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

Introduction Diabetes mellitus is a pandemic disease and is one of the main threats to human health. Figure 1. Global projection for diabetes epidemic: 2011-2030: source Reference: Ansari et al. 2015. International Journal of Diabetes Research 4(1): 7-12

Introduction Pathophysiology GLUT4: Glucose transporter Adapted from: https://www.pinterest.pt/pin/161707442848089173/

Introduction Hyperglicaemia Source: http://www.giostar.com/Therapy/diabetes-type-2/

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

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

α-Glucosidase With acarbose Without acarbose α-Glucosidase SGLT1 Intestinal villy Enterocyte α-Glucosidase SGLT1 Carbohydrates Glicose Acarbose Source: Adapted from http://www.servier.com/Powerpoint-image-bank 12

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

Introduction Flavonoids Flavone Isoflavone Flavanone Anthocyanin Flavonol Flavanol

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

Introduction a

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

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

Methods Chemical structures of the tested flavonoids a

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

Methods Inhibitor Abs. 405 nm a

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) 40 - 60 min, 37˚C Abs= 405 nm a

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

Variation of substrate concentration Results Variation of substrate concentration α-glucosidase = 0.050 U/mL a

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

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

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).

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

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

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

Results Molecular docking calculations a

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

Methods Inhibition type of the most active flavonoids a

Results Mixed Inhibition a

Competitive Inhibition Results Competitive Inhibition a

Non-Competitive Inhibition Results Non-Competitive Inhibition a

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

Conclusions A microanalysis tecnhique was implemented for the evaluationof the inhibitory effect of flavonoids against α-glucosidase. Enzyme concentration: 0.050 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

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Aknowlgements a