| Tumor is one of the most harmful diseases to human health, as WHO reports, up to seven million people die from cancer per year the entire world, and the new patient are about eight million, which is gradually increasing. In 2020, the new incidence will be twenty million per year all the world, and the cancer will be the first killer for human health. Chemistry drug, operation and radiotherapy had successfully cured many cancers, but the side-effect and drug-resistance were obvious, so founding new drugs is urgent.In recent years, anti-tumor drugs from plant have aroused people's attention. Asparagus officinalis is a perennial herb plant; it has abundant nutrition and many active components such as polysaccharide, saponin, flavonoids, tissue protein, and trace element. Many scientific studies showed Asparagus officinalis had anti-tumor effect and enhanced people's immunity. The research is increasing gradually around the would, especially on cancer cure and prevention, but the present work mainly focus on asparagus officinalis juice or extracts, so the exact anti-tumor active components in'Asparagus officinalis and anti-tumor mechanism are unclear. Based on the vain field, we choose asparagus polysaccharide and saponin as study object, and identify anti-tumor active components in Asparagus officinalis and anti-tumor mechanism.There are substantial connections between tumor occurrence and development with organism immunity. Red blood cells is the most cells in blood cycle, it can adjust many immune functions, which play an important role in organism immunity. The change of red blood cells immunity may affect tumor occurrence and development; there are many immune molecules on red blood cells surface, which are the most important material basis for its immune function. Many anti-tumor drugs cured the patients by enhancing their immunity, and polysaccharide from plant had the advantage to enhance organism immunity. So based on this feature, we determined asparagus polysaccharide anti-tumor effect for the first time, and study the mechanism via red blood cells immunity.There are substantial connections between tumor occurrence and development with cell apoptosis. Apoptosis is the process of programmed cell death that may occur in multi-cellular organisms. Use cell apoptosis induction as tumor treatment method has been hotspot for anti-tumor drug discovery. So based on this feature, we determined HepG2 cell apoptosis induced by asparagus saponin for the first time, and study the mechanism via mitochondrial pathway, such as apoptosis induction factor, apoptosis death switch, apoptosis starting molecule, apoptosis executing molecule and so on.The experiments include three parts:1. Study on anti-tumor effect of Asparagus polysaccharide and Asparagus saponins; 2. Study on the effect of Asparagus polysaccharide on immune and biochemistry function of erythrocyte in S180 mice; 3. Study on apoptosis of HepG2 cell induced by Asparagus saponins via mitochondrial pathway.1 Study on anti-tumor effect of Asparagus polysaccharide and Asparagus saponins1.1 Effect of Asparagus polysaccharide and Asparagus saponins on tumor weight and survival time of tumor model miceOBJECTIVE:To determine the effect of Asparagus polysaccharide and Asparagus saponins on tumor weight and survival time of tumor model mice, confirm the anti-tumor effect in vitro.METHODS:Tumor mice model was made, and they were treated with different dosage of Asparagus polysaccharide and Asparagus saponins for 7 days, the anti-tumor rate and life-lengthening rate was calculated. RESULTS:①Asparagus polysaccharide inhibited tumor growth of S180 mice, which the tumor weight was reduced remarkably compared with control group (P<0.05). Asparagus polysaccharide improved survival time of H22 mice, which the survival time was lengthened remarkably compared with control group (P<0.05).②Asparagus saponins inhibited tumor growth of S180 mice, which the tumor weight was reduced remarkably compared with control group (P<0.05). Asparagus saponins improved survival time of H22 mice, which the survival time was lengthened remarkably compared with control group (P<0.05).CONCLUSION:Asparagus polysaccharide and Asparagus saponins had anti-tumor effect in vitro.1.2 Effect of Asparagus polysaccharide and Asparagus saponins on human tumor cell proliferation OBJECTIVE:To determine the effect of Asparagus polysaccharide and Asparagus saponins on human tumor cell proliferation, confirm the anti-tumor effect in vivo. METHODS: HepG2 and SGC-7901 cells were cultured, and they were treated with different dosage of Asparagus polysaccharide and Asparagus saponins, the cell proliferation inhibitory rate was measured by MTT and SRB methods. RESULTS:①The result of MTT showed Asparagus polysaccharide had little inhibitory effect on cell proliferation, which was unremarkably compared with control group (P>0.05).②Result of MTT showed Asparagus saponins had inhibitory effect on cell proliferation, and the IC50 for HepG2 and SGC-7901 was 101.15μg/mL and 177.5μg/mL. The result of SRB showed Asparagus saponins had inhibitory effect on cell proliferation, and the GI50 for HepG2 and SGC-7901 was 107.53μg/mL and 157.44μg/mL. CONCLUSION:Asparagus polysaccharide had no anti-tumor effect in vitro, while Asparagus saponins had anti-tumor effect in vitro.2 Study on the effect of Asparagus polysaccharide on immune and biochemistry function of erythrocyte in S180 mice2.1 Effect of Asparagus polysaccharide on the number and activity of erythrocyte complement receptor 1(CD35) in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on the number and activity of erythrocyte complement receptor 1 in S180 mice. METHODS:Red blood cells from mice venous blood were labeled by rat anti-mouse CD35 monoclonal antibody and FITC-conjugated goat anti-mouse antibody. Using flow cytometry, we determined the number of ECR1. Using microscope, we studied the the adherence between erythrocyte immunity and C3b receptor or tumor-cell by RBC-C3bRR and DTER. Suspensions of lymphocytes and plasma were separated from anticoaguted whole blood of normal mice. Suspensions of erythrocytes were separated from anticoaguted whole blood of normal mice and S180 mice treated by Asparagus polysaccharide. Usnig S180 cells as activating antigen, plasma as reactive medium, lymphocytes as effective cell, we established two immuno-response systems of lymphocytes regulated by erythrocytes with or without antigen activation. The expression of CD25 on lymphocytes was measured by flow cytometry. RESULTS:①The number of CR1 in S180 mice group was remarkably low compared with that in normal mice group (P<0.01), and Asparagus polysaccharide could increase CR1 number remarkably compared with control group (P<0.05).②RBC-C3bRR and DTER in S180 mice group was remarkably low compared with that in normal mice group (P<0.01), and Asparagus polysaccharide could increase RBC-C3bRR and DTER remarkably compared with control group (P<0.05).③Under antigen activation, the erythrocytes could positively regulate immunological reaction of lymphocytes, the expression of CD25 on lymphocytes regulated by erythrocytes in normal group was much higher than that in tumor groups, and in tumor groups treated by Asparagus polysaccharide was much higher than that in tumor control group; Under no antigen activation, the erythrocytes could also positively regulate immunological reaction of lymphocytes and increase the expression of CD25, but the effect was weaker than that under antigen activation.CONCLUSION:Asparagus polysaccharide can improve the erythrocyte immune function of S180 mice, which may be one of its most important anti-tumor mechanisms.2.2 Effect of Asparagus polysaccharide on erythrocyte membrane components and fluidity in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on erythrocyte membrane components and fluidity in S180 mice. METHODS:S180 mice were administrated with Asparagus polysaccharide by i.p. for 7d, and we determined band 3 and glycophorin A content by SDS-PAGE, spectrophotometer with test kit was used to measure phospholipids, cholesterol and sialic acid, DPH dye and fluorescence spectrophotometry was used to determine membrane fluidity. RESULTS:①The content of phospholipids in S180 mice group was remarkably low and cholesterol in S180 mice group was remarkably high compared with that in normal mice group, and Asparagus polysaccharide could increase phospholipids content and decrease cholesterol content remarkably compared with control group (P<0.05,P<0.01).②The content of band 3 and glycophorin A in S180 mice group was remarkably low compared with that in normal mice group, and Asparagus polysaccharide could increase both proteins content remarkably compared with control group (P<0.05,P<0.01).③The content of sialic acid in S180 mice group was remarkably low compared with that in normal mice group, and Asparagus polysaccharide could increase the content remarkably compared with control group (P<0.05,P<0.01).④The erythrocyte membrane fluidity in S180 mice group was remarkably low compared with that in normal mice group, and Asparagus polysaccharide could improve membrane fluidity remarkably compared with control group (P<0.05,P<0.01). CONCLUSION:Asparagus polysaccharide can adjust the abnormality of S180 mice, and improve membrane fluidity, restore erythrocyte membrane structure and function.2.3 Effect of Asparagus polysaccharide on erythrocyte membrane potential in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on erythrocyte membrane potential in S180 mice. METHODS:Tumor mice model was made and treated with different dosage of Asparagus polysaccharide for 7 days, suspensions of erythrocytes were separated from anticoaguted whole blood and dyed with DiBAC4(3), flow cytometry was used to determine erythrocyte membrane potential. RESULTS:The erythrocyte fluorescence intensity in S180 mice group was remarkably high compared with that in normal mice group (P<0.01), which showed erythrocyte membrane potential in S180 mice group was high, Asparagus polysaccharide could decrease erythrocyte fluorescence intensity and lower erythrocyte membrane potential remarkably compared with control group (P<0.05).CONCLUSION:Asparagus polysaccharide can adjust erythrocyte membrane potential and maintain cell inner environment and ion transmembrance diffusion.2.4 Effect of Asparagus polysaccharide on erythrocyte ion channel in S180 miceOBJECTIVE:To study the effect of Asparagus polysaccharide on erythrocyte ion channel in S180 mice. METHODS:Tumor mice model was made and treated with different dosage of Asparagus polysaccharide for 7 days, and suspensions of erythrocytes were separated from anticoaguted whole blood, spectrophotometer with test kit was used to measure Na+,K+-ATPase, Ca2+,Mg2+-ATPase activity, erythrocytes were dyed with Fluo-3/AM, MQAE, BCECF-AM and laser scanning confocal microscope and fluorescence spectrophotometry were used to determine [Ca2+],[CI-],pH. RESULTS:①Asparagus polysaccharide could increase erythrocyte Na+,K+-ATPase, Ca2+,Mg2+-ATPase activity, lower [Ca2+] concentration, improve cation channel transport activity.②Asparagus polysaccharide could lower [CI-] concentration, improve anion channel transport activity.③Asparagus polysaccharide could increase intra-cellular pH, maintain acid-base balance. CONCLUSION:Asparagus polysaccharide can improve erythrocyte ion channel transport activity, maintain cell inner environment and protect cell structure, enhance erythrocyte immune function. 2.5 Effect of Asparagus polysaccharide on complex mobility of erythrocyte in mice OBJECTIVE:To study the effect of Asparagus polysaccharide on complex mobility of erythrocyte in S180 mice. METHODS:Tumor mice model was made and treated with different dosage of Asparagus polysaccharide for 7 days, and suspensions of erythrocytes were separated from anticoaguted whole blood, high performance capillary electrophoresis was used to determine complex mobility of erythrocyte. Experimental conditions included the following:capillaries,75μm×50cm; buffer for electrophoresis, phosphate solution containing hydroxypropylmethyl cellulose (0.1mol/L, pH7.4); injection pressure,3.448kPa; injection time,10s; separation voltage,20kV; column temperature,25℃. RESULTS:The migration time of erythrocyte in S180 mice group was longer than that in normal mice group, and Asparagus polysaccharide could remarkably shorten migration time of erythrocyte compared with control group (P<0.05). The complex mobility of erythrocyte in S180 mice group was longer than that in normal mice group, and Asparagus polysaccharide could remarkably increase complex mobility of erythrocyte compared with control group (P<0.05). CONCLUSION: Asparagus polysaccharide improved complex mobility of erythrocyte in S180 mice, which is possibly related with the change of charges density on erythrocytes surface. It is believed that HPCE can be used as an auxiliary tool for determining the physiological state and functions of erythrocytes.3. Study on apoptosis of HepG2 cell induced by Asparagus saponins via mitochondrial pathway3.1 Determination of apoptosis of HepG2 cell Induced by Asparagus saponins. OBJECTIVE:To study apoptosis of HepG2 cell inducted by Asparagus saponins.METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 48h and 72h.Cells were dyed with Hoechst 33258 and observed under fluorescence microscope, and the ultrastructural changes of cells were observed by transmission electron microscopy. Cells were dyed with Annexin V-FITC/PI and PI, flow cytometry was used to determine apoptosis rate and cell cycle. RESULTS:①Under fluorescence microscope, cells in control group showed integrated and clear edge image. After treated with Asparagus saponins for 48h, cells appeared apoptosis image, as concentration increased, the cells morphology changed irregular, and the number of apoptotic bodies increased.②Under transmission electron microscopy, cells in control group showed prominent nuclear and abundant organella. After treated with Asparagus saponins for 72h, cells appeared apoptosis image, as concentration increased, the apoptosis morphology was gradually obvious and apoptotic bodies appeared.③After treated with Asparagus saponins for 48h, cells apoptosis rate was gradually high. As concentration increased, the early apoptosis rate decreased and the late apoptosis rate increased.④After treated with Asparagus saponins for 72h, cell cycle changed much, and the ratio of cells increased in S phase and decreased in G2/M phase. As concentration increased, apoptotic peak gradually appeared.CONCLUSION: Asparagus saponins can induce HepG2 apoptosis.3.2 Effect of Asparagus saponins on HepG2 intracellular reactive oxygen species (ROS), Ca2+, pHOBJECTIVE:To study the effect of Asparagus saponins on HepG2 intracellular ROS, Ca2+, pH. METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 24h.Cells were dyed with DCFH-DA, Fluo-3/AM, BCECF/AM, flow cytometry and laser scanning confocal microscope were used to determine intracellular ROS, Ca2+, pH.RESULTS:①After treated with Asparagus saponins for 24h, the cells fluorescence intensity intensified, the intracellular ROS level increased remarkably compared with control group (P<0.01).②After treated with Asparagus saponins for 24h, the cells fluorescence intensity intensified, the intracellular Ca2+ level increased remarkably compared with control group (P<0.01).③After treated with Asparagus saponins for 24h, the cells fluorescence intensity weakened, the intracellular pH decreased remarkably compared with control group (P<0.01).CONCLUSIONAsparagus saponins can adjust HepG2 intracellular ROS,Ca2+,pH level and induce downstream apoptosis event happen.3.3 Effect of Asparagus saponins on HepG2 mitochondrial permeability transition pore (MPTP) and Mitochondrial membrane potential (MMP)OBJECTIVE:To study the effect of Asparagus saponins on HepG2. mitochondrial permeability transition pore (MPTP) and mitochondrial membrane potential (MMP). METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 24h.Cells were dyed with MPTP fluorescence kit and Rhodamine 123, laser scanning confocal microscope was used to observe MPTP, MMP. RESULTS:①After treated with Asparagus saponins for 24h, the cells fluorescence intensity weakened, the MPTP activity increased remarkably compared with control group (P<0.05).②After treated with Asparagus saponins for 24h, the cells fluorescence intensity weakened, the MMP level decreased remarkably compared with control group (P<0.05). CONCLUSION:Asparagus saponins can open mitochondrial permeability transition pore, turn on the death switch and decrease mitochondrial membrane potential, then induce cell apoptosis.3.4 Effect of Asparagus saponins on apoptosis related protein expression in HepG2OBJECTIVE:To study the effect of Asparagus saponins on apoptosis related protein expression in HepG2. METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins. Cells were incubated with Bcl-2,Bax,Cyt-c,Caspase-9,Caspase-3 antibody respectively, then incubated with FITC-conjugated antibody, flow cytometry was used to determine protein expression. RESULTS:①After treated with Asparagus saponins for 24h, the intracellular Cyt-c expression increased, which was in a dosage dependent manner.②After treated with Asparagus saponins for 48h, the intracellular activated Caspase-9, Caspase-3 expression increased, which was in a dosage dependent manner.③After treated with Asparagus saponins for 24h, the intracellular Bcl-2 expression decreased and Bax expression decreased. CONCLUSION:Asparagus saponins can adjust apoptosis related protein expression in HepG2, and induce cell apoptosis via mitochondrial pathway.3.5 Effect of Asparagus saponins on Caspase-9 and Caspase-3 activity in HepG2 OBJECTIVE:To study the effect of Asparagus saponins on Caspase-9 and Caspase-3 activity in HepG2. METHODS:HepG2 cells were cultured and treated with different dosage of Asparagus saponins for 24h and 48h. Caspase kit and enzyme-labeled instrument were used to determine Caspase-9 and Caspase-3 activity. RESULTS:①After treated with Asparagus saponins, pNA production increased, Caspase-9 activity increased remarkably compared with control group (P<0.01), and the activity at 48h was higher than that at 24h. The activity at 24h was increased by 48.21%,63.89%,102.11%, and the activity at 48h was increased by 55.52%, 93.83%,135.89%.②After treated with Asparagus saponins, pNA production increased, Caspase-3 activity increased remarkably compared with control group (P<0.01), and the activity at 48h was higher than that at 24h. The activity at 24h was increased by 46.79%, 55.03%,74.24% and the activity at 48h was increased by 80.03%,143.32%,218.66%.CONCLUSION:Asparagus saponins can increase Caspase-9 and Caspase-3 activity in HepG2, and induce cell apoptosis via caspase dependent mitochondrial pathway. Asparagus officinalis had anti-tumor effect, the active component and the mechanism as follows:1. Asparagus polysaccharide can improve immune function of S180 mice:①Asparagus polysaccharide can increase CR1 number, increase RBC-C3bRR and DTER, enhance the regulation of erythrocyte CR1 on immunological reaction of lymphocyte.②Asparagus polysaccharide can increase band 3 protein and glycophorin A content on erythrocyte membrane, increase phospholipids content and decrease cholesterol content, increase sialic acid content, adjust membrane fluidity.③Asparagus polysaccharide can decrease erythrocyte fluorescence intensity and lower erythrocyte membrane potential.④Asparagus polysaccharide can increase erythrocyte Na+,K+-ATPase, Ca2+,Mg2+-ATPase activity, lower [Ca2+] concentration, improve cation channel transport activity; lower [CI-] concentration, improve anion channel transport activity; lower [H+] concentration, increase intra-cellular pH, maintain acid-base balance.⑤Asparagus polysaccharide can shorten erythrocyte migration time, increase erythrocyte complex mobility.2. Asparagus saponins can induce HepG2 cell apoptosis via mitochondrial pathway:①Cell morphology under fluorescence microscope and transmission electron microscope showed Asparagus saponins can induce HepG2 cell apoptosis, and apoptotic bodies appear. Asparagus saponins can change cell cycle and induce S phase arrest, increase the ratio of cells in S phase and decrease in G2/M phase.②Asparagus saponins can increase intracellular ROS level, increase intracellular Ca2+ level, decrease intracellular pH, so induce HepG2 cell apoptosis.③Asparagus saponins can open mitochondrial permeability transition pore, turn on the death switch and decrease mitochondrial membrane potential, then induce cell apoptosis via un-reversible mitochondrial pathway.④Asparagus saponins can adjust apoptosis related protein expression in HepG2 and induce cell apoptosis. It increases intracellular Cyt-C, activated Caspase-9 and Caspase-3, Bax expression, decreases intracellular Bcl-2 expression.⑤Asparagus saponins can increase Caspase-9 and Caspase-3 activity in HepG2 in a dosage and time dependent manner, and induce cell apoptosis via caspase dependent mitochondrial pathway.
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