枳椇肉质果梗多糖的分离纯化、结构分析及生物活性研究
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摘要
枳椇(Hovenia dulcis)为鼠李科(Rhamnaceae)高大落叶乔木,在我国分布广泛。枳棋属植物中的肉质果梗作为中药使用已有上千年的历史,其具有清热利尿,止渴除烦和解酒毒等功效。现代药理证明,枳棋肉质果梗具有解酒护肝、抗脂质过氧化以及抗疲劳等功能活性。
目前对枳棋肉质果梗的研究主要集中在其粗提物和小分子化合物,而关于肉质果梗多糖的研究则未见报道。本论文以枳椇肉质果梗为原料,主要包括提取条件优化、分离纯化、理化性质、初步结构分析、抗氧化活性、抗急性酒精性肝损伤活性、免疫调节活性以及抑制肿瘤细胞增殖活性。主要结果如下：
1枳椇肉质果梗多糖提取条件优化
选择提取温度、提取时间和液料比3个因素分别进行单因素实验,在此基础上设计三因素五水平的响应面法实验进行提取条件的优化,通过Design-Expert软件对数据进行处理,得到枳椇肉质果梗多糖最佳提取条件为：提取温度96℃,提取时间200min,液料比21mL/g,提取次数3次。在此条件下多糖的得率为10.47%。
按最优提取条件提取得到的枳椇肉质果梗多糖,经过冻融法去除淀粉和S-8大孔吸附树脂法脱色后,溶解性提高,颜色变浅,最终的粗多糖得率为3.3%。
2枳椇肉质果梗多糖分离纯化、理化性质及初步结构分析
采用DEAE-纤维素阴离子交换柱色谱法和葡聚糖G-100凝胶过滤柱色谱法对枳棋肉质果梗粗多糖进行两步纯化,获得三个纯化组分HDPS-1、HDPS-2和HDPS-3,得率分别是29.64%、10.93%和18.20%。采用HPLC法对HCPS-1、HCPS-2和HCPS-3的纯度进行进一步鉴定,结果表明HDPS-1、HDPS-2和HDPS-3三者都是均一的多糖组分。其相对重均分子量分别为：234.75,69.72和52.50kD。
采用苯酚硫酸法、间羟联苯法、氯化钡-明胶法、考马斯亮蓝法对粗多糖、HDPS-1、 HDPS-2和HDPS-3中总糖含量、糖醛酸含量、硫酸基含量、蛋白质含量进行了测定。结果表明,粗多糖、HDPS-1、HDPS-2和HDPS-3中总糖含量分别是68.13%、86.94%、73.62%和69.41%；糖醛酸含量分别是16.54%、2.10%、8.80%和43.95%；硫酸基含量分别是8.45%,2.68%,2.98%和3.53%。另外,蛋白质含量测定结果表明粗多糖含有1.50%蛋白质,而纯化各组分则不含有蛋白质。GC分析表明,粗多糖由7种单糖组成,分别为鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖和半乳糖,其摩尔比是19.50：31.4：1.53：3.68：1.58：12.88：29.25；HDPS-1,HDPS-2和HDPS-3的主要单糖组成为鼠李糖,阿拉伯糖和半乳糖,三个样品中这三种单糖所占的摩尔百分比分别是HDPS-1:42.04%,17.00%和30.91%；HDPS-2:5.32%,48.76%和44.14%;HDPS-3:30.77%,37.76%和22.32%。
红外光谱扫描表明,HDPS-1、HDPS-2和HDPS-3含有多糖的特征吸收峰。高碘酸氧化和Smith降解结果表明,三个纯化组分HDPS-1,HDPS-2,HDPS-3均是以1→3或1→3,6或1→3,4或1→3,4,6或1→2,3或1→2,4或1→2,4,6或1→2,3,4键连接糖基为主,分别占各自总摩尔数比例为59%,54%和61%。
3枳棋肉质果梗多糖抗氧化活性和抗急性酒精性肝损伤活性
采用化学分析的方法评价了枳棋肉质果梗粗多糖的体外抗氧化活性。结果表明,枳棋肉质果梗粗多糖具有清除超氧阴离子、抗脂质过氧化以及螯合金属离子的能力。在此基础上,采用酒精灌胃制备小鼠急性酒精性肝损伤模型,评价了枳棋肉质果梗粗多糖体内抗急性酒精性肝损伤的效果。结果表明,提前给药枳棋肉质果梗粗多糖20天,能显著减弱因肝损伤引起的血液中ALT和AST含量的升高,显著增强酒精性肝损伤小鼠肝脏中抗氧化酶SOD和GSH-Px的活性、减少脂质过氧化产物MDA的生成。结果表明,枳棋肉质果梗粗多糖可能是通过增强体内抗氧化酶的活性、直接清除ROS、抑制脂质过氧化以及螯合金属离子等途径来拮抗氧化应激引起的急性酒精性肝损伤。
4枳棋肉质果梗多糖免疫调节活性
首先采用体外细胞培养模型初步评价了枳棋肉质果梗多糖三个纯化组分的免疫调节活性。并根据体外实验的筛选结果,对于具有相对最强体外免疫调节活性的纯化组分,进一步研究了其在小鼠体内的免疫调节活性。
体外免疫实验结果显示,HDPS-1,HDPS-2和HDPS-3均能在不同程度上刺激脾细胞增殖、促进巨噬细胞NO的合成、增强巨噬细胞酸性磷酸酶活性和巨噬细胞吞噬中性红的能力,说明HDPS-1,HDPS-2和HDPS-3均具有不同程度的体外免疫调节活性。对实验结果的对比分析发现,免疫调节活性在三个纯化组分之间存在显著差异,从强到弱依次是：HDPS-1>HDPS-2>HDPS-3。
HDPS-1的体内免疫实验结果显示,不同剂量的HDPS-1均可以拮抗环磷酰胺导致的小鼠脾脏萎缩,提高脾脏中的溶菌酶活性,一定剂量的HDPS-1还能显著的提高免疫抑制小鼠血清中IL-6和IFN-γ水平。此外,HDPS-1体内发挥免疫调节活性具有很强的剂量依赖性,即在中、低浓度时具有促进免疫的作用,而在高浓度时具有一定程度的免疫抑制作用。这些结果表明,HDPS-1可能是通过调控与细胞免疫和体液免疫相关细胞因子的分泌来发挥其双向免疫调节活性。
5枳棋肉质果梗多糖抑制肿瘤细胞增殖活性
采用MTT法考察了HDPS对三种肿瘤细胞(A549、HepG2和BGC-823)的抑制增殖作用,并选取其中对HDPS最敏感的肿瘤细胞株,进一步研究HDPS对该细胞株细胞形态以及细胞凋亡的影响。
实验结果表明,HDPS-1,HDPS-2和HDPS-3对肿瘤细胞A549和Hep G2均无明显的增殖抑制作用,但对肿瘤细胞BGC-823细胞抑制增殖效果显著。三个组分对肿瘤细胞BGC-823的增殖抑制作用均呈显著的量效和时效关系,在给药时间72h、给药浓度320μg/mL时,HDPS-1,HDPS-2和HDPS-3的抑制率分别为：93.94%,84.38%和78.33%。其中HDPS-1具有最强的增殖抑制作用,在给药时间为24,48和72h时,HDPS-1的半数抑制浓度分别为：207.05,44.13和5.02μg/mL.
细胞形态学研究证明HDPS-1可以显著的抑制肿瘤细胞BGC-823的生长,且随剂量的增长细胞密度逐渐下降,细胞萎缩变形直至发生凋亡；Hoechst33258染色和流式细胞仪检测同时证明,HDPS-1诱导的细胞凋亡是其发挥抑制肿瘤细胞BGC-823增殖的主要原因。
Hovenia dulcis, belongs to a small genus of Rhamnaceae, is a tall deciduous tree that widely distribute in China. The peduncles of Hovenia dulcis have been used as a traditional Chinese herbal medicine for more than a millennium. The main effect including clear away heat, diuretic, slak thirst, and detoxify alcoholic intoxication. Modern pharmaceutical researches revealed that the peduncles of Hovenia dulcis could reduce alcohol toxicity, protect liver injury, resist fatigue and have anti-lipidperoxidation activities.
Recent studies about the peduncles of Hovenia dulcis were mainly concentrated on its' crude extracts and some of low molecular weight chemical constituents, there is no report about the water-soluble polysaccharide from peduncles of Hovenia dulcis (HDPS). In this thesis, HDPS was studied, including optimization of extraction parameters, isolation and purification, chemical characterization, preliminary analysis of structures, antioxidant activity, hepatoprotective activity against acute alcohol-induced liver injury, immunomodulatory activity and inhibition activity of tumor proliferation. Main results are listed as follows:
1. Optimization of extraction parameters of HDPS
Extraction temperature, extraction time and liquid-raw ratio were selected in single-factor experiments. Based on the single-factor experiments, response surface methodology (RSM) experiments were applied for the optimization of extraction parameters of HDPS. Then the data was analyzed by Design-Expert Software, and the optimum extraction parameters were obtained as follows:extraction temperature96℃, extraction time200min, liquid-raw ratio21mL/g and extraction times3. Under optimum extraction conditions, the real extraction yield of HDPS was10.47%.
HDPS obtained with the optimum extraction conditions, was treated by freeze-thaw action and S-8macroporous resin to remove starches and decolor, respectively. After two step treatment, HDPS with a little color and high soluble, the yield of crude HDPS was methods. The results showed that crude HDPS could scavenge O2· free radicals, inhibit lipid-peroxidation and chelate Fe2+in vitro. Based on its' antioxidant activities in vitro, hepatoprotective activity against acute alcohol-induced liver injury were evaluated by acute alcohol administration mice model. The results showed that crude HDPS pretreatment for20days could remarkably prevent alcohol-induced elevation of ALT and AST, enhance the activity of antioxidant enzymes (SOD and GSH-Px) and decrease the generation of lipid-peroxidation production (MDA). These results suggested that crude HDPS possessed a hepatoprotective effect against acute alcohol-induced liver injury via enhancing activity of antioxidant enzymes, scavenging reactive oxygen species, inhibiting lipid-peroxidation and chelating metal irons against the oxidative stress.
4. Immunomodulatory activity of HDPS
Firstly, Immunomodulatory activities in vitro of HDPS-1, HDPS-2and HDPS-3were evaluated by using the cell model experiments. Then according to the screening results of in vitro experiments, the purified fraction possessing best in vitro immunomodulatory activity was further studied for its' immunomodulatory activity in vivo.
Immunomodulatory activities in vitro results demonstrated that all the three fractions had potent immunostimulatory activity, resulting in stimulating proliferation of splenocytes, enhancing phagocytosis, nitric oxide production and acid phosphatase activity of peritoneal macrophages. Furthermore, the three polysaccharide fractions had significant differences in stimulatory effect at every evaluation item, and their effects were in the order of HDPS-1> HDPS-2>HDPS-3(from high to low).
Immunomodulating activities in vivo results showed that different dose HDPS-1could resist mice spleen weight decrease induced by Cyclophosphamide and enhance activity of LZM in spleen, and a certain dose HDPS-1could significantly enhance the content of IL-6and IFN-y in serum of immune suppress mice. In addition, HDPS-1plays an immunomodulatory activity in vivo with a high dose-dependent manner, that is, the low or medium dose could promote the immune function, and in high dose has an immunosuppressive activity to a certain degree. These results suggested that HDPS-1possessing a double immunomodulatory activity via regulating the generation of cytokine related with cellular and humoral immunity.
5. Proliferation inhibitory activities of HDPS on tumor cells
Proliferation inhibitory activities of HDPS for three tumor cell lines (A549, HepG2and BGC-823) were evaluated by MTT assay. For the most sensitive cell line to HDPS, the effects of HDPS on its'morphology and apoptosis were further studied.
Results showed that HDPS-1, HDPS-2and HDPS-3have no significant inhibitory effect on the proliferation of tumor cell line A549and Hep G2, but have significant inhibitory effect for BGC-823. Three purified fractions have proliferation inhibition on BGC-823at significant time and dose dependent manner. Under drug administration time72h and dose320μg/mL, the inhibition rates of HDPS-1, HDPS-2and HDPS-3on BGC-823proliferation were93.94%,84.38%and78.33%,respectively. Among them HDPS-1showed the strongest inhibitory effect on the proliferation of BGC-823. At drug administration time24,48and72h,50%concentration of inhibition of HDPS-1was207.05,44.13and5.02μg/mL.
Study on the cell morphology showed that HDPS-lcould significantly inhibit proliferation of BGC-823,the cell density decreased with the increase of sample dose, cellular atrophy and even apoptosis. Hoechst33258stain and flow cytometry detection confirmed that HDPS-1-induced cell apoptosis plays a main effect on its' proliferation inhibitory effect to BGC-823.
目前对枳棋肉质果梗的研究主要集中在其粗提物和小分子化合物,而关于肉质果梗多糖的研究则未见报道。本论文以枳椇肉质果梗为原料,主要包括提取条件优化、分离纯化、理化性质、初步结构分析、抗氧化活性、抗急性酒精性肝损伤活性、免疫调节活性以及抑制肿瘤细胞增殖活性。主要结果如下：
1枳椇肉质果梗多糖提取条件优化
选择提取温度、提取时间和液料比3个因素分别进行单因素实验,在此基础上设计三因素五水平的响应面法实验进行提取条件的优化,通过Design-Expert软件对数据进行处理,得到枳椇肉质果梗多糖最佳提取条件为：提取温度96℃,提取时间200min,液料比21mL/g,提取次数3次。在此条件下多糖的得率为10.47%。
按最优提取条件提取得到的枳椇肉质果梗多糖,经过冻融法去除淀粉和S-8大孔吸附树脂法脱色后,溶解性提高,颜色变浅,最终的粗多糖得率为3.3%。
2枳椇肉质果梗多糖分离纯化、理化性质及初步结构分析
采用DEAE-纤维素阴离子交换柱色谱法和葡聚糖G-100凝胶过滤柱色谱法对枳棋肉质果梗粗多糖进行两步纯化,获得三个纯化组分HDPS-1、HDPS-2和HDPS-3,得率分别是29.64%、10.93%和18.20%。采用HPLC法对HCPS-1、HCPS-2和HCPS-3的纯度进行进一步鉴定,结果表明HDPS-1、HDPS-2和HDPS-3三者都是均一的多糖组分。其相对重均分子量分别为：234.75,69.72和52.50kD。
采用苯酚硫酸法、间羟联苯法、氯化钡-明胶法、考马斯亮蓝法对粗多糖、HDPS-1、 HDPS-2和HDPS-3中总糖含量、糖醛酸含量、硫酸基含量、蛋白质含量进行了测定。结果表明,粗多糖、HDPS-1、HDPS-2和HDPS-3中总糖含量分别是68.13%、86.94%、73.62%和69.41%；糖醛酸含量分别是16.54%、2.10%、8.80%和43.95%；硫酸基含量分别是8.45%,2.68%,2.98%和3.53%。另外,蛋白质含量测定结果表明粗多糖含有1.50%蛋白质,而纯化各组分则不含有蛋白质。GC分析表明,粗多糖由7种单糖组成,分别为鼠李糖、阿拉伯糖、岩藻糖、木糖、甘露糖、葡萄糖和半乳糖,其摩尔比是19.50：31.4：1.53：3.68：1.58：12.88：29.25；HDPS-1,HDPS-2和HDPS-3的主要单糖组成为鼠李糖,阿拉伯糖和半乳糖,三个样品中这三种单糖所占的摩尔百分比分别是HDPS-1:42.04%,17.00%和30.91%；HDPS-2:5.32%,48.76%和44.14%;HDPS-3:30.77%,37.76%和22.32%。
红外光谱扫描表明,HDPS-1、HDPS-2和HDPS-3含有多糖的特征吸收峰。高碘酸氧化和Smith降解结果表明,三个纯化组分HDPS-1,HDPS-2,HDPS-3均是以1→3或1→3,6或1→3,4或1→3,4,6或1→2,3或1→2,4或1→2,4,6或1→2,3,4键连接糖基为主,分别占各自总摩尔数比例为59%,54%和61%。
3枳棋肉质果梗多糖抗氧化活性和抗急性酒精性肝损伤活性
采用化学分析的方法评价了枳棋肉质果梗粗多糖的体外抗氧化活性。结果表明,枳棋肉质果梗粗多糖具有清除超氧阴离子、抗脂质过氧化以及螯合金属离子的能力。在此基础上,采用酒精灌胃制备小鼠急性酒精性肝损伤模型,评价了枳棋肉质果梗粗多糖体内抗急性酒精性肝损伤的效果。结果表明,提前给药枳棋肉质果梗粗多糖20天,能显著减弱因肝损伤引起的血液中ALT和AST含量的升高,显著增强酒精性肝损伤小鼠肝脏中抗氧化酶SOD和GSH-Px的活性、减少脂质过氧化产物MDA的生成。结果表明,枳棋肉质果梗粗多糖可能是通过增强体内抗氧化酶的活性、直接清除ROS、抑制脂质过氧化以及螯合金属离子等途径来拮抗氧化应激引起的急性酒精性肝损伤。
4枳棋肉质果梗多糖免疫调节活性
首先采用体外细胞培养模型初步评价了枳棋肉质果梗多糖三个纯化组分的免疫调节活性。并根据体外实验的筛选结果,对于具有相对最强体外免疫调节活性的纯化组分,进一步研究了其在小鼠体内的免疫调节活性。
体外免疫实验结果显示,HDPS-1,HDPS-2和HDPS-3均能在不同程度上刺激脾细胞增殖、促进巨噬细胞NO的合成、增强巨噬细胞酸性磷酸酶活性和巨噬细胞吞噬中性红的能力,说明HDPS-1,HDPS-2和HDPS-3均具有不同程度的体外免疫调节活性。对实验结果的对比分析发现,免疫调节活性在三个纯化组分之间存在显著差异,从强到弱依次是：HDPS-1>HDPS-2>HDPS-3。
HDPS-1的体内免疫实验结果显示,不同剂量的HDPS-1均可以拮抗环磷酰胺导致的小鼠脾脏萎缩,提高脾脏中的溶菌酶活性,一定剂量的HDPS-1还能显著的提高免疫抑制小鼠血清中IL-6和IFN-γ水平。此外,HDPS-1体内发挥免疫调节活性具有很强的剂量依赖性,即在中、低浓度时具有促进免疫的作用,而在高浓度时具有一定程度的免疫抑制作用。这些结果表明,HDPS-1可能是通过调控与细胞免疫和体液免疫相关细胞因子的分泌来发挥其双向免疫调节活性。
5枳棋肉质果梗多糖抑制肿瘤细胞增殖活性
采用MTT法考察了HDPS对三种肿瘤细胞(A549、HepG2和BGC-823)的抑制增殖作用,并选取其中对HDPS最敏感的肿瘤细胞株,进一步研究HDPS对该细胞株细胞形态以及细胞凋亡的影响。
实验结果表明,HDPS-1,HDPS-2和HDPS-3对肿瘤细胞A549和Hep G2均无明显的增殖抑制作用,但对肿瘤细胞BGC-823细胞抑制增殖效果显著。三个组分对肿瘤细胞BGC-823的增殖抑制作用均呈显著的量效和时效关系,在给药时间72h、给药浓度320μg/mL时,HDPS-1,HDPS-2和HDPS-3的抑制率分别为：93.94%,84.38%和78.33%。其中HDPS-1具有最强的增殖抑制作用,在给药时间为24,48和72h时,HDPS-1的半数抑制浓度分别为：207.05,44.13和5.02μg/mL.
细胞形态学研究证明HDPS-1可以显著的抑制肿瘤细胞BGC-823的生长,且随剂量的增长细胞密度逐渐下降,细胞萎缩变形直至发生凋亡；Hoechst33258染色和流式细胞仪检测同时证明,HDPS-1诱导的细胞凋亡是其发挥抑制肿瘤细胞BGC-823增殖的主要原因。
Hovenia dulcis, belongs to a small genus of Rhamnaceae, is a tall deciduous tree that widely distribute in China. The peduncles of Hovenia dulcis have been used as a traditional Chinese herbal medicine for more than a millennium. The main effect including clear away heat, diuretic, slak thirst, and detoxify alcoholic intoxication. Modern pharmaceutical researches revealed that the peduncles of Hovenia dulcis could reduce alcohol toxicity, protect liver injury, resist fatigue and have anti-lipidperoxidation activities.
Recent studies about the peduncles of Hovenia dulcis were mainly concentrated on its' crude extracts and some of low molecular weight chemical constituents, there is no report about the water-soluble polysaccharide from peduncles of Hovenia dulcis (HDPS). In this thesis, HDPS was studied, including optimization of extraction parameters, isolation and purification, chemical characterization, preliminary analysis of structures, antioxidant activity, hepatoprotective activity against acute alcohol-induced liver injury, immunomodulatory activity and inhibition activity of tumor proliferation. Main results are listed as follows:
1. Optimization of extraction parameters of HDPS
Extraction temperature, extraction time and liquid-raw ratio were selected in single-factor experiments. Based on the single-factor experiments, response surface methodology (RSM) experiments were applied for the optimization of extraction parameters of HDPS. Then the data was analyzed by Design-Expert Software, and the optimum extraction parameters were obtained as follows:extraction temperature96℃, extraction time200min, liquid-raw ratio21mL/g and extraction times3. Under optimum extraction conditions, the real extraction yield of HDPS was10.47%.
HDPS obtained with the optimum extraction conditions, was treated by freeze-thaw action and S-8macroporous resin to remove starches and decolor, respectively. After two step treatment, HDPS with a little color and high soluble, the yield of crude HDPS was methods. The results showed that crude HDPS could scavenge O2· free radicals, inhibit lipid-peroxidation and chelate Fe2+in vitro. Based on its' antioxidant activities in vitro, hepatoprotective activity against acute alcohol-induced liver injury were evaluated by acute alcohol administration mice model. The results showed that crude HDPS pretreatment for20days could remarkably prevent alcohol-induced elevation of ALT and AST, enhance the activity of antioxidant enzymes (SOD and GSH-Px) and decrease the generation of lipid-peroxidation production (MDA). These results suggested that crude HDPS possessed a hepatoprotective effect against acute alcohol-induced liver injury via enhancing activity of antioxidant enzymes, scavenging reactive oxygen species, inhibiting lipid-peroxidation and chelating metal irons against the oxidative stress.
4. Immunomodulatory activity of HDPS
Firstly, Immunomodulatory activities in vitro of HDPS-1, HDPS-2and HDPS-3were evaluated by using the cell model experiments. Then according to the screening results of in vitro experiments, the purified fraction possessing best in vitro immunomodulatory activity was further studied for its' immunomodulatory activity in vivo.
Immunomodulatory activities in vitro results demonstrated that all the three fractions had potent immunostimulatory activity, resulting in stimulating proliferation of splenocytes, enhancing phagocytosis, nitric oxide production and acid phosphatase activity of peritoneal macrophages. Furthermore, the three polysaccharide fractions had significant differences in stimulatory effect at every evaluation item, and their effects were in the order of HDPS-1> HDPS-2>HDPS-3(from high to low).
Immunomodulating activities in vivo results showed that different dose HDPS-1could resist mice spleen weight decrease induced by Cyclophosphamide and enhance activity of LZM in spleen, and a certain dose HDPS-1could significantly enhance the content of IL-6and IFN-y in serum of immune suppress mice. In addition, HDPS-1plays an immunomodulatory activity in vivo with a high dose-dependent manner, that is, the low or medium dose could promote the immune function, and in high dose has an immunosuppressive activity to a certain degree. These results suggested that HDPS-1possessing a double immunomodulatory activity via regulating the generation of cytokine related with cellular and humoral immunity.
5. Proliferation inhibitory activities of HDPS on tumor cells
Proliferation inhibitory activities of HDPS for three tumor cell lines (A549, HepG2and BGC-823) were evaluated by MTT assay. For the most sensitive cell line to HDPS, the effects of HDPS on its'morphology and apoptosis were further studied.
Results showed that HDPS-1, HDPS-2and HDPS-3have no significant inhibitory effect on the proliferation of tumor cell line A549and Hep G2, but have significant inhibitory effect for BGC-823. Three purified fractions have proliferation inhibition on BGC-823at significant time and dose dependent manner. Under drug administration time72h and dose320μg/mL, the inhibition rates of HDPS-1, HDPS-2and HDPS-3on BGC-823proliferation were93.94%,84.38%and78.33%,respectively. Among them HDPS-1showed the strongest inhibitory effect on the proliferation of BGC-823. At drug administration time24,48and72h,50%concentration of inhibition of HDPS-1was207.05,44.13and5.02μg/mL.
Study on the cell morphology showed that HDPS-lcould significantly inhibit proliferation of BGC-823,the cell density decreased with the increase of sample dose, cellular atrophy and even apoptosis. Hoechst33258stain and flow cytometry detection confirmed that HDPS-1-induced cell apoptosis plays a main effect on its' proliferation inhibitory effect to BGC-823.
引文
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