Study On Isolation And Identification Of Active Ingredients From Hymenoleana Nana And Their Bioactivities

Hymenoleana nana is a plant of Umbelliferae family.It is mainly grown in Xinjiang of China at an altitude of 4300-4900 m.It is a local herbal medicine,and can prevent from coronary heart disease,cerebral thrombosis,diabetes and hypertension.The bioactive chemicals in H.nana are responsible for the pharmacological functions.However,publications available on the chemical composition of this plant are very limited.It is required to conduct a phytochemistry work and to reveal the leading bioactive chemicals.Therefore,in this work,a systematic study was carried out on the purification,structure identification,biological activity of the bioactive chemicals in H.nana.The shells,stems,flowers and whole plants of dried H.nana were pulverized and extracted with 80% ethanol at room temperature to obtain crude extracts.The crude extracts were sequentially extracted with petroleum ether,chloroform and ethyl acetate to obtain four fractions.The contents of total flavonoids in the four fractions were determined.The flavonoid contents in the whole plant and shell tissues were relatively high,followed by the flowers and stem parts.The contents of total flavonoids change in the sequence of the four fractions is chloroform fraction > ethyl acetate fraction> petroleum ether fraction > aqueous fraction.The total phenol contents in the whole plant and shell were higher than those in the stem and flower tissues.The contents of total phenol change in the sequence of the four fractions is chloroform fraction > ethyl acetate fraction > petroleum ether fraction > aqueous fraction.Anti-oxidation and anti-tumor experiments were performed on four crude extracts and four fractions.The results showed that the anti-oxidation and anti-tumor effects of the whole plant and shell were better than other crude samples tested.The ethyl acetate fraction and the chloroform fraction have better antioxidant and anti-tumor effects than the other two fractions.As these two fractions contained more active substances,the two fractions were selected for further separation and purification of the active chemicals and evaluation of the bioactivity of active chemicals.Through purification by silica gel column,ODS column,and semipreparative HPLC,six compounds were isolated and purified.Their chem-ical structures and molecular weights were identified by 1H and 13 C NMR,and MS.The six compounds were luteolin(C1),luteolin-7-O-glucoside(C2),kaempferol-7-O-rhamnoside(C3),?-sitosterol(C4),bergapten(C5),Isooxypeucedamin(C6).These compounds were isolated for the first time from H.nana.These compounds were evaluated for their antioxidant activitiese by using DPPH,ABTS,ORAC assays in vitro.C1 and C4 performed better abilities to scavenge free radicals than the other compounds tested.The human breast cancer cells MCF-7 and MDA-MB-231 cells were used to evaluate the anti-tumor activity.C1(IC50 74.05 ?g/m L)and C4(IC50 66.83 ?g/m L)have significant inhibitory effects on the proliferation of MCF-7 cancer cells.C5 have a weaker inhibitory effect than C1 and C4.At the maximum concentration of 100 ?g/m L,the inhibition rate of MCF-7 cells was 36.58%.C2,C3 and C6 were the less sensitive to the proliferation of MCF-7 cells in the experimental concentration range.For MDA-MB-231 cells,C5(IC50 44.92 ?g/m L)has the most significant inhibitory effect on the proliferation,and C1(IC50 64.58 ?g/m L)also has a moderate inhibitory effect.The IC50 value of C6 was 85.89 ?g/m L.C3 has no inhibitory effect on MDA-MB-231 cells in the experimental concentration range.When a high concentration was used,a promotional effect was observed.