Flavonoids are phenolic substances naturally distributed in plants with potential antidiabetic activity through the inhibition of α-glucosidase. Therefore, the present study investigated the inhibitory effects of α-glucosidase activity by purified flavonoids (PF) isolated from Moringa oleifera leaves before and after simulated gastric digestion in the absence or presence of nitrate. Furthermore, the combined effects of PF, chlorogenic acid and acarbose to reduce the acarbose dosage that caused 50% inhibition of α-glucosidase activity were studied by using CompuSyn software. Five flavonoids (rutin, kaempferol acetyl glycoside, kaempferol 3-O-glucoside, quercetin-3-glucoside and quercetin-3-acetyl-glucoside) were identified by HPLC-ESI-MS. The results showed that at a concentration of 100 μg/mL, flavonoids and acarbose inhibited α-glucosidase activity by 54.41% and 69.17%, respectively. Lineweaver-Burk plots revealed uncompetitive inhibition of α-glucosidase by PF. Flavonoids caused an 8.17 times reduction in acarbose concentration which was required to inhibit α-glucosidase. In the absence or presence of nitrate, the inhibitory activity decreased by 48.44% and 72.12%, respectively, after simulated gastric digestion when compared with undigested flavonoids. Rutin and kaempferol 3-O-glucoside showed the highest stability in the simulated gastric liquid, while quercetin-3-acetyl-glucoside and kaempferol 3-O-glucoside exhibited the highest stability in the simulated gastric liquid in the presence of nitrate. •Inhibition of purified flavonoids (PF) from Moringa oleifera leaf on α-glucosidase was investigated.•PF inhibited α-glucosidase non-competitively at 800 μg/mL by 99.1%.•Combination of PF with acarbose reduced the amount of acarbose required to reduce α-glucosidase activity by 50%.•Inhibitory activity of PF on α-glucosidase decreased after in vitro digestion.