| Urticafissa E.Pritz is a perennial herb belonging to genus Urtica in Urticaceae family.The medicinal part of the plant is a whole herb.It has the effects of removing rheumatism,activating blood and relieving pain.The plants of Urtica are widely distributed all over the world and there are many varieties.In the current trend of increasing incidence of diabetes,traditional Chinese medicines have been widely used by scholars in the development and research of hypoglycemic drugs due to its advantages of abundant resources,low toxicity,et al.Studies have shown that crude extracts of Urtica fissa E.Pritz have hypoglycemic effect.however,its active fraction and constituents of hypoglycemic effect are not clear.In this study,the detection of α-glucosidase inhibitory activity is used as tracking method,and combined with antioxidant activity and molecular docking technology to elucidate the active fraction and constituents of hypoglycemic effect in Urtica fissa.First,the method of α-glucosidase activity detection was used to screen inhibitory activities of roots(IC50=11.809mg/mL)、stems(IC50=12.402mg/mL)and leaves(IC50=12.402mg/mL)of Urtica fissa,it shows that the Urtica fissa roots have a high inhibitory activity in different parts of plants.The solution was acquired by extracting a certain amount of roots of Urtica fissa coarse powder with 70%ethanol at room temperature,soaking for 3 times,and 48h every time,the extract was obtained after vacuum concentration.The crude extract was suspended in the hot water,and then successively extracted with petroleum ether,ethyl acetate,and n-butanol,The solvents were recovered by vacuum concentration to obtain petroleum ether fraction 7.7%,ethyl acetate fraction 5.2%,n-butanol fraction 48.9%and water fraction 29.3%.The detection ofα-glucosidase inhibitory activity of four fractions showed that petroleum ether fraction had almost no activity relative to α-glucosidase,and the order of the remaining three fractions were ethyl acetate fraction(IC50=8.499mg/mL)>n-butanol fraction(IC50=13.241 mg/mL)>water fraction(IC50=19.774mg/mL).The total flavonoid contents of the four fractions were ethyl acetate fraction(70.62mg/g)>n-butanol fraction(45.48mg/g)>water fraction(18.76mg/g)>petroleum fraction(10.23mg/g).In the antioxidant activity test,the order of scavenging rates of DPPH radicals by the four fractions were ethyl acetate fraction(IC50=40.361 μg/mL)>n-butanol fraction(IC50=268.710μg/mL)>water fraction(IC50=788.757μg/mL)>petroleum ether fraction(IC50=5539.13μg/mL);the order of scavenging rates of hydroxyl radicals by four fractions were ethyl acetate fraction(IC50=3.313mg/mL)>n-butanol fraction(IC50=4.559mg/mL)>water fraction(IC50=1 5.782mg/mL)>petroleum ether fraction(IC50=22.862mg/mL);the order of total reducing power of four fractions were ethyl acetate fraction>n-butanol fraction>water fraction>petroleum ether fraction.The anti-oxidation ability of four fractions was consistent with the trend of total flavonoid content of each fraction.From these,it can be seen that the active fraction of the strongest hypoglycemic effect is ethyl acetate fraction,followed by n-butanol fraction.Second,the compounds from activity part of ethyl acetate fraction were isolated and purified by silica column,fifteen monomer compounds were isolated fron part,the structures were identified by NMR,ESI-HR-MS and comparison spectral data with known literatures.Fifteen compounds are n-Tetratriacont-20,23-dienoic acid(1),β-sitosterol(2),Methyl p-hydroxycinnamate(3),Palmitic acid(4),Anisic aldehyde(5),Quercetin(6),Kaempferitrin(7),Di-n-butyl phthalate(8),Emodin(9),p-Coumaric acid(10),Succinic acid(11),Daucosterol(12),Scopoletin(13),Adenosine(14),Uridine(15).N-butanol fraction was also coarsely separated to obtain one inorganic compound,it was identified by IR as Potassium nitrate(16),Compounds 3,4,7,9 and 10 were isolated from Urtica fissa for the first time,compounds 1 and 5 were isolated from Urtica for the first time.Third,the structure of α-glucosidase as the receptor protein,the fifteen compounds isolated by ethyl acetate fraction as the ligand molecules,the interaction energy scores of ligand and receptor were calculated by molecular docking technology,it showed twelve compounds had good binding activity with α-glucosidase;Using acarbose(IC50=4.254mg/mL)as a positive control,the activities of these twelve compounds were verified by α-glucosidase inhibitory activity,it showed that the compounds with strong inhibitory activity onα-glucosidase are daucosterin(IC50=1.693mg/mL),behenosin(IC50=1.725mg/mL),and n-Tetratriacont-20,23-dienoic acid(IC50=2.940mg/mL).It was concluded that daucosterin,behenosin and n-Tetratriacont-20,23-dienoic acid are the basis for the hypoglycemic activity of Urtica fissa. |
