Abstract
BackgroundIn Chinese traditional medicine, Agrimonia pilosa Ledeb (APL) exhibits great effect on treatment of type 2 diabetes mellitus (T2DM), however its mechanism is still unknown. Considering that T2DM are correlated with postprandial hyperglycemia and oxidative stress, we investigated the α-glucosidase inhibitory activity and the antioxidant activity of flavonoid compound (FC) and triterpenoid compound (TC) from APL.MethodsEntire plants of APL were extracted using 95% ethanol and 50% ethanol successively. The resulting extracts were partitioned and isolated by applying liquid chromatography using silica gel column and Sephadex LH 20 column to give FC and TC. The content of total flavonoids in FC and the content of total triterpenoids in TC were determined by using UV spectrophotometry. HPLC analysis was used to identify and quantify the monomeric compound in FC and TC. The α-glucosidase inhibitory activities were determined using the chromogenic method with p-nitrophenyl-α-D-glucopyranoside as substrate. Antioxidant activities were assessed through three kinds of radical scavenging assays (DPPH radical, ABTS radical and hydroxyl radical) & β-carotene-linoleic acid assay.ResultsThe results indicate FC is abundant of quercitrin, and hyperoside, and TC is abundant of 1β, 2β, 3β, 19α-tetrahydroxy-12-en-28-oic acid (265.2 mg/g) and corosolic acid (100.9 mg/g). The FC & the TC have strong α-glucosidase inhibitory activities with IC50 of 8.72 μg/mL and 3.67 μg/mL, respectively. We find that FC show competitive inhibition against α-glucosidase, while the TC exhibits noncompetitive inhibition. Furthermore, The FC exhibits significant radical scavenging activity with the EC50 values of 7.73 μg/mL, 3.64 μg/mL and 5.90 μg/mL on DPPH radical, hydroxyl radical and ABTS radical, respectively. The FC also shows moderate anti-lipid peroxidation activity with the IC50 values of 41.77 μg/mL on inhibiting β-carotene bleaching.ConclusionThese results imply that the FC and the TC could be responsible for the good clinical effects of APL on T2MD through targeting oxidative stress and postprandial hyperglycaemia. So APL may be good sources of natural antioxidants and α-glucosidase inhibitors exhibiting remarkable potential value for the therapy of T2DM.
Highlights
In Chinese traditional medicine, Agrimonia pilosa Ledeb (APL) exhibits great effect on treatment of type 2 diabetes mellitus (T2DM), its mechanism is still unknown
The percentage scavenging values were calculated from the absorbance of the blank (A0) and of the sample (AS) using the following equation: Inhibition assay for α-glucosidase activity The α-glucosidase inhibitory activities of triterpenoid compound (TC) and the flavonoid compound (FC) were determined according to the chromogenic method with slight modifications [19]
Despite a great of efforts have been made in treatment of T2DM in clinic, poor effect accounted for a single target is still a major challenge
Summary
Chemicals Butylated hydroxyl toluene (BHT), gallic acid, β-carotene, linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH·), ρ-nitrophenyl α-D-glucopyranoside(PNPG), 3,5-dinitro salicylic acid, soluble potato starch and 1-deoxyrojirimycine, αGlucosidase (from Saccharomyces cerevisiae), HPLC grade methanol and acetonitrile were purchased from Sigma Chemical Co. The percentage scavenging values were calculated from the absorbance of the blank (A0) and of the sample (AS) using the following equation: Inhibition assay for α-glucosidase activity The α-glucosidase inhibitory activities of TC and the FC were determined according to the chromogenic method with slight modifications [19]. The reaction mixture contained 10 μL of 0.02 U/μL α-glucosidase solution in 0.2 M Na-phosphate buffer (pH6.8), 10 μL of a sample in DMSO (or DMSO itself as blank control) with the concentration of 20.0, 50.0, 100.0, 200.0, 400.0, 500.0 μg/mL, respectively, 50 μL of Na-phosphate buffer (pH6.8), which were mixed and incubated at 37°C for 20 min. 100 μL of sample in methanol at different concentration of 5.0, 10.0, 50.0, 100.0, 500.0, 1000.0, 2000.0 μg/mL respectively, and 880 μL of above solution (incubated at 37°C for 30 min) was dispensed to test tubes and 20 μL of H2O2 (0.01%) were added. Difference with a value of p < 0.01 were considered statistically significant
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