染料木黄酮钙配合物的合成、表征、性质研究及其在异黄酮纯化中的应用
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摘要
配位作用在天然药物中普遍存在,研究发现,根据协同效应,某些天然药物中有效成分的金属配合物的生物活性明显强于单一有效成分。
染料木黄酮具有多种生物活性和生理功能。它能抗氧化、抗炎、抑菌,有弱雌激素活性,能有效地预防和抑制各种肿瘤、骨质疏松症、妇女更年期综合症及心血管疾病等多种疾病的发生。染料木黄酮的结构具有较高的超离域度,完整的大π键共轭体系,强配位氧原子和合适的空间构型,是金属离子良好的螯合配体,易与金属离子进行螯合反应。因此金属离子能够促进人体的许多生理功能。根据协同效应,药物中加入金属离子不仅可以改善药物的理化性质,促进药物吸收,更重要的是还可增强药效。配合物的形成对金属元素的吸收和代谢都起重要作用。因此,研究染料木黄酮与金属形成的配合物,有助于该类化合物新型金属基药物的开发利用。
为了设计高效、低毒的抗氧化性染料木黄酮配合物,论文选用更年期女性大多缺少的钙元素为中心原子。以染料木黄酮和氢氧化钙为原料合成了染料木黄酮钙配合物。首先优选配合物形成的条件,条件实验结果显示：除了反应温度和反应时间以外,反应体系的pH是决定配合物能否形成的关键因素,合成的最优工艺条件为pH 8.5、反应温度65℃、搅拌时间8.0h,此时染料木黄酮-钙配合物的产率为77.42%。
采用红外光谱、紫外光谱、热重分析及核磁共振等手段对其结构进行了表征。结果表明：染料木黄酮以分子中的4位羰基O和5位羟基O与Ca2+配位而形成分子式为[Ca(C15H9O5)2]·2H2O的配合物。
论文通过对染料木黄酮钙配合物抗氧化活性的分析,探讨了配合物构效关系及协同作用。采用邻苯三酚自氧化法、Feton反应-邻二氮菲光度法和DPPH自由基方法分别测定了配体与配合物清除超氧自由基、羟自由基和DPPH自由基的能力。结果表明染料木黄酮及其钙配合物清除超氧自由基能力随浓度的增大逐渐增大；在相同浓度下,钙配合物的清除能力明显强于配体染料木黄酮,提高了20%。染料木黄酮具有一定的清除羟基自由基的能力,当其形成钙配合物后,其清除能力也大大增强,最高可达到65%,提高了近50%。同时配体本身及配合物抗羟自由基的活性随浓度升高有一极大值,随后则下降。染料木黄酮及其配合物均具有一定的清除DPPH自由基的能力。随着浓度的增加,二者清除能力仅略有增强,在相同浓度下,配合物对DPPH自由基的清除率要高于染料木黄酮,但这种能力的提高并不明显,最高仅1%左右。
论文又考察了染料木黄酮及其配合物的抑菌性质,测定了其抑制大肠杆菌、绿脓杆菌、李斯特氏菌的能力。结果表明染料木黄酮及其配合物对大肠杆菌、李斯特氏菌、绿脓杆菌都有一定的抑菌效果。随着浓度的增大,抑菌效果明显。二者均对绿脓杆菌的抑制效果最强。相比较而言,染料木黄酮配合物的抑菌性仅略高于染料木黄酮,这可能与钙离子对细菌的生长有促进作用有关。
通过单因素及二次正交旋转设计试验,并按实际生产需要进行了修正,确立了最佳提取条件为：液固比42:1,提取时间3.7h,提取温度49℃。在此修正条件下实际测得提取率为93.12%。
应用染料木黄酮配合物合成的原理,对30%的大豆异黄酮粉进行了纯化,成功的将异黄酮粉中的主要成分染料木苷和黄豆苷进行分离,得到高纯度的染料木苷和黄豆苷。对于进一步制备活性更高的染料木黄酮和黄豆苷元具有重要意义。以期为染料木黄酮的进一步开发利用及设计低毒、高效的抗癌药物提供有价值的参考信息。
Complexing action is universal in natural druggery,and it is reported that the biological activities of metallic complex is obvious stronger than single component due to the concordance effection.
The genistein is a kind of effective component from natural druggery, which has many pharmacological actions and biological activities such as antioxidation, anti-flammatory,antiviral. It have weak estrogen activeness and it can prevent and suppress each kind of tumor, the osteoporosis the woman menopause syndrome and cardiovascular disease effectively. Due to a convenient position of the 5-OH and 4-CO groups in a molecule,the genistein's structure composes a largeπ-bond conjugation system,of which the oxygen atoms has a potential activity to chelate with metal ions. It will contribute to be a new pharmacological effect or to increase their efficacy when genistein chelated with some microelements that have bioactivity and pharmacological activity themselves. Therefore, the research of genistein metal complexes, will help us to develop and utilize genistein from a new viewpoint of novel natrue compound and metal-based drug.
To design some new type antioxidants of metallic complex of genistein with higher activity, using genistein and calcium hydroxide as raw material, the calcium-genistein complex was synthesized. Besides the temperature of reaction system and the reaction time the pH value of reaction solution was a key factor of the form of coordination complexes. And the optimal pH ranges were pH 8.5, the optimal reaction temperature were 65℃, the optimal reaction time were 8h. The yields of synthesizing Gen-Ca complexes were 77.42%.
The calcium-genistein complex was synthesized and characterized with the methods of IR spectra, UV-spectra, thermal analysis and NMR spectrum etc. The result showed that in calcium-genistein complex Ca2+ was coordinated by 5-OH and 4-CO groups of genistein and the molecular formula is [Ca(C15H9O5)2]·2H2O
In this paper, structure-bioactivity and cooperative action for coordination complexes had been studied through analyzing the antioxidant activity of Gen-Ca complexes. The biological activities of the complex were studied by means of pyrogallol autoxidation, orthophenanthroline photometry and DPPH free radical. The result showed that at the same concentration, the scavenging effects of Gen-Ca on superoxide radical was stronger than Gen. The scavenging effects of the ligand itself and the complex increased along with the concentration increasing, and the anti-hydroxy free radical activeness elevated along with the concentration but had a maximum value, but anti-DPPH free radical ability was not so obvious.
And also the bacteriostasis activities of the ligand and its complex against three different types of bacteria namely Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes were studied. The result showed that the ligand and its complex had certain bacteriostasis effect, and the complex showed an increased activity compared to the respective ligand. Along with concentration increased, the bacteriostasis effect was obvious, and the Listeria monocytogenes was the most.
Using the principle of genistein-Ca complex, we carried on the purification to 30% soybean isoflavone powder, simultaneously inspected that the optimum condition of solvent regurgitant extraction established through the single factors and quadratic rotary combination design. The optimal was that material rate was 42:1, the extraction time was 3.7h, the extraction temperature was 49℃, and the yield was 93.12%. We successfully separated the genistin and daidzin which was the main component of 30% soybean isoflavone powder. It had further important significance for preparing genistein and daidzein.It will supply the valuable referenced information for the further development and utilization of genistein and exploiting some new type anticancer medicine.
染料木黄酮具有多种生物活性和生理功能。它能抗氧化、抗炎、抑菌,有弱雌激素活性,能有效地预防和抑制各种肿瘤、骨质疏松症、妇女更年期综合症及心血管疾病等多种疾病的发生。染料木黄酮的结构具有较高的超离域度,完整的大π键共轭体系,强配位氧原子和合适的空间构型,是金属离子良好的螯合配体,易与金属离子进行螯合反应。因此金属离子能够促进人体的许多生理功能。根据协同效应,药物中加入金属离子不仅可以改善药物的理化性质,促进药物吸收,更重要的是还可增强药效。配合物的形成对金属元素的吸收和代谢都起重要作用。因此,研究染料木黄酮与金属形成的配合物,有助于该类化合物新型金属基药物的开发利用。
为了设计高效、低毒的抗氧化性染料木黄酮配合物,论文选用更年期女性大多缺少的钙元素为中心原子。以染料木黄酮和氢氧化钙为原料合成了染料木黄酮钙配合物。首先优选配合物形成的条件,条件实验结果显示：除了反应温度和反应时间以外,反应体系的pH是决定配合物能否形成的关键因素,合成的最优工艺条件为pH 8.5、反应温度65℃、搅拌时间8.0h,此时染料木黄酮-钙配合物的产率为77.42%。
采用红外光谱、紫外光谱、热重分析及核磁共振等手段对其结构进行了表征。结果表明：染料木黄酮以分子中的4位羰基O和5位羟基O与Ca2+配位而形成分子式为[Ca(C15H9O5)2]·2H2O的配合物。
论文通过对染料木黄酮钙配合物抗氧化活性的分析,探讨了配合物构效关系及协同作用。采用邻苯三酚自氧化法、Feton反应-邻二氮菲光度法和DPPH自由基方法分别测定了配体与配合物清除超氧自由基、羟自由基和DPPH自由基的能力。结果表明染料木黄酮及其钙配合物清除超氧自由基能力随浓度的增大逐渐增大；在相同浓度下,钙配合物的清除能力明显强于配体染料木黄酮,提高了20%。染料木黄酮具有一定的清除羟基自由基的能力,当其形成钙配合物后,其清除能力也大大增强,最高可达到65%,提高了近50%。同时配体本身及配合物抗羟自由基的活性随浓度升高有一极大值,随后则下降。染料木黄酮及其配合物均具有一定的清除DPPH自由基的能力。随着浓度的增加,二者清除能力仅略有增强,在相同浓度下,配合物对DPPH自由基的清除率要高于染料木黄酮,但这种能力的提高并不明显,最高仅1%左右。
论文又考察了染料木黄酮及其配合物的抑菌性质,测定了其抑制大肠杆菌、绿脓杆菌、李斯特氏菌的能力。结果表明染料木黄酮及其配合物对大肠杆菌、李斯特氏菌、绿脓杆菌都有一定的抑菌效果。随着浓度的增大,抑菌效果明显。二者均对绿脓杆菌的抑制效果最强。相比较而言,染料木黄酮配合物的抑菌性仅略高于染料木黄酮,这可能与钙离子对细菌的生长有促进作用有关。
通过单因素及二次正交旋转设计试验,并按实际生产需要进行了修正,确立了最佳提取条件为：液固比42:1,提取时间3.7h,提取温度49℃。在此修正条件下实际测得提取率为93.12%。
应用染料木黄酮配合物合成的原理,对30%的大豆异黄酮粉进行了纯化,成功的将异黄酮粉中的主要成分染料木苷和黄豆苷进行分离,得到高纯度的染料木苷和黄豆苷。对于进一步制备活性更高的染料木黄酮和黄豆苷元具有重要意义。以期为染料木黄酮的进一步开发利用及设计低毒、高效的抗癌药物提供有价值的参考信息。
Complexing action is universal in natural druggery,and it is reported that the biological activities of metallic complex is obvious stronger than single component due to the concordance effection.
The genistein is a kind of effective component from natural druggery, which has many pharmacological actions and biological activities such as antioxidation, anti-flammatory,antiviral. It have weak estrogen activeness and it can prevent and suppress each kind of tumor, the osteoporosis the woman menopause syndrome and cardiovascular disease effectively. Due to a convenient position of the 5-OH and 4-CO groups in a molecule,the genistein's structure composes a largeπ-bond conjugation system,of which the oxygen atoms has a potential activity to chelate with metal ions. It will contribute to be a new pharmacological effect or to increase their efficacy when genistein chelated with some microelements that have bioactivity and pharmacological activity themselves. Therefore, the research of genistein metal complexes, will help us to develop and utilize genistein from a new viewpoint of novel natrue compound and metal-based drug.
To design some new type antioxidants of metallic complex of genistein with higher activity, using genistein and calcium hydroxide as raw material, the calcium-genistein complex was synthesized. Besides the temperature of reaction system and the reaction time the pH value of reaction solution was a key factor of the form of coordination complexes. And the optimal pH ranges were pH 8.5, the optimal reaction temperature were 65℃, the optimal reaction time were 8h. The yields of synthesizing Gen-Ca complexes were 77.42%.
The calcium-genistein complex was synthesized and characterized with the methods of IR spectra, UV-spectra, thermal analysis and NMR spectrum etc. The result showed that in calcium-genistein complex Ca2+ was coordinated by 5-OH and 4-CO groups of genistein and the molecular formula is [Ca(C15H9O5)2]·2H2O
In this paper, structure-bioactivity and cooperative action for coordination complexes had been studied through analyzing the antioxidant activity of Gen-Ca complexes. The biological activities of the complex were studied by means of pyrogallol autoxidation, orthophenanthroline photometry and DPPH free radical. The result showed that at the same concentration, the scavenging effects of Gen-Ca on superoxide radical was stronger than Gen. The scavenging effects of the ligand itself and the complex increased along with the concentration increasing, and the anti-hydroxy free radical activeness elevated along with the concentration but had a maximum value, but anti-DPPH free radical ability was not so obvious.
And also the bacteriostasis activities of the ligand and its complex against three different types of bacteria namely Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes were studied. The result showed that the ligand and its complex had certain bacteriostasis effect, and the complex showed an increased activity compared to the respective ligand. Along with concentration increased, the bacteriostasis effect was obvious, and the Listeria monocytogenes was the most.
Using the principle of genistein-Ca complex, we carried on the purification to 30% soybean isoflavone powder, simultaneously inspected that the optimum condition of solvent regurgitant extraction established through the single factors and quadratic rotary combination design. The optimal was that material rate was 42:1, the extraction time was 3.7h, the extraction temperature was 49℃, and the yield was 93.12%. We successfully separated the genistin and daidzin which was the main component of 30% soybean isoflavone powder. It had further important significance for preparing genistein and daidzein.It will supply the valuable referenced information for the further development and utilization of genistein and exploiting some new type anticancer medicine.
引文
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Axel K, Hans L, Edda K.2006.Alternative Pathways as Mechanism for the Negative Effects Associated with Overexpression of Superoxide Dismutase.J Theor Bio.238:828～840.
Blois M.S.1958.Antioxidant Determinations by the Use of a Stable Free Radical.Nature. 181:1199～1200.
Bovee Oudenhiven, I.M., M.L.Wissink, J.T.Woyters, R.V.der.1999. Meer.Dietary Calcium Phosphate Stimulates Intestinal Lactobacilli and Decreases the Severty of a Salmonella Infection in Rats.Nutr.129:607～612.
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Chen C T, Snslick K S.1993.One Dimensional Coordination Polymers:Applications to Material Science.Coord Chem Rev.128:293-322.
Chiang L Y, Lu F J, Lin J T.1995.Free Radical Scavenging Activity of Water-Soluble Fullerens.J.Chem Soc.(1):283～284.
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Fan J., Shen R., Tang N.2004.Synthesis,Characterization and Antioxidative Activity of Lanthanide Complexes with Genistein. Journal of Rare Earths.22,Suppl:25～28.
GT.Castro, S.E.Blanco.2004. Structural and Spectroscopic Study of 5,7-Dihydroxy Flavone and its Complex with Aluminum. Spectrochimica Acta Part A.60(10):2235～2241.
Jacek Z., Jerzy G., Grzegorz G.2003.Radical Scavenging Properties of Genistein. Free Radical Biology&Medicine.35(8):958～965.
Jeffrey B.Harborne, Christine A.2000.Williams.Advances in Flavonoid Research Since 1992. Phytochemistry.55(6):481～504. Jing Zhou, Liufang Wang, Jinyi Wang, et al.2001. Synthesis, Characterization, Antioxidativeand Antitumor Activities of Solid Quercetin Rare Earth(Ⅲ) Complexes. Journal of Inorganic Biochemistry.83(1):41～48.
Jovanovic S.V., Steenken S., Tosic M.,et al.1994.Flavonoids as Antioxidants.J.Am.Chem.Soc. 116:4846～4851.
Kelloff GJ, Crowell JA, Hank EJ et al.1996.Strategy and Planning for Chemopreventive Drug Development:Clinical Development Plans Ⅱ. Cellular Bioch.26(suppl):54.
Kostyuk V.A., Potapovich A.I., Strigunova E.N.,etal.2004. Experimental Evidence that Flavonoid Metal Complexes May Act as Mimics of Superoxide Dismutase. Archives of Biochemistry and Biophysics.428:204～208.
Laetitia Dangleterre, Jean-Paul Cornard.2005. Interaction of Lead(Ⅱ) Chloride with Hydroxyilavones in Methanol:A Spectroscopic Study. Polyhedron.24(12):1593～1598.
Majid Y.Moridani, Jalal Pourahmad, Hoang Bui, et al.2003.Dietary Flavonoid Iron Complexes Ascytoprotective Superoxide Radical Scavengers. Free Radical Biologry & Medicine.34(2):243～253.
MarianV., Dieter L., Jan M. et al.2007.Free Radicals and Antioxidants in Normal Physiological Functions and Human Disease.Int.J. Biochem.Cell.Boil.39:44～84.
Poovaiah BW, Reddy ASN.1993. Calcium and Signal Transduction in Plants. J.Crit. Rev. Plant Sci,12:185～221.
Rao CV, Wang C.X., Simi B,etal.1997.Cancer Res.57:3717.
Rubens F.V.de Souza, Wagner F.De Giovani.2005.Synthesis, Spectral and Electrochemical Properties of Al(Ⅲ) and Zn(Ⅱ) Complexes with Flavonoids. Spectrochimica Acta Part A.61(9):1985～1990.
Setchell K D.1998. Phytoestrogens:the Biochemistry, Physiloogy, and Implications for Human Health of Soy Isoflavones.Am J Clin Nutr.68:333～1346.
Tawa R, Sakurai H.1997. Determination of Four Active Oxygen Species such as H2O2,·OH O2 and ·O2 by Luminol and Clachemiluminescence Methods and Evaluation of Antioxidative Effects of Hydroxy-Bengoic Acids. Anal Let.30(15):2811～2825.
Y.F.M.Kishk, H.M.A.A1-Sayed.2007.Free Radical Scavenging and Antioxidative Activities of some Polysaccharides in Emulsions. LWT.40:270～277.
Yokozawa T., Dong E., Natagawa T., et al.1998.In Vitro and in Vivo Studies on the Radical Scavenging Activity of Tea.J Agric Food Chem.46(6):2143～2150.