Synthesis and bioactive evaluation of hesperidin derivatives

Cyclodextrin glycosyltransferase (CGTase) catalyzes an intermolecular transglycosylation reaction to produce hesperidin glycosides, which can be used in several industries. The recombinant CGTase was purified up to 5-fold by 5% (w/v) starch adsorption and the specific activity of the enzyme was 3.315 x 10^3 units/mg with a 46% yield. The 72-kDa relative molecular mass of the purified enzyme was determined by 10% SDS-PAGE. The preliminary structure of hesperidin glycosides (HGs) was confirmed by glucoamylase treatment and analyzed on thin-layer chromatography (TLC). The LC-MS/MS profiles of HGs showed the important fragments at m/z ratios of 345.21 (added glucose to glucose of rutinose in HG1) and 687.28 (added maltose to glucose of rutinose in HG2), confirming that the structures of HG1 and HG2 were α-glucosyl hesperidin and α-maltosyl hesperidin, respectively. The MIC and MBC studies showed that transglycosylated HG1 and HG2 had better antibacterial and bactericidal activities than hesperidin and diosmin, and were more active against Staphylococcus aureus than Escherichia coli. Hesperidin, HG1, HG2, and diosmin inhibited α-glucosidase with IC50 values of 2.75 ± 1.57, 2.48 ± 1.61, 2.36 ± 1.48, and 2.99 ± 1.23 mg/mL, respectively. In addition, the in vitro anti-viability effect of hesperidin glycosides was determined. The trypan blue exclusion assay of MRC-5 normal cells and A549 cancer cells showed that hesperidin, diosmin, hesperidin + diosmin, HG1 have the potential to inhibit MRC-5 normal cells with an IC50 value of >150 µg/mL, while HG2, HG1 + diosmin, and HG2 + diosmin treatment showed that their IC50 values were 139.67 ± 3.18, 110.39 ± 3.55, and 104.43 ± 4.07 µg/mL at 72 hours, respectively, while A549 cells viability were inhibit with a lower IC50 value of hesperidin, diosmin, hesperidin + diosmin, HG1, HG2, HG1 + diosmin and HG2 + diosmin were 92.90 ± 4.53, 97.66 ± 4.23, 96.22 ± 2.03, 88.85 ± 5.48, 87.35 ± 5.73, 57.72 ± 6.78 and 47.33 ± 6.98 µg/mL, at 72 hours, respectively. Similarly, the MTS assay of cells showed that hesperidin, diosmin, hesperidin + diosmin, HG1, HG2, HG1 + diosmin, and HG2 + diosmin have the potential to inhibit MRC-5 cells with an IC50 value of 137.58 ± 5.39, 149.69 ± 6.32, 147.68 ± 6.69, 149.44 ± 5.48, 143.32 ± 5.53, 128.81 ± 7.13 and 130.84 ± 7.25 µg/mL, respectively, while A549 cells proliferation were inhibited with a lower IC50 value of 58.66 ± 3.02, 106.31 ± 3.52, 94.19 ± 7.02, 54.57 ± 7.08, 49.44 ± 6.28, 45.99 ± 4.35 and 42.74 ± 2.99 µg/mL at 72 hours, respectively. The inhibitory effects on the scratch assay and Matrigel invasion assay of A549 cells showed that hesperidin, diosmin, HG1, and HG2 inhibited cell migration and invasion in a time- and dose-dependent manner. The anti-cancer effect of HGs showed significantly higher compared with the original hesperidin (* P < 0.05). Thus, hesperidin glycosides have the potential to be developed into antibacterial drugs, treatments for treating α-glucosidase-associated type 2 diabetes, and anticancer drugs.