茶叶浸提物对功能性酶活性和二级结构的影响研究
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摘要
本文研究了绿茶、铁观音、红茶和六堡茶浸提物及茶多酚对血管紧张素转化酶(ACE)、胰脂肪酶(PPL)和胰α-淀粉酶(PPA)等三种功能性酶活性的影响,并选取PPL和PPA,研究其与茶源物质相互作用后紫外差示光谱、荧光猝灭光谱和圆二色谱的变化。
首先比较了紫外可见光法和高效液相色谱法对研究茶多酚ACE活性的适用性,得出一种直接进样、等梯度洗脱便能达到良好分析效果的RP-HPLC测定方法。以马尿酰-组氨酰-亮氨酸(HHL)为反应底物,ACE为催化剂,反应所生成的马尿酸为测定指标,未加酶抑制剂的反应为空白对照。使用Intersil ODS-3色谱柱(4.6mm i.d.×250mm,5μm),柱温35℃,流动相为甲醇-超纯水(体积比35：65,各含0.05%(v/v)三氟乙酸及0.1%(v/v)三乙胺),流速0.8 mL/min,检测波长228nm。在马尿酸浓度为5-50μg/mL时,马尿酸浓度与其峰面积呈良好的线性关系(R2=0.9997),马尿酸的回收率为93.55%-101.50%,相对标准偏差(RSD)为2.95%。采用此法测得绿茶茶多酚对ACE具有一定的抑制作用,抑制率随着茶多酚浓度的升高呈现先升高再降低的总体趋势,最大的抑制率为29.44%,此时茶多酚浓度为2mg/mL。
其次研究了茶多酚对PPL和PPA活性的影响。结果表明其对PPL和PPA均有显著的抑制作用,茶多酚对PPL和PPA的半抑制浓度IC50分别为1.23和0.82mg/mL,为可逆性抑制,可逆抑制反应的类型均为非竞争性抑制,抑制常数Ki分别为2.49和1.08mg/mL。通过茶浸提物对PPA抑制活性的研究发现,绿茶、铁观音、红茶和六堡茶浸提物的半抑制浓度IC50分别为8.99,8.89,7.90和40.8mg/mL。抑制动力学结果表明四种茶浸提物对PPA的抑制反应均为可逆抑制,可逆抑制的抑制类型均为非竞争性抑制,抑制常数Ki分别为：11.59,10.37,9.52和43.29mg/mL。
紫外差示光谱结果表明,不同浓度的茶多酚和茶浸提物均能引起PPL和PPA紫外吸收光谱的变化,光谱谱峰升高,峰位红移,浓度越高,变化幅度越大。四种茶浸提液相比,红茶的影响程度最大,六堡茶最小。以278nm和295nm波长光进行激发,研究了四种茶浸提物和茶多酚与PPL和PPA相互作用后的荧光猝灭光谱,结果显示随着茶源物质浓度的增加,PPL和PPA的荧光发射强度逐渐降低,峰位发生红移。结合Stern-Volmer方程,推断四种茶浸提液和茶多酚对PPL和PPA的荧光猝灭均为以非共价键结合的某种不发光的配合物引起的静态猝灭。
通过圆二色谱技术,研究了四种茶浸提液对PPL和PPA圆二色谱及二级结构的影响,结果显示四种茶浸提物均能促使PPA和PPL双负峰往单一负峰转变,茶叶发酵程度越低,即茶多酚的含量越高,转变越明显,峰高变化也越显著。与茶浸提物相互作用后PPA和PPL均变现出α-螺旋含量降低,而β-折叠含量增多,此种结构变化与酶的失活具有相关性。
The effects of green tea, Oolong tea, black tea, liupao tea and tea polyphenols(TP) on the activity of angiotensin converting enzyme(ACE), porcine pancreas lipase(PPL) and porcine pancreasα-amylase(PPA) were studied in this paper. PPL and PPA were selected to study the changes of UV difference spectra, fluorescence spectra and circular dichroism(CD) after treatment with those tea source materials.
Firstly, UV-visible spectrophotometry and HPLC method were compared to verify their applicability. An simple, reliable and precise RP-HPLC method was established for the analysis of ACE inhibitory activity of green tea polyphenols(GTP) with N-hippuryl-His-Leu (HHL) tetrahydrate as the reaction substrate and hippurid acid as the reaction product. The chromatographic conditions were as follows:Column, Intersile ODS-3(4.6mm i.d.×250mm, 5μm); column temperature,35℃; mobile phase, methanol-ditilled water(35:65, v/v, both containing 0.05%(v/v) trifluoroacetic acid and 0.1% triethylamine); flow rate,0.8mL/min; detection wavelength,228nm. An excellent linearity over the range of 5-50μg/mL(R2=0.9997) was observed. The recoveries of hippuric acid were 93.55%-101.50% with a relative standard deviation(RSD) as 2.95%. Measured by this method, GTP showed ACE inhibitory activity The maximum inhibitory rate reached to 29.44% at 2mg/mL of GTP concentration.
Secondly, the effects of TP to the activity of PPL and PPA were studied. Results showed that TP had a significant inhibitory effects to both PPL and PPA, the IC5o were 1.23 and 0.82mg/mL respectively. The inhibitory types were both reversible via non-competitive inhibition with the Ki as 2.49 and 1.08mg/mL respectively. When comes to the four types of tea, the content of TP and solids were reduced with their degree of fermention and their IC50 to PPA were gained through inhibitory research. Meantime, the kinetics equations were established to analyse the inhibition type. Results showed that they were all reversible via non-competitive with the Ki as 11.59,10.37,9.52 and 43.29mg/mL.
Thirdly, the results of UV difference spectra suggested that the UV spectra of PPL and PPA could be changed by different concentration of TP and tea extracts with the peak height increasing and the peak location red shifting, the higher the concentration the more distinctive the change. Compared with the four types of tea extracts, the black tea had the greatest influence while the liupao tea had the smallest. This phenomenon maybe caused from the change of the spacial structure and microenvironment of aromatic amino-acid residues in PPL and PPA by tea sources, which led to the change of PPL and PPA enzyme molecular conformation so as to affect their catalytic activity. What's more, the bindings of TP and the four types of tea to PPL and PPA were studied by fluorescence quenching spectra with 278nm and 295nm wavelength light as stimulated emission, results showed the fluorescent intensity reduced with the concentration increasement and the peak location red shiftment. And the quenching mechanism was researched according to the characteristic feature of Stern-Volmer equation, from which we concluded it was static quenching led by some non-luminous complexes combined with non-covalent bond.
Finally, the CD spectra and secondary structure of PPL and PPA after treament with the four types of tea extracts and TP were obtained using CD technique. Results exhibited that all the tea sources could lead to the change of CD peak height, shape and location, the higher the concentration the more distinctive the change. The two characteristic negative peak of PPL and PPA turned to single by the impact of tea sources and the peak height changed remarkably with the content of TP. As for the secondary structure, the content ofα-helix reduced while theβ-sheet increased after interaction with tea sources. The reduction ofα-helix indicated that the orientation of hydrogen bond changed and the increasement ofβ-sheet caused the unnatural sheet mistakes come up, which two aspects made the nature conformation of enzyme protein can't form so as to the activity of enzyme inactivated.
首先比较了紫外可见光法和高效液相色谱法对研究茶多酚ACE活性的适用性,得出一种直接进样、等梯度洗脱便能达到良好分析效果的RP-HPLC测定方法。以马尿酰-组氨酰-亮氨酸(HHL)为反应底物,ACE为催化剂,反应所生成的马尿酸为测定指标,未加酶抑制剂的反应为空白对照。使用Intersil ODS-3色谱柱(4.6mm i.d.×250mm,5μm),柱温35℃,流动相为甲醇-超纯水(体积比35：65,各含0.05%(v/v)三氟乙酸及0.1%(v/v)三乙胺),流速0.8 mL/min,检测波长228nm。在马尿酸浓度为5-50μg/mL时,马尿酸浓度与其峰面积呈良好的线性关系(R2=0.9997),马尿酸的回收率为93.55%-101.50%,相对标准偏差(RSD)为2.95%。采用此法测得绿茶茶多酚对ACE具有一定的抑制作用,抑制率随着茶多酚浓度的升高呈现先升高再降低的总体趋势,最大的抑制率为29.44%,此时茶多酚浓度为2mg/mL。
其次研究了茶多酚对PPL和PPA活性的影响。结果表明其对PPL和PPA均有显著的抑制作用,茶多酚对PPL和PPA的半抑制浓度IC50分别为1.23和0.82mg/mL,为可逆性抑制,可逆抑制反应的类型均为非竞争性抑制,抑制常数Ki分别为2.49和1.08mg/mL。通过茶浸提物对PPA抑制活性的研究发现,绿茶、铁观音、红茶和六堡茶浸提物的半抑制浓度IC50分别为8.99,8.89,7.90和40.8mg/mL。抑制动力学结果表明四种茶浸提物对PPA的抑制反应均为可逆抑制,可逆抑制的抑制类型均为非竞争性抑制,抑制常数Ki分别为：11.59,10.37,9.52和43.29mg/mL。
紫外差示光谱结果表明,不同浓度的茶多酚和茶浸提物均能引起PPL和PPA紫外吸收光谱的变化,光谱谱峰升高,峰位红移,浓度越高,变化幅度越大。四种茶浸提液相比,红茶的影响程度最大,六堡茶最小。以278nm和295nm波长光进行激发,研究了四种茶浸提物和茶多酚与PPL和PPA相互作用后的荧光猝灭光谱,结果显示随着茶源物质浓度的增加,PPL和PPA的荧光发射强度逐渐降低,峰位发生红移。结合Stern-Volmer方程,推断四种茶浸提液和茶多酚对PPL和PPA的荧光猝灭均为以非共价键结合的某种不发光的配合物引起的静态猝灭。
通过圆二色谱技术,研究了四种茶浸提液对PPL和PPA圆二色谱及二级结构的影响,结果显示四种茶浸提物均能促使PPA和PPL双负峰往单一负峰转变,茶叶发酵程度越低,即茶多酚的含量越高,转变越明显,峰高变化也越显著。与茶浸提物相互作用后PPA和PPL均变现出α-螺旋含量降低,而β-折叠含量增多,此种结构变化与酶的失活具有相关性。
The effects of green tea, Oolong tea, black tea, liupao tea and tea polyphenols(TP) on the activity of angiotensin converting enzyme(ACE), porcine pancreas lipase(PPL) and porcine pancreasα-amylase(PPA) were studied in this paper. PPL and PPA were selected to study the changes of UV difference spectra, fluorescence spectra and circular dichroism(CD) after treatment with those tea source materials.
Firstly, UV-visible spectrophotometry and HPLC method were compared to verify their applicability. An simple, reliable and precise RP-HPLC method was established for the analysis of ACE inhibitory activity of green tea polyphenols(GTP) with N-hippuryl-His-Leu (HHL) tetrahydrate as the reaction substrate and hippurid acid as the reaction product. The chromatographic conditions were as follows:Column, Intersile ODS-3(4.6mm i.d.×250mm, 5μm); column temperature,35℃; mobile phase, methanol-ditilled water(35:65, v/v, both containing 0.05%(v/v) trifluoroacetic acid and 0.1% triethylamine); flow rate,0.8mL/min; detection wavelength,228nm. An excellent linearity over the range of 5-50μg/mL(R2=0.9997) was observed. The recoveries of hippuric acid were 93.55%-101.50% with a relative standard deviation(RSD) as 2.95%. Measured by this method, GTP showed ACE inhibitory activity The maximum inhibitory rate reached to 29.44% at 2mg/mL of GTP concentration.
Secondly, the effects of TP to the activity of PPL and PPA were studied. Results showed that TP had a significant inhibitory effects to both PPL and PPA, the IC5o were 1.23 and 0.82mg/mL respectively. The inhibitory types were both reversible via non-competitive inhibition with the Ki as 2.49 and 1.08mg/mL respectively. When comes to the four types of tea, the content of TP and solids were reduced with their degree of fermention and their IC50 to PPA were gained through inhibitory research. Meantime, the kinetics equations were established to analyse the inhibition type. Results showed that they were all reversible via non-competitive with the Ki as 11.59,10.37,9.52 and 43.29mg/mL.
Thirdly, the results of UV difference spectra suggested that the UV spectra of PPL and PPA could be changed by different concentration of TP and tea extracts with the peak height increasing and the peak location red shifting, the higher the concentration the more distinctive the change. Compared with the four types of tea extracts, the black tea had the greatest influence while the liupao tea had the smallest. This phenomenon maybe caused from the change of the spacial structure and microenvironment of aromatic amino-acid residues in PPL and PPA by tea sources, which led to the change of PPL and PPA enzyme molecular conformation so as to affect their catalytic activity. What's more, the bindings of TP and the four types of tea to PPL and PPA were studied by fluorescence quenching spectra with 278nm and 295nm wavelength light as stimulated emission, results showed the fluorescent intensity reduced with the concentration increasement and the peak location red shiftment. And the quenching mechanism was researched according to the characteristic feature of Stern-Volmer equation, from which we concluded it was static quenching led by some non-luminous complexes combined with non-covalent bond.
Finally, the CD spectra and secondary structure of PPL and PPA after treament with the four types of tea extracts and TP were obtained using CD technique. Results exhibited that all the tea sources could lead to the change of CD peak height, shape and location, the higher the concentration the more distinctive the change. The two characteristic negative peak of PPL and PPA turned to single by the impact of tea sources and the peak height changed remarkably with the content of TP. As for the secondary structure, the content ofα-helix reduced while theβ-sheet increased after interaction with tea sources. The reduction ofα-helix indicated that the orientation of hydrogen bond changed and the increasement ofβ-sheet caused the unnatural sheet mistakes come up, which two aspects made the nature conformation of enzyme protein can't form so as to the activity of enzyme inactivated.
引文
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