| 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 alkaloidsOBJECTIVE: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-11.2 Study on antitumor activities of different polar fractions of Capparisspinosa L. alkaloidsOBJECTIVE: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. alkaloidsOBJECTIVE: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 alkaloidsOBJECTIVE: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 alkaloids2.1 Inhibited growth of SGC-7901 by component A10 in Capparis spinosa L. polar alkaloidsOBJECTIVE: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 onSGC-7901 apoptosisOBJECTIVE: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 alkaloids3.1 Effect of component A10 in Capparis spinosa L. polar alkaloids on key things of mitochondrial pathwayOBJECTIVE: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 pathwayOBJECTIVE: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. CONCLUSIONSThe 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 RESEARCH1. 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.
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