银条多糖分离纯化、抗肿瘤和免疫调节活性研究
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摘要
银条(Stachys floridana Schuttl. ex Benth)是唇形科水苏属多年生蓄根草本植物,分布于华北、华南和新疆等地,多处于半野生状态。人工栽培主要集中在河南偃师,占全国栽培总量的95%以上。然而目前对银条的研究主要涉及其繁育、栽培及贮藏保鲜等方面,而对银条中活性物质研究开发较少,尤其多糖方面的系统研究几乎没有。鉴于此,本研究以偃师银条为原料,分别对其多糖提取工艺及超滤浓缩、分离纯化、理化性质、抗肿瘤活性和免疫调节活性进行了研究。主要结果如下：
1银条多糖提取工艺及超滤浓缩的研究
选择提取温度、提取时间、液料比及提取次数4个因素分别进行单因素实验,在单因素实验基础上,选择提取温度、提取时间及液料比3个参数进行三因素三水平的Box-Behnken实验(BBD),对此3个参数的实验组合进行优化,通过Design-Expert软件对数据进行处理,得出银条多糖的最优提取条件为提取温度94℃,提取时间4h,液料比19mL/g。在此条件下银条多糖的提取率为9.98±0.60%。
选择50kDa超滤膜,以膜通量为指标,首先研究了操作压力、超滤温度和料液pH值3个工艺参数对银条提取液超滤效果的影响,并在此基础上通过正交实验确定超滤浓缩的最佳工艺参数,最后通过测定清洗前后膜通量的变化确定最佳的超滤膜清洗方法。结果表明：最佳超滤工艺参数为操作压力0.35MPa,操作温度30℃,料液pH6.5；最佳清洗方法为40℃下用pH值为8.5的NaOH溶液清洗0.5h；超滤浓缩效果优于常规浓缩。
2银条多糖分离纯化、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银条多糖进行分级纯化,获得一个主要纯化组分SFPSA,得率是12.57%。采用HPLC法对SFPSA的纯度进行进一步鉴定,结果表明SFPSA是均一的多糖组分,其相对平均分子量为：168.30kDa。
采用苯酚硫酸法,以混合糖为标准品测定SFPSA的总糖含量78.41±5.11%,比单用葡萄糖为标准品测定的结果(56.17%±3.94%)要高,而且混标法测定SFPSA中总糖含量重现性好,平均回收率可达97.85%,12h内结果稳定。
分别采用间羟联苯法、氯化钡-明胶法、考马斯亮蓝法对SFPSA中糖醛酸含量、硫酸基含量、蛋白质含量进行了测定。结果表明,SFPSA中糖醛酸含量25.20±1.88%,硫酸基含量1.98±0.21%和蛋白质含量分别为1.28±0.04%。
分别采用糖腈乙酰化法和PMP柱前衍生化法测定SFPSA单糖组成。结果表明,糖腈乙酰化法测定的SFPSA中含鼠李糖,阿拉伯糖,葡萄糖和半乳糖等单糖组分,其摩尔百分比为8.19：37.74：3.46：50.61；PMP柱前衍生化法测定的SFPSA主要由六种单糖组成,分别是鼠李糖(Rha)、葡萄糖醛酸(GlcA)、半乳糖醛酸(GlaA)、葡萄糖(Glc)、半乳糖(G1a)和阿拉伯糖(Ara),其摩尔百分含量比为7.75：1.65：14.92：1.87：33.17：40.64,该结果说明SFPSA是一种主要由半乳糖、半乳糖醛酸和阿拉伯糖组成的杂多糖。而且,PMP柱前衍生化法能够更加全面的反映样品的单糖组成。
红外光谱扫描表明,SFPSA具有多糖特征吸收峰,而且具有β-吡喃糖苷键的特征吸收峰。紫外光谱扫描结果显示,SFPSA在280nn处没有显著的吸收峰,这表明SFPSA中蛋白质含量很低。
3银条多糖抗结肠癌活性及其机理的初步研究
采用MTT法考察了SFPSA对三种肿瘤细胞株(BGC-823、HGC-27和HT-29)增殖的抑制作用,并选取其中对SFPSA最敏感的肿瘤细胞株,进一步研究SFPSA对该细胞株增殖抑制作用的机理。实验结果表明,SFPSA能抑制这三种肿瘤细胞的增殖,且这种抑制作用均呈现一定的浓度和时间的依赖性。另外,给药72h后,SFPSA对三种肿瘤细胞的半数抑制浓度(IC50)分别为3662.24μg/mL、1136.73μg/mL、407.52μg/mL,由此可见,人结肠癌HT-29细胞对SFPSA最为敏感。
细胞形态学研究证明SFPSA可以显著的抑制人结肠癌HT-29细胞的生长,且随剂量的增长细胞密度逐渐下降,细胞间隙变大,失去“群集”特性,最后失去粘附性而死亡。Hoechst33258染色后,荧光显微镜观察显示其细胞核内出现凋亡碎片,即呈现显著的凋亡特征,这表明SFPSA处理可以引起人结肠癌HT-29细胞凋亡。
FITC-Annexin V/PI双染色和流式细胞仪检测结果表明,经SFPSA处理HT-29细胞48h后,随着SFPSA处理浓度的增加,HT-29细胞的凋亡率从11.3±2.07%上升到47.4±0.98%。细胞周期分析结果显示,经SFPSA处理HT-29细胞48h后,随着SFPSA处理浓度的增加,HT-29细胞中G0/G1期细胞比例从42.35±3.21%下降到24.38±4.01%,而G2、M期细胞比例从31.57±2.18%上升到56.84±4.12%,表明SFPSA可以通过在HT-29细胞中产生G2/M阻断,使其无法进入有丝分裂过程而促使其进入凋亡过程。
采用荧光定量PCR分析SFPSA对HT-29细胞中凋亡相关基因Bax、Bcl-2和p53的mRNA表达水平的影响。结果表明,SFPSA处理HT-29细胞48h后,随着SFPSA处理浓度的增加,促凋亡成员Bax和p53的mRNA相对表达水平上升,与对照相比,前者分别增加了1.21倍和1.67倍,后者分别增加了1.26倍和1.74倍。而抑凋亡成员Bcl-2的mRNA相对表达水平下降,与对照相比,分别下降了3.79倍和11.3倍。另外,分光光度法研究Caspase-3活化度的结果表明,HT-29细胞经SFPSA处理48h后,细胞中Caspase-3被活化。
根据以上结果,我们可以推断SFPSA处理HT-29细胞后,可能通过上调p53表达,进而诱导Bax表达增加和Bcl-2表达减少,最后引起Caspases的级联放大反应而最终导致细胞凋亡。
4银条多糖免疫调节活性
运用体外细胞模型实验研究了SFPSA的体外免疫调节活性。结果表明：SFPSA能促进小鼠脾淋巴细胞增殖,促其释放IFN-y,且能活化小鼠腹腔巨噬细胞,促其释放免疫活性分子NO和IL-6,增强其酸性磷酸酶活性和吞噬中性红的能力。而且SFPSA的这种体外免疫调节能力具有双向性,即在低浓度下具有免疫促进作用,而在高浓度下则开始表现为不同程度的免疫抑制作用。
采用环磷酰胺诱导的免疫抑制小鼠模型研究了SFPSA的体内免疫调节活性。结果显示,SFPSA可以使因环磷酰胺(Cy)诱导产生的免疫功能低下得以恢复,具体表现为SFPSA处理可以显著地提高脾脏指数、迟发型超敏反应(DTH)的耳肿胀度、脾脏和血清中溶菌酶(LZM)的活性和血清中溶血素水平,且这种变化与SFPSA呈现剂量依赖关系。这些结果表明,SFPSA不仅能够提高免疫抑制小鼠的非特异性免疫能力,还可以提高其特异性免疫能力。
Stachys floridana Schuttl. ex Benth, also called as "yintiao", is a perennial herbaceous plant belonging to the Stachys genus of Lamiaceae and distributes mainly in North of China, South of China and Xinjiang uygur autonomous region of China in half-wild state. It is artificial cultivated mainly in Yanshi City Henan province, accounting for more than95%of the total cultivation. However, recent studies about "yintiao" were mainly concentrated on its breeding, cultivation and fresh-keeping and so on, and there is no report about active substances such as polysaccharides from the rhizome of Stachys floridana Schuttl. ex Benth (SFPS). In this thesis, SFPS was studied systemically, including optimization of extraction condition and parameters of ultrafiltration concentration, isolation and purification, physicochemical characterization, antitumor and immunomodulatory activity. Main results are listed as follows:
1Research on extraction condition and ultrafiltration concentration of SFPS
Extraction temperature, extraction time, ratio of water to raw material and extraction times were selected in single-factor experiments. Based on the single-factor experiments, combination of the extraction parameters (time, temperature and ratio of water to raw material) was optimized by using three-factor-three-level Box-Behnken design (BBD). Then the data was analyzed by Design-Expert Software, and the optimum extraction parameters were obtained as follows:extracting temperature94℃, extracting time4h, ratio of water to raw material19ml/g. Under optimum extraction conditions, the real extraction yield of SFPS was10.02%.
The effect of operation pressures, ultrafiltration temperature and pH value on membrane flux were investigated through the single factor experiment and multi-factor orthogonal experiment design in sequence, and the optimized conditions were obtained, which as follows:operation pressure was0.35Mpa, temperature was30℃and pH value was6.5. Subsequently, the best washing method of membrane was determined by measuring the change of membrane flux before and after cleaning, and the results showed that the cleaning effect was best after washing for0.5h using pH8.5NaOH solution at40℃. The results showed ultrafiltration concentration is superior to conventional concentration.
2Isolation, purification, physicochemical characterization and preliminary structure analysis of SFPSA
The crude SFPS were purified by DEAE-cellulose anion-exchange column chromatography and Sephadex G-100gel-filtration column chromatography successively. One mainly fraction, named as SFPSA, was obtained. The recovery rates of SFPSA based on the amount of crude HCPS were12.57%. Purity of SFPSA was further confirmed by using HPLC, and the results showed that it was homogenerous polysaccharide and the average molecular weight is168.30kDa.
The total sugar content of SFPSA was detected by sulfuric acid-phenol coloration method, and the results were78.41±5.11%and56.17±3.94%, using glucose and sugar mixture as standard solution respectively. And results of the latter was stable in12h, had good reproducibility, and the average recovery was97.85%.
Uronic acid content, sulfuric radical content and protein content of SFPSA were determined by m-hydroxybiphenyl assay, gelatin-barium chloride assay and coomassie brilliant blue coloration method, respectively. The results were as follows:uronic acid content was25.20±1.88%, sulfuric radical content was1.98±0.21%, protein content in SFPSA was1.28±0.04%, respectively.
Monosaccharide composition of SFPSA was determined using sugar nitriles acylation method and PMP pre-column derivatization method respectively. The result of the former showed SFPSA was mainly composed of rhamnose, arabinose, glucose and galactose with the molar ratio of8.19:37.74:3.46:50.61, and the result of the latter showed SFPSA mainly consisted of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose with the molar ratio of7.75:1.65:14.92:1.87:33.17:40.64. PMP pre-column derivatization method can comprehensively reflect the monosaccharide composition of SFPSA.
In FTIR spectrum, characteristic absorptions of polysaccharides, carboxyl group and beta pyranoid glycosidic bond were all observed in SFPSA. Results of Uv spectroscopy scanning showed that SFPSA contained a few protein, as evident by no significant absorption peaks in260-280nm place.
3Proliferation inhibitory activities of SFPSA on human colon cancer cell HT-29and preliminary research on its mechanism
Proliferation inhibitory activities of SFPSA for three tumor cell lines (BGC-823、 HGC-27and HT-29) were evaluated by MTT assay, and the most sensitive cell line to SFPSA was selected and used as the research object of anti-cancer mechanism. Results showed SFPSA could inhibit proliferation of three cell lines in dose-and time-dependent manner. In addition, after drug administration for72h, half inhibition concentration of SFPSA on BGC-823、HGC-27and HT-29was3662.24μg/mL、1136.73μg/mL、407.52μg/mL respectively, and obviously human colon cancer HT-29cells was more sensitive to SFPSA than BGC-823and HGC-27.
Study on the cell morphology showed that SFPSA could significantly inhibit proliferation of HT-29, and the concrete performance was that the cell density decreased, intercellular increased, and "cluster" characteristics lost with the increase of sample dose, and finally died because of losing adhesion. After Hoechst33258staining, apoptosis characteristics of HT-29cells were observed with fluorescence microscopy. The results showed apoptosis pieces appeared in the nucleus of HT-29cells, indicating SFPSA administration could induce apoptosis of human colon cancer HT-29cells.
Quantification of apoptotic cells was performed using an Annexin-V-FITC Apoptosis Detection Kit according to the protocols provided by the manufacturer, and apoptotic cells was analyzed using flow cytometry. The results showed that after SFPSA treatment for48h, apoptosis rate in HT-29cell increased from11.3±2.07%to47.4±0.98%along with the increase of SFPSA concentration. The effect of SFPSA on HT-29colon cancer cell cycle phase distribution was also assessed using flow cytometry. And the results showed that after SFPSA treatment for48h, the proportion of cells in the G0/G1phase decreased from42.35±3.21%to24.38±4.01%, while the G2/M phase cells increased from31.57±2.18%to56.84±4.12%, indicating that SFPSA might induced cell apoptosis by arresting G2/M phase of HT-29cell division.
The effect of SFPSA on mRNA relative expression levels of apoptosis-related genes such as Bax, Bcl-2and p53in HT-29cells were analyzed using real-time quantitative PCR. The results showed that after SFPSA treatment for48h, mRNA relative expression levels of the Bax gene and p53gene as the pro-apoptotic members increased with the SFPSA concentration increased, and compared with the control, the former increased by1.21times and1.67times respectively, while the latter increased by1.26times and1.74times respectively. In contrast, mRNA relative expression levels of Bcl-2gene as one of antiapoptotic members markedly decreased at3.79times and11.3times respectively with the incresment of SFPSA concentration. In addition, Caspase-3activation degree was studied by spectrophotometry, and the result showed the activity of Caspase-3in HT-29cells increased because of SFPSA administration.
Above results showed SFPSA might induce apoptosis of HT-29cells through up-regulation of p53mRNA expression, thereby inducing up-regulation of Bax mRNA expression and down-regulation of Bcl-2mRNA expression in SFPSA-treated cells, and finally causing Caspases amplification cascade.
4Immunomodulatory activity of SFPSA
Immunomodulating activities in vitro of SFPSA were evaluated by using the cell model experiments. Results showed that SFPSA could promote proliferation of spleen lymphocyte and the release of IFN-y, and also activate the peritoneal macrophages, urging them to release the immune reactive molecules such as NO and IL-6, and enhancing the acidic phosphatase activity and the ability of devouring neutral red of peritoneal macrophages. These results showed SFPSA had bidirectional immunomodulatory ability, that is, it could promote the immune function in the low or medium dose, and had some degree of immunosuppressive activity in high dose.
Immunomodulating activities in vivo of SFPSA were measured by using the animal model experiments. Results showed that administration of SFPSA could significantly increase the index of spleen, activity of LZM in spleen and serum, the swelling rate of ear in DTH and hemolysin levels in serum of immune suppress mice in a dose-dependent manner. These results suggested that SFPSA could improve nonspecific immunity and specific immune ability of Cy-immunosuppressed mice.
1银条多糖提取工艺及超滤浓缩的研究
选择提取温度、提取时间、液料比及提取次数4个因素分别进行单因素实验,在单因素实验基础上,选择提取温度、提取时间及液料比3个参数进行三因素三水平的Box-Behnken实验(BBD),对此3个参数的实验组合进行优化,通过Design-Expert软件对数据进行处理,得出银条多糖的最优提取条件为提取温度94℃,提取时间4h,液料比19mL/g。在此条件下银条多糖的提取率为9.98±0.60%。
选择50kDa超滤膜,以膜通量为指标,首先研究了操作压力、超滤温度和料液pH值3个工艺参数对银条提取液超滤效果的影响,并在此基础上通过正交实验确定超滤浓缩的最佳工艺参数,最后通过测定清洗前后膜通量的变化确定最佳的超滤膜清洗方法。结果表明：最佳超滤工艺参数为操作压力0.35MPa,操作温度30℃,料液pH6.5；最佳清洗方法为40℃下用pH值为8.5的NaOH溶液清洗0.5h；超滤浓缩效果优于常规浓缩。
2银条多糖分离纯化、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银条多糖进行分级纯化,获得一个主要纯化组分SFPSA,得率是12.57%。采用HPLC法对SFPSA的纯度进行进一步鉴定,结果表明SFPSA是均一的多糖组分,其相对平均分子量为：168.30kDa。
采用苯酚硫酸法,以混合糖为标准品测定SFPSA的总糖含量78.41±5.11%,比单用葡萄糖为标准品测定的结果(56.17%±3.94%)要高,而且混标法测定SFPSA中总糖含量重现性好,平均回收率可达97.85%,12h内结果稳定。
分别采用间羟联苯法、氯化钡-明胶法、考马斯亮蓝法对SFPSA中糖醛酸含量、硫酸基含量、蛋白质含量进行了测定。结果表明,SFPSA中糖醛酸含量25.20±1.88%,硫酸基含量1.98±0.21%和蛋白质含量分别为1.28±0.04%。
分别采用糖腈乙酰化法和PMP柱前衍生化法测定SFPSA单糖组成。结果表明,糖腈乙酰化法测定的SFPSA中含鼠李糖,阿拉伯糖,葡萄糖和半乳糖等单糖组分,其摩尔百分比为8.19：37.74：3.46：50.61；PMP柱前衍生化法测定的SFPSA主要由六种单糖组成,分别是鼠李糖(Rha)、葡萄糖醛酸(GlcA)、半乳糖醛酸(GlaA)、葡萄糖(Glc)、半乳糖(G1a)和阿拉伯糖(Ara),其摩尔百分含量比为7.75：1.65：14.92：1.87：33.17：40.64,该结果说明SFPSA是一种主要由半乳糖、半乳糖醛酸和阿拉伯糖组成的杂多糖。而且,PMP柱前衍生化法能够更加全面的反映样品的单糖组成。
红外光谱扫描表明,SFPSA具有多糖特征吸收峰,而且具有β-吡喃糖苷键的特征吸收峰。紫外光谱扫描结果显示,SFPSA在280nn处没有显著的吸收峰,这表明SFPSA中蛋白质含量很低。
3银条多糖抗结肠癌活性及其机理的初步研究
采用MTT法考察了SFPSA对三种肿瘤细胞株(BGC-823、HGC-27和HT-29)增殖的抑制作用,并选取其中对SFPSA最敏感的肿瘤细胞株,进一步研究SFPSA对该细胞株增殖抑制作用的机理。实验结果表明,SFPSA能抑制这三种肿瘤细胞的增殖,且这种抑制作用均呈现一定的浓度和时间的依赖性。另外,给药72h后,SFPSA对三种肿瘤细胞的半数抑制浓度(IC50)分别为3662.24μg/mL、1136.73μg/mL、407.52μg/mL,由此可见,人结肠癌HT-29细胞对SFPSA最为敏感。
细胞形态学研究证明SFPSA可以显著的抑制人结肠癌HT-29细胞的生长,且随剂量的增长细胞密度逐渐下降,细胞间隙变大,失去“群集”特性,最后失去粘附性而死亡。Hoechst33258染色后,荧光显微镜观察显示其细胞核内出现凋亡碎片,即呈现显著的凋亡特征,这表明SFPSA处理可以引起人结肠癌HT-29细胞凋亡。
FITC-Annexin V/PI双染色和流式细胞仪检测结果表明,经SFPSA处理HT-29细胞48h后,随着SFPSA处理浓度的增加,HT-29细胞的凋亡率从11.3±2.07%上升到47.4±0.98%。细胞周期分析结果显示,经SFPSA处理HT-29细胞48h后,随着SFPSA处理浓度的增加,HT-29细胞中G0/G1期细胞比例从42.35±3.21%下降到24.38±4.01%,而G2、M期细胞比例从31.57±2.18%上升到56.84±4.12%,表明SFPSA可以通过在HT-29细胞中产生G2/M阻断,使其无法进入有丝分裂过程而促使其进入凋亡过程。
采用荧光定量PCR分析SFPSA对HT-29细胞中凋亡相关基因Bax、Bcl-2和p53的mRNA表达水平的影响。结果表明,SFPSA处理HT-29细胞48h后,随着SFPSA处理浓度的增加,促凋亡成员Bax和p53的mRNA相对表达水平上升,与对照相比,前者分别增加了1.21倍和1.67倍,后者分别增加了1.26倍和1.74倍。而抑凋亡成员Bcl-2的mRNA相对表达水平下降,与对照相比,分别下降了3.79倍和11.3倍。另外,分光光度法研究Caspase-3活化度的结果表明,HT-29细胞经SFPSA处理48h后,细胞中Caspase-3被活化。
根据以上结果,我们可以推断SFPSA处理HT-29细胞后,可能通过上调p53表达,进而诱导Bax表达增加和Bcl-2表达减少,最后引起Caspases的级联放大反应而最终导致细胞凋亡。
4银条多糖免疫调节活性
运用体外细胞模型实验研究了SFPSA的体外免疫调节活性。结果表明：SFPSA能促进小鼠脾淋巴细胞增殖,促其释放IFN-y,且能活化小鼠腹腔巨噬细胞,促其释放免疫活性分子NO和IL-6,增强其酸性磷酸酶活性和吞噬中性红的能力。而且SFPSA的这种体外免疫调节能力具有双向性,即在低浓度下具有免疫促进作用,而在高浓度下则开始表现为不同程度的免疫抑制作用。
采用环磷酰胺诱导的免疫抑制小鼠模型研究了SFPSA的体内免疫调节活性。结果显示,SFPSA可以使因环磷酰胺(Cy)诱导产生的免疫功能低下得以恢复,具体表现为SFPSA处理可以显著地提高脾脏指数、迟发型超敏反应(DTH)的耳肿胀度、脾脏和血清中溶菌酶(LZM)的活性和血清中溶血素水平,且这种变化与SFPSA呈现剂量依赖关系。这些结果表明,SFPSA不仅能够提高免疫抑制小鼠的非特异性免疫能力,还可以提高其特异性免疫能力。
Stachys floridana Schuttl. ex Benth, also called as "yintiao", is a perennial herbaceous plant belonging to the Stachys genus of Lamiaceae and distributes mainly in North of China, South of China and Xinjiang uygur autonomous region of China in half-wild state. It is artificial cultivated mainly in Yanshi City Henan province, accounting for more than95%of the total cultivation. However, recent studies about "yintiao" were mainly concentrated on its breeding, cultivation and fresh-keeping and so on, and there is no report about active substances such as polysaccharides from the rhizome of Stachys floridana Schuttl. ex Benth (SFPS). In this thesis, SFPS was studied systemically, including optimization of extraction condition and parameters of ultrafiltration concentration, isolation and purification, physicochemical characterization, antitumor and immunomodulatory activity. Main results are listed as follows:
1Research on extraction condition and ultrafiltration concentration of SFPS
Extraction temperature, extraction time, ratio of water to raw material and extraction times were selected in single-factor experiments. Based on the single-factor experiments, combination of the extraction parameters (time, temperature and ratio of water to raw material) was optimized by using three-factor-three-level Box-Behnken design (BBD). Then the data was analyzed by Design-Expert Software, and the optimum extraction parameters were obtained as follows:extracting temperature94℃, extracting time4h, ratio of water to raw material19ml/g. Under optimum extraction conditions, the real extraction yield of SFPS was10.02%.
The effect of operation pressures, ultrafiltration temperature and pH value on membrane flux were investigated through the single factor experiment and multi-factor orthogonal experiment design in sequence, and the optimized conditions were obtained, which as follows:operation pressure was0.35Mpa, temperature was30℃and pH value was6.5. Subsequently, the best washing method of membrane was determined by measuring the change of membrane flux before and after cleaning, and the results showed that the cleaning effect was best after washing for0.5h using pH8.5NaOH solution at40℃. The results showed ultrafiltration concentration is superior to conventional concentration.
2Isolation, purification, physicochemical characterization and preliminary structure analysis of SFPSA
The crude SFPS were purified by DEAE-cellulose anion-exchange column chromatography and Sephadex G-100gel-filtration column chromatography successively. One mainly fraction, named as SFPSA, was obtained. The recovery rates of SFPSA based on the amount of crude HCPS were12.57%. Purity of SFPSA was further confirmed by using HPLC, and the results showed that it was homogenerous polysaccharide and the average molecular weight is168.30kDa.
The total sugar content of SFPSA was detected by sulfuric acid-phenol coloration method, and the results were78.41±5.11%and56.17±3.94%, using glucose and sugar mixture as standard solution respectively. And results of the latter was stable in12h, had good reproducibility, and the average recovery was97.85%.
Uronic acid content, sulfuric radical content and protein content of SFPSA were determined by m-hydroxybiphenyl assay, gelatin-barium chloride assay and coomassie brilliant blue coloration method, respectively. The results were as follows:uronic acid content was25.20±1.88%, sulfuric radical content was1.98±0.21%, protein content in SFPSA was1.28±0.04%, respectively.
Monosaccharide composition of SFPSA was determined using sugar nitriles acylation method and PMP pre-column derivatization method respectively. The result of the former showed SFPSA was mainly composed of rhamnose, arabinose, glucose and galactose with the molar ratio of8.19:37.74:3.46:50.61, and the result of the latter showed SFPSA mainly consisted of rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose with the molar ratio of7.75:1.65:14.92:1.87:33.17:40.64. PMP pre-column derivatization method can comprehensively reflect the monosaccharide composition of SFPSA.
In FTIR spectrum, characteristic absorptions of polysaccharides, carboxyl group and beta pyranoid glycosidic bond were all observed in SFPSA. Results of Uv spectroscopy scanning showed that SFPSA contained a few protein, as evident by no significant absorption peaks in260-280nm place.
3Proliferation inhibitory activities of SFPSA on human colon cancer cell HT-29and preliminary research on its mechanism
Proliferation inhibitory activities of SFPSA for three tumor cell lines (BGC-823、 HGC-27and HT-29) were evaluated by MTT assay, and the most sensitive cell line to SFPSA was selected and used as the research object of anti-cancer mechanism. Results showed SFPSA could inhibit proliferation of three cell lines in dose-and time-dependent manner. In addition, after drug administration for72h, half inhibition concentration of SFPSA on BGC-823、HGC-27and HT-29was3662.24μg/mL、1136.73μg/mL、407.52μg/mL respectively, and obviously human colon cancer HT-29cells was more sensitive to SFPSA than BGC-823and HGC-27.
Study on the cell morphology showed that SFPSA could significantly inhibit proliferation of HT-29, and the concrete performance was that the cell density decreased, intercellular increased, and "cluster" characteristics lost with the increase of sample dose, and finally died because of losing adhesion. After Hoechst33258staining, apoptosis characteristics of HT-29cells were observed with fluorescence microscopy. The results showed apoptosis pieces appeared in the nucleus of HT-29cells, indicating SFPSA administration could induce apoptosis of human colon cancer HT-29cells.
Quantification of apoptotic cells was performed using an Annexin-V-FITC Apoptosis Detection Kit according to the protocols provided by the manufacturer, and apoptotic cells was analyzed using flow cytometry. The results showed that after SFPSA treatment for48h, apoptosis rate in HT-29cell increased from11.3±2.07%to47.4±0.98%along with the increase of SFPSA concentration. The effect of SFPSA on HT-29colon cancer cell cycle phase distribution was also assessed using flow cytometry. And the results showed that after SFPSA treatment for48h, the proportion of cells in the G0/G1phase decreased from42.35±3.21%to24.38±4.01%, while the G2/M phase cells increased from31.57±2.18%to56.84±4.12%, indicating that SFPSA might induced cell apoptosis by arresting G2/M phase of HT-29cell division.
The effect of SFPSA on mRNA relative expression levels of apoptosis-related genes such as Bax, Bcl-2and p53in HT-29cells were analyzed using real-time quantitative PCR. The results showed that after SFPSA treatment for48h, mRNA relative expression levels of the Bax gene and p53gene as the pro-apoptotic members increased with the SFPSA concentration increased, and compared with the control, the former increased by1.21times and1.67times respectively, while the latter increased by1.26times and1.74times respectively. In contrast, mRNA relative expression levels of Bcl-2gene as one of antiapoptotic members markedly decreased at3.79times and11.3times respectively with the incresment of SFPSA concentration. In addition, Caspase-3activation degree was studied by spectrophotometry, and the result showed the activity of Caspase-3in HT-29cells increased because of SFPSA administration.
Above results showed SFPSA might induce apoptosis of HT-29cells through up-regulation of p53mRNA expression, thereby inducing up-regulation of Bax mRNA expression and down-regulation of Bcl-2mRNA expression in SFPSA-treated cells, and finally causing Caspases amplification cascade.
4Immunomodulatory activity of SFPSA
Immunomodulating activities in vitro of SFPSA were evaluated by using the cell model experiments. Results showed that SFPSA could promote proliferation of spleen lymphocyte and the release of IFN-y, and also activate the peritoneal macrophages, urging them to release the immune reactive molecules such as NO and IL-6, and enhancing the acidic phosphatase activity and the ability of devouring neutral red of peritoneal macrophages. These results showed SFPSA had bidirectional immunomodulatory ability, that is, it could promote the immune function in the low or medium dose, and had some degree of immunosuppressive activity in high dose.
Immunomodulating activities in vivo of SFPSA were measured by using the animal model experiments. Results showed that administration of SFPSA could significantly increase the index of spleen, activity of LZM in spleen and serum, the swelling rate of ear in DTH and hemolysin levels in serum of immune suppress mice in a dose-dependent manner. These results suggested that SFPSA could improve nonspecific immunity and specific immune ability of Cy-immunosuppressed mice.
引文
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