Studies Of Tea Extracts On The Activity And Secondary Structure Of Functional Enzymes

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.