Extraction Of Main Active Compositions From Kumquat And Their Antioxidation And Bacteriostasis In Vitro

Kumquat belongs to rutaceae Fortunella. It has many kinds of characteristic such as antioxidation, bacteriostasis, anticancer effect, regulation of immunity, prevention and treatment of cardiovaxcular and cerebralvascular diseases, and etc. At present, the researches focus on cultivation, food processing and chemical analysis. In this reseach, the technical conditions of Microwave Extraction and Ultrasonic Extraction on flavones and limonins were optimized. Moreover, the activities of antioxidation on oil and DNA, free radical scavenging and bacteriostasis of kumquat crude extracts were studied through experiment in vitro.In the research of flavones which come from kumquat by Microwave Extraction, the reduced regression equation of extraction process was determined by the quadratic regression general rotation design with four factors. It was as follows:Y= 2.08314+ 0.031 25X1-0.038 33X2+0.038 17X3+0.031 25X4+0.057 25X1X2+0.062 50X1X3-0.105 85X12-0.032 97X22-0.027 72X32. All the tested factors affected the yield significantly, with the order as enthanol concentration> extraction time> extraction temperature> ratio of solvents and material. Predicting the model through the Aprogram solution of Excel, and according to practical situation, the optimal process conditions for Microwave Extraction was as follows:enthanol concentration at 76.7%, extraction temperature at 57℃, extraction time at 37 min, ratio of solvents and material at 30:1. The yield of kumquat flavones was 2.181±0.007%, the retrieving rate was 98.46±1.05%, RSD= 1.07%(n= 5)In the study of limonins which come from kumquat by Ultrasonic Extraction, the reduced regression equation of extraction process was determined through the quadratic regression general rotation design with four factors. It was as follows:Y= 2.83229+ 0.257 17X1-0.238 00X2+0.181 00X3+0.085 00X4-0.154 00X1X3-0.183 57X12-0.084 20X22-0.095 32X32. All the tested factors affected the yield significantly, with the order as ultrasonic treatment time> ultrasonic treatment power> soaking temperature> ratio of solvents and material. Predicting the model through the Aprogram solution of Excel, and according to practical situation, the optimal process conditions for Ultrasonic Extraction was as follows:ultrasonic treatment time at 20 min, ultrasonic treatment power at 440 W, soaking temperature at 59℃, ratio of material and solvents at 30:1, soaking time at 60 min. The yield of kumquat limonins was 3.262±0.028‰, the retrieving rate was 98.03±1.55%, RSD= 1.58%(n= 5)The results of experimental study on the antioxidation effect on oil, showed that kumquat crude extracts had antioxidation characteristic. The effect of crude extracts of flavones was superior to extracts of limonins. The crude extracts of flavones had synergistic action with Vc, VE. The crude extracts of limonins showed weak antioxidation on oil, when the dosage was 0.08%. The crude extracts of flavones had not synergistic action with Vc, VE, and less stability of it.The experimental results of study on the the antioxidation effect on DNA, showed that the crude extracts of flavones had antioxidation on DNA, when the test sample at concentration of 0.16 mg/mL. When the concentration of crude extracts of limonins was more than 2.50 mg/mL, it showed that crude extracts had antioxidation on DNA. The effect of antioxidation on DNA was increased along with the increasing of the test sample concentration.In the experiment of scavenging on free radical in vitro, the crude extracts of flavones showed the scavenging effect on hydroxyl radical(·OH), superoxide anion radical (-OH) and DPPH, the scavenging order was DPPH>·OH> O2·, the half scavenging concentration(IC50) were 0.45 mg/mL,0.53 mg/mLand 41.28 mg/mL, respectively. The scavenging effect of the crude extracts of limonins was not as good as flavones. It showed that the extracts had scavenging effect on DPPH and hydroxyl radical (·OH). The IC50 were 1.76 mg/mL and 18.37 mg/mL, respectively. The effect of scavenging on superoxide anion radical (O2·) was not distinctive.The antibacterial experiment in vitro showed that the crude extracts of flavones had inhibitory effect on Staphyloclccus aureus, Bacillus subtilis, Excherichia coli and Mucor racemosus. The inhibiting order of the crude extracts of kumquat flavone was S. aureus> B. subtilis> E. coli> M. racemosus. The minimum inhibition concentrations (MIC) were 0.63 mg/mL,1.25 mg/mL,1.25 mg/mL and 2.50 mg/mL, respectively. The half inhibiting time(IT5o) were 4.65 h,5.70 h,6.74 h and 9.71 h, respectively. The crude extracts of limonins had inhibitory effect on Staphyloclccus aureus, Bacillus subtilis, Excherichia coli, Aspergillus niger.The inhibiting order was B. subtilis> S. aureus> E. coli> A.s niger. MIC were 1.25 mg/mL,2.50 mg/mL,2.50 mg/mL and 2.50 mg/mL, respectively. IT50 were 4.20 h,5.61 h,6.38 h and 7.65 h, respectively.In summary, Microwave Extraction and Ultrasonic Extraction have benefits to the extraction of the active compositions of kumquat; they are high-efficient, fast and have protective effect to active compositions of kumquat. They would be the optimize choice for extraction of active compositions of kumquat. Moreover, the crude extracts of kumquat have antioxidation on oil and DNA, free radical scavenging and bacteriostasis. They could be applied in medical industry and health food. The research would offer broad approach for the utilizational of kumquat.