五没食子酰葡萄糖锗的合成及其体外抗肿瘤活性
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摘要
以单宁酸为原料,通过甲醇醋酸溶液降解、乙酸乙酯萃取、葡聚糖凝胶色谱柱纯化,制得1,2,3,4,6-O-五没食子酰葡萄糖(PGG),得率为18%,经HPLC分析纯度为99.90%。采用FT-IR、~1H-NMR、~(13)C-NMR及ESI-MS等分析手段对PGG的结构进行了表征。利用PGG与锗离子在溶液中进行络合反应,反应液经冷冻沉淀、离心、冷冻干燥得到PGG-Ge络合物,采用FT-IR对络合物的结构进行了表征。采用CCK8法研究了PGG-Ge络合物对人结肠癌细胞HCT116、人宫颈癌细胞Hela、人胰腺癌细胞Bx PC3、人小细胞肺癌细胞A549、人胃癌细胞SGC7901及人乳腺癌细胞MCF7增殖的影响。结果表明:PGG-Ge络合物对6种肿瘤细胞的增殖均具有明显的抑制作用,且随着浓度的增加,抑制作用也逐渐增强。当浓度达到160μg/m L时,对6种肿瘤细胞增殖的抑制率均达到了80%左右。而相同浓度下对照品顺铂对受试肿瘤细胞增殖的抑制率只有4.30%～18.54%。PGG-Ge络合物对各肿瘤细胞增殖的IC_(50)值均小于90μg/m L,表现出良好的抗肿瘤活性。
Organic germanium( Ge) compound has wide applications as additives in food and cosmetic products due to its antitumor property. In this study,1,2,3,4,6-Pentagalloylglucose( PGG),which is present at relatively low levels in tannic acid and at even lower levels in plants,was prepared from the degradation of tannic acid with methanolic acetic acid. PGG was then extracted with ethyl acetate,and purified with a Sephadex LH-20 chromatography column.The yield of PGG could reach 18% and the purity was up to 99.90%,which determined by HPLC analysis. The chemical structure of PGG was determined with FT-IR,~1 H-NMR,~(13)C-NMR and ESI-MS. PGG-Ge complex was prepared by complexation reaction of PGG with germanium ions followed by cry precipitation,centrifugation,and cryodesiccation.The chemical structure of the complex was characterized by FT-IR. The effects of PGG-Ge on the proliferation of human colon cancer cell HCT116,human cervical cancer Hela,human pancreatic cancer cell BxPC3,human small cell lung cancer cell A549,human gastric cancer cell SGC7901 and human breast cancer cell MCF7 were examined by using CCK8 method. The results showed that the PGG-Ge complex had a significant inhibitory effect on the proliferation of the aforementioned six tumor cells. In addition,the inhibitory effect gradually enhanced with the increase of the PGG-Ge concentration. When the concentration reached 160 μg/mL,the inhibition rate of six tumor cells reached about80%. As a comparison,the inhibition rate for tumor cells of cisplatin was calculated to be only 4.30%-18.54%at the same concentration. The minimum half-inhibiting concentration of proliferation( IC_(50)) of PGG-Ge to each tumor cell was less than 90 μg/mL,indicating good inhibitory effect on proliferation for all aforementioned tumor cells. The result reported in this study is a new polyphenol( PGG)-germanium complex synthesized for the first time.
Organic germanium( Ge) compound has wide applications as additives in food and cosmetic products due to its antitumor property. In this study,1,2,3,4,6-Pentagalloylglucose( PGG),which is present at relatively low levels in tannic acid and at even lower levels in plants,was prepared from the degradation of tannic acid with methanolic acetic acid. PGG was then extracted with ethyl acetate,and purified with a Sephadex LH-20 chromatography column.The yield of PGG could reach 18% and the purity was up to 99.90%,which determined by HPLC analysis. The chemical structure of PGG was determined with FT-IR,~1 H-NMR,~(13)C-NMR and ESI-MS. PGG-Ge complex was prepared by complexation reaction of PGG with germanium ions followed by cry precipitation,centrifugation,and cryodesiccation.The chemical structure of the complex was characterized by FT-IR. The effects of PGG-Ge on the proliferation of human colon cancer cell HCT116,human cervical cancer Hela,human pancreatic cancer cell BxPC3,human small cell lung cancer cell A549,human gastric cancer cell SGC7901 and human breast cancer cell MCF7 were examined by using CCK8 method. The results showed that the PGG-Ge complex had a significant inhibitory effect on the proliferation of the aforementioned six tumor cells. In addition,the inhibitory effect gradually enhanced with the increase of the PGG-Ge concentration. When the concentration reached 160 μg/mL,the inhibition rate of six tumor cells reached about80%. As a comparison,the inhibition rate for tumor cells of cisplatin was calculated to be only 4.30%-18.54%at the same concentration. The minimum half-inhibiting concentration of proliferation( IC_(50)) of PGG-Ge to each tumor cell was less than 90 μg/mL,indicating good inhibitory effect on proliferation for all aforementioned tumor cells. The result reported in this study is a new polyphenol( PGG)-germanium complex synthesized for the first time.
引文
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[16]王俊杰,赫春香.槲皮素-锗配合物的合成及清除氧自由基活性的研究[J].高师理科学刊,2010,30(1):71-74.DOI:10.3969/j.issn.1007-9831.2010.01.023.WANG J J,HE C X.Study on synthesis and scavenging radical activity of complex of Quercetin-Germanium(Ⅳ)[J].Journal of Science of Teachers'College and University,2010,30(1):71-74.
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[18]PI J,ZENG J,LUO J J,et al.Synthesis and biological evaluation of Germanium(IV)-polyphenol complexes as potential anti-cancer agents[J].Bioorganic&Medicinal Chemistry Letters,2013,23(10):2902-2908.DOI:10.1016/j.bmcl.2013.03.061.
[2]姬丙艳,许光,姚振,等.锗的研究进展及开发前景[J].中国矿业,2016,25(S1):22-24.DOI:10.3969/j.issn.1004-4051.2016.z1.006.JI B Y,XU G,YAO Z,et al.The advancement and development prospect of germanium[J].China Mining Magazine,2016,25(S1):22-24.
[3]PI J,ZENG J,LUO J J,et al.Synthesis and biological evaluation of Germanium(IV)-polyphenol complexes as potential anti-cancer agents[J].Bioorganic&Medicinal Chemistry Letters,2013,23(10):2902-2908.DOI:10.1016/j.bmcl.2013.03.061.
[4]PATAI S,RAPPOPORT Z.The chemistry of organic germanium,tin and lead compounds[J].American Journal of Sociology,1995,105(3):1485-1520.DOI:10.1002/0470857188.ch20.
[5]黄河宁,胡晓慧,黄河清,等.质谱技术研究儿茶素及儿茶素-锗多聚体特性[J].分析化学,2006,34(1):52-56.DOI:10.3321/j.issn:0253-3820.2006.01.012.HUANG H N,HU X H,HUANG H Q,et al.Polymer characteristics of catechin and catechin-Ge4+measured with a technology of mass spectrometry[J].Chinese Journal of Analytical Chemistry,2006,34(1):52-56.
[6]黄河宁,陈东仕,胡晓慧,等.茶多酚及茶多酚锗的组成、结构和抑制肺癌细胞生长速率的研究[J].厦门大学学报(自然科学版),2007,46(2):258-261.DOI:10.3321/j.issn:0438-0479.2007.02.025.HUANG H N,CHEN D S,HU X H,et al.Composition,structure,and inhibiting growth rate of lung-cancer cell by tea polyphenol and tea polyphenol-germanium[J].Journal of Xiamen University(Natural Science),2007,46(2):258-261.
[7]翟广玉,渠文涛,朱玮,等.芦丁锗的合成、表征及生物活性研究[J].天然产物研究与开发,2013,25(2):166-169,176.DOI:10.3969/j.issn.1001-6880.2013.02.004.ZHAI G Y,QU W T,ZHU W,et al.Study on synthesis,characterization and biological activities ofrutin-germanium[J].Natural Product Research and Development,2013,25(2):166-169,176.
[8]LI S P,XIE W L,CAI H H,et al.Hydroxyl radical scavenging mechanism of human erythrocytes by quercetin-germanium(IV)complex[J].European Journal of Pharmaceutical Sciences,2012,47(1):28-34.DOI:10.1016/j.ejps.2012.04.019.
[9]李惠晨,胡伟,丁井永.五没食子酰基葡萄糖抗肿瘤的作用研究进展[J].现代肿瘤医学,2014,22(9):2255-2258.DOI:10.3969/j.issn.1672-4992.2014.09.78.LI H C,HU W,DING J Y.The research progress of Penta-Ogalloyl-β-D-glucose in anti-tumor effect[J].Journal of Modern Oncology,2014,22(9):2255-2258.
[10]KRAAL P,JANSEN B,NIEROP K G J,et al.Copper complexation by tannic acid in aqueous solution[J].Chemosphere,2006,65(11):2193-2198.DOI:10.1016/j.chemosphere.2006.05.058.
[11]BAYAT S,AGHAZADEH S,NOAPARAST M,et al.Germanium separation and purification by leaching and precipitation[J].Journal of Central South University,2016,23(9)2214-2222.DOI:10.1007/s11771-016-3279-6.
[12]CRUZ B H,DIAZ-CRUZ J M,ARINO C,et al.Heavy metal binding by tannic acid:a voltammetric study[J].Electroanalysis,2000,12(14):1130-1137.DOI:10.1002/1521-4109(200010)12:14<1130::AID-ELAN1130>3.0.CO;2-7.
[13]刘瑜琛,张亮亮,汪咏梅,等.分光光度法测定单宁酸-Fe3+反应结合常数及其化学计量比[J].林业工程学报,2018,32(2):47-52.DOI:10.13360/j.issn.2096-1359.2018.02.008.LIU Y C,ZHANG L L,WANG Y M,et al.Spectrophotometric determination of formation constants and stoichiometry of tannic acid complexes with Fe3+[J].Journal of Forestry Engineering,2018,32(2):47-52.
[14]HAGERMAN A E,ZHAO Y,JOHNSON S.Methods for determination of condensed and hydrolyzable tannins[M]//HAGERMANA E,ZHAO Y,JOHNSON S.ACS Symposium Series.Washington,DC:American Chemical Society,1997:209-222.DOI:10.1021/bk-1997-0662.ch012.
[15]张晓燕,王金辉,李铣.白芍的化学成分研究[J].沈阳药科大学学报,2001,18(1):30-32.DOI:10.3969/j.issn.1006-2858.2001.01.010.ZHANG X Y,WANG J H,LI X.A study on the chemical constituents of Paeonia lactiflora Pall.[J].Journal of Shenyang Pharmaceutical University,2001,18(1):30-32.
[16]王俊杰,赫春香.槲皮素-锗配合物的合成及清除氧自由基活性的研究[J].高师理科学刊,2010,30(1):71-74.DOI:10.3969/j.issn.1007-9831.2010.01.023.WANG J J,HE C X.Study on synthesis and scavenging radical activity of complex of Quercetin-Germanium(Ⅳ)[J].Journal of Science of Teachers'College and University,2010,30(1):71-74.
[17]翁鹭娜.茶多酚锰的组成、结构、络合、稳定性和诱导肿瘤细胞凋亡的研究[D].厦门:厦门大学,2007.WENG L N.Tea polyphenols-manganes and tea polyphenols-germanium composition,structure,complex,complexation,stability and effects of anticancer[D].Xiamen:Xiamen University,2007.
[18]PI J,ZENG J,LUO J J,et al.Synthesis and biological evaluation of Germanium(IV)-polyphenol complexes as potential anti-cancer agents[J].Bioorganic&Medicinal Chemistry Letters,2013,23(10):2902-2908.DOI:10.1016/j.bmcl.2013.03.061.