螺旋藻的优化培养与抗肿瘤作用初探
摘要
螺旋藻因其富含蛋白质、多糖、不饱和脂肪酸、维生素、矿物质等多种营养成分,而被FAO和WHO分别誉为“21世纪最理想的食品”和“人类21世纪的最佳保健品”。现今的工业化生产仅以螺旋藻的生物量为指标,各种活性成分的产量低,远不能满足市场需求。而螺旋藻代谢产物与培养环境密切相关,故本课题研究了营养盐、光照、温度和植物生长调节剂对螺旋藻生物量、蛋白、胞内多糖(intracellular polysaccharide,IPS)、胞外多糖(extracellular polysaccharide,EPS)和超氧化物歧化酶(superoxide dismutases,SOD)含量的影响,以期找到高产各种成分的螺旋藻的最佳培养条件。同时,初步研究了螺旋藻EPS、IPS和PC对Hela细胞的抑制作用和机理。
研究结果表明,当C、N、P、NaCl的浓度(g/L)配比为A(18,2.5,0.5,1.0),生物量提高了0.69倍;B(14,2.5,0.5,0.5),EPS提高了0.49倍;C(10,2.5,0.8,1.5)时,IPS提高了0.71倍;D(18,2.5,0.2,1.5),蛋白含量提高了0.33倍。极大螺旋藻438的最适培养条件为:光强4000lux～5000lux,温度30～35℃,蛋白和IPS积累的条件与螺旋藻最适生长条件一致。对极大螺旋藻生长不利的温度、光照条件,会促进螺旋藻EPS的分泌和SOD的活性,即高温和弱光更利于胞外多糖的积累,低温和强光显著提高SOD酶活力。低浓度植物生长调节剂显著促进了螺旋藻的生长和蛋白的含量,高浓度对其生长有抑制作用。但高浓度有利于EPS的产生,提高了SOD酶活力。此研究结果确定了螺旋藻生长和有利于多糖、蛋白质和SOD累积的最佳培养条件,为通过优化培养条件,调控活性物质提供了理论依据。
体外实验证明IPS、EPS和PC对Hela细胞增殖都有抑制作用,且存在明显的剂量效应。三者抑制率随浓度的增加而升高,在最佳剂量80μg/mL、100μg/mL、120μg/mL的作用下,抑制率分别达到为47.92%、42.95%、31.22%。采用Caspase-3试剂盒检测它们对Hela细胞Caspase-3的活性的影响,发现经过36h、24h、12h后Caspase-3活性达到最高,分别为对照组的4.06倍、6.64倍、5.70倍。推断其抗肿瘤的机制可能是通过启动Caspase途径从而诱导HeLa细胞的凋亡。
Spirulina were hailed as "the perfect food in the 21st century"and "the optimal health product in the 21st century" by FAO and WHO due to its nutritional value and the ability to produce valuable chemical compounds,such as protein,polysaccharide, unsaturated fatty acids,vitamins,etc.In recent years,there is a great market demand for Spirulina.The biomass was consideed as the sole indicator in the current culture, so the output of active ingredient is far from satisfying the needs of the market. Microalgal biomass and metabolites are close related to its culture conditions. Therefore,it is significant to optimize the cultural conditions of spirulina for realizing the high biomass and achieving nutritional ingredients from Spirulina for different purpose.The effects of medium,temperature,light intensity and phytohormones on the growth and the metabolites of protein,intracellular polysaccharide(IPS), extracellular polysaccharide(EPS),superoxide dismutases(SOD) were studied in a systematic way.At the same time,the anti-tumor effect of EPS,IPS and PC was preliminary studied.
The results showed that,when the concentration of sodium bicarbonate,nitrogen sources,phosphate,and sodium chloride in the medium was proportioned as A(g/L)(18,2.5,0.5,1.0),B(14,2.5,0.5,0.5),C(10,2.5,0.8,1.5),D(18,2.5,0.2,1.5), the metabolites of biomass,EPS,IPS and protein in the Spirulina was as 0.69,0.49, 0.71,0.33 times those of original medium respectively.It was evident that the biomass,IPS,EPS and protein of Spirulina all increased outstandingly through optimization of the medium ingredient.It was found that the light intensity was at the range of 4000 lux to 5000 lux and the temperature was in the range of 30～35℃was the optimum condition for the growth of Spirulina.The optimum temperature and light for excreting of IPS were consistent with optimum condition for the growth of Spirulina.Illumination and temperture unsuitable for the gowth of Spirulina would raise EPS content and SOD activity.That is,high temperature and feeble illumination were favorable to the excreting of EPS.SOD activity was increased notably under the condition of low temperature and strong light.Lower concentration of phytohormones significantly promoted the growth and protein content of Spirulina,higher concentrations inhibited its growth but accelerated EPS and enhanced SOD activity. The optimized cultural conditions were ascertained,so as to provid the theory and practical parameters of scale-up exploitation protein,polysaccharides and SOD form Spirulina in the functional food and pharmacy.
The antitumor function of IPS,EPS and PC by MTT assay indicated that the optimum dosage were 80μg/mL,100μg/mL,120μg/mL and the inhibition rates were achieved 47.92%,42.95%and 31.22%respectively.Caspase-3 activity was measured by Caspase-3 kit.Caspase-3 activity achieved to the highest,that was as 4.06,6.64, 5.70 times as the control group respectively at 36h,24h,12h after being added the IPS, EPS and PC.It can be inferred that the mechanism of antitumor effect of three extracts of Spirulina was through activating the Caspase-3 protease family,thus inducing cell apoptosis.
研究结果表明,当C、N、P、NaCl的浓度(g/L)配比为A(18,2.5,0.5,1.0),生物量提高了0.69倍;B(14,2.5,0.5,0.5),EPS提高了0.49倍;C(10,2.5,0.8,1.5)时,IPS提高了0.71倍;D(18,2.5,0.2,1.5),蛋白含量提高了0.33倍。极大螺旋藻438的最适培养条件为:光强4000lux～5000lux,温度30～35℃,蛋白和IPS积累的条件与螺旋藻最适生长条件一致。对极大螺旋藻生长不利的温度、光照条件,会促进螺旋藻EPS的分泌和SOD的活性,即高温和弱光更利于胞外多糖的积累,低温和强光显著提高SOD酶活力。低浓度植物生长调节剂显著促进了螺旋藻的生长和蛋白的含量,高浓度对其生长有抑制作用。但高浓度有利于EPS的产生,提高了SOD酶活力。此研究结果确定了螺旋藻生长和有利于多糖、蛋白质和SOD累积的最佳培养条件,为通过优化培养条件,调控活性物质提供了理论依据。
体外实验证明IPS、EPS和PC对Hela细胞增殖都有抑制作用,且存在明显的剂量效应。三者抑制率随浓度的增加而升高,在最佳剂量80μg/mL、100μg/mL、120μg/mL的作用下,抑制率分别达到为47.92%、42.95%、31.22%。采用Caspase-3试剂盒检测它们对Hela细胞Caspase-3的活性的影响,发现经过36h、24h、12h后Caspase-3活性达到最高,分别为对照组的4.06倍、6.64倍、5.70倍。推断其抗肿瘤的机制可能是通过启动Caspase途径从而诱导HeLa细胞的凋亡。
Spirulina were hailed as "the perfect food in the 21st century"and "the optimal health product in the 21st century" by FAO and WHO due to its nutritional value and the ability to produce valuable chemical compounds,such as protein,polysaccharide, unsaturated fatty acids,vitamins,etc.In recent years,there is a great market demand for Spirulina.The biomass was consideed as the sole indicator in the current culture, so the output of active ingredient is far from satisfying the needs of the market. Microalgal biomass and metabolites are close related to its culture conditions. Therefore,it is significant to optimize the cultural conditions of spirulina for realizing the high biomass and achieving nutritional ingredients from Spirulina for different purpose.The effects of medium,temperature,light intensity and phytohormones on the growth and the metabolites of protein,intracellular polysaccharide(IPS), extracellular polysaccharide(EPS),superoxide dismutases(SOD) were studied in a systematic way.At the same time,the anti-tumor effect of EPS,IPS and PC was preliminary studied.
The results showed that,when the concentration of sodium bicarbonate,nitrogen sources,phosphate,and sodium chloride in the medium was proportioned as A(g/L)(18,2.5,0.5,1.0),B(14,2.5,0.5,0.5),C(10,2.5,0.8,1.5),D(18,2.5,0.2,1.5), the metabolites of biomass,EPS,IPS and protein in the Spirulina was as 0.69,0.49, 0.71,0.33 times those of original medium respectively.It was evident that the biomass,IPS,EPS and protein of Spirulina all increased outstandingly through optimization of the medium ingredient.It was found that the light intensity was at the range of 4000 lux to 5000 lux and the temperature was in the range of 30～35℃was the optimum condition for the growth of Spirulina.The optimum temperature and light for excreting of IPS were consistent with optimum condition for the growth of Spirulina.Illumination and temperture unsuitable for the gowth of Spirulina would raise EPS content and SOD activity.That is,high temperature and feeble illumination were favorable to the excreting of EPS.SOD activity was increased notably under the condition of low temperature and strong light.Lower concentration of phytohormones significantly promoted the growth and protein content of Spirulina,higher concentrations inhibited its growth but accelerated EPS and enhanced SOD activity. The optimized cultural conditions were ascertained,so as to provid the theory and practical parameters of scale-up exploitation protein,polysaccharides and SOD form Spirulina in the functional food and pharmacy.
The antitumor function of IPS,EPS and PC by MTT assay indicated that the optimum dosage were 80μg/mL,100μg/mL,120μg/mL and the inhibition rates were achieved 47.92%,42.95%and 31.22%respectively.Caspase-3 activity was measured by Caspase-3 kit.Caspase-3 activity achieved to the highest,that was as 4.06,6.64, 5.70 times as the control group respectively at 36h,24h,12h after being added the IPS, EPS and PC.It can be inferred that the mechanism of antitumor effect of three extracts of Spirulina was through activating the Caspase-3 protease family,thus inducing cell apoptosis.
引文
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