A wide variety of natural and synthetic compounds have been screened to modulate DM pathways upon inhibition of the carbohydrate hydrolyzing enzymes, α-amylase and α-glucosidase [19, 29,30,31]. Among these, some chromones has been studied regarding inhibition of both enzymes, varying the type and number of substituents attached to their skeleton [19]. As far as known, this is the first report on α-amylase and α-glucosidase inhibitory activity by a range of synthetic 2-SC, which includes twelve derivatives with different substitution patterns (Fig. 1). Thus, compounds are numbered 1-3, in which compounds of set 1 possess a 3′,4′-(OH)2 in B-ring, compounds of set 2 bear a 4′-OH in B-ring and compounds of set 3 presents no substitution in B-ring. The variation in A-ring involves the presence in both 5- and 7-hydroxy groups (derivatives A), 7-OH substitution (derivatives B), 5-OH substitution (derivatives C) and without substitution in A-ring (derivatives D).

Following the obtained results, the inhibitory effects on pancreatic α-amylase activity by 2-SC seem to depend particularly on the OH-substitution pattern at the styryl aromatic ring, B-ring. Compounds of set 1, with a catechol unit [3′,4′-(OH)2] in B-ring, presented a considerably higher effect than those lacking this feature as it can be confirmed by the comparison of the results from set 1 with the those from sets 2 and 3. In set 1 we found the most active compounds of this work, 2-SC 1C, with a 5-hydroxy group of A-ring, presenting an IC50 value of 25.9 ± 0.9 μM and 2-SC 1A, with 5- and 7-hydroxy groups of A-ring, exhibiting an IC50 value of 29 ± 2 μM. Moreover, low efficiency was observed for compounds of set 3 (unsubstituted in B-ring), where it was not possible to achieve the IC50 values of the compounds at the highest tested concentration. Accordingly, it was previously described in in vitro studies that the absence of substituents in B-ring in the several flavonoids was disadvantageous for the inhibitory activity against α-amylase [21, 32, 33].

The presence of the hydroxy substituents in the A-ring also contributes positively to the inhibitory efficiency of 2-SC 1 and 2. Thus, 2-SC 1A (IC50 = 29 ± 2 μM) and 2A (IC50 = 62 ± 3 μM), which possess 5,7-(OH)2 substitution were shown to be more effective inhibitors than 2-SC 1D (IC50 = 88 ± 2 μM) and 2D (33% inhibition at 200 μM), that are unsubstituted in A-ring. A similar conclusion was taken by our group when a panel of structurally-related flavones was tested, lacking the Cα = Cβ double bond of 2-SC. Luteolin [5,7,3′,4′-(OH)4 flavone] (IC50 = 78 ± 3 μM) and apigenin [5,7,4′-(OH)3 flavone] (IC50 = 122 ± 7 μM) were more active than 3′,4′-(OH)2 flavone (46% inhibition at 200 μM) and 4′-OH flavone (no inhibitory effect) [21]. Even more, comparing the results of the above-referred 2-SC with the related flavones we can notice that 2-SC are more active against α-amylase than flavones, leading to infer that the presence of the Cα = Cβ double bond is relevant to enhance the inhibitory effect. This fact points to a likely contribution of the styryl moiety to molecular stabilization, increasing the compound’s inhibitory activity [34].

The most active compounds were 2-SC 1A-C and 2A, with inhibitory order potency as 2-SC 1C (IC50 = 25.9 ± 0.9 μM) ≈ 2-SC 1A (IC50 = 29 ± 2 μM) > 2-SC 2A (IC50 = 62 ± 3 μM) ≈ 2-SC 1B (IC50 = 68 ± 3 μM). Nevertheless, the positive control, acarbose (IC50 = 0.62 ± 0.07 μM), was a noticeably more efficient inhibitor of α-amylase than the tested 2-SC.

The mechanism explaining α-amylase inhibition and the kinetic parameters of all active compounds and the positive control acarbose were further assessed using the generalized Michaelis-Menten model and its simplifications. Both strategies revealed that compounds 1A-D, 2A and 2B act via the competitive type of inhibition i.e., compete directly for the catalytic site of α-amylase. In this circumstance, higher amounts of substrate would be required to generate similar reaction product concentrations in the same period. Acarbose displayed a mixed-type inhibition mechanism, as previously described by other authors for the inhibition of pancreatic α-amylase enzymatic activity [11, 21, 33, 35].

Concerning the hydrolytic catalysis promoted by α-glucosidase and regarding its inhibition by the 2-SC tested, whatever the considered sets 1-3, derivatives A (possessing 5,7-(OH)2 substitution) were the most active compounds. Even so, 2-SC 1A (IC50 = 19 ± 3 μM) was 1.5 times more active than 2-SC 2A (IC50 = 29 ± 3 μM) and 4 times more active than 2-SC 3A (IC50 = 76 ± 3 μM). These results lead us to infer the importance, simultaneously, of the 5-OH and 7-OH groups for the high inhibitory activity towards α-glucosidase. This is consistent with previous findings pinpointing the presence of hydroxy groups in the A-ring as pivotal to the inhibitory profile of flavones. Accordingly, luteolin [5,7,3′,4′-(OH)4 flavone] (IC50 = 46 ± 6 μM) and apigenin [5,7,4′-(OH)3 flavone] (IC50 = 82 ± 6 μM) were more efficient α-glucosidase inhibitors than the corresponding flavones with a single hydroxyl group in A-ring and even more active than those lacking this substitution on A-ring [22].

In contrast, derivatives D (without OH groups in A-ring) were the less active of their groups, as can be observed by comparison of the IC50 values of 2-SC 1D (IC50 = 149 ± 12 μM), 2-SC 2D (IC50 = 136 ± 10 μM) and 2-SC 3D (24% inhibition at 200 μM) with those of the respective derivatives A. A similar conclusion was drawn by us and other groups when studied the inhibitory effects of flavonoids substituted with hydroxy groups [22, 36, 37]. A dramatic loss of the α-glucosidase inhibitory efficiency was verified for flavones missing any hydroxy group on A-ring, as noticed for 3′,4′-(OH)2 flavone (32% inhibition at 200 μM), 4′-OH flavone and parent flavone that were not active for the maximum tested concentration of 200 μM [22].

The presence of a 7-OH group in the A-ring of 2-SC contributed positively to the α-glucosidase inhibitory activity since all derivatives B were able to inhibit this enzyme, with inhibitory order potency as 2-SC 2B (IC50 = 56 ± 7 μM) > 2-SC 1B (IC50 = 86 ± 7 μM) > 2-SC 3B (IC50 = 143 ± 6 μM). The presence of the 5-OH group in the A-ring also influenced the inhibitory effects by 2-SC and was more notorious in derivative 1C (IC50 = 47 ± 2 μM), twice more active than derivative 2C (IC50 = 105 ± 2 μM).

Another important feature can be taken from the comparison of the results from the inhibitory activity of 2-SC and those of the corresponding flavones. Analyzing the IC50 values of the most active 2-SC 1A (IC50 = 19 ± 3 μM), 2A (IC50 = 29 ± 3 μM), 1C (IC50 = 47 ± 2 μM) and 2B (IC50 = 56 ± 7 μM) we can state that were considerably lower than those of the corresponding flavones: luteolin [5,7,3′,4′-(OH)4 flavone] (IC50 = 46 ± 6 μM); apigenin [5,7,4′-(OH)3 flavone] (IC50 = 82 ± 6 μM); 5,3′,4′-(OH)3 flavone (IC50 = 66 ± 7 μM) and 7,4′-(OH)2 flavone (IC50 ≈ 200 μM), clear evidence of the importance of Cα = Cβ double bond for the high inhibitory profile of 2-SC [22, 34].

Unlike the α-amylase inhibitory assay, all active 2-SC were able to inhibit α-glucosidase in a very efficient manner, with corresponding IC50 values ranging from 19 to 149 μM, being the most active 2-SC 1A (IC50 = 19 ± 3 μM), more than 25 times more active than the positive control, acarbose (IC50 = 528 ± 9 μM). A strong inhibition of α-glucosidase combined with a mild inhibitory activity against α-amylase will avoid a prolonged inhibition of starch hydrolysis and the accumulation of undigested carbohydrates in the colon, responsible for the severe gastrointestinal complications registered in type 2 DM patients when using this therapeutic strategy [13, 19].

The type of α-glucosidase inhibition and the kinetic constants of all active 2-SC and of the positive control, acarbose, were also evaluated, using Lineweaver–Burk plots and Solver supplement of Excel Microsoft Office™. The two methods were in accordance and showed that 2-SC 1A behaved as a competitive inhibitor, meaning that this compound competes directly with the substrate for the activity of the α-glucosidase enzyme. The mixed type of inhibition exhibited by derivative 2B is representative that the compound can bind to the free enzyme or to enzyme-substrate complex but has a greater affinity for one state or the other. The remaining tested compounds displayed a non-competitive type of inhibition on α-glucosidase activity, meaning that the compounds can bind with the same affinity to free enzyme and enzyme-substrate complex, resulting in a decrease of the enzymatic activity that cannot be overcome by increasing the substrate concentration.

Concerning the positive control acarbose, a competitive type of inhibition mechanism was observed, which is corroborated by other authors [22, 35].