1. College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, China
2. Department of Hematology, Jiangsu Hospital of Traditional Chinese Medicine，Nanjing 210029, China
3. Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, Nanjing 210023, China
4. National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing 210023, China
摘要： 研究泽泻醇类化合物23-乙酰泽泻醇B(alisol B 23-acetate, 23B)、24-乙酰泽泻醇A(alisol A 24-acetate, 24A)混合物(24A∶23B含量比=1∶1)与抑癌基因p53DNA的作用机理，探讨泽泻醇类化合物抗肿瘤作用的分子机制。紫外-可见吸收光谱法、荧光光谱法与分子模拟联用探讨23B，24A及24A-23B混合物与p53DNA的作用方式。紫外光谱显示泽泻醇单体与其混合物部分嵌插入p53DNA，他们使p53DNA紫外吸收降低的程度为：24A∶23B(1∶1)>23B>24A。荧光光谱显示泽泻醇单体及其混合物与p53DNA的相互作用模式均为嵌插结合，结合强度为：24A∶23B(1∶1)>23B>24A。分子模拟显示，泽泻醇单体及其混合物与p53DNA结合能的大小顺序为：24A∶23B (1∶1)>23B>24A，23B与p53DNA f 链的腺嘌呤脱氧核苷酸(DA4)形成1个氢键，24A-23B复合物与p53DNA的DA4、胸腺嘧啶脱氧核苷酸(DT19)形成4个氢键。24A，23B及其混合物与p53DNA结合的强度顺序：24A∶23B (1∶1)>23B>24A，表明24A和23B对抗癌靶点p53DNA具有协同作用，三者与p53DNA的作用方式均为部分嵌插结合。同时，泽泻醇化合物母环C14-和结构中的空间位阻，p53DNA f 链的DA4中磷酸上的氧原子为泽泻醇类化合物与p53DNA相互作用的关键结合位点，是该类泽泻醇发挥抗肿瘤作用的活性中心。24A侧链C19-上的羟基，p53DNA f 链的DA4中腺嘌呤上的氮原子和氧原子，e链的DT19中胸腺嘧啶上的氧原子为泽泻醇类化合物协同增效作用的关键。
关键词：泽泻醇单体；泽泻醇混合物；p53DNA；光谱法；分子模拟
Abstract：The paper aims to study the interactive mechanism of alisol B 23-acetate (23B), alisol A 24-acetate(24A) and content of 23B∶24A=1∶1 mixture with p53DNA which is tumor-suppressor gene, and explore the molecular mechanism of the antitumor effects of alisol acetates. The interaction of 23B, 24A and 24A-23B mixture with p53DNA was investigated by Ultraviolet and Visible Spectroscopy (UV-Vis), Fluorescence spectroscopy and Molecular simulation. UV-Vis showed that p53DNA was partially inserted by alisol mononers and its mixture, in which the decrease degree between p53DNA’s UV-Vis absorbance was 24A∶23B (1∶1)>23B>24A. Fluorescence spectroscopy demonstrated that the mode of interaction of p53DNA and alisol mononers and its mixture was inserted, in which the bonding strength was 24A∶23B (1∶1)>23B>24A. Molecular simulation illustrated that the sequence of binding of alisol mononers and its mixture with p53DNA was 24A∶23B (1∶1)>23B>24A, in which 23B formed a hydrogen bond with Adenine nucleotides (DA4) of p53DNA while 24A-23B mixture formed four hydrogen bonds with the DA4 and Thymine Nucleotides (DT19). The sequence of binding of alisol mononers and its mixture with p53DNA was 24A∶23B(1∶1)>23B>24A, in which the mode of interaction between them and p53DNA were all partial insertion, indicating that 24A and 23B had synergistic effects on anticancer target p53DNA. In addition, the parental rings C14- of alisol acetates and their steric hindrance and the oxygen in phosphoric acid of DA4 in the f chain in p53DNA were the binding site of the interaction between alisol acetates and p53DNA, which was the active center of the antitumor effects of alisol acetates. The key to the synergy of alisol acetates is that the the hydroxyl group on the side chainn C19- of 24A and the nitrogen and oxygen on adenine of DA4 in the f chain in p53DNA and the oxygen atom in thymine of DT19 in the e chain in p53DNA.
[1] Zhang L L, Xu Y L, Tang Z H, et al. Phytomedicine, 2016, 23(8): 800.
[2] Xu W, Li T, Qiu J F, et al. Anti-Cancer Agents in Medicinal Chemistry, 2015, 15(2): 228.
[3] Ma Q, Han L, Bi X, et al. Phytochemistry, 2016, 131: 150.
[4] Meek D W. Biochemical Journal, 2015, 469(3): 325.
[5] Liao J C C, Lam R, Brazda V, et al. Structure, 2011, 19(3): 418.
[6] Dehkordi M N, Bordbar A K, Lincoln P, et al. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 2012, 90: 50.
[7] ZHANG Qiu-ju, LIU Bao-sheng, LI Gai-xia, et al(张秋菊, 刘保生, 李改霞, 等). Spectroscopy and Spectral Analysis(光谱学与光谱分析), 2016, 36(9): 2879.
[8] Shen H Y, Shao X L, Xu H, et al. International Journal of Electrochemical Science, 2011, 6: 532.
[9] Li F, Cao L, Li X, et al. Toxicology Letters, 2015, 232(2): 533.
[10] Ezhuthupurakkal P B, Polaki L R, Suyavaran A, et al. Materials Science and Engineering: C, 2017, 74: 597.
[11] Pakravan P, Masoudian S. Iranian Journal of Pharmaceutical Research: IJPR, 2015, 14(1): 111.
[12] Tarushi A, Polatoglou E, Kljun J, et al. Dalton Transactions, 2011, 40(37): 9461.
[13] Carr M I, Jones S N. Translational Cancer Research, 2016, 5(6): 707.
[14] XU Fei, CHEN Jun, GU Wei, et al(徐 飞, 陈 军, 谷 巍, 等). Chinese Journal of Pharmaceatical Analysis(药物分析杂志), 2011, 31(11): 2035.
[15] Xu Fei, Wu Qinan, Chen Jun, et al. Bioorganic & Medicinal Chemistry Letters, 2014, 24(17): 4099.
[16] XU Fei, ZHANG Lin-qun, HE Li-wei, et al(徐 飞, 张林群, 何立巍, 等). Acta Chimica Sinica(化学学报), 2011, 69(19): 2228.
[17] XU Fei, YU Hui, LU Cai, et al(徐 飞, 于 慧, 陆 彩, 等). Journal of Nanjing University of Traditional Chinese Medicine(南京中医药大学学报), 2016，(5): 451.
[18] XU Fei, LU Cai, WU Qi-nan, et al(徐 飞, 陆 彩, 吴启南, 等). Traditional Chinese Drug Research and Clinical Pharmacology(中药新药与临床药理), 2016, 27(6): 737.
[19] Xu Fei, Yu Hui, Lu Cai, et al. Evidence-Based Complementary and Alternative Medicine, 2016, 2016: 4753852.
