2.3.1. Structure-Activity Relationship Assessment of Flavones as α-Glucosidase Inhibitors

Tested flavone compounds **1**, **2**, **3**, and **4**, along with flavone standard aglycones, i.e., apigenin and luteolin, exhibited strong α-glucosidase inhibition, where IC50 values were determined at 613, 328, 439, and 390 μM, respectively, exceeding that of acarbose determined at 662 μM, a commercial α-glucosidase inhibitor anti-diabetic drug. The order of activity of the isolated flavone glycosides was as such, with IC50 values at 327.9, 390.4, 439.2, and 612.9 μM for compounds, viz., **2**, **4**, **3**, and **1**, which are much higher than those of acarbose. However, flavone glycosides were less potent than their corresponding aglycones, i.e., apigenin and luteolin, with IC50 values at 85.6 and 48.2 μM, respectively (Figure 4A,B, Table 5).

Among glycosides, compound **2**, identified as luteolin 6-C-α-L-rhamnopyranosyl-(1- 2)-β-D-fucopyranoside, showed the highest inhibitory activity among all isolated flavone glycosides in line with its aglycone, suggestive for the improved efficacy of C-glycosyl flavone against α-glucosidase enzyme, which is in agreement with reports that sugar moiety attached at C-6 position improved efficacy against pancreatic lipase inhibitory activity [40], extended herein to include the α-glucosidase inhibition effect (Figure 4B).

In contrast, compound **1**, identified as apigenin 6-C-(2-deoxy-β-D-galactopyranoside)- 7-O-β-D-quinovopyranoside, showed the weakest inhibitory activity among all isolated flavone glycosides, with IC50 612.9 μM, likely attributed to the glycosylation of hydroxy group at the C-7 position [41] (Figure 4B) and absent in compounds **2**, **3**, and **4**. Compounds **3** and **4** exhibited strong inhibition with an IC50 value of 439.2 μM and 390.4 μM, respectively, in line with previously published data on the efficacy of apigenin 6-*C*-(2- O-α-rhamnopyranosyl)-β-fucopyranoside in lowering the glucose level in hyperglycemic rats [40]. Standard apigenin and luteolin showed the strongest inhibitory activity, with IC50 values at 85.6 and 48.2 μM, respectively, compared to that of acarbose (661.6 μM), with luteolin showing the stronger inhibitory activity compared to that of acarbose, which is in

accordance with the previously reported α-glucosidase inhibitory activity [42]. In line with our findings, hydroxylation at C-3 of the B-ring of apigenin, in particular, was reported to enhance the α-glucosidase inhibition activity [41], whereas glycosylation affected it negatively compared to the aglycones [43].

**Figure 4.** (**A**) IC50 (μM) of tested compounds against α-glucosidase enzymes in vitro. (**B**) The potential sites of flavone *C-*glycosides affecting α-glucosidase inhibitory potential. (**C**) The potential sites of dihydrochalcone *C*-glycosides affecting the α-glucosidase inhibitory potential. The up arrows represent increased inhibition, whereas the down arrows represent decreased inhibition activity. Results are expressed as mean ± SE (*n* = 3).

**Table 5.** IC50 of tested compounds using in vitro α-glucosidase inhibition assay. (-) indicates inactive compounds.


2.3.2. Structure-Activity Relationship of Dihydrochalcones and Their Glycosides as α-Glucosidase Inhibitors

The isolated dihydrochalcone glycosides, viz., compounds **5**, **6**, **7**, **8**, **9**, **10**, **11**, and **12**, were assessed for their α-glucosidase inhibitory activity in comparison to acarbose, together with two standard dihydrochalcones, phloretin and its glucoside phloretin-2 -glucose, commonly named phloridzin. All dihydrochalcone glycosides, viz., **5**, **6**, **7**, **8**, **9**, **10**, and **11**, were found inactive except for compound **12**, which showed relatively strong activity with IC50 at 323.6 μM (Figure 4A, Table 5). The reported weak α-glucosidase inhibitory activity of diglycosylated chalcones [41] clarified the inactivity of all isolated compounds and suggested that with regards to α-glucosidase inhibition, glycosylated forms of flavones are more active than dihydrochalcones. Further study is warranted, to carefully assess the α-glucosidase enzyme kinetic analysis of the dihydrochalcone carambolaside Q.

Regarding standard dihydrochalcones, phloretin was reported as a strong α-glucosidase inhibitor [44] and as a glucose transporter inhibitor [45], with a measured IC50 value at 110.4 μM (Figure 4A). Further, phloridzin revealed moderate inhibitory activity, with an IC50 of 853.1 μM (Figure 4A), in accordance with the reported dose-dependent α-glucosidase inhibition [46] and confirming that the inhibitory activity of monoglycosyl chalcones is lower than its aglycones [41] (Figure 4C). These results suggests that α-glucosidase inhibitory activity of *A. carambola* L. extract is mainly mediated by flavone glycosides composition, with a smaller contribution coming from dihydrochalcone glycosides being less active, except for compound **12**.

#### **3. Materials and Methods**
