J Food Bioact

**Table 1.** Trypsin apparent catalytic parameters (V ^app_max, K^app_M, K_i and K_i′) and inhibition type for the hydrolysis of BAPNA in the presence of the flavonoids
Flavonoid	Concentration (μM)	V ^app_max (10⁻² mM/min)	K^app_M (mM)	Inhibition type	K_i and K′_i (mM)
Data are represented as mean value ± standard deviation of triplicate analysis. Different letters in the same column indicate statistically significant values (Fisher's least significant difference analysis, p < 0.05) respect to control, or between treatments for free enzyme or enzyme substrate dissociation constants (K_i and K_i′, respectively). V_max and K_M correspond to maximal rate and Michaelis-Menten constant, respectively. n.d. means not determined.
CONTROL	0.00	3.75 ± 0.20^a	0.35 ± 0.05^fg	None	0.00	0.00
HES	12.55	3.60 ± 0.10^a	0.35 ± 0.01^g	Mixed	73.15 ± 1.90^a	78.05 ± 2.50^a
25.10	3.60 ± 0.05^a	0.40 ± 0.05^fg
50.09	3.40 ± 0.70^b	1.00 ± 0.10^d
LUT	12.70	3.05 ± 0.50^ab	1.00 ± 0.00^d	Mixed	42.05 ± 0.70^d	44.50 ± 0.85^c
25.45	2.10 ± 0.00^e	2.40 ± 0.15^b
50.20	1.40 ± 0.70^f	5.60 ± 0.95^a
QUE	12.56	3.60 ± 0.15^a	0.40 ± 0.00^f	Mixed	58.90 ± 0.10^c	61.00 ± 1.30^b
25.08	3.40 ± 0.10^b	0.80 ± 0.18^d
50.32	2.65 ± 0.20^d	1.25 ± 0.05^c
CAT	12.49	3.80 ± 0.25^a	0.35 ± 0.00^g	None	n.d.	n.d.
25.07	3.70 ± 0.00^a	0.35 ± 0.00^g
50.91	3.70 ± 0.15^a	0.35 ± 0.05^g
RUT	12.56	3.70 ± 0.05^a	0.40 ± 0.10^fg	Mixed	68.20 ± 2.10^b	75.00 ± 8.00^a
25.00	3.40 ± 0.20^a	0.50 ± 0.00^e
50.54	3.00 ± 0.05^c	1.05 ± 0.30^cd

Table 1. Trypsin apparent catalytic parameters (V ^app_max, K^app_M, K_i and K_i′) and inhibition type for the hydrolysis of BAPNA in the presence of the flavonoids

Flavonoid

Concentration (μM)

V ^app_max (10⁻² mM/min)

K^app_M (mM)

Inhibition type

K_i and K′_i (mM)

Data are represented as mean value ± standard deviation of triplicate analysis. Different letters in the same column indicate statistically significant values (Fisher's least significant difference analysis, p < 0.05) respect to control, or between treatments for free enzyme or enzyme substrate dissociation constants (K_i and K_i′, respectively). V_max and K_M correspond to maximal rate and Michaelis-Menten constant, respectively. n.d. means not determined.

CONTROL

0.00

3.75 ± 0.20^a

0.35 ± 0.05^fg

None

0.00

HES

12.55

3.60 ± 0.10^a

0.35 ± 0.01^g

Mixed

73.15 ± 1.90^a

78.05 ± 2.50^a

25.10

3.60 ± 0.05^a

0.40 ± 0.05^fg

50.09

3.40 ± 0.70^b

1.00 ± 0.10^d

LUT

12.70

3.05 ± 0.50^ab

1.00 ± 0.00^d

Mixed

42.05 ± 0.70^d

44.50 ± 0.85^c

25.45

2.10 ± 0.00^e

2.40 ± 0.15^b

50.20

1.40 ± 0.70^f

5.60 ± 0.95^a

QUE

12.56

3.60 ± 0.15^a

0.40 ± 0.00^f

Mixed

58.90 ± 0.10^c

61.00 ± 1.30^b

25.08

3.40 ± 0.10^b

0.80 ± 0.18^d

50.32

2.65 ± 0.20^d

1.25 ± 0.05^c

CAT

12.49

3.80 ± 0.25^a

0.35 ± 0.00^g

None

n.d.

25.07

3.70 ± 0.00^a

0.35 ± 0.00^g

50.91

3.70 ± 0.15^a

0.35 ± 0.05^g

RUT

12.56

3.70 ± 0.05^a

0.40 ± 0.10^fg

Mixed

68.20 ± 2.10^b

75.00 ± 8.00^a

25.00

3.40 ± 0.20^a

0.50 ± 0.00^e

50.54

3.00 ± 0.05^c

1.05 ± 0.30^cd

**Table 2.** Fluorescence quenching parameters (K^app_D, K_sv, k_q, n and K_a) of trypsin in the presence of flavonoids
Flavonoid	K^app_D (µM)	K_sv (10⁻¹ mM⁻¹)	k_q (10⁻¹² mM⁻¹ s⁻¹)	n	K_a (10⁻¹ mM⁻¹)
Data are the mean value ± standard deviation of triplicate analysis. Different letters in the same column indicate statistically significant values (Fisher's least significant difference analysis, p < 0.05) respect to control. n.d. means not determined.
HES	38.00 ± 4.02^a	0.96 ± 0.05^b	0.10 ± 0.01^c	0.70 ± 0.15^b	0.60 ± 0.00^d
LUT	16.10 ± 3.00^c	1.87 ± 0.10^a	9.86 ± 1.05^a	0.90 ± 0.01^a	1.60 ± 0.22^a
QUE	17.50 ± 1.50^c	1.22 ± 0.30^b	6.43 ± 0.40^b	0.80 ± 0.20^ab	0.90 ± 0.14^b
CAT	n.d.	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^d	0.00 ± 0.00^e
RUT	31.00 ± 2.67^b	0.51 ± 0.00^c	6.61 ± 0.15^b	0.60 ± 0.03^c	0.70 ± 0.01^c

Table 2. Fluorescence quenching parameters (K^app_D, K_sv, k_q, n and K_a) of trypsin in the presence of flavonoids

Flavonoid

K^app_D (µM)

K_sv (10⁻¹ mM⁻¹)

k_q (10⁻¹² mM⁻¹ s⁻¹)

K_a (10⁻¹ mM⁻¹)

Data are the mean value ± standard deviation of triplicate analysis. Different letters in the same column indicate statistically significant values (Fisher's least significant difference analysis, p < 0.05) respect to control. n.d. means not determined.

HES

38.00 ± 4.02^a

0.96 ± 0.05^b

0.10 ± 0.01^c

0.70 ± 0.15^b

0.60 ± 0.00^d

LUT

16.10 ± 3.00^c

1.87 ± 0.10^a

9.86 ± 1.05^a

0.90 ± 0.01^a

1.60 ± 0.22^a

QUE

17.50 ± 1.50^c

1.22 ± 0.30^b

6.43 ± 0.40^b

0.80 ± 0.20^ab

0.90 ± 0.14^b

CAT

n.d.

0.00 ± 0.00^d

0.00 ± 0.00^e

RUT

31.00 ± 2.67^b

0.51 ± 0.00^c

6.61 ± 0.15^b

0.60 ± 0.03^c

0.70 ± 0.01^c

**Table 3.** Gibbs free energy (ΔG°, kCal/mol), the trypsin amino acid residues interacting with flavonoids, and the distance (Å) for the possible enzyme-flavonoid conformations
Flavonoids	Amino acid residues lining the binding site, distance and binding energy interaction
HES	Tyr¹⁵¹ (3.6; 3.8)	Asp⁷⁴ (2.8), Tyr¹⁵¹ (3.2)	−6.7
LUT	Tyr¹⁵¹ (3.8; 3.7; 3.7; 3.8)	Asp⁷⁴ (3.1), Gln¹⁹² (2.4)	−7.3
QUE	Tyr¹⁵¹ (3.9; 3.7; 4.0; 3.9)	Gly¹⁹³ (3.1)	−6.5
CAT	Ninguna	Asn³⁴ (3.1), Arg⁶² (2.8), Gln⁶⁴ (3.1)	−7.5
RUT	Ninguna	Asn⁹⁷ (2.9; 3.1), Ser¹⁹⁵ (2.8), Gln¹⁹² (2.7), Gly²¹⁶ (2.7)	−7.0

Table 3. Gibbs free energy (ΔG°, kCal/mol), the trypsin amino acid residues interacting with flavonoids, and the distance (Å) for the possible enzyme-flavonoid conformations

Flavonoids

Amino acid residues lining the binding site, distance and binding energy interaction

Hydrophobic

Hydrogen Binding

ΔG°

HES

Tyr¹⁵¹ (3.6; 3.8)

Asp⁷⁴ (2.8), Tyr¹⁵¹ (3.2)

−6.7

LUT

Tyr¹⁵¹ (3.8; 3.7; 3.7; 3.8)

Asp⁷⁴ (3.1), Gln¹⁹² (2.4)

−7.3

QUE

Tyr¹⁵¹ (3.9; 3.7; 4.0; 3.9)

Gly¹⁹³ (3.1)

−6.5

CAT

Ninguna

Asn³⁴ (3.1), Arg⁶² (2.8), Gln⁶⁴ (3.1)

−7.5

RUT

Ninguna

Asn⁹⁷ (2.9; 3.1), Ser¹⁹⁵ (2.8), Gln¹⁹² (2.7), Gly²¹⁶ (2.7)

−7.0