Extraction,Separation And Application Of Active Components From Green Peel Of Juglans Mandshurica Maxim

Juglans mandshurica Maxim is a very important medicinal source plant in China.Its bark,leaves,and fruits and green peel can be used as medicines,which have significant anti-oxidant,anti-inflammatory,anti-cancer and antibacterial effects.In this paper,the extraction and separation of active ingredients from the green peel of Juglans mandshurica,the optimization of the extraction process of triterpenoids,and the research and application of its biological activity were studied,which provided a theoretical basis for the development of more comprehensive utilization value of Juglans mandshurica.80%ethanol was used to extract the green peel of Juglans mandshurica Maxim,and then extracted with petroleum ether,chloroform and ethyl acetate in this order.After the extracted phases were separated by thin-layer chromatography and column chromatography,the active ingredients in each phase were detected by gas chromatography(GC)and gas chromatography-mass spectrometry(GC-MS).Six active ingredients were separated of petroleum ether extraction phase with eluent petroleum ether:ethyl acetate=20:80.Seven active ingredients were separated of chloroform extraction phase with eluent chloroform:ethyl acetate=40:60.Six active ingredients were separated of chloroform extraction phase with eluent chloroform:ethyl acetate=20:80.Four active ingredients were separated of ester extraction phase with eluent chloroform:methanol=80:20.Based on the single-factor experiment,a multi-factor experiment was designed based on the Box-Behnken central combination method.The effects of ethanol volume fraction,ultrasonic temperature,ultrasonic time and liquid-to-material ratio on the extraction rate of total triterpenes from the green peel of Juglans mandshurica were explored to determine the optimal extraction process conditions by using response surface methodology.The total triterpenes in leaves of Juglans mandshurica were extracted in the same way as a comparison.The optimal extraction conditions of total triterpenes from the green peel of Juglans mandshurica were ethanol volume fraction 80%,ultrasonic temperature 44℃,ultrasonic time 46 min,and liquid-to-material ratio 20:1.At this time,the maximum extraction rate of total triterpenoids in the green peel was 2.70 mg/g.The optimal extraction conditions of total triterpenes from leaves of Juglans mandshurica were ethanol volume fraction 80%,ultrasonic temperature 50℃,ultrasonic time 34 min,and liquid-to-material ratio 16:1.At this time,the maximum extraction rate of total triterpenoids in the leaves was 1.53 mg/g.The application of total triterpenoids extracted from the green peel of Juglans mandshurica was mainly manifested in two aspects:antioxidant activity and antibacterial activity.Through the elimination of four kinds of free radicals by the triterpenoids from the green peel of Juglans mandshurica,the clearance rates of hydroxyl free radicals,nitrite ions,superoxide anions and DPPH free radicals were 46.72%,58.31%,62.82%and 71.74%respectively.The order of antioxidant activity of the four free radicals was determined as follows:DPPH radical>superoxide anion>nitrite ion>hydroxyl radical.Based on the inhibitory effects of triterpenoids from the green peel of Juglans mandshurica on Phanerochaete chrysosporium Burdsall and Trichoderma viridey the minimum bacteriostatic concentration of Phanerochaete chrysosporium Burdsall was 11～22 μg·mL-1,and the minimum bacteriostatic concentration of Trichoderma viride was 5.5～11 μg·mL-1.Its antibacterial activity against Trichoderma viride was stronger than that against Phanerochaete chrysosporium Burdsall.