制作用优于阳性药顺铂。结果评定：合成化合物或植物提取纯品的IC5010 μg/mL或植物提取物的IC5020 μg/mL时,则判断样品在体外对肿瘤细胞有杀灭作用。2不同浓度的ailanthone作用MCF-7人乳腺癌细胞24 h、48 h以及72 h后,MCF-7人乳腺癌细胞的存活率分别逐渐降低,表明ailanthone对人乳腺癌MCF-7细胞的生长有明显的抑制作用,而且呈现时间和浓度的双重依赖性。而不同浓度的2-dihydroailanthone对U251人神经胶质瘤细胞的抑制作用也同样很明显。流式细胞仪检测不同浓度的ailanthone和2-dihydroailanthone分别作用于MCF-7人乳腺癌细胞和U251入神经胶质瘤细胞48 h后的凋亡实验结果表明,凋亡率明显高于空白对照组,且随药物浓度增高细胞凋亡率明显增高,差异有统计学意义(P0.05)。而不同浓度的药物作用于肿瘤细胞48h后的细胞周期实验结果表明,G0/G1期的细胞百分比逐渐增加,而S期和G2/M期的细胞百分比逐渐降低,表明细胞阻滞发生在G0/G1期。通过RT-PCR检测发现,不同浓度的ailanthone和2-dihydroailanthone分别作用于人乳腺癌MCF-7细胞和U251人神经胶质瘤细胞48 h后,Bax/gapdh的百分比逐渐升高,而Bcl-2/gapdh的百分比逐渐降低,实验结果表明,Bax mRNA的表达随药物浓度的增加而上升,Bcl-2 mRNA的表达随药物浓度的增加而降低,呈现一定的剂量依赖性,说明这两种药物能增加Bax mRNA的表达,并抑制Bcl-2 mRNA的表达,且具有一定的量效关系。Western blot法检测Bax及Bcl-2蛋白表达结果。不同浓度的ailanth one和-dihydroailanthone分别作用人乳腺癌MCF-7和U251人神经胶质瘤细胞48 h后,检测Bax. Bcl-2蛋白表达,bax/β-actin的百分比逐渐升高,而bcl-2/β-actin百分比则逐渐降低,实验结果表明,与对照组比较,不同剂量的药物可使Bcl-2表达降低,而Bax表达增加,且呈现一定的剂量依赖性关系。第三部分椿皮中二氯甲烷提取部位分散片制备工艺与质量标准的研究分散片具有稳定性好、便于携带、服用方便、崩解和溶出速度快、生物利用度较高等优点,可直接口服或投入水中分散后服用,尤其适于吞服困难的患者,并且口感良好,提高了患者的依从性,日益受到广大患者的青睐。目的：以椿皮中二氯甲烷提取部位所提取、分离的有效部位为研究对象,利用现代化的中药药剂学技术手段和方法,使其具有现代剂型的各种特征,对其进行药剂学研究。在具体的研究中制备快速释放和稳定性好的臭椿苦酮提取物分散片,着重研究制备过程中的影响因素和最优工艺,并对分散片进行质量评价。此研究的意义在于通过药剂学研究,将难溶于水溶液的臭椿苦酮提取物,制备成为分散片,从而为椿皮在临床中的广泛应用打下基础。方法：采用紫外分光光谱法建立臭椿苦酮提取物体外分析方法,以测定其含量,为臭椿苦酮提取物分散片的处方筛选、工艺研究、质量研究及体外分析奠定基础。利用单因素考查和正交设计法,以12小时累计溶出度为指标,对臭椿苦酮提取物分散片的处方进行研究,筛选最佳制备方法。结果：根据处方工艺研究结果,拟定了臭椿苦酮提取物分散片的处方,进行了分散片的小试制备,片剂各检查项以及脆碎度、分散均匀性和含量均满足质量标准要求。参考中国药典2010版中有关分散片的相关规定,制定了本品的质量标准,并对含量测定和体外溶出度进行了方法学研究。以紫外分光光度法测定含量。方法学研究结果表明,臭椿苦酮在1.5μg/mL-30 μg/mL的浓度范围内,其吸收度与浓度呈良好的线性关系,相关系数为0.9997。重现性及稳定性良好,精密度实验RSD为0.15%,符合精密度要求,回收率值为99.02%。结论：1本研究对椿皮(Ailanthus altissima bark)的化学成分中二氯甲烷部位进行了系统的研究,分离并鉴定了9个化合物。结合本研究所得到的化合物的波谱数据及查阅文献,总结了不同取代基的苦木素类化合物的核磁共振波谱特征,为此类化合物的快速结构鉴定提供了依据。2本研究对椿皮的化学成分中二氯甲烷部位中的各个有效部位和单体化合物进行了抗肿瘤作用研究,发现各个有效部位和单体化合物基本上都有一定的抗肿瘤活性,甚至有的化合物的活性超过了阳性对照药(顺铂)。以ailanthone和2-dihydroailanthone为代表化合物,通过肿瘤细胞凋亡检测、肿瘤细胞周期检测、RT-PCR和蛋白印迹等实验方法研究了ailanthone和2-dihydroailanthone对MCF-7人乳腺癌细胞和U251人神经胶质细胞的抗肿瘤作用机制,实验结果发现,这两种药物所表现的抗肿瘤作用与下调Bcl-2的基因表达,同时上调Bax基因表达有关。为有针对性的分离、纯化化合物以及进行下一步的药剂学研究提供了指导原则。3以臭椿苦酮提取物为原料,按照中国药典对分散片的有关规定,进行处方组成与制备工艺的研究,完成了分散片的制备工艺,建立了臭椿苦酮提取物分散片的质量评价方法,并进行了方法学和稳定性研究,各项指标均达到中国药典2010版对分散片的要求。
[Abstract]:The first part is about the chemical constituents of dichloromethane extracted from the bark of Chinese toon. Ailanthus altissima Swingle is a deciduous tree of the genus Ailanthus. It is named for its peculiar odor after the rupture of the epifoliar oil gland. It is a native tree species in China and widely distributed in China except Heilongjiang, Jilin and Hainan provinces. As an ornamental plant, Ailanthus altissima was introduced into North America and Europe in the 18th century. Up to now, it has been widely distributed in Europe, and is now one of the seven major tree species in the United States. Recent studies have shown that the extract of Chinese toon bark can be used as an insecticide because of its anti-amoeba activity. At the same time, it has been proved that it has obvious anti-cancer effect and can be used in the treatment of colon cancer, cervical cancer and rectum cancer. The main chemical constituents of Chinese toon bark are bitter wood picrin, volatile constituents, terpenoids, alkaloids, etc. The most striking chemical constituents are bitter wood picrin, and bitter wood picrins are mostly tetracyclic diterpenoid lactones and pentacyclic diterpenoids. Aim: To study the extracting parts of dichloromethane from the bark of Ailanthus altissima. The chemical constituents, especially bitter lignans, were systematically studied by various methods, including 13C-NMR, 1H-NMR, HMQC, COSY, HMBC and NOESY. The planar and stereoscopic structures of the isolated monomers were identified by spectroscopic and spectroscopic methods, including 13C-NMR, 1H-NMR, HMQC, COSY, HMBC and NOESY. METHODS: The dried Chinese toon bark was extracted by heat reflux with 95% ethanol and concentrated to obtain the total extract. Dextran gel, preparation of thin layer chromatography and preparation of high performance liquid chromatography were used to separate the chemical constituents of the above dichloromethane extracts. The monomers were identified by modern analytical methods such as 13C-NMR,'H-NMR, HMQC, COSY, HMBC and NOESY. The results were as follows: vanillin (1), stigmast-4-en-3-one (2), isoailanthone (3), shinjudilactone (4), ailanthone (5), shinjulactone B (6), 6a-tigloyloxy chaparrin (7), 2-dihydroailanthone (8), 12-dihydroisoanthone (9). Part II The extraction site of dichloromethane from Chinese toon bark and the antitumor activity of the compounds were studied. In the first Chinese Pharmacopoeia , it is a traditional Chinese medicinal material. The bark of Ailanthus altissima used in ancient times refers to the dry skin or root bark of Chinese toon and Ailanthus altissima, especially the bark of Ailanthus altissima. Slip, hemorrhoids and diarrhea of blood more than recorded in the use of the root of Ailanthus altissima and Chinese pepper roast, plus the root of onion with the decoction after fumigation, can treat postpartum intestinal detachment of women, with honey decoction can treat hemorrhoids, single use of the decoction of the skin of Chinese toon can treat sores and tinea, etc. Modern pharmacological research found that in addition to the above-mentioned traditional efficacy, but also have anti-tuberculosis. Tumor, anti-tuberculosis bacteria, anti-EB virus, anti-amoeba, anti-malaria and other effects, especially anti-tumor effect is obvious, its chemical constituents bitter wood picrin has the effect of treating colon cancer, cervical cancer, rectal cancer, and so on, is currently one of the hot spots of pharmacological research. Objective: To screen the antitumor activity of the extracts and the compounds separated, purified and prepared from the bark of Ailanthus altissima by modern screening techniques of tumor cell activity. Inhibitory rate of tumor cell proliferation of each component was analyzed. The antitumor mechanism was studied by using ailanthone and 2-dihydroailanthone as representative compounds. Methods: 1 The antitumor activity of different extracts and compounds was detected by MTT method. The antitumor mechanisms of 2-dihydroailanthone and 2-Ailanthone on MCF-7 human breast cancer cells and U251 human glioma cells at different concentrations were studied. One and 2-dihydroailanthone were used to detect the inhibitory rate of proliferation, apoptosis, cell cycle, Bax and Bcl-2 mRNAd expression by RT-PCR and Bax and Bcl-2 protein expression by Western blot respectively. The results showed that for 13 tumor cell lines, the unused extracts and compounds had different antitumor activities, while compounds 2 and 4 had relatively low antitumor activities at high, medium and low concentrations. The IC50 values of compounds 5 and 6 on 13 tumor cells were lower than those of the positive drug cisplatin, suggesting that their inhibitory effect on 13 tumor cells was better than that of the positive control drug cisplatin; compound 3 on U251, Hela, AsPC-1, HCT116, AGS, SMMC-7721, U-2OS, CCRF-CEM The IC50 values of the cells were lower than those of the positive control drug cisplatin, indicating that the inhibitory effect of compound 3 on the 9 tumor cells was better than that of the positive control drug cisplatin. The IC50 values of compound 1 on U251, Hela, HCT116, AGS, SMMC-7721, U-2OS, CCRF-CEM cells were lower than those of the positive control drug cisplatin, indicating that compound 1 had better inhibitory effect on the 7 tumor cells than that of the positive control drug cisplatin. Results: When the IC5010 ug/mL of synthetic compound or plant extract or IC5020 ug/mL of plant extract were used, it was judged that the sample could kill tumor cells in vitro. 2 Different concentrations of ailanthone could reduce the survival rate of MCF-7 breast cancer cells 24 h, 48 h and 72 h after treatment, respectively. The results showed that the growth of human breast cancer MCF-7 cells was significantly inhibited by ailanthone in a time-and concentration-dependent manner. The inhibitory effect of 2-dihydroailanthone on U251 glioma cells was also evident. Different concentrations of ailanthone and 2-dihydroailanthone were detected by flow cytometry. The results showed that the apoptosis rate of MCF-7 breast cancer cells and U251 glioma cells 48 hours after they were treated with MCF-7 and U251 respectively was significantly higher than that of the blank control group. The apoptosis rate of MCF-7 breast cancer cells and U251 glioma cells was significantly higher than that of the blank control group (P 0.05). The results showed that the percentage of cells in G0/G1 phase increased gradually, while that in S phase and G2/M phase decreased gradually, indicating that cell block occurred in G0/G1 phase. The results showed that the expression of Bax mRNA increased with the increase of drug concentration, and the expression of Bcl-2 mRNA decreased with the increase of drug concentration. The expression of Bcl-2 mRNA showed a dose-dependent manner, indicating that the two drugs could increase the expression of Bax mRNA and inhibit the expression of Bcl-2 mRNA. Western blot was used to detect the expression of Bax and Bcl-2 protein. After 48 hours of treatment with different concentrations of ailanth one and dihydroailanthone respectively, the expression of Bax.Bcl-2 protein was detected in human breast cancer MCF-7 and U251 glioma cells. The percentage of bax/beta-actin increased gradually, while the percentage of bcl-2/beta-actin decreased gradually. The results showed that the expression of Bcl-2 was decreased and the expression of Bax was increased in a dose-dependent manner when compared with the control group. Part III Study on preparation technology and quality standard of dichloromethane extract dispersible tablets from Chinese toon bark It has the advantages of high speed, high bioavailability and so on. It can be taken orally or dispersed in water directly. It is especially suitable for patients with dysphagia. It has a good taste and improves the compliance of patients. It is increasingly favored by the majority of patients. Pharmaceutical studies on the dispersible tablets of Toona bitter ketone extract with rapid release and good stability were carried out. The influencing factors and optimum technology in the preparation process were emphatically studied, and the quality of dispersible tablets was evaluated. The significance of this study is to prepare a dispersible tablet from the extract of Ailanthus altissima which is difficult to dissolve in aqueous solution by pharmaceutics research, so as to lay a foundation for its wide application in clinic. Methods: A method for in vitro analysis of the extract of Ailanthus altissima was established by ultraviolet spectroscopy to determine the content of the extract. The formulation, technology, quality and in vitro analysis of the dispersible tablets were studied by single factor test and orthogonal design. The 12-hour cumulative dissolution was used as the index to select the best preparation method. The formulation of the dispersible tablets was prepared in a small scale. The tablets were tested and the fragility, uniformity and content of the dispersible tablets met the requirements of the quality standards. The results showed that the absorbance of Toona bitter ketone was linear with the concentration in the range of 1.5 ug/mL-30 ug/mL. The correlation coefficient was 0.9997. The RSD of precision test was 0.15%, which met the precision requirement and the recovery rate was 99.02%. Nine compounds were isolated and identified from the chemical constituents of Ailanthus altissima bark. The NMR spectroscopic characteristics of different substituents of bitter lignin compounds were summarized based on the spectroscopic data and references. In this study, we studied the anti-tumor effects of various active parts and monomers of dichloromethane in the chemical constituents of Chinese toon bark. It was found that the active parts and monomers of Chinese toon bark basically had certain anti-tumor activities, and even some of them were more active than the positive control drug (cisplatin). (2) Using ailanthone and 2-dihydroailanthone as representative compounds, we detected apoptosis, cell cycle, RT-PCR and protein of tumor cells.
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R96

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