摘要
白藜芦醇(Resveratrol)是植物体内一种天然的二苯烯类多酚物质,是在外来病菌侵入、紫外线照射等不利因素影响下,由植物产生的一种植物抗毒素。具有明显抗菌、抗炎、抗癌、抗高血压脂症、抗脂质过氧化等多方面的活性。利用现代植物组培技术直接获得次生代谢产物用于药物生产,具有保护植物资源、生产周期短、不受地区和季节限制、个体差异小、便于工业化生产等优点。然而,大多数有用的次生代谢物质在植物细胞中含量极低,为此,在工业化生产中首先需对植物细胞进行选育和改良,以提高植物细胞中次生物质含量。利用称重法称量,采用有机溶剂法提取,HPLC法分析测定,从而考察了外源添加物、不同碳源、光照条件、紫外处理及不同收获时间对葡萄愈伤组织生长及其中白藜芦醇生成量的影响。结果表明,苯丙氨酸、肉桂酸、水杨酸、β-环糊精、秋水仙素、硝酸银的添加均可有效提高该细胞内白藜芦醇的含量,而且有明显的剂量效应,在所试范围内,其适宜浓度分别为30、30、40、40、5、30 mg·L-1;除秋水仙素外,其他几种添加物对愈伤组织的增殖无显著的促进作用,肉桂酸、硝酸银和水杨酸还有一定的抑制作用。就几种不同碳源来说,增殖效果是蔗糖的优于其他两种糖,而这两种糖对白藜芦醇的累积作用又优于蔗糖的,但是随着渗透压的增加,仅蔗糖对愈伤组织生长和白藜芦醇积累有促进作用,而葡萄糖和麦芽糖却有一定的抑制作用;几种光质对该愈伤组织的增殖效果由高到低依次为黄光、绿光、红光、蓝光、白光,对白藜芦醇的累积作用由强到弱依次为白光、黄光、红光、蓝光,绿光下生长的愈伤组织中未检测到白藜芦醇。经紫外处理的愈伤组织中白藜芦醇含量随处理时间的延长先增加后减少,处理10 min的(104μg·g~(-1))远高于处理时间大于15 min的,然而随着处理强度的增强呈现上升趋势,4.0 W·m~(-2)强度下处理过的愈伤组织中其含量比未处理的高出约4.2倍,比在0.05 W·m~(-2)下处理的高出3.3倍。在接种后的第9 d,白藜芦醇的生成量最大,达64.5μg·g~(-1),之后开始逐渐下降,45 d后只检测到3.7μg·g~(-1)。
Resveratrol, a known antioxidant compound naturally produced in plants as a self-defense agent in response to exogenous stimuli such as pathogen attack, ozone treatment, pre-harvest and post-harvest UV-C irradiation, etc, belongs to the phenolic phytoalexins group and stilbenes. Resveratrol has many biological activities, i.e. estrogenic activity, cardiovascular protective effect, neuroprotective capacity, and cancer chemopreventive activity. The treatment of plant cells with biotic and abiotic elicitors has been one of the most effective means to improve the yields of secondary metabolites in plant cell cultures. In our study, weighing method for growth, organic solvent method for extraction and HPLC method for determination were used to study the influences of supplementary materials (phenylalanine, cinnamic acid, salicylic acid,β-cyclodextrin, colchicines and silver nitrate), carbon sources (sucrose, glucose and maltose), light quality (red, yellow, blue, green and white light), UV-C treatment and harvest time on the growth of the callus from Pinot Noir and its resveratrol accumulation. The results showed that 30, 30, 40, 40, 5, 30 mg·L-1 are the optimal concentrations in turn for resveratrol-forming; except colchicines, the other additions did not show any stimulative effect on the multiplication, and cinnamic acid, salicylic acid and silver nitrate inhabited its growth. Sucrose was superior to the other two for the multiplication but inferior to those for resveratrol accumulation, however, as the osmotic pressure increased, only sucrose can enhance the callus growth and resveratrol-forming which was suppressed by glucose and maltose. The order of multiplication effects from the high to low was yellow, green, red, blue, white light, and that of resveratrol accumulation was white, yellow, red, blue, green, but resveratrol was not detected in the callus growing under green light. With the treatment of UV-C, longer periods of irradiation considerably weakened the capacity of the calli to synthesis resveratrol, and with exposure time of 10min, maximal induction took place (104μg·g~(-1)); as the intensity of the light increased, the content of resveratrol was enhanced obviously, 0.4 W·m~(-2) made the content of resveratrol increase to 170μg·g~(-1), 4.2 and 3.3 times more than the control and 0.05 W·m~(-2) did respectively. 9 days after the inoculation of the callus, 64.5μg·g~(-1) resveratrol formed, which was the highest content in the whole growth period, and only 3.7μg·g~(-1) was detected on the 45th day.
引文
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