The mechanism of interactions between tea polyphenols and porcine pancreatic alpha‐amylase: Analysis by inhibition kinetics, fluorescence quenching, differential scanning calorimetry and isothermal titration calorimetry

Abstract

ScopeThis study aims to use a combination of biochemical and biophysical methods to derive greater mechanistic understanding of the interactions between tea polyphenols and porcine pancreatic α‐amylase (PPA).Methods and resultsThe interaction mechanism was studied through fluorescence quenching (FQ), differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) and compared with inhibition kinetics. The results showed that a higher quenching effect of polyphenols corresponded to a stronger inhibitory activity against PPA. The red‐shift of maximum emission wavelength of PPA bound with some polyphenols indicated a potential structural unfolding of PPA. This was also suggested by the decreased thermostability of PPA with these polyphenols in DSC thermograms. Through thermodynamic binding analysis of ITC and inhibition kinetics, the equilibrium of competitive inhibition was shown to result from the binding of particularly galloylated polyphenols with specific sites on PPA. There were positive linear correlations between the reciprocal of competitive inhibition constant (1/K ic), quenching constant (K FQ) and binding constant (K itc).ConclusionThe combination of inhibition kinetics, FQ, DSC and ITC can reasonably characterize the interactions between tea polyphenols and PPA. The galloyl moiety is an important group in catechins and theaflavins in terms of binding with and inhibiting the activity of PPA.

Soluble polysaccharides reduce binding and inhibitory activity of tea polyphenols against porcine pancreatic α-amylase

“…For the respective polyphenols, there were negative linear correlations between both the values of 1/K ic and K FQ with that of IC 50 in the absence and presence of three polysaccharides (Table 5). In addition, there was a linear relationship between K FQ and 1/K ic in the absence of polysaccharides (Table 6), consistent with both constants reflecting the binding of polyphenols with PPA (Sun et al, 2017). Interestingly, in the presence of polysaccharides, although both the values of 1/K ic and K FQ were decreased, the slopes remained about the same (Table 6).…”

“…Similarly, in the Different letters in the same line represent significantly different mean values for the corresponding parameters (P < 0.05). The values of K FQ and k q are from our previous study (Sun et al, 2017). The polyphenols used to calculate each correlation included EGCG, ECG, TA, TF, TF1 and TF2.…”

“…GTE and ECG were able to quench the PPA fluorescence through both the dynamic (collision-controlled quenching) and static (complex-controlled quenching) mechanism. Taking into account the high values of K FQ and k q , TF2 was suggested to quench the PPA fluorescence through the formation of complex with the enzyme (the static mechanism) (Sun et al, 2017). As shown in Table 2, although the three polysaccharides decreased the coefficients or slope of Stern-Volmer equations, they did not alter the quenching mechanism of tea polyphenols as the types of equations remained unchanged.…”

“…However, limited work has been conducted on the effects of soluble polysaccharides on the inhibitory activity of polyphenols against a-amylase. The competitive or uncompetitive inhibition of an enzyme is attributed to the reversible (solution state) binding of the inhibitor with the enzyme or enzymesubstrate complex, rather than insoluble aggregate formation (Sun, Gidley, & Warren, 2017). Therefore, PPA inhibition, IC 50 values, kinetics of inhibition and fluorescence quenching can be applied to characterize the inhibition of enzymes and the effects of additional components.…”

Effect of in vitro gastrointestinal digestion on the composition and bioactivity of anthocyanins in the fruits of cultivated Lycium ruthenicum Murray

“…Recently, the practice of using phytochemicals to inhibit carbohydrate-hydrolyzing enzymes (e.g. α-amylase and αglucosidase) has attracted widespread interests, as the enzyme inhibition can be a potential therapeutic substitute to insulin injection for type II diabetes (Sun, Gidley, & Warren, 2017). α-Glucosidase is a key digestive enzyme hydrolyzing terminal non-reducing (1→4)-linked short chain reducing sugars (e.g.…”