|| STUDIES ON THE CULTURE SYSTEM OF SCUTELLARIA BAICALENSIS GEORGI HAIRY ROOTS Ⅱ|
|| Scutellaria baicalensis Georgi, hairy root culture, baicalin, carbohydrate, elicitor, hormone|
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。
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%。
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.