【作者】 苏东林；

【导师】 单杨；

【作者基本信息】 湖南农业大学 ， 食品科学， 2007， 硕士

【摘要】 柑桔类水果是全世界产量最大的水果之一、也是一种重要的经济植物，皮渣作为其主要副产物占柑桔总量的20％～40％，具有较大的利用价值；据研究，柑桔皮中含有丰富的生物活性物质—黄酮类化合物。黄酮类药物具有抗脑缺血作用、抗心肌缺血作用、抗心律失常作用、镇痛作用、保肝护肝作用、对消化性溃疡的保护作用、抗病毒、抗肿瘤作用等。此外，还有大量研究表明黄酮类天然产物有效成分有降压、降血脂、抑制血小板聚集等多种药理作用，说明天然产物黄酮类有效成分确有多种生物学活性。为此，受到各国政府和医药、食品等行业的高度重视和关注。而黄酮类有效成分的提取又是其研发和应用的关键技术和阶段之一，但到目前为止，对柑桔皮黄酮资源提取方面的研究报道还不多，纯化工艺和化学成分研究鲜见报道。为开发利用该资源，论文对其提取、分离、纯化及化学成分结构鉴定进行了研究。现将主要结果报告如下：1、建立以芦丁为标准品的柑桔皮黄酮类含量测定方法，采用3种紫外—可见分光光度法对不同条件处理下的柑桔皮中总黄酮含量进行了测定和比较分析。研究表明，3种分光光度测定方法中，黄酮含量以硝酸铝显色法（345 nm）为最高，直接测定法（270 nm）次之，氯化铝显色法（415 nm）最低；经过HPLC检测后，选定氯化铝（275 nm）为显色系统的含量测定方法。2、为确定最优的提取条件，利用中心组合设计对柑桔皮总黄酮纤维素酶法辅助提取工艺中的浸提时间（X₂）、浸提温度（X₄）、pH值（X₃）和酶用量（X₁）4因子的最优化组合进行了定量研究，建立并分析了各因子与黄酮得率（Y）关系的数学模型：Y=-17.41531250+7.14583333X₁+0.11956250X₂+2.52125000X₃+0.25337500X₄-6.36458333X₁^*X₁-0.00064896X₂^*X₂+0.00862500X₂^*X₃-0.32958333X₃^*X₃-0.01275000X₃^*X₄-0.00204583X₄^*X₄。结果表明：最佳的工艺条件为酶用量0.55％、时间123 min、pH值4.4、温度48℃。经试验验证，在此条件下，黄酮得率（3.51±0.13）％（n=3），与理论计算值3.621669％基本一致。回归模型能较好地预测柑桔皮中总黄酮的提取得率。3、对柑桔皮中的总黄酮进行微波提取，采用L₁₆（4⁴）正交优化试验，以探讨微波功率、液料比、辐照时间、pH值和提取次数对柑桔皮黄酮提取率的影响。正交试验结果表明，微波法提取总黄酮的最佳条件是：微波功率400 W，辐照时间180 s，液料比50，pH值5。在此条件下提取两次，所得总黄酮含量为3.37％。4、采用二次通用旋转组合设计研究了柑桔皮中总黄酮的超声波辅助提取工艺，并对建立的数学模型Y=-10.20174246+0.19594606X₁+1.32168206X₂+0.19143333X₃+0.00024257X₁^*X₄-0.00125581X₂^*X₄-0.00144333X₁^*X₁-0.10808333X₂^*X₂-0.00363833X₃^*X₃-0.00006954X₄^*X₄进行了规划求解及试验验证。结果表明：pH值（X₂）对总黄酮得率（Y）有极显著影响，乙醇浓度（X₁）、液料比（X₃）以及时间（X₄）同乙醇浓度之间和时间同pH值之间的交互项对总黄酮得率有显著影响，超声时间对总黄酮得率无明显影响。经试验验证，当pH值为5.6、乙醇浓度为75％、液料比为26、超声时间为80 min时总黄酮得率最高，达（3.314±0.063）％（n=3），与理论计算值3.348449％基本一致。回归模型可较好地预测柑桔皮中总黄酮的提取得率。5、采用反相高效液相色谱测定了不同提取条件下柑桔皮中橙皮苷和柚皮苷的含量。色谱柱为ODS C₁₈柱（150 mm×4.6 mm i．d．），流动相为甲醇—水（体积比为40／60），流速为1.0 mL／min，检测波长270 nm，柱温25℃。橙皮苷和柚皮苷的线性范围分别为0.007168g／L～0.28g／L和0.012288g／L～0.48 g／L；二者含量分别为17.5～21.9 mg／g（RSD=2.2～3.1％）和2.14～2.39 mg／g（RSD=2.8～3.7％）；其平均回收率分别为96.5～101.4％（RSD=2.3～3.3％）和97.3～103.6％（RSD=2.8～3.6％）。6、采用95％（V／V）、70％（V／V）醇提柑桔皮粉，经石油醚、乙酸乙酯、正丁醇依次萃取后，再利用硅胶柱层析、聚酰胺柱吸附、Sephadex LH—20等方法分离纯化，得到36个组分。更多还原

【Abstract】 Citrus fruit is one of the biggest yield of fruit in the word and has important economic value,peel is their mainly byproduct and has 20％～40％output in the total citrus fruit. Citrus peel isrich in many biologically active compounds and good sources of flavonoids. Flavone drugs havethe effects of anti—cerebra ischemia, anti—myocardial ischemia, anti—cardiac amhyth,analgesic, protecting liver, protecting alimentary ulcer, anti—virus, anti—cancer, and so on.Otherwise, many researches indicate that flavone natural drugs have the pharmacologicals ofblood pressure depressing, lowering blood lipide, inhibiting platelet aggregation. So it was takeninto account by governments, pharmaceutical and food industries, and the researches of flavonenatural produce in food. The extraction of flavone natural produce was the key point of itsresearch and development. Extraction, purification, separation and identification of flavonoids incitrus peel were studied in this thesis for better developing this resource. The main results weredescribed as follows:1、Did establish a method which acted rutin as standard sample to measure determination oftotal flavonoids in citrus peel. Meanwhile, the contents of the total flavonoids in citrus peel thatdisposed through different conditions were measured and compared and analyzed by using theultraviolet—visible spectrophotometry with three methods. The results showed that, of the threemethods, the concentration of the total flavonoids was the highest by using the Al（NO₃）₃ （345nm）, and the coloration method with direct measurement （270 nm） and AlCl₃ （415 nm） took thesecond and the third place. The method of assaying the total flavonoids was determined by theultraviolet—visible spectrophotometry with AlCl₃ （275 nm） system after being studied by HPLC.2、The influences of extraction time （X₂）, extraction temperature （X₄）, pH value （X₃） and theratio of cellulase to material （X₁, w/w） which affiliated with the enzyme—assisted extractiontechnique of total flavonoids from citrus peel on the yield （Y） were studied according to thecentral composite design in order to make sure the optimized extraction conditions. Onemathematical model （Y=-17.41531250+7.14583333X₁+0.11956250X₂+2.52125000X₃+0.25337500X₄-6.36458333X₁^*X₁-0.00064896X₂^*X₂+0.00862500X₂^*X₃-0.32958333X₃^*X₃-0.01275000X₃^*X₄-0.00204583X₄^*X₄） established and analyzed by response surface methodology （RSM）was adequate to describe the relationships between the studied factors and the response of totalflavonoids extraction yield. Based on the canonical analysis, the optimum conditions wereobtained as follows: the additived ratio of cellulose was 0.55％, the time was 123 minutes, pHvalue was 4.4 and the temperature was 48℃. With the technological conditions, the extractionrate was （3.51±0.13）％（n=3）, which was close to the estimated value 3.621669％attained byusing regression model. This model can be used to predict the experiment results well.3、The total flavonoids in citrus peel were extracted by microwave treatment. The effects ofaverage microwave output power, liquid—solid ratio, irradiation time, pH value and extractionfrequency on the extraction yield were investigated. And the orthogonal test design L₁₆（4⁴） was used to optimize the conditions of microwave treatment. The optimum conditions of microwavetreatment were as follows: microwave output power 400 W, irradiation time 180 seconds, theliquid—solid ratio 50, pH value 5. Under the best extraction conditions whose frequency ofextraction was 2, the total content of flavonoids was 3.37%.4、Ultrasonic wave—assisted extraction technique of total flavonoids （Y） from citrus peel wasstudied according to a current, rotational and combinational design of quadratic regression. Then,one quadratic regression model （Y=-10.20174246+0.19594606X₁+1.32168206X₂+0.19143333X₃+0.00024257X₁^*X₄-0.00125581X₂^*X₄-0.00144333X₁^*X₁-0.10808333X₂^*X₂-0.00363833X₃^*X₃-0.00006954X₄^*X₄） for the total flavonoids yield was established, the extraction conditionswere optimized by using above model and the optimized results were verified by experiments.Results shows that the pH value had a very significant effect on the extraction rate of totalflavonoids. Ethanol concentration （X₁）, the ratio of solution to material （X₃） and the interactionbetween time （X₄） and ethanol concentration, also time and pH value （X₂） had significant effectson the extraction rate. Extraction time had no significant effects on the extraction rate. Researchresults indicate that the optimized extraction conditions for obtaining maximum yield of totalflavonoids are pH value 5.6, ethanol concentration 75%, ratio of solution to material 26,extraction time 80 minutes. Under the optimized conditions, the experimental value of yield was（3.314+0.063）% （n=3）, which was close to the estimated value 3.348449% attained by usingregression model. The model for the total flavonoids yield have better prediction effects.5、An RP—HPLC method was established for the determination of hesperidin and naringin inthe citrus peel under different extracted conditions. Hesperidin and naringin were separated on anODS C₁₈ column （150 mm×4.6 mm i.d.） with methanol—water （volume ratio of 40/60） as themobile phase and detected at 270 nm. The flow rate was 1.0 mL/min. The column temperaturewas set at 25℃. The calibration curves were linear in the ranges of 0.007168 g/L～0.28 g/L and0.012288 g/L～0.48 g/L for hesperidin and naringin respectively. Determination of hesperidinwas 17.5～21.9 mg/g （RSD=2.2～3.1%） and naringin’s determination was 2.14～2.39 mg/g（RSD=2.8～3.7%）. The average recoveries were 96.5～101.4% （RSD=2.3～3.3%） forhesperidin and 97.3～103.6% （RSD=2.8～3.6%） for naringin. The method is simple, rapid,accurate and reliable.6、The powder of citrus peel was extracted by 95% （V/V） and 70% （V/V） ethanol. Theextraction was successively partitioned with petroleum ehter, ethyl acetate, n—butyl alcohol.Then the three fractions were separated by silica, polyamide, Sephadex LH—20 columnchromatographies, thirty—six chemical constituents were isolated.更多还原