The Study On Extraction,Separation,Antiinflammatory And Immunoumodulatory Activity Of Total Flavonoids From Passiflora Edulis

Objective: Passiflora edulis is a herbaceous vine of the Passiflora genus Passifloraceae.Studies have shown that the main ingredient of Passiflora edulis is flavonoids.The purpose of this study was to extract the total flavonoids from Passiflora edulis,and to establish the preliminary quality standard of the extraction of Passiflora edulis,to investigate the anti-inflammatory and immunomodulatory effects of the extraction of Passiflora edulis,and to further provide a experimental foundation for the development and utilization of the extraction of Passiflora edulis.Methods:(1)After raw materials screening,extraction solvent screening and single factor test,using ethanol concentration,water bath temperature,extraction time and ratio of material to liquid as indicators,using orthogonal test to optimize extraction process.(2)Using Ultraviolet and Visible spectrophotometry to establish the method for the determination of total flavonoids in the extraction of Passiflora edulis.Using thin layer chromatography to establish the method for identification of orientin in the extraction of Passiflora edulis.Using high performance liquid chromatography to establish the method for the determination of orientin in the extraction of Passiflora edulis.(3)The acute anti-inflammatory model of BALB/c mice was established by carrageenan-induced paw oedema.The effects of the extraction of Passiflora edulis(250,500 and 1000 mg/kg·bw)on the swelling and inhibition ratio of the paw oedema were investigated.The levels of TNF-α,IL-1β,IL-2,IL-4,IL-6 and IL-10 in the serum were determined by ELISA,and pathological sections of toe were made for histopathological observation.The immunosuppressive model of mice was established by cyclophosphamide-induced immunosuppression.The effects of the extraction of Passiflora edulis(250,500 and 1000 mg/kg·bw)on spleen and thymus index in mice were investigated.The levels of Ig A,Ig G,Ig M,TNF-α,IL-2,IFN-γ and IL-10 in serum were determined by ELISA,and pathological sections of spleen and thymus were made for histopathological observation.Results:(1)The best process for extracting the extraction of Passiflora edulis by ethanol hot reflux method was ethanol concentration of 80 %,water bath temperature of 90 ℃,extraction time of 60 min and ratio of material to liquid 1∶8 g/m L,the preparation process was stable and the transfer rate of orientin was 55.06 %-56.08 %;(2)The method for the determination of total flavonoids in the extraction of Passiflora edulis was successfully established by Ultraviolet and Visible spectrophotometry.The identification method of orientin in the extraction of Passiflora edulis was successfully established by thin layer chromatography.The method for the determination of orientin in the extraction of Passiflora edulis was successfully established by high performance liquid chromatography.(3)The anti-inflammatory model on BALB/c mice by carrageenan-induced acute paw edma was successfully established.The extraction of Passiflora edulis exerted obvious anti-inflammatory effect in this model,which can be significantly reduced the swelling rate and the contents of TNF-α,IL-1β and IL-6 in the serum,as well as the levels of IL-2,IL-4,IL-10 and IFN-γ in the serum were significantly increased in this model.The immunosuppression model on BALB/c mice by cyclophosphamide-induced was successfully established.The extraction of Passiflora edulis showed obvious immunomodulatory effects in this model,which can significantly increase the thymus index,spleen index and the level of Ig A,Ig G,Ig M,IL-2,IL-10,TNF-α and IFN-γ in the serum of immunosuppressed mice.Conclusion:(1)The optimal extraction process of the extraction of Passiflora edulis was successfully established.The process was stable and reproducible.(2)The method for determining the content of total flavonoids in the extraction of Passiflora edulis was successfully established,and the identification method and content determination method of orientin in the extraction of Passiflora edulis was successfully established,which provided a methodological basis for formulating the quality standard of the extraction of Passiflora edulis.(3)The extraction of Passiflora edulis has obvious anti-inflammatory activity on carrageenan-induced paw oedema and immunomodulatory activity on cyclophosphamide-induced immunosuppression in BALB/c mice.