1. Marisa Freitas, Adelaide Sousa, Daniela Ribeiro, Eduarda Fernandes
Prediction of anti-diabetic activity of flavonoids targeting α-glucosidase
Marisa Freitas, Adelaide Sousa, Daniela Ribeiro, Eduarda Fernandes 1

2. Introduction
Diabetes mellitus is a pandemic disease and is one of the main threats to human health.
Figure 1. Global projection for diabetes epidemic: : source
Reference: Ansari et al International Journal of Diabetes Research 4(1): 7-12

3. Introduction Pathophysiology GLUT4: Glucose transporter
Adapted from:

4. Introduction Hyperglicaemia
Source:

5. Pharmacological treatment
Introduction
Pharmacological treatment
Insulin secretagogues
Biguanides
Thiazolidinediones
α-glucosidase
Sulfonylureas
Meglitidines
Tolbutamide
Chloroprapamide
Glyburide
Repaglinide
Metmorfin
Rosiglitazone
Acarbose
Miglitol

6. Maltase-glucoamylase
Introduction
Starch
α-amylase
Maltose
α-glucosidase
Glucose
Sucrase-isomaltase
Maltase-glucoamylase

7. α-Glucosidase With acarbose Without acarbose α-Glucosidase SGLT1
Intestinal villy
Enterocyte
α-Glucosidase
SGLT1
Carbohydrates
Glicose
Acarbose
Source: Adapted from 12

8. Introduction Side effects of anti-diabetic drugs Flatulence;
Abdominal discomfort;
Hepatic disturbances;
Contraindicated in patients with inflammatory diseases

9. Introduction Flavonoids Flavone Isoflavone Flavanone Anthocyanin
Flavonol
Flavanol

10. Introduction Biological activities of flavonoids
Anti-diabetic
Anti-inflamamtory
Antioxidant
Antimicrobicide
Anti-tumoral
Inhibition of α-glucosidase 12

11. Introduction a

12. Structure activity relationship SAR
Introduction
Experimental variables
Enzyme concentration
0.3 U/mL
1.7 U/mL
Substrate concentration
1 mM
20 mM
Kinetic time
5 min
120 min
Structure activity relationship SAR a

13. AIM
Optimization of a microanalysis tecnhique for the evaluation of the α-glucosidase activity
Study of the inhibitory activity of a panel of flavonoids against the α-glucosidase activity
Inhibition type of the most active flavonoids 1 2 3 a

14. Methods
Chemical structures of the tested flavonoids a

15. Methods Chemical structures of the tested flavonoids a
In the group D we fixed the OH group in positions 5 and 7 and vary the substituents in the 3’ and 4’ of the B ring and the positon 3 of the C ring.
With the E group we want to test the relevance of the presence or absence of the C2=C3 double bond in the C-ring, comparing with other groups of flavonoids. a

16. Methods
Inhibitor
Abs. 405 nm a

17. Methods Optimization of the microanalysis tecnhique DMSO
Phosphate buffer, pH=6.8
α-glucosidase from Saccharomyces cerevisiae (0 – 0.4 U/mL)
5 min, 37˚C
pNPG (150 – 2400 µM)
min, 37˚C
Abs= 405 nm a

18. Variation of α-glucosidase concentration
Results
Variation of α-glucosidase concentration
pNPG = 600 µM a

19. Variation of substrate concentration
Results
Variation of substrate concentration
α-glucosidase = U/mL a

20. Methods Study of the inhibitory activity of a panel of flavonoids
Flavonoids/Acarbose/DMSO
Phosphate buffer, pH=6.8
α-glucosidase from Saccharomyces cerevisiae (0.05 U/mL)
5 min, 37˚C
pNPG (600 µM)
30 min, 37˚C
Abs= 405 nm a

21. Results
Table 1. Structures and in vitro α-glucosidase inhibition by the studied flavonoids (IC50 µM, mean ± SEM).
Compound
Structure
R2’ R3
R3’
R4’ R6 R7 R8
IC50 (μM) A1
(Flavone) - H
<20%*200 μM a A2 OH
<20%* 200 μM a A3 A4
32 ± 4%* 200 μM a A5
54 ± 3 A6
OM e
<20%* 100 μM a B1 B2
31 ± 4%* 200 μM a B3
66 ± 2 B4
66 ± 7
a - Inhibitory activity (mean ± SEM %) at the highest tested concentration (in superscript). a

22. Results
Table 1. Structures and in vitro α-glucosidase inhibition by the studied flavonoids (IC50 µM, mean ± SEM).
Compound
Structure
R2’ R3
R3’
R4’ R6 R7 R8
IC50 (μM) C1 - H OH
<20%* 200 μM a C2
53 ± 4 C3
≈ 200 C4
42 ± 4 C5
96 ± 10 C6
95 ± 7 C7
7.6 ± 0.4 C8
OMe
22 ± 2%* 100 μM a C9
C10
86 ± 6
C11
31 ± 3%* 200 μM a
C12
OBn
C13
32 ± 3%* 200 μM a
a - Inhibitory activity (mean ± SEM %) at the highest tested concentration (in superscript).

23. Results
Table 1. Structures and in vitro α-glucosidase inhibition by the studied flavonoids (IC50 µM, mean ± SEM).
Compound
Structure
R2’ R3
R3’
R4’ R6 R7 R8
IC50 (μM) D1
(Chrysin) H -
<20%* 50 μM a D2
(Galangin) OH
21 ± 3%* 200 μM a D3
(Baicalein)
44 ± 3 D4
89 ± 3 D5
(Apigenin)
82 ± 6 D6
(Kaempferol)
32 ± 3 D7
(Luteolin)
46 ± 6
D8 (Quercetin)
15 ± 3 D9
(Morin)
32 ± 2 a

24. Results
Table 1. Structures and in vitro α-glucosidase inhibition by the studied flavonoids (IC50 µM, mean ± SEM).
Compound
Structure
R2’ R3
R3’
R4’ R6 R7 R8
IC50 (μM) E1
(Naringenin) - H
45 ± 3%* 200 μM a E2
(Eriodictyol) OH
35 ± 4%* 200 μM a E3
(Taxifolin)
≈200
Positive control:
Acarbose
607 ± 56
a - Inhibitory activity (mean ± SEM %) at the highest tested concentration (in superscript). a

25. Results
The most active flavonoids:   A5
D8 (quercetin) C7 a

26. Results
Molecular docking calculations a

27. Methods Inhibition type of the most active flavonoids
Enzyme: 0.05 U/mL
pNPG: 300, 600 e 1200 µM
Michaelis-Menten Equation:
V 0 = V máx [S] K m +[S]
Lineweaver-Burk Equation:
1 V 0 = K m V máx × 1 [S] + 1 V máx a

28. Methods
Inhibition type of the most active flavonoids a

29. Results
Mixed Inhibition a

30. Competitive Inhibition
Results
Competitive Inhibition a

31. Non-Competitive Inhibition
Results
Non-Competitive Inhibition a

32. Results a Flavonoid Type of Inhibition Ki (μM) A5 Mixed 41.0 B3 127.0
Table 2. Ki values (lM) for the inhibition of yeast a-glucosidase by the selected flavonoids.
Flavonoid
Type of Inhibition
Ki (μM) A5
Mixed
41.0 B3
127.0 C7
Competitive
6.5
D8 (quercetin)
6.8
E3 (taxifolin)
Non- competitive
347.1
Positive control:
Acarbose
457.3 a

33. Conclusions
A microanalysis tecnhique was implemented for the evaluationof the inhibitory effect of flavonoids against α-glucosidase.
Enzyme concentration: U/mL
Substrate concentration: 600 µM
Kinetic time: 30 minutes
The substitution pattern of flavonoids significatively affects their inhibitory activity.
The flavonoid structure, the position and number of OH groups are determinant factors for the intended effect. a

34. a

35. Aknowlgements a