【作者】 李鑫；

【导师】 季宇彬；

【作者基本信息】 哈尔滨商业大学 ， 药物分析， 2018， 硕士

【摘要】 朱顶红(Hippeastrum vittatum)是单子叶亚纲石蒜科(Amaryllidaceae)植物,为朱顶红属所有种类总称,原产于墨西哥加勒比海以及阿根廷的北部。朱顶红花形优美、色泽艳丽。通过查阅文献了解到,朱顶红有较高的药用价值,其中化学成分含量丰富,包括生物碱,挥发油,糖类等多种物质,当中中的挥发油含量丰富并具有多种药理活性,具有抗菌,抗氧化,抗肿瘤,促透等功效。但挥发油这种物质由于沸点较低,提取工艺相对复杂,因而目前对朱顶红叶片中挥发油的提取与抗肿瘤等药理活性研究较少。因此,本课题以此为切入点,对朱顶红叶片中挥发油的提取进行工艺进行筛选和优化,并对提取出的挥发油进行抗肿瘤活性研究。为朱顶红叶片中挥发油提取提供理论数据也对未来拟做抗肿瘤新药的研发提供理论基础。由于挥发油的提取率通常较低,目前对朱顶红叶片中挥发油提取的研究较少,本课题首先对朱顶红叶片中挥发类成分的提取工艺进行筛选和优化。水蒸气蒸馏法、萃取法与蒸馏萃取法三种方法提取率分别为0.39%、0.89%、0.59%。萃取法提取率最高、通过正交试验可得知萃取法的最优工艺是液料比为2:1,提取温度70℃,提取时间3.5 h。将三种方法所得挥发油经气相色谱与质谱仪分析,萃取法共得到20种相似度较高的化学成分,水蒸气蒸馏13种,蒸馏萃取法14种。以萃取法提取率最高且所得化学成分最多,故后续文章所需挥发油均以萃取法来提取。在前半部分通过大量实验对提取工艺进行筛选和优化后,后半部分对朱顶红叶片中挥发类成分的抗肿瘤活性进行筛选。本课题后半部分首先采用CCK-8来检测朱顶红叶片中片中总挥发油对肝癌HepG-2细胞的增殖抑制作用。发现在2.5 mg/L-80 mg/L的范围内,朱顶红总挥发油对肝癌HepG-2细胞的增殖抑制作用随着给药浓度的增加而增强,具有增值抑制作用。在对朱顶红总挥发油进行气相色谱与质谱联用技术进行成分分析时发现,总挥发油中以棕榈酸含量最高,通过查阅文献得知,棕榈酸也具有多种药理活性。因此推测朱顶红叶片中挥发类成分的抗肿瘤活性由棕榈酸所体现,所以本课题后续对棕榈酸的抗肿瘤活性进行筛选。本课题继续采用CCK-8来检测朱顶红叶片中片总挥发油中的棕榈酸对肝癌HepG-2细胞的增殖抑制作用。发现在10umol/L-60 umol/L的范围内,朱顶红总挥发油对肝癌HepG-2细胞的增殖抑制作用随着给药浓度的增加而增强,具有增值抑制作用。根据SPSS数据分析,确定了棕榈酸对肝癌HepG-2细胞的半数抑制率IC50为42 umol/L。以所确定的半数抑制率为后续实验给药的中浓度,高浓度为80umol/L,低浓度为20 umol/L。根据课题组的前期实验可知,阳性药羟基喜树碱的半数抑制率IC50为40 umol/L。根据CCK-8实验所确定的高中低三个浓度对培养24 h后的肝癌HepG-2细胞给药,给药24 h后取出对细胞形态进行观察。在倒置显微镜下观察给药24 h后的细胞可发现给药组细胞随浓度的增大,细胞逐渐出现边界模糊,形态变圆,细胞结构破碎,细胞碎片化的现象,且浓度越大,作用时间越久,现象越明显;给药24 h的肝癌HepG-2细胞经过染色后在荧光显微镜下观察凋亡小体依次增多,细胞形态逐渐变得不规则,可见随着浓度的增加,越来越多的细胞核破碎,并随着浓度的增加细胞周围呈现出弥散状的荧光光晕。浓度越大,凋亡作用越明显。判断了棕榈酸会使肝癌HepG-2细胞产生凋亡现象后,本课题采用流式细胞仪对不同浓度给药后的肝癌HepG-2细胞进行凋亡率的检测。阳性组凋亡率为(27.746±0.13)%,空白组凋亡率为(1.174±0.27)%,高浓度组凋亡率为(36.155±0.02)%,中浓度组凋亡率为(20.316±0.18)%,低浓度组凋亡率为(5.275±0.11)%。与空白组的细胞相比,凋亡率随给药组浓度的增大而升高,具有统计学意义。综上所述,在朱顶红叶片挥发油的提取工艺研究中,萃取法有最大提取率,并且能分析出最多的组分。判断萃取法为提取朱顶红叶片中挥发油的最好方法。细胞实验中验证朱顶红叶片中总挥发油对肝癌HepG-2细胞具有增值抑制作用,朱顶红叶片总挥发油中棕榈酸对肝癌HepG-2细胞具有抗肿瘤活性。更多还原

【Abstract】 Hippeastrum vittatum is a monocotyledonous Amaryllidaceae plant of the genus Hippeastrum vittatum,native to the Mexican Caribbean sea and northern Argentina.Hippeastrum vittatum has beautiful flowers and bright colors.Through consulting the literature,we know that Hippeastrum vittatum has medicinal value,among which the chemical substances are rich,including alkaloids,volatile oil,sugar and other substances.The volatile oil in the leaves of Hippeastrum vittatum is rich in content and has various pharmacological activities,and has the effects of resisting bacteria,resisting oxidation,promoting penetration and the like.However,because of the low boiling point and the complicated extraction process,there are few studies on the extraction and anti-tumor activity of volatile oil from Hippeastrum vittatum leaves.Therefore,in this study,the extraction process of volatile oil from Hippeastrum vittatum leaves was optimized and the anti-tumor activity of the extracted volatile oil was studied.Provide theoretical data for large-scale industrial extraction of volatile oil from leaves of Hippeastrum vittatum and provide theoretical basis for future research and development of new antitumor drugs.In this paper,the extraction process of volatile components from Hippeastrum vittatum leaves was optimized.The extraction rate of steam distillation,extraction and distillation extraction were 0.39%,0.89%and 0.59%,respectively.The extraction rate was the highest,and the optimum extraction process was obtained by orthogonal test as follows:the ratio of liquid to material was 2,the extraction temperature was 70℃,and the extraction time was 3.5 h.The volatile oils obtained by the three methods were analyzed by gas chromatography and mass spectrometry,and 20 kinds of chemical components with high similarity were obtained by extraction,13 kinds by steam distillation and 14 kinds by distillation extraction.The extraction rate was the highest and the chemical components were the most,so the essential oil needed in subsequent articles was extracted by extraction.In the first half of this paper,after a large number of experiments to screen and optimize the process,in the second half of the Hippeastrum vittatum leaf volatile components of anti-tumor activity screening.In the second half of this thesis,CCK-8 was used to detect the inhibitory effect of total volatile oil in Hippeastrum vittatum leaf on the proliferation of HepG-2 cells.It was found that in the range of 2.5 mg/L-80 mg/L,the inhibitory effect of total volatile oil of Hippeastrum vittatum on proliferation of HepG-2 cells was enhanced with the increase of administration concentration,and the inhibitory effect of total volatile oil of on proliferation of HepG-2 cells was increased.The content of palmitic acid in the total volatile oil of Hippeastrum vittatum was the highest by GC-MS,and the literature showed that palmitic acid also had a variety of pharmacological activities.Therefore,it is speculated that the antitumor activity of volatile compounds in Hippeastrum vittatum leaves is reflected by palmitic acid.therefore,the subject will screen the antitumor activity of palmitic acid in the future.In this study,CCK-8 was used to detect the inhibitory effect of palmitic acid in the total volatile oil of Hippeastrum vittatum leaf on the proliferation of HepG-2 cells.It was found that in the range of 100 umol/L-600 umol/L,the inhibitory effect of total volatile oil from Hippeastrum vittatum on proliferation of HepG-2 cells was enhanced with the increase of administration concentration,and the inhibitory effect of total volatile oil on proliferation of HepG-2 cells was increased.umol/L。According to SPSS data analysis,the IC50 of palmitic acid to HepG-2 cells was determined to be 42 umol/L.umol/LThe determined median lethal dose was 80 umol/L at high concentration and 40 umol/L at low concentration 20 umol/L.The IC50 of half lethal dose of hydroxycamptothecin was 40 umol/L.The HepG-2 cells of liver cancer cultured for 24 h were treated with CCK-8 at three concentrations of high and low,and the morphology of HepG-2 cells was observed after 24 h.After 24 h,the cells in the treatment group were observed under the inverted microscope.with the increase of the concentration,the cells gradually appeared the phenomenon of fuzzy boundary,round shape,broken cell structure,cell fragmentation,and the larger the concentration,the longer the action time,the more obvious the phenomenon;HepG-2 cells of liver cancer after 24 h administration were stained by fluorescence microscope to observe the apoptotic bodies increased in turn,and the cell morphology gradually became irregular.it was observed that with the increase of concentration,more and more nuclei were broken,and with the increase of concentration,diffuse fluorescent halo appeared around the cells.umol/LThe higher the concentration,the more obvious the apoptosis.Finally,the apoptosis rate of HepG-2 cells was detected by flow cytometry.umol/LThe apoptosis rate of positive group was(27.746±0.13)%,that of blank group was(1.174±0.27)%,that of high concentration group was(36.155±0.02)%,that of middle concentration group was(20.316±0.18)%,and that of low concentration group was(5.275±0.11)%.umol/LCompared with the control group,the apoptosis rate increased with the increase of concentration in the administration group,which was statistically significant.In summary,the total volatile oil in Hippeastrum vittatum leaves has a value-added inhibitory effect on HepG-2 cells,and palmitic acid in Hippeastrum vittatum leaves has anti-tumor activity on HepG-2 cells.更多还原