RESUMO
Twenty-eight compounds were isolated and purified from Clinopodium chinense by Sephedax LH-20, ODS, MCI and preparative HPLC. Their structures were identified as apigenin (1), apigenin-7-O-β-D-glucopyranoside (2), apigenin-7-O-β-D-glucuronopyranoside (3), thellungianol (4), apigenin-7-O-β-D-rutinoside (5), luteolin (6), luteolin-4'-O-β-D-glucopyranoside (7), apigenin-7-O-β-D-pyranglycuronate butyl ester (8), luteolin-7-O-β-D-rutinoside (9), luteolin-7-O-β-D-noehesperidoside (10), acacetin (11), acacetin-7-O-β-D-glucuronopyranoside (12), buddleoside (13), naringenin (14), pruning (15), nairutin (16), isosakuranetin (17), isosakuranin (18), didymin (19), hesperidin (20), kaempferol (21), quercetin (22), kaempferol-3-O-α-L-rahmnoside (23), p-hydroxycinnamic acid (24), caffeic acid (25), cis-3-[2-[1-(3,4-dihydroxy-phenyl)-1 -hydroxymethyl]-1,3-ben-zodioxol-5-yl]-(E)-2-propenoic acid (26), mesaconic acid (27), gentisic acid 5-O-β-D-(6'-salicylyl)-glucopyranoside (28). Among them, compounds 7, 9-10, 12, 23, 26-28 were isolated from the Clinopodium for the first time. The protective effects of compounds 1-6, 8-17 and 19 against H2O2-induced H9c2 cardiomyocyte injury were tested, compounds 15 exhibited significantly protective effects. Compared with the cell viability of (62.12±6.18)% in the model, pruning exhibited viabilities of (84.25±7.36)% at 25.0 mg•L⁻¹, respectively, using quercetin as a positive control [cell viability of (84.55±8.26)%, 20 mg•L⁻¹].
RESUMO
Objective: In this study, we specifically deleted apigenin (AP) from the active fraction of Clinopodium chinense (CCE), and aimed at identifying the effect of AP on how CCE exerted its amelioration on high glucose-induced injury in human umbilical vein endothelial cells (EA.hy926). Methods: By using Sephadex LH-20 gel column chromatography, AP was specifically deleted from CCE, and the apigenin-depleted sample of CCE (CCEAP-)was obtained. The cultured endothelial cells were divided into five groups: normal control group, high glucose model group, CCE group, CCEAP- group, and AP group. The cell viability was assayed by MTT assay. Flow cytometry was used to measure the intracellular reactive oxygen species (ROS). Morphology of cell apoptosis was determined by fluorescence microscopy with Hoechst staining. The rate of apoptosis was measured by flow cytometry staining with AnnexinV-FITC. Caspase-3 activity was measured using caspase-3 colorimetric assay kit. The expression of Bax was detected by Western blotting. Results: CCE and AP could significantly improve the cell viability, reduce the generation of intracellular ROS in EA.hy926 induced by high glucose. Meanwhile, CCE and AP can reduce the ratio of apoptosis, activity of caspase-3, and expression of Bax, while CCE knocked out apigenin (CCEAP-) had a slight improvement on the high glucose-induced endothelial cell injury. Conclusion: AP is one of the principal components improving the high glucose-induced endothelial cell injury, and its anti-apoptosis effect may be related to anti-oxidative stress.