藤黄可乐化学成分及其抗氧化活性研究  

Chemical Constituents from Garcinia kola Seeds and Their Anti-Oxidant Activity

作　　者：王丽仙 于元元 宋孟 王琪 邓长生[1] 黄新安[1] 吴青业[1] 宋健平[1] WANG Lixian;YU Yuanyuan;SONG Meng;WANG Qi;DENG Changsheng;HUANG Xin'an;SONG Jianping(Guangzhou University of Chinese Medicine,Guangzhou 510405 Guangdong,China;Science and Technology Park,Guangzhou University of Chinese Medicine,Guangzhou 510445 Guangdong,China)

机构地区：[1]广州中医药大学,广东广州510405 [2]广州中医药大学科技产业园,广东广州510445

出　　处：《中药新药与临床药理》2020年第10期1133-1140,共8页Traditional Chinese Drug Research and Clinical Pharmacology

基　　金：广东省科技厅“扬帆计划”引进创新创业团队专项(2014YT02S008);广东省中医药局科研项目(粤中医办函〔2019〕43号);广州市科技计划项目(201807010007);科技部新药创制重大专项(2018ZX09303008)。

摘　　要：目的研究藤黄可乐种子的化学成分及其抗氧化活性。方法采用正相硅胶柱色谱、Sephadex LH-20柱色谱以及高效液相色谱(HPLC)等多种手段对藤黄可乐种子乙酸乙酯萃取相进行分离纯化,并根据波谱数据及理化性质鉴定化合物结构。采用2,2-联苯基-1-苦基肼基(DPPH)法和2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)法对所得化合物进行抗氧化活性测试。采用分子对接方法预测C1化合物(Garcinianin)与核因子E2相关因子2(NRF2)的相互结合能力。以终浓度为0、0.78、1.56、3.125、6.25、12.5、25、50、100、200μmol·L^-1的Garcinianin对人急性单核细胞白血病细胞(THP-1)细胞进行干预,以CCK8法检测THP-1细胞活力。采用脂多糖(LPS,1μg·mL^-1)诱导THP-1细胞,并以25、50、100μmol·L^-1Garcinianin进行干预;采用ELISA法测定THP-1细胞谷胱甘肽(GSH)含量;q RT-PCR法检测NRF2、血红素氧合酶1(HO-1)m RNA表达;Western Blot法检测NRF2蛋白表达;DCFH-DA荧光探针法测定活性氧(ROS)的含量。结果从乙酸乙酯萃取相分离得到6个单体化合物,分别为C1(Garcinianin)、C2(Kolaflavanone)、C3(GB1a)、C4(GB2)、C5(Panciflavanon)、C6(山奈酚)。DPPH法测定不同化合物抗氧化活性强弱为C1>C5>C4>C6>C2>C3,ABTS法测定不同化合物抗氧化活性强弱为C1>C5>C3>C6>C2>C4,后续选择抗氧化活性最佳的C1化合物(Garcinianin)进行相关实验。Garcinianin与NRF2分子对接显示二者亲密结合。Garcinianin浓度≤100μmol·L^-1时,无明显细胞毒性。与空白组比较,LPS组的THP-1细胞NRF2、HO-1 mRNA表达及NRF2蛋白表达明显下调(P<0.05),细胞上清液中GSH含量显著降低(P<0.01),ROS含量明显升高;与LPS组比较,Garcinianin各浓度组的THP-1细胞NRF2、HO-1 mRNA表达及NRF2蛋白表达均明显上调(P<0.05),细胞上清液中GSH含量显著升高(P<0.01),ROS含量降低。结论藤黄可乐中的6个化合物都具有不同程度的抗氧化作用,Garcinianin可能是通过调节NRFObjective To study the chemical constituents and antioxidant activities of Garcinia kola seeds.Methods Separation and purification were carried out by various means such as silica gel column chromatography,Sephadex LH-20 column chromatography and HPLC,identifying structures by spectral data and physicochemical properties.The antioxidant activity of the compounds was tested by 2,2-biphenyl-1-picrylhydrazyl(DPPH)method and ABTS method.The molecular docking method was used to predict the mutual binding ability of C1 compound(Garcinianin)and nuclear factor NF-E2 related factor(NRF2).Garcinianin with final concentrations of 0,0.78,1.56,3.125,6.25,12.5,25,50,100,200μmol·L^-1 was used to intervene THP-1 cells,and the activity of THP-1 cells was detected by CCK8 method.Lipopolysaccharid(LPS,1μg·mL^-1)induced THP-1 cells were interfered with 25,50 and 100μmol·L^-1 garcinianin;ELISA kit was used for detection of glutathione(GSH)content.QRT-PCR was carried to detect mRNA expression of NRF2 and heme oxygenase 1(HO-1);and Western Blot was used to detect protein expression of NRF2.The content of reactive oxygen species(ROS)was determined by DCFH-DA fluorescent probe.Results Six compounds were isolated from the ethyl acetate extract phase,named C1(Garcinianin),C2(Kolaflavanone),C3(GB1a),C4(GB2),C5(Panciflavanon)and C6(kaempferol).Antioxidant activities of different compounds determined by DPPH method were as C1>C5>C4>C6>C2>C3;and the antioxidant activities of different compounds determined by ABTS method were as C1>C5>C3>C6>C2>C4.The compound with the best antioxidant activity(C1,Garcinianin)was subsequently selected for relevant experiments.The docking of garcinianin and NRF2 molecule showed that they were closely bound.No cytotoxicity was observed when garcinianin concentration≤100μmol·L^-1.Compared with blank group,the mRNA and protein expression of NRF2 significantly decreased(P<0.05)in LPS model group,the GSH content in the supernatant of the cells decreased(P<0.01),and the ROS content increased.Compared with the

关 键 词：藤黄可乐 化学成分 结构鉴定 抗氧化活性 Garcinianin THP-1细胞 NRF2信号通路 

分 类 号：R284.1[医药卫生—中药学] R285.5[医药卫生—中医学]