摘要
野西瓜(Capparis spinosa L)为山柑科山柑属植物,又称刺山柑、老鼠瓜、槌果藤等,味辛苦,性温,具有祛风除湿、止瘀消肿、止痛活血之功效。文献报道和前期实验显示,野西瓜具有抗肿瘤作用,且其活性成分为其生物碱类成分。本文对野西瓜生物碱的抗肿瘤活性组分进行了筛选,找出其敏感肿瘤细胞株为人胃癌细胞株SGC-7901,得到具有较强抑制肿瘤细胞增殖的组分——野西瓜极性生物碱A10组分,并应用HPLC法和UV法测定其含量。在此基础上,研究了野西瓜极性生物碱A10组分对SGC-7901细胞的细胞毒作用,通过MTT实验、SRB实验和集落形成率实验发现野西瓜极性生物碱A10组分对SGC-7901具有生长抑制作用；然后利用凋亡形态学方法即荧光显微镜法和投射电镜法对SGC-7901凋亡形态进行定性研究；经PI单染法和AnnexinV-FITC/PI双染法,利用流式细胞仪和激光共聚焦显微镜测定了野西瓜极性生物碱A10组分对SGC-7901细胞凋亡率和周期的影响；然后对野西瓜极性生物碱A10组分所诱导SGC-7901细胞凋亡线粒体通路的关键事件进行研究,包括MPTP孔开放程度、膜电位丧失、Cyt-c释放、Caspase-9及Caspase-3激活；在确定了其凋亡的线粒体途径后,又对线粒体凋亡调控因子如活性氧水平、Ca2+浓度进行检测,判断ROS积聚和钙超载是否参与野西瓜极性生物碱A10组分所诱导线粒体凋亡通路的调控。最后,对线粒体凋亡通路调控基因Bcl-2和Bax在蛋白表达水平和mRNA基因转录水平上分别利用Vestern Blot法、流式细胞术和RT-PCR法进行检测,以期揭示野西瓜极性生物碱A10组分诱导SGC-7901细胞凋亡的线粒体通路。
1野西瓜生物碱抗肿瘤活性组分的研究
1.1野西瓜总生物碱含量测定的研究
目的：测定野西瓜中盐酸水苏碱含量和总生物碱的含量。方法：HPLC方法和UV方法。结果：应用HPLC测定野西瓜中盐酸水苏碱的含量,得到盐酸水苏碱线性回归方程为Y=100.58X-9.3751(r=0.9999),说明其在1.5-30μg范围内呈良好线性关系,测得野西瓜中盐酸水苏碱的含量为8.2257 mg·g-’。同时以雷氏铵盐剩余比色法测定野西瓜中总生物碱(以盐酸水苏碱计)的含量,得到盐酸水苏碱线性回归方程分别为Y=0.621 7X+0.0108(r=0.9986),说明在其0.060-0.301 mg·mL-1范围内线性关系良好,测得野西瓜总生物碱含量(以盐酸水苏碱计)为104.7749 mg·g-1。通过方法学考察证实两个含量测定方法准确可靠。结论：HPLC法测得盐酸水苏碱的含量为8.2257 mg·g-1,雷氏铵盐剩余比色法(以盐酸水苏碱计)测定野西瓜中总生物碱的含量为104.7749 mg·g-1。
1.2野西瓜生物碱不同极性部位抗肿瘤活性的研究
目的：寻找野西瓜抗肿瘤的活性部位。方法：采用渗漉法对野西瓜果实进行初提取,然后将渗漉液通过阳离子交换树脂,待树脂床饱和后,对树脂进行碱化,干燥,再进一步对树脂床进行溶剂分级萃取。萃取顺序为氯仿、正丁醇、70%乙醇萃取,得到三个部位氯仿层、正丁醇层和70%乙醇层。采用MTT法对三个部位进行活性筛选。结果：野西瓜生物碱的70%乙醇极性部位有明显的抗肿瘤作用,IC50为33.437μg·mL-1,为野西瓜生物碱抑制SGC-7901细胞的活性部位。野西瓜生物碱的氯仿萃取部位、正丁醇萃取部位对SGC-7901细胞也具有一定的抑制作用,但作用不如70%乙醇极性部位部位明显。因此野西瓜生物碱的70%乙醇极性部位初步确定为野西瓜生物碱抑制SGC-7901细胞的抗肿瘤活性部位。结论：人胃癌SGC-7901细胞是野西瓜总生物碱的敏感细胞株,野西瓜生物碱70%乙醇层是其抗肿瘤的活性部位。
1.3野西瓜生物碱活性部位不同组分抗肿瘤活性的研究
目的：对野西瓜生物碱抗肿瘤活性部位——70%乙醇极性部位进行进一步分离,继续采用MTT方法进行活性组分的追踪。方法：柱层析和MTT法。结果：采用硅胶柱对野西瓜生物碱抗肿瘤活性部位——70%乙醇部位进行进一步的分离,用氯仿-甲醇梯度洗脱,TCL跟踪合并,得到A1～A12共12个组分,经改良碘化铋钾显色实验证实A1-A5、A11和A12组分中不含有生物碱成分,而A6～A10组分含有生物碱成分。经MTT实验筛选A6～A10组分的抗肿瘤活性。结果显示,A6～A10组分对SGC-7901细胞增殖均具有一定的抑制作用,其中A10组分的抑制作用最强,IC50为31.529μg·mL-1。称之为野西瓜极性生物碱A10组分。结论：A10组分是野西瓜生物碱抗肿瘤活性组分,称之为野西瓜极性生物碱A10组分。
目的：测定野西瓜极性生物碱A10组分的含量。方法：HPLC法和UV法。
结果：应用HPLC测定野西瓜极性生物碱A10组分中盐酸水苏碱含量,得到盐酸水苏碱线性回归方程为Y=1004.2X-5.3806(r=0.9999),结果表明：盐酸水苏碱在3-30μg范围内呈良好线性关系。测得野西瓜极性生物碱A10组分中盐酸水苏碱含量为367.7296 mg·g-1,方法学考察证明该含量测定方法准确可靠。采用本部分实验一所述UV法测得野西瓜极性生物碱A10组分中生物碱含量(以盐酸水苏碱计)为784.2096 mg·g-1。结论：HPLC法测得野西瓜极性生物碱A10组分中盐酸水苏碱含量为367.7296 mg·g-1。UV法测得野西瓜极性生物碱A10组分中生物碱含量(以盐酸水苏碱计)为784.2096 mg·g-1。
2野西瓜极性生物碱A10组分对SGC-7901细胞的生长抑制和诱导凋亡作用
2.1野西瓜极性生物碱A10组分对SGC-7901细胞生长的抑制作用
目的：对野西瓜极性生物碱A10组分对SGC-7901细胞生长的抑制作用进行研究。方法:MTT法、SRB法和肿瘤细胞克隆原形成法。结果：MTT实验得出野西瓜极性生物碱A10组分对SGC-7901细胞IC50为31.529μg·mL-1。从SRB实验结果可以看出,低浓度的野西瓜极性生物碱A10组分通过抑制细胞生长而起到抗肿瘤作用,而高浓度时则主要通过杀死肿瘤细胞起到抗肿瘤作用,经计算GI50为31.785μg·mL-1,LC50为37.210μg·mL-1,TGI为45.864 gg·mL-1。肿瘤细胞克隆原实验结果显示,野西瓜极性生物碱A10组分可以剂量依赖性抑制SGC-7901细胞集落的形成,其IC50为37.47μg·mL-1。结论：三个实验结果基本一致,更加客观的说明野西瓜极性生物碱A10组分对SGC-7901细胞具有生长抑制作用。根据以上结果,选择野西瓜极性生物碱A10组分为后续药理实验的受试药,且低、中、高剂量分别为15、30、60μg·mL-1。
2.2野西瓜极性生物碱A10组分对SGC-7901细胞凋亡的影响
目的：观察野西瓜极性生物碱A10组分作用后SGC-7901细胞形态学的改变和细胞凋亡率的测定。方法：荧光显微镜观察法、透射电镜观察法、激光共聚焦显微镜、PI单染法和Annexin V-FITC/PI双染经流式细胞仪进行检测。结果：荧光显微镜下、透射电镜和激光共聚焦显微镜下均可观察到典型的细胞凋亡形态,SGC-7901细胞随着给药剂量的增大,凋亡细胞特征性形态越明显。SGC-7901细胞经不同浓度的野西瓜极性生物碱A10组分作用48 h后,流式细胞仪直方图上出现亚二倍体峰,表明肿瘤细胞发生晚期凋亡。不同质量浓度的野西瓜极性生物碱A10组分作用24 h后,与空白对照组比较,各给药组SGC-7901细胞均发生不同程度的早期凋亡,且出现凋亡的比例逐渐增加。结论：野西瓜极性生物碱A10组分可以诱导SGC-7901细胞发生凋亡。
3野西瓜极性生物碱A10组分诱导SGC-7901凋亡线粒体通路的研究
3.1野西瓜极性生物碱A10组分对SGC-7901细胞凋亡线粒体通路关键事件的影响
目的：研究野西瓜极性生物碱A10组分对SGC-7901细胞凋亡线粒体通路关键事件的影响,判断是否启动了线粒体凋亡通路。方法：Reagent A染色、罗丹明123染色、Western Blot法和酶标仪法。结果：在经过野西瓜极性生物碱A10组分作用后,SGC-7901细胞膜孔道不同程度的开放,膜电位发生了明显下降,并导致Cyt-c由线粒体释放至细胞浆,活化Caspase-9并启动Caspase级联反应,激活下游的Caspase-3从而诱导肿瘤细胞发生凋亡。结论：野西瓜极性生物碱A10组分诱导肿瘤细胞凋亡是由于启动了线粒体凋亡途径。
3.2野西瓜极性生物碱A10组分对SGC-7901细胞凋亡线粒体通路调控因子的影响
目的：探讨ROS和钙超载是否参与了野西瓜极性生物碱A10组分诱导的细胞凋亡过程。阐明Bcl-2和Bax基因对野西瓜极性生物碱A10组分所诱导细胞凋亡线粒体通路的调控作用。方法：DCFH-DA染色、Fluo-3/AM探针染色、Western Blot法、流式细胞术和RT-PCR法。结果：SGC-7901细胞内活性氧水平随着野西瓜极性生物碱A10组分质量浓度的升高而升高,细胞内钙离子浓度也相应的增大。给药组Bcl-2蛋白表达水平与对照组相比有所下降,而Bax蛋白表达量随之增加。野西瓜极性生物碱A10组分能够降低Bcl-2基因的mRNA表达,上调Bax基因的mRNA表达,并且具有一定的剂量依赖性。结论：经野西瓜极性生物碱A10组分作用后,活性氧的聚集和钙超载激活了细胞凋亡线粒体机制。野西瓜极性生物碱A10组分通过调节基因的转录水平下调抗凋亡蛋白Bcl-2的表达,上调促凋亡蛋白Bax的表达来调控线粒体凋亡通
本论文通过HPLC法测得野西瓜中盐酸水苏碱含量为8.2257 mg·g-1,雷氏铵盐剩余比色法(以盐酸水苏碱计)测定野西瓜中总生物碱含量为104.7749mg·g-1。MTT法筛选出人胃癌SGC-7901细胞是野西瓜生物碱的敏感细胞株,野西瓜生物碱70%乙醇层是其抗肿瘤的活性部位,A10组分为野西瓜生物碱抗肿瘤活性组分,称之为野西瓜极性生物碱A10组分。并经HPLC法测得野西瓜极性生物碱A10组分中盐酸水苏碱含量为367.7296 mg·g-1。UV法测得野西瓜极性生物碱A10组分中生物碱含量(以盐酸水苏碱计)为784.2096 mg·g-1。实验发现,野西瓜极性生物碱A10组分对SGC-7901细胞生长增殖具有抑制作用,并通过诱导细胞凋亡的方式发挥其抗肿瘤作用。野西瓜极性生物碱A10组分迫使MPTP孔开放,促使线粒体膜电位的崩溃,并促进Cyt-c的释放,触动Caspase-9,启动Caspase级联反应,并最终激活了凋亡执行分子Caspase-3而诱导SGC-7901细胞凋亡。野西瓜极性生物碱A10组分通过钙稳态失衡,ROS积聚来调控SGC-7901细胞线粒体凋亡。野西瓜极性生物碱A10组分通过调节基因的转录水平下调Bcl-2蛋白表达,上调胞浆内Bax蛋白的表达,降低Bcl-2/Bax比例,进而调控凋亡的线粒体通路。
本研究的创新点
1.采用活性跟踪的方法进行野西瓜生物碱抗肿瘤活性组分的筛选
2.从定性和定量两个方面揭示野西瓜极性生物碱A10组分诱导SGC-7901细胞凋亡。
3.首次发现野西瓜极性生物碱A10组分通过启动线粒体通路诱导SGC-7901细胞凋亡。
Capparis spinosa L. is the plant of Capparis in Capparidaceae, also known as cishangan, laoshugua, zuiguoteng, and so on. It has the efficacies of hard tasted, warm-natured, dispelling wind and eliminating dampness, stasis swelling, relieving pain and activating blood circulation. Reports in the literatures and preliminary experiments show that Capparis spinosa L. has the anti-tumor effect, and its active ingredient is alkaloid. In this paper, the antitumor activity fractions of Capparis spinosa L. alkaloids are filtrated. As a result, SGC-7901 is the susceptive cell. And the component A10 in Capparis spinosa L. alkaloids is gained with strongest growth inhibition effect. The content of the component A10 is determined by HPLC and UV methods. On this basis, the component A10 in Capparis spinosa L. polar alkaloids has the activity of cytotoxicity in vitro human gastric cancer SGC-7901 tumor cells. The results of MTT test, SRB test and colony formation rate also show that the component A10 in Capparis spinosa L. polar alkaloids has the strongest growth inhibition on the SGC-7901. The qualitative researches on apoptosis morphology of SGC-7901 are studied by fluorescence microscopy and transmission electron microscopy. Through PI staining and Annexin V-FITC/PI double staining, the influence on the apoptosis rate of SGC-7901 cells induced by the component A10 in Capparis spinosa L. polar alkaloids is determined via flow cytometry and confocal laser microscopy. Then the key things of mitochondrial apoptotic pathway induced by the component A10 in Capparis spinosa L. polar alkaloids are studied, including MPTP hole opening, membrane potential losing, cytochrome C releasing, Caspase-9 and Caspase-3 activating. After determination of mitochondrial apoptotic pathway, the level of reactive oxygen species and Ca2+ concentration are detected to explore weather ROS accumulation and Ca2+ overload play roles in regulation on mitochondrial pathway of apoptosis induced by the component A10 in Capparis spinosa L. polar alkaloids. After that, the level of protein expression and gene transcription of the apoptosis related genes:Bcl-2 and Bax were detected respectively by Western Blot, immunofluorescence and RT-PCR in order to reveal the mitochondrion pathway of SGC-7901 apoptosis induced by the component A10 in Capparis spinosa L. polar alkaloids.
1. Studies on anti-tumor active alkaloids from Capparis spinosa L.
1.1 Studies on content determination of Capparis spinosa L. total alkaloids
OBJECTIVE:To determine the contents of Stachydrine hydrochloride and total alkaloids in Capparis spinosa L.. METHODS:HPLC and UV methods. RESULTS:The content of Stachydrine hydrochloride in Capparis spinosa L. was determined by HPLC. The regression equation of Stachydrine hydrochloride was Y=100.58X-9.3751 (r=0.9999), showing fine linear relationship in 1.5-30μg. The content of Stachydrine hydrochloride in Capparis spinosa L. was 8.2257 mg·g-1. Also the concent of total alkaloids in Capparis spinosa L. was determined by the method of ammonium tetrathiocyanodiaminochromate remaining colorimetric technique (in terms of Stachydrine hydrochloride), and the regression equations was Y=0.621 7X+0.0108 (r=0.998 6), showing fine linear relationship in 0.060-0.301 mg·mL-1. The content of total alkaloids in Capparis spinosa L. was 104.7749 mg·g-1. The methods were exact and credible through methodology evaluation. CONCLUSION:From HPLC method, the content of Stachydrine hydrochloride in Capparis spinosa L. was 8.2257 mg·g-1. From the ammonium tetrathiocyanodiaminochromate remaining colorimetric technique (in terms of Stachydrine hydrochloride), the content of total alkaloids in Capparis spinosa L. was 104.7749 mg·g-1
1.2 Study on antitumor activities of different polar fractions of Capparis
spinosa L. alkaloids
OBJECTIVE:To find the anti-tumor activity fraction from Capparis spinosa L..
METHODS:The percolation method was used for the early extract from Capparis spinosa L, and then the percolation liquid was passed through acid cation exchange resin. Until the resin bed was saturated, it was alkalified, dried, and then further extracted by different polar solvents, which were chloroform extraction, butanol and 70% ethanol extraction, After three fractions were obtained, such as chloroform layer, n-butanol layer and 70% ethanol layer, MTT method was used to screen out their activities. RESULTS:70% alcohol fraction of Capparis spinosa L. alkaloids had the significant anti-tumor effect, which was the active fraction of Capparis spinosa L. alkaloids, and its IC50 was 33.437μg·mL-1. Chloroform and n-butanol extraction fractions in Capparis spinosa L. also had some inhibitory effects on SGC-7901 cells, but not as the fraction of 70% ethanol significantly. Therefore,70% alcohol fraction of Capparis spinosa L. alkaloids was identified initially as anti-tumor part on SGC-7901 cells. CONCLUSION:Human gastric cancer SGC-7901 cells are the sensitive cell line of Capparis spinosa L. alkaloids, while 70% alcohol fraction is the anti-tumor part.
1.3 Study on anti-tumor activities of different components in active fraction of Capparis spinosa L. alkaloids
OBJECTIVE:The anti-tumor fraction of Capparis spinosa L. alkaloids——70% ethanol fraction was further separated with tracking active component by MTT method. METHODS: The column chromatography and MTT method. RESULTS:The 70% ethanol part was separated by Silica column with chloroform-methanol gradient elution and TCL tracking mergers. As a result,12 components named A1～A12 were obtained. The improved Potassium Heptaiodobismuthate stain test indicated that A1～A5, A11 and A12 components did not contain alkaloids and A6～A10 components had the alkaloids. The results of MTT test showed that A6～A10 components had inhibition on SGC-7901 cells growth. The component A10 had the strongest inhibited activity, and its IC50 was 31.529μg·mL-1, which was named the component A10 in Capparis spinosa L. polar alkaloids. CONCLUSION:The component A10 is the anti-tumor component, which is known as the component A10 in Capparis spinosa L.polar alkaloids.
1.4 Studies on content determination of Capparis spinosa L. polar alkaloids
OBJECTIVE:To determine the content of component A10 in Capparis spinosa L. polar alkaloids. METHODS:HPLC and UV method. RESULTS:The content of Stachydrine hydrochloride in component A10 in Capparis spinosa L. polar alkaloids was determinated by HPLC, and the regression equation was Y=1004.2X-5.380 6 (r=0.999 9), showing fine linear relationship in 3～3Oμg. From HPLC method, the content of Stachydrine hydrochloride in component A10 in Capparis spinosa L. polar alkaloids was 367.7296 mg·g-1. The method was exact and credible through methodology evaluation. From UV method described as test one in this chapter, the content of alkloids in component A10 in Capparis spinosa L. polar alkaloids was 784.2096 mg·g-1.CONCLUSION:From HPLC test, the content of Stachydrine hydrochloride in component A10 in Capparis spinosa L. polar alkaloids is 367.7296 mg·g-1 The content of alkloids in component A10 in Capparis spinosa L. polar alkaloids was 784.2096 mg·g-1 through UV method.
2 Inhibited growth and induced apoptosis of SGC-7901 component A10 in Capparis spinosa L. polar alkaloids
2.1 Inhibited growth of SGC-7901 by component A10 in Capparis spinosa L. polar alkaloids
OBJECTIVE:To study the inhibited effect of component A10 in Capparis spinosa L. polar alkaloids on SGC-7901 tumor cell growth. METHODS:MTT test, SRB test and Formation of clonolgenic tumor cell test. RESULTS:From MTT test,IC50 of component A10 in Capparis spinosa L. polar alkaloids on SGC-7901 was 31.529μg-mL-1. From SRB test, low dosage component A10 in Capparis spinosa L. polar alkaloids had the anti-tumor effect by inhibiting growth of SGC-7901 cells, while high dosage by killing tumor cells. GI50 was 31.785μg·mL-1. LC50was 37.210μg·mL-1. TGI was 45.864μg·mL-1. The colony formation of SGC-7901 cell could be inhibited significantly by component A10 in Capparis spinosa L. polar alkaloids in dose-dependent manner. The IC50 value was 37.47μg·mL-1. CONCLUSION: All results of three tests are consistent. The inhibited effect of component A10 in Capparis spinosa L. polar alkaloids on SGC-7901 tumor cell growth is approved impersonality. The component A10 in Capparis spinosa L. polar alkaloids is selected as the tested drug, and the dosages are 15,30 and 60μg·mL-1 respectively. 2.2 Effect of component A10 in Capparis spinosa L. polar alkaloids on
SGC-7901 apoptosis
OBJECTIVE:The purpose was to study apoptotic morphology and apoptotic rate induced by component A10 in Capparis spinosa L. polar alkaloids. METHODS:Fluorescence microscope, Transmission electron microscope, confocal microscopy, PI single-staining method and Annexin V-FITC/PI double staining with flow cytometry. RESULTS:Typical apoptotic morphology was observed under fluorescence microscope, ransmission electron microscope and confocal microscopy. Hypodiploid peak appeared in flow cytometry histogram of SGC-7901 after treated with component A10 in Capparis spinosa L. polar alkaloids for 48 h, which showed late apoptosis was happened in a dose-dependent relationship. Compared with control group, different concentrations of component A10 in Capparis spinosa L. polar alkaloids could induce different degrees of early apoptosis in SGC-7901. The increased ratio of early apoptosis (D4 area) could be tested in flow cytometry with a dose-dependent relationship. As the concentration of component A10 in Capparis spinosa L. polar alkaloids was increased, the proportion of SGC-7901 cells undergoing early apoptosis was gradually increasing. CONCLUSION:Characteristic apoptosis can be caused by component A10 in Capparis spinosa L. polar alkaloids.
3 Study on of mitochondrial apoptosis pathway in SGC-7901 induced by component A10 in Capparis spinosa L. polar alkaloids
3.1 Effect of component A10 in Capparis spinosa L. polar alkaloids on key things of mitochondrial pathway
OBJECTIVE:The purpose was to detect the key things of mitochondrial pathway and judge whether the pathway of mitochondrial apoptosis in SGC-7901 was started. METHODS: Reagent A staining, Rhodamine 123 staining, Western Blot and Enzyme-labeling instrument.
RESULTS:After treated by component A10 in Capparis spinosa L. polar alkaloids, cell membrane channels in SGC-7901 were opened and membrane potential was decreased sharply. Then, Cyt-c was released from mitochondria to cytoplasm. The component A10 in Capparis spinosa L. polar alkaloids can active Caspase-9, and then activate the Caspase cascade reaction. In the end, Caspase-3 in the downstream is activated to induce SGC-7901 cell apoptosis. CONCLUSION:Apoptosis induced by component A10 in Capparis spinosa L. polar alkaloids is caused by activating mitochondrial apoptosis pathway.
3.2 Effect of component A10 in Capparis spinosa L. polar alkaloids on regulating factors of mitochondrial pathway
OBJECTIVE:To explore whether ROS and Ca2+ overload were involved in apoptosis process induced by component A10 in Capparis spinosa L. polar alkaloids and to detect the regulation effects of Bcl-2 and Bax protein on the mitochondrial pathway of apoptosis induced by component A10 in Capparis spinosa L. polar alkaloids. METHODS:DCFH-DA staining, Fluo-3/AM probes staining, Western Blot, immunofluorescence method and RT-PCR. RESULTS:ROS levels were enhanced as concentration of polar alkaloids increased, while [Ca2+] was increased with a dose-dependent relationship. Bcl-2 expression level of drug groups were decreased compared with the control group. The protein expression of Bax in SGC-7901 was increased with the increase of the drug concentration, following a certain dose-dependent relationship. The component A10 in Capparis spinosa L. polar alkaloids reduced the gene mRNA expressions of Bcl-2, and up-regulated gene mRNA expression of Bax in a dose-dependent relationship. CONCLUSION:Accumulation of ROS and Ca2+ overload activate SGC-7901 apoptosis after treated by component A10 in Capparis spinosa L. polar alkaloids. The component A10 in Capparis spinosa L. polar alkaloids down-regulates anti-apoptotic protein Bcl-2 expression and up-regulates pro-apoptotic protein Bax expression by regulation of gene transcription to regulate the mitochondria apoptosis pathway. CONCLUSIONS
The content of Stachydrine hydrochloride in Capparis spinosa L. was 8.2257 mg·g-1 determined by HPLC. Through the ammonium tetrathiocyanodiaminochromate remaining colorimetric technique (use Stachydrine hydrochloride count), the content of total alkaloids in Capparis spinosa L. was 104.7749 mg·g-1. Human gastric cancer SGC-7901 cells are sensitive to Capparis spinosa L. total alkaloids. The 70% ethanol extraction of Capparis spinosa L. alkaloids is the anti-tumor active fraction, and the component A10, named the component A10 in Capparis spinosa L. polar alkloids, is the anti-tumor active one. In addition, the content of Stachydrine hydrochloride in component A10 in Capparis spinosa L. polar alkaloids is 367.7296 mg·g-1 by HPLC method, while the content of alkloids in component A10 is 784.2096 mg·g-1through UV method. The component A10 in Capparis spinosa L. polar alkaloids has an inhibition effects on the growth of SGC-7901 cells proliferation and express its anti-tumor effects by the way of apoptosis induction. The component A10 in Capparis spinosa L. polar alkaloids promotes the opening of mitochondrial MPTP pore and decreases mitochondrial membrane permeability, which will induce Cyt-c release from mitochondria directly. Caspase-9 is activated and Caspase reaction is started. Then the downstream Caspase-3 is activated, while SGC-7901 cells apoptosis are caused. The component A10 in Capparis spinosa L. polar alkaloids regulates SGC-7901 cells mitochondria apoptosis via ROS excess and Ca2+ overload. The component A10 in Capparis spinosa L. polar alkaloids may down-regulate anti-apoptosis Bcl-2 protein express and up-regulate protein content of pro-apoptosis Bax via regulation of gene transcription to regulate the mitochondria apoptosis pathway. In the end, Bcl-2 and Bax gene co-regulate the mitochondrial apoptosis pathway induced by component A10 in Capparis spinosa L. polar alkaloids.
INNOVATIONS OF THE RESEARCH
1. Using the method of activity tracking to isolate the anti-tumor active component from Capparis spinosa L. alkaloids.
2. To reveal from both qualitative and quantitative hand on SGC-7901 cells apoptosis induced by component A10 in Capparis spinosa L. polar alkaloids.
3. It is found for the first time that component A10 in Capparis spinosa L. polar alkaloids induces SGC-7901 cells apoptosis via mitochondrial pathway.
引文
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