Li Huaizhi, Cheng Yan, Dong Hongjing, Wang Xiao, Li Jia, Gao Qianshan
Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China; College of Pharmacy, Shandong University of Traditional Chinese Medicine, 4655 Daxue Street, Jinan, Shandong 250355, China.
Key Laboratory of TCM Quality Control Technology, Shandong Analysis and Test Center, Shandong Academy of Sciences, 19 Keyuan Street, Jinan, Shandong 250014, China.
J Chromatogr A. 2016 Oct 14;1468:42-48. doi: 10.1016/j.chroma.2016.09.039. Epub 2016 Sep 20.
Salvianolic acid A is the major bioactive compound in Danshen, however, due to the chemical instability and low content in Danshen, it is difficult to extract amount of salvianolic acid A. Therefore, this study was to establish an effective strategy for obtaining adequate amount of salvianolic acid A, subcritical water extraction was used to degrade salvianolic acid B and prepare salvianolic acid A. Different reaction conditions including temperature, time, concentration and pH value in subcritical water were investigated. Under 40mg/mL of reactant concentration, 180°C of temperature, 4.0 of pH value and 60min of reaction time, the highest yield rate of salvianolic acid A reached 34.86%. Then, the degradation products were successfully separated by pH-zone-refining counter-current chromatography with the solvent system Pet-EtAc-n-BuOH-HO (2:3:1:9, v/v), where 10mM TFA was added in stationary phase and 10mM NH·HO in mobile phase. As a result, a total of 227.3mg of salvianolic acid A at 98.2% purity, 38.9mg of danshensu at 99.3% purity, 9.5mg of salvianolic acid D at 92.7% purity, and 32.8mg of protocatechuic aldehyde at 93.1% purity were obtained from 1.2g degradation products of salvianolic acid B by one-step purification. The results demonstrated that the combinative application of subcritical water and pH-zone-refining counter-current chromatography is a potential technique for the preparative separation of salvianolic acid A from salvianolic acid B.
丹酚酸A是丹参中的主要生物活性成分，然而，由于其化学稳定性差且在丹参中含量较低，难以提取到足量的丹酚酸A。因此，本研究旨在建立一种获取足量丹酚酸A的有效策略，采用亚临界水萃取法降解丹酚酸B并制备丹酚酸A。考察了亚临界水中不同的反应条件，包括温度、时间、浓度和pH值。在反应物浓度为40mg/mL、温度为180°C、pH值为4.0、反应时间为60min的条件下，丹酚酸A的最高产率达到34.86%。然后，采用pH-区带精制逆流色谱法，以Pet-EtAc-n-BuOH-H2O（2:3:1:9，v/v）为溶剂体系，在固定相中加入10mM三氟乙酸，流动相中加入10mM氨水，成功分离了降解产物。结果，从1.2g丹酚酸B降解产物中一步纯化得到了纯度为98.2%的丹酚酸A共227.3mg、纯度为99.3%的丹参素38.9mg、纯度为92.7%的丹酚酸D 9.5mg以及纯度为93.1%的原儿茶醛32.8mg。结果表明，亚临界水与pH-区带精制逆流色谱法的联合应用是从丹酚酸B中制备分离丹酚酸A的一种潜在技术。
