Professor Ming Zhou of Shanghai Institute of Technology: The ability of rutin and quercetin in buckwheat to inhibit starch-digesting enzymes

With the improvement of people's quality of life and the improvement of medical standards, the global average healthy life expectancy has increased
steadily.
However, the alarming increase in the incidence of metabolic diseases such as diabetes has become a major public health problem
in many countries.
Tartary buckwheat has shown good market potential
due to its high nutritional and medicinal value.
Studies have found that buckwheat is rich in starch, especially resistant starch, which can be an ideal food
for diabetics.
At the same time, buckwheat contains rich flavonoids, including rutin, quercetin, kaempferol and other components, which are the main functional factors for lowering blood sugar, of which rutin is the main component of buckwheat flavonoids, accounting for more than
70% of flavonoids.

Therefore, Zhou Ming, Ma Sijia, Zhou Xiaoli*, etc.
of the School of Fragrance and Flavor Technology and Engineering of Shanghai University of Technology discussed the individual inhibitory effect and mechanism of the main flavonoids (rutin and quercetin) on starch-digesting enzymes in buckwheat by simulating the in vitro digestion system of buckwheat starch, as well as the inhibitory effect on starch-digesting enzyme activity when present, to provide a certain theoretical basis for the deep processing of buckwheat products.
In order to regulate the function of buckwheat and maximize the hypoglycemic effect of buckwheat
.

1.
The ability of rutin and quercetin to inhibit starch-digesting enzymes alone

From Figure 1A, the IC50 values of rutin and quercetin for α-amylase were 0.
36 mg/mL and 0.
22 mg/mL, respectively.
From Figure 1B, the IC50 values of rutin and quercetin for α-glucosidase were 1.
3 mg/mL and 0.
362 mg/mL
, respectively.
Quercetin has a stronger inhibitory ability than rutin because the hydroxyl group on the C ring of quercetin is replaced by a glycan group to become rutin, and the conversion of hydroxyl radicals to glycosyls may occur steric hindrance, weakening the binding of rutin to enzymes, thereby reducing the inhibitory ability
of rutin.

2.
Analysis of the inhibitory kinetics of rutin and quercetin on starch-digesting enzymes

As can be seen from Figure 2, both rutin and quercetin at different mass concentrations can obtain a straight line through the origin, and the slope decreases
with the increase of mass concentration.
It can be seen that rutin and quercetin have reversible inhibitory effects on α-amylase and α-glucosidase
.

As shown in Figures 3A and C, the double reciprocal curve of rutin and quercetin against α-amylase inhibition, all straight lines intersect the longitudinal axis, and as the mass concentration of rutin and quercetin increases, the Km value increases while the v maximum remains constant
.
Therefore, rutin and quercetin are competitive inhibition of α-amylase, and by binding to the active center of α-amylase, occupying the binding site of the substrate, thereby inhibiting the hydrolysis
of the substrate.
According to the competitive inhibition formula, the Ki values of rutin and quercetin for α-amylase were 806.
29 and 793.
92 μg/mL, respectively, indicating that the binding of quercetin to α-amylase was stronger and had a stronger inhibitory effect, which was consistent with
the experimental results of quercetin inhibiting α-amylase stronger than rutin in section 2.
1.

As shown in Figure 3B, all straight lines intersect in quadrant 3, and the Km value and v maximum decrease at the same time, indicating that rutin has a mixed inhibition of α-glucosidase, where the Ki value is 9.
61 μmol/L and the Kis value is 3.
38 μmol/L, indicating that rutin is more inclined to bind
to enzyme-substrate complexes 。 As shown in Figure 3D, all straight lines intersect in quadrant 2, and the Km value gradually increases, while the maximum v gradually decreases, indicating that quercetin is a mixed inhibition of α-glucosidase, with a Ki value of 3.
72 μmol/L and a Kis value of 9.
74 μmol/L, indicating that quercetin is more inclined to bind
to free α-glucosidase.

3.
Fluorescence spectroscopy analysis of starch-digesting enzymes by rutin and quercetin

As shown in Figures 4A and B, the λmax of α-amylase and α-glucosidase moved from 340 nm to 335 nm and 332 nm, respectively, as the concentration of rutin increased; As shown in Figures 4C and D, the λmax of α-amylase and α-glucosidase shifted from 342 nm and 340 nm to 338 nm
, respectively, as quercetin concentrations increased.
This phenomenon suggests that there is an interaction between rutin and quercetin and α-amylase and α-glucosidase, resulting in the gradual exposure of amino acid residues to aqueous solutions and reduced polarity in the environment
.

4.
Analysis of binding properties of rutin and quercetin to starch-digesting enzymes

From Figure 5, it can be seen that rutin and quercetin have a good linear relationship
with the Stern-Volmer curves of α-amylase and α-glucosidase.
It can be seen from Table 1 that with the increase of temperature, the Ksv values of rutin for α-amylase and α-glucosidase decreased by 2.
58×10 4 L/mol and 1.
97×10 4 L/mol, respectively, and the Ksv values of quercetin for α-amylase and α-glucosidase decreased by 1.
60×10 4 L/mol and 2.
12×10⁴L/mol, respectively.
The Ksv values decreased with the increase of temperature, and the Kq values were 10¹³ and 10¹² orders of magnitude, which was much greater than the maximum scattering collision quenching constant of 2.
0×^{10 10}L/(mol·s) of biological macromolecules, indicating that rutin and quercetin and α-amylase and α-glucosidase were all statically quenched
by forming complexes.

As can be seen from Figure 6, the bilogarithmic curves of rutin and quercetin interaction between α-amylase and α-glucosidase have a good linear relationship, and the slope and intercept of the fitted straight line are combined with the static quenching formula, from which the Ka value and n
can be calculated.
As shown in Table 1, the Ka value of quercetin to α-amylase and α-glucosidase was higher than that of rutin, indicating that quercetin had stronger binding to α-amylase and α-glucosidase.
The Ka value of rutin and quercetin to α-amylase was higher than that of α-glucosidase, indicating that rutin and quercetin had stronger binding to α-amylase
.
In addition, the n-value is approximately 1, indicating that rutin and quercetin have only one (or class) binding site on α-amylase and α-glucosidase
.

As shown in Table 1, both ΔG are negative, indicating that the binding of rutin and quercetin to α-amylase and α-glucosidase is a spontaneous process
.
When rutin and quercetin react with α-amylase, both ΔH and ΔS are positive, indicating that this binding is an entropy-driven exothermic process, and hydrophobic interaction forces and hydrogen bonds are the main drivers for the stabilization of rutin and quercetin with α-amylase
complexes.
When rutin and quercetin react with α-glucosidase, both ΔH and ΔS are negative, indicating that the main driving force is hydrogen bonds
.

5.
The joint inhibitory effect of rutin and quercetin on starch digestive enzymes