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黄芩毛状根培养体系的研究-Ⅱ-郭乐康-2009

2013年06月17日 11:52 郭乐康 点击:[]

 

 作者姓名  郭乐康
 中文论文题名  黄芩毛状根培养体系的研究-Ⅱ
 外文论文题名  STUDIES ON THE CULTURE SYSTEM OF SCUTELLARIA BAICALENSIS GEORGI HAIRY ROOTS Ⅱ
 论文提交日期  2009-05-31
 学位年度  2009
 中文论文关键词  黄芩,毛状根培养,黄芩苷,碳源,诱导子,外源激素
 外文论文关键词  Scutellaria baicalensis Georgi, hairy root culture, baicalin, carbohydrate, elicitor, hormone

 

中文论文文摘


    黄芩(Scutellaria baicalensis Georgi)为唇形科草本植物,是一味传统的常用中药。主产于河北、辽宁、陕西、山东、内蒙古等地。其根性寒,味苦,有清热燥湿、泻火解毒、止血、安胎等功效。现代药理研究表明其主要有效成分黄芩苷(baicalin)具有抗肿瘤、抗HIV等作用。以其开发新的药物前景将十分广阔,但因黄芩为多年生草本植物,生长较慢,限制黄芩产品深度开发。利用现代生物技术建立黄芩毛状根培养体系是解决中药资源可持续发展的有效途径之一。
    本论文主要对黄芩毛状根培养、黄芩苷积累规律、黄芩毛状根放大培养及高效液相法测定黄芩毛状根中黄芩苷含量等相关内容进行了研究。研究结果如下:
    1. 高效液相色谱法测定黄芩毛状根中黄芩苷含量的色谱条件为:色谱柱:化学键合型十八烷基柱(SciC18色谱柱);流动相:甲醇:水:磷酸=50:50:0.2;检测波长274nm;柱温:室温;流速:1mL/min。在该条件下,黄芩苷的保留时间为9min,在12~72μg/mL内线性关系良好(r=0.9992),平均加样回收率为99.32%,RSD=1.36%。
    2. 分别以蔗糖、葡萄糖和果糖为碳源研究了碳源对黄芩毛状根的生长和黄芩苷积累的影响。研究表明:三种碳源,黄芩毛状根生长曲线均呈“S”型;蔗糖最有利于黄芩毛状根生物量的积累,生长量(干重)最高达13.1g/L,黄芩苷含量为8.16%;果糖最适合黄芩苷的合成,黄芩苷含量最高达10.66%,生长量为11.4g/L。
    3. 温度对黄芩毛状根培养的影响研究表明:28℃培养,有利于生物量积累,最高达12.80g/L(干重),20℃培养明显有利于黄芩苷的积累,黄芩苷含量最高达8.80%。毛状根的最大生物量和次生代谢产物积累并不在同一温度。通过对变温培养条件优化研究结果显示,毛状根的最大生物量和次生代谢产物积累均有较大提高,结果:毛状根生长量(干重)为13.3g/L,黄芩苷含量为12.58%,黄芩苷产量为1.62g/L,其产量比28℃恒温培养提高了66.34%。
    4. 分别研究了诱导子、激素、前体及补加培养基对黄芩毛状根中黄芩苷产量的影响。研究表明:以黑曲霉诱导子和米曲霉诱导子浓度为40mg/L和20mg/L时,Co2+浓度为15.3×10-5 mmol/L时,黄芩苷产量较对照培养分别提高了9.18%、10.20%和13.27%;生长素NAA和细胞分裂素6-BA的浓度为0.4mg/L和0.2mg/L时,黄芩苷产量较对照培养分别提高了25.74%和24.75%;添加0.4mmol/L的苯丙氨酸,黄芩苷产量较对照培养提高了36.63%;补加培养基后毛状根培养至70d,黄芩苷产量较对照培养提高了56.44%。
    5. 黄芩毛状根放大培养的初步研究表明,毛状根在气升反应装置中能迅速生长,并有效富集黄芩苷。气升培养55d时,黄芩苷产量较摇床培养下降了22.41%,提示培养方式的改变对黄芩苷的的积累有一定的影响,需对放大培养条件进行深入研究。
    通过对黄芩毛状根生长和黄芩苷积累的研究,建立了黄芩毛状根培养体系,为规模化培养黄芩毛状根生产黄芩苷等药用成分奠定了理论基础。

 

外文论文文摘


    Scutellaria baicalensis Georgi is one kind of traditional Chinese medicines, a herb of the Labiatae genus, widely distributes in Hebei, Liaoning, Shaanxi, Shandong and Inner Mongolia, and the medical functions of it are cleaning the heat and humidity, detoxifying, stanching and soothing fetus. The modern pharmacological studies indicated that baicalin, the major functional component of S.baicalensis, has abilities of anti-tumor and anti-human immunodeficiency virus (HIV). It also has potential application value to develop baicalin to be new drugs, but for the reason of slow growth of S.baicalensis, a perennial herb, which limites development of the products made from S.baicalensis. One effective way to solve the problem of sustainable development is to establish a culture system for culturing S.baicalensis hairy roots based on some modern biotechnologies.
    In this research, the culture conditions, baicalin accumulation pattern, amplification culture and determination of baicalin by high performance liquid chromatography (HPLC) were mainly studied. The results are as follows:
    1. The determination of baicalin in hairy roots by HPLC was performed on SciC18 column with a mobile phase of methanol water (50:50), contains 0.2% phosphate. The flow rate is 1 mL/min, the temperature is room temperature and the detective wave length is 274 nm. The retention time of baicalin is 9 min and the good linearity in the range of 12~72μg/mL (r=0.9992) is got under those conditions, and the average recovery is 99.32% with RSD of 1.36%.
    2. Effects of different carbohydrates on hairy roots growth and baicalin synthesis were investigated in this study. The results showed that the growth curve all turn on an “s” appearance using sucrose, glucose and fructose as carbon sourse respectively. Sucrose is benefit to the growth of hairy roots and in the case the highest dry weight reaches to 13.1g/L and baicalin content is 8.16%. Fructose makes the accumulation of baicalin to 10.66% with the dry weight of 11.4 g/L.
    3. Temperature is an important factor affecting the hairy root culture. At 28℃, the highest dry weight is 12.80g/L, while at 20℃ the maximum baicalin content is 8.80%. The culture temperature giving the maximum baicalin content is not consistent with that giving the maximum biomass, so the method of two-step culture with different temperature was taken, and the dry weight and baicalin content are 13.3g/L and 12.58% and the yield of baicalin achieves 1.62g/L which increases by 66.34% compared to the conference (28℃).
     4. The effects of elicitors, hormones, precursor and replenish medium on the production of baicalin were studied. The results show that the yield of baicalin increases by 9.18%, 10.20% and 13.27% respectively, while concentrations of elicitors of Asp.niger, Asp.oryzae and Co2+ are 40mg/L, 20mg/L and 15.3×10-5 mmol/L respectively. The yield of baicalin increases by 25.74% and 24.75%, while concentrations of NAA and 6-BA are 0.4mg/L and 0.2 mg/L. The baicalin yield increases by 36.63% is achieved, when 0.4mmol/L phenylanine is added. The baicalin yield increases by 56.44% on 70 days after the medium was replenished.
    5. The research of amplification culture shows that the hairy roots of S.baicalensis grew rapidly and accumulated baicalin efficiently in air-lift reactor. The baicalin production decreased 22.41% compared to rotational culture implying the culture style influences the accumulation of baicalin in some extent.
    The culture system of hairy root of S.baicalensis was established by studying different factors affecting on biomass and baicalin accumulation, which builds the theoretical foundation for production of baicalin by hairy roots of S.baicalensis in a large-scale.

    

 

 

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