Study On Extraction,Purification, Structural Identification And Biological Activities Of Limonoids From Kumquat

Kumquat had more than300years cultivation histories in the Youxi. It’s nutrition is rich, including the Vitamin, mineral substance, amino acid, fructose, pectin and so on. The peel and the fruit pulp may edible together. It has very high watching, edible and medicinal value. Currently, the Youxi kumquat active ingredient research mainly focuses on flavonoids, volatile oils, polysaccharides and so on, while limonoids in kumquat rarely reported. In recent years, limonoids has become a hot research, the substance has the activity of anticancer, antibacterial and antioxidant.In this paper, Fujian Youxi kumquat as raw material, the limonoids extraction, purification, detection, analysis and identification, the oxidation kinetics and the antibacterial activity were studied. The studies provide a theoretical basis for the development of the health and pharmacological functions of kumquat.Results of the study are as follows,(1) UPLC detection system of limonin and nomilin was established. The method had good linear relationship in the range of1μg/mL～1780μg/mL for limonin(R2=0.9999) and1μg/mL～1720μg/mL for nomilin(R2=0.9999). Recovery ratios were97.19%-100.06%and95.35%-99.83%, and relative standard deviations were0.74%-2.25%and0.80%-3.36%, detection limit were1.65μg/g and1.28μg/g respectively. UPLC detection system greatly reduced the analysis time, the solvent consumption, the cost analysis in the condition of guarantee of the degree of seperation. The method possesses merits of efficient, reasonable, accuracy and resproducibility, it can be used for the detection of limonin and nomilin in kumquat, orange, grapefruit and other plants.(2) Effect on the limonin yield by the cosolvent type, cosolvent concentration, dosage of cosolvent, extraction pressure, extraction time, extraction temperature and CO2flow rate were studied. Extraction pressure, extraction time, extraction temperature were selected as main effect factors. Response surface methodology (RSM) was applied to optimize the process conditions for supercritical carbon dioxide extraction of limonin in kumquat. The optimal process conditions for supercritical carbon dioxide extraction were as follows:extraction pressure34MPa, extraction time2.5h, extraction temperature50℃. Under the certain condition, the yield of limonin was up to1.2g/kg.(3) The kumquat supercritical carbon dioxide extracts were isolated and purified by the recrystallization method, two high purity monomer substances were obtained. The substances identified as limonin and nomilin by infrared absorption spectrometry (IR), one-dimensional NMR (1D NMR), two-dimensional nuclear magnetic resonance (2D NMR), and with UPLC-MS/MS and UPLC-QTOF-MS furth verification.(4) Kumquat limonin showed good antioxidant effect on lard when the dosage is0.04%. Compared with antioxidants PG, BHT, VC, VE, the antioxiant activity of the limonin is low, but it still can make the shelf life of lard life extension for nearly40%. Oil oxidation kinetics studies showed that the oxidation reactions process of lard which adding the limonin, changing from one-level reaction to zero-level reaction, the constant of reaction rate is0.0135h-1,1/10of the blank lard approximately. The results showed that the kumquat limonin which added to lard reduced the reaction rate, changed the oxidizing reaction course and played the oxidation resistance role.(5) The antibacterial experiment in vitro showed that the kumquat limonin had inhibitory effect on Staphylococcus aures, Salmonella, Shigella, Escherichia coli, Bacillus subtilis, Aspergillus niger and Saccharomyces cervisiae. The inhibiting order of the kumquat limonin was Bacillus subtilis> Staphylococcus aures> Escherichia coli> Aspergillus niger> Shigella> Salmonella> Saccharomyces cervisiae. The minimum inhibition concentrations(MIC) were1.25mg/mL、1.25mg/mL、1.25mg/mL、2.50mg/mL、2.50mg/mL、2.50mg/mL、5.00mg/mL respectively. The minimum bactericidal concentration(MBC) were given as below:1.25mg/mL、2.50mg/mL、2.50mg/mL、2.50mg/mL、2.50mg/mL、5.00mg/mL、5.00mg/mL. The antibacterial activity of kumquat limonin remained stable at different temperatures, but pH affected its antibacterial activity significantly. The kumquat limonin had strong antibacterial activity under the acidic condition, but unstable under alkaline conditions. Under strong alkaline conditions, its had almost no antibacterial activity.