Isolation And Identification Of Chemical Constituents From Pimpinella Brachycarpa (Kom.) Nakai And Study On Its Antioxidant Activity In Vitro

Pimpinella brachycarpa is umbelliferae perennial herbaceous plant. Pimpinella brachycarpa has high nutrition value, including vitamins, amino acids, iron, copper, zinc, selenium and other trace elements. Pimpinella brachycarpa can be used as a medicine.It has efficacy of activating blood, lowering blood pressure, heat-clearing and detoxifying, removing dampness, analgesia etc. It can be used for the treatment of diseases such as hepatitis, neuralgia and leucorrhea increase. It also can be used to prevent hypertension and stroke. At present, the research of Pimpinella brachycarpawas observed in chemical composition analysis of essential oil of rape seed and nutrient analysis of whole grass, there was rarely report on its functional composition and activity, limited the development on health value and high added value. This experiment did a system research on chemical composition and anti-oxidation activity of the vegetative part of Pimpinella brachycarpa.[Objective]:Pimpinella brachycarpa is one of the popular wild vegetables. This research extracted its active components, studied its antioxidation effect, isolated and identified its composition, then studied antioxidant mechanism on one of the components. This research provided theoretical basis for Pimpinella brachycarpa developing into a new product.[Methods]:The effective components were extracted by alcohol (90%), then extracted by petroleum ether, ethyl acetate, n-butyl alcohol and water. Total phenolic contents, total flavonoid contents and antioxidant effects of different polar extracts on H2O2-induced oxidative damage were examined. Scavenging activity of DPPH, ABTS radical and inhibitory effects were analyzed. Extracts of ethyl acetate and n-butanol were separated and purificated, then the structures of chemical constituents were identified by spectroscopic analyses including nuclear magnetic resonance (NMR) and fourier transform infrared (FTIR). The cytotoxicity of quercitrin was determined by MTT method. Antioxidant effects, intracellular ROS,[Ca2+]i, cAMP and cGMP levels of quercitrin on H2O2-induced oxidative damage were examined.[Results]:The n-butanol extraction layer had the highest TPC of29.0567±0.27014mg of gallic acid equivalents (GAE)/g and the ethyl acetate extraction layer had the highest TFC of872.422±12.0185μg of equivalents(QE)/g. The cell viability loss induced by H2O2was attenuated after co-incubation with four layer extracts (0.5-2mg/mL, except petroleum ether).It has a significantly high DPPH radical scavenging activity by ethyl acetate extraction layer (EC50=0.629193±0.125684mg/mL) and ABTS+radical scavenging activity by n-butanol extraction layer (EC50=2.938682±0.003569mg/mL) Six compounds were isolated and identified. They are myristic acid (1),24ζ-methyl-5R-lanosta-25-one (2),β-sitoserol (3), pregnenolone (4),β-daucosterol (5), quercitrin (6). Compounds1,2,3,4, and5were isolated from ethyl acetate extraction layer. Compound6was isolated from n-butanol extraction layer. They were identified from Pimpinella brachycarpa for the first time. Quercitrin had no cytotoxicity in the concentration of1-50μg/mL, it could protect the cell, inhibit the content of ROS and [Ca2+]i, recovered the concentration of cAMP damaged by H2O2.[Conclusion]:Different polar extracts of Pimpinella brachycarpa had antioxidant activity, the ethyl acetate extraction layer and n-butanol extraction layer were obvious. The antioxidant effect of quercitrin may via a cAMP-dependent pathway.