【作者】 黎继烈；

【导师】 李忠海； 钟海雁；

【作者基本信息】 中南林业科技大学 ， 林产化学加工工程， 2007， 博士

【摘要】 金橘属芸香科,富含挥发油、黄酮、柠檬苦素、果胶等生物活性成分,具有抗氧化、抑菌、抗癌、调节免疫、降脂及防治心脑血管疾病等多种功效,是一种具有很大开发潜力药用保健资源。本文以金橘为原料,分析其挥发性成分；探讨金柑黄酮类化合物和柠檬苦素类化合物的微波辅助萃取及超声波辅助萃取工艺；通过体外活性评价试验,探索金橘黄酮类粗提物及柠檬苦素类粗提物体外抗油脂氧化、抑制DNA氧化损伤、清除自由基和抑菌活性；通过小白鼠体内活性评价,研究金橘黄酮类粗提物抗衰老、降血脂、降血糖及对免疫功能和胃肠吸收功能的影响。试验旨在初步探明金橘的化学成分组成及其生理活性成分,为金橘的保健功能及药理功能开发提供依据。采用顶空-固相微萃取(HS-SPME)技术、气相色谱／质谱／计算机联用(GC/MS/DS)技术对金橘挥发性成分进行分析,研究了金橘用量、萃取时间及萃取温度对金橘中挥发性物质萃取的影响。优化后得到的萃取条件为：金橘果浆用量为5.0 g,萃取时间为25 min,萃取温度为45℃。本试验确定了45种化合物,三个品种的金橘挥发性成分中都分别含有烯、醇、酮、酯等化合物,以萜烯类化合物为主。在金橘黄酮微波辅助萃取工艺中,通过四元二次回归通用旋转组合试验构建模型。试验中所选参试因子对微波辅助萃取效率均有显著影响,其顺序依次为乙醇浓度>萃取时间>萃取温度>液料比。通过规划求解,对回归模型方程进行预测,并结合试验仪器参数设定要求,确定金橘黄酮微波辅助萃取的最优工艺条件为：乙醇浓度75%,萃取温度57℃,萃取时间37 min,液料比30：1。以试验验证,此条件下,金橘黄酮微波辅助萃取得率达到2.18±0.01%,平均回收率为98.46±1.05%,RSD=1.07%(n=5)。在金橘柠檬苦素超声波辅助萃取工艺中,通过四元二次回归通用旋转组合试验构建模型。试验中所选参试因子对超声波辅助萃取金橘柠檬苦素效率影响显著性顺序为超声波处理时间>超声波处理功率>浸提温度>液料比。经规划求解并结合实际,确定最优工艺条件为：超声波处理时间20 min,处理功率440 W,浸提温度59℃,液料比30：1,浸提60 mmin。验证试验证明,在此条件下萃取,柠檬苦素超声波辅助萃取得率达到3.26±0.03‰,平均回收率为98.03±1.55%,RSD=1.58%(n=5)。以金橘黄酮类及柠檬苦素类粗提物为原料,进行体外活性研究,结果显示：(1)金橘黄酮类粗提物在低浓度(0.16 mg/mL)时已表现出较好的抑制DNA氧化损伤活性；柠檬苦素类粗提物则在较高浓度(> 2.50 mg/mL)时,表现出抑制DNA氧化作用。(2)金橘黄酮类粗提物对羟自由基·OH、超氧阴离子自由基O2·和DPPH清除效果顺序为DPPH>·OH> O2·,半清除率浓度(IC50)分别为0.45mg/mL、0.53mg/mL和41.28 mg/mL.金橘柠檬苦素类粗提物对DPPH及·OH有清除作用,半清除率浓度分别为1.76 mg/mL和18.37 mg/mL。(3)金橘黄酮类及柠檬苦素类粗提物均有一定的抗油脂氧化作用,黄酮类粗提物与Vc及VE有协同增效作用；柠檬苦素类粗提物添加量为0.08%时,有微弱的抗油脂氧化活性,与Vc及VE无显著的协同增效作用。(4)金橘黄酮类粗提物对金黄色葡萄球菌、枯草杆菌、大肠杆菌及毛霉有一定的抑制作用,最小抑菌浓度(MIC)分别为0.63 mg/mL、1.25mg/mL、1.25 mg/mL和2.50 mg/mL;半抑菌率时间(IT50)分别为4.65 h、5.70 h、6.74 h和9.71 h。柠檬苦素类粗提物对枯草杆菌、金黄色葡萄球菌、大肠杆菌及黑曲霉有抑制作用；最小抑菌浓度(MIC)分别1.25 mg/mL、2.50 mg/mL、2.50 mg/mL和2.50 mg/mL;半抑菌率时间(IT50)分别为4.20 h、5.61 h、6.38 h和7.65 h。以金橘黄酮粗提物为原料,以小白鼠为试验对象,探讨不同产地金橘(湖南浏阳和江西遂川)对其生理活性的影响。结果显示：(1)试验组能够明显延长小鼠的生存寿命。80 mg/kg-bw剂量对血清SOD, MDA含量和平均寿命的影响具有显著性(P<O.05),160 mg/kg-bw剂量的影响具有极显著性(P<0.01)。(2)20mg/kg-bw剂量可显著降低小鼠血中的血脂(P<0.05),80 mg/kg-bw剂量降血脂作用极显著(P<0.01)。(3)160 mg/kg-bw剂量可显著降低糖尿病小鼠第6天血糖(P<0.05),对第17天血糖降低效果非常显著(P<0.01); 80 mg/kg-bw剂量可显著降低糖尿病小鼠第17天血糖(P<0.05),对正常小鼠血糖的影响不明显。(4)80 mg/kg-bw剂量可显著提高小鼠免疫能力和胃肠吸收功能(P<0.05),160mg/kg-bw剂量可极显著性提高小鼠免疫能力和胃肠吸收功能(P<0.01)。更多还原

【Abstract】 Kumquat which belongs to rutaceae Fortunella is a quite potential medical resource due to its rich in easy volatile, flavones, limonin and pectin, and etc. In this study, the technical conditions of Microwave Extraction and Ultrasonic Extraction on flavones and limonin were discussed. Furthermore, the activities of antioxidation on oil and DNA, free radical scavenging and bacteriostasis of kumquat crude extracts were also identified through the experiment in vitro. The whole research was to prove up the chemical constituents and biological activities of kumquat so as to provide basis for exploiting the physiological and pharmacological function of kumquat. The main study contents and results are given as follows:The volatile composition of fresh kumquat (F. Crassifolia Swingle, F. Japonica Swingle and F. margarita Swingle) separated from Liuyang, Suichuan and Gui Lin was analyzed by Solid Phase Micro Extraction (SPME) coupled with GC/MS/DS. The optimal experiment parameters for SPME were:5.0g juice,25 min at 45℃. The experiment identified forty-five volatile components in total in the three kumquat samples. It contained alkenes, alcohols, ketones, esters and etc. Most of volatile components were Alkenes.In the research of flavones which come from kumquat by Microwave Extraction, the reduced regression equation of extraction process was determined by four factors quadratic regression rotation combination design. All the tested factors affected the yield significantly, with the order as ethanol 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 were as follows:ethanol concentration 76.7%, extraction temperature 57℃, extraction time 37 min, and ratio of solvents and material 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 limonin 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. 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 were as follows:ultrasonic treatment time 20 min, ultrasonic treatment power 440 W, soaking temperature 59℃, ratio of material and solvents 30:1, and soaking time 60 min. The yield of kumquat limonin was 3.262±0.028%. The retrieving rate was 98.03±1.55%, RSD=1.58%(n =5).Experiments on in-vitro biological activity were made using the crude extracts of flavones and limonin as materials showed the results as follows:The experimental results of study on the antioxidation effect on DNA showed that the crude extracts of flavones had antioxidation on DNA, while the test sample was at concentration of 0.16 mg/mL. When the concentration of crude extracts of limonin was more than 2.50 mg/mL, it showed that crude extracts had antioxidation on DNA.In the experiment of scavenging on free radical in vitro, the crude extracts of flavones showed scavenging effect on hydroxyl radical(·OH), superoxide anion radical (O2·) and DPPH. The scavenging order was DPPH>·OH> O2·The half scavenging concentration (IC50) were 0.45 mg/mL,0.53 mg/mL, and 41.28 mg/mL respectively. It showed that the extracts had scavenging effect on DPPH and hydroxyl radical (·OH). The IC5o were 1.76 mg/mL and 18.37 mg/mL respectively.The results of experimental study on the antioxidation effect on oil showed that kumquat crude extracts had antioxidation characteristic. The crude extracts of flavones had synergistic action with Vc, VE The crude extracts of limonin showed weak antioxidation on oil, when the dosage was 0.08%. The crude extracts of flavones had not synergistic action with VC, VE.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 flavones 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 (IT50) was given as below:4.65 h,5.70 h,6.74 h and 9.71 h. The crude extracts of limonin also had inhibitory effect on Staphyloclccus aureus, Bacillus subtilis, Excherichia coli, and Aspergillus niger. The inhibiting order was B. subtilis> S. aureus> E. coli> A.s niger. MICs were given as below: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.In this paper, research was also made which used crude flavones extracts from kumquat as materials while mice was used as experimental objects, in order to discussing the effect of different locality kumquat (Liuyang and Suichuan the breed is Fortunella Crassifolia Swingle) to the physiological effects of kumquat total flavones. The main study contents and results are given as follows:Compared with model group, the experimental groups could obviously prolong the life-span of mice. The 80 mg/kg·bw doses had markness (P<0.05) effect to SOD, MDA and average life-span; the 160 mg/kg·bw doses had greatly markness (P<0.01) effect.The 20 mg/kg·bw doses of Kumquat had markness effect (P<0.05) in lowering blood lipid; and the 80 mg/kg·bw doses of Kumquat had greatly markness effect (P<0.01) in lowering blood lipid.The regulating blood sugar effect of kumquat total flavones was as below: compared with model group, after 6 days, the 160 mg/kg·bw doses had markness effect (P<0.05) to the diabetes mice’s blood sugar; after 17 days, this doses had great markness effect (P<0.01). The 80 mg/kg·bw doses had markness effect (P<0.05) to the diabetes mice’s blood sugar after 17 days,however it had no obvious effect to the normal mice.The effect of kumquat total flavones on the immunity system was tested, through evaluating the phagocytic function of monocyte macrophage, the ear tumefaction reduced by DNFB, swimming time, capacity of lacking of Oxygen, the index of thymus and spleen. The results were showed as below:compared with model group, the two middle doses had markness effect (P<0.05); the two higher doses had great markness effect (P<0.01).The effect of kumquat total flavones on the gastrointestinal absorption function was studied, the 80 mg/kg·bw doses had markness effect (P<0.05) to the diabetes mice’s blood sugar doses had markness effect (P<0.05) while the 160 mg/kg-bw doses had great markness effect (P<0.01) on improving the gastrointestinal absorption function.更多还原