柑桔果实主要类黄酮成分检测及含量分析
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摘要
我国是柑桔的原产地，品种资源丰富，柑桔产量居世界第二。流行病学和动物试验研究表明，柑桔类水果中类黄酮、类胡萝卜素、类柠檬苦素等活性物质，具有抗氧化活性、抗癌、预防循环系统疾患、抗炎症、抗过敏及抗菌等功效，有望应用于功能性食品的开发中。因此，开展我国不同种类柑桔果实中类黄酮化合物的含量检测及其成分分析研究，意义重大。
本文研究了利用高效液相色谱法同时检测柑桔中柚皮素-7-β-芸香糖苷、柚皮苷、橙皮苷、新橙皮苷、香风草甙、柚皮苷元、橙皮素等7种黄烷酮和甜橙黄酮、川皮苷、桔黄酮等3种多甲氧基黄酮的分析方法，测定了我国甜橙、宽皮柑桔、柚、柠檬及杂柑等主要种类的124个果汁样品(柑桔原汁)和81个果皮样品(干果皮)中的黄烷酮和多甲氧基黄酮的含量，并初步探讨了不同种类柑桔果汁和果皮中类黄酮的分布规律。主要研究结果如下：
1、在国内首次建立了同时检测柑桔中橙皮苷、柚皮苷、柚皮素-7-β-芸香糖苷、甜橙黄酮等10种类黄酮化合物的高效液相色谱法。果汁样品用70％甲醇提取，果皮样品用50％二甲基甲酰胺提取，Waters Symmetry C_(18)分析柱(250×4.6mm，5μm)，0.2％乙酸水溶液和乙腈为流动相，梯度洗脱，二极管阵列检测器(287 nm，330nm)检测，40min内能将10种类黄酮完全分离，7种黄烷酮的平均回收率在94.20％～104.7％之间，相对标准偏差为1.48％～5.41％，精密度0.54％～4.05％，最低检测限0.005 mg／L～0.01mg／L；3种多甲氧基黄酮的平均回收率在97.37％～106.58％之间，相对标准偏差为1.95％～2.48％，精密度0.31％～0.78％，最低检测限均为0.01 mg／L。结果表明该方法操作简单，方便快速，具有较高的灵敏度和精密度，可满足柑桔类水果的质量控制和品质快速检测的需要。
2、系统分析了甜橙、宽皮柑桔、杂柑和柠檬等种类的124个柑桔果汁样品。结果表明，不同种类柑桔果汁中黄烷酮化合物的组成不同。甜橙、宽皮柑桔、杂柑和柠檬汁中的黄烷酮化合物主要为橙皮苷，介于68.39mg／L～233.88mg／L之间，其次是柚皮素-7-β-芸香糖苷和香风草甙，柚皮苷、新橙皮苷、柚皮苷元和橙皮素很少检出；柚汁中的黄烷酮化合物以柚皮苷为主，平均含量为166.41mg／L，几乎为7种黄烷酮总含量的90％，少量为柚皮素-7-β-芸香糖苷和橙皮苷；葡萄柚汁中的黄烷酮化合物主要为柚皮苷和柚皮素-7-β-芸香糖苷，平均含量分别为753.34mg／L和199.23mg／L，以及微量的橙皮苷(4.75mg／L)。在所测果汁样品中，以湖南产冰糖橙果汁中的橙皮苷、柚皮素-7-β-芸香糖苷和香风草甙含量最高，分别为482.18mg／L、612.88 mg／L和186.41 mg／L。
同样，不同种类柑桔果汁中多甲氧基黄酮化合物的组成也不同。甜橙黄酮、川皮苷和桔黄酮主要存在于甜橙、宽皮柑桔和杂柑果汁中，而柚、葡萄柚和柠檬汁均未检出。主栽宽皮柑桔果汁中的多甲氧基黄酮总含量平均为6.89 mg／L，橙汁为5.64 mg／L，杂柑果汁为3.26mg／L。而国家种质资源圃中扁桔汁中的川皮苷、桔黄酮和甜橙黄酮含量最高，分别为16.98mg／L、7.89 mg／L和3.12mg／L。
3、对81个柑桔果皮样品中的10种类黄酮化合物的含量测定表明，果皮中的类黄酮含量丰富，其黄烷酮几乎是果汁中的50～100倍，多甲氧基黄酮含量远远高于果汁。
不同种类柑桔果皮中黄烷酮和多甲氧基黄酮含量不同，甜橙果皮中的类黄酮以黄烷酮为主，约为类黄酮总含量的73.99％，而宽皮柑桔中的类黄酮则以多甲氧基黄酮为主，约为类黄酮总含量的53.19％，其多甲氧基黄酮的含量是甜橙果皮中的5.24倍。
宽皮柑桔果皮中的类黄酮含量大小依次为川皮苷＞橙皮苷＞桔黄酮＞柚皮素-7-β-芸香糖苷＞甜橙黄酮＞香风草甙，而甜橙果皮为橙皮苷＞川皮苷＞柚皮素-7-β-芸香糖苷＞甜橙黄酮＞香风草甙＞桔黄酮。
椪柑果皮中多甲氧基黄酮含量远远高于其它品种，其中又以浙江产椪柑果皮中含量最高，高达1.5％(以干重计)，可作为提取多甲氧基黄酮化合物的优质原料，为柑桔皮渣综合利用开辟了新途径。
4、通过比较甜橙和宽皮柑桔中柚皮素-7-β-芸香糖苷、橙皮苷、香风草甙等黄烷酮以及甜橙黄酮、川皮苷等多甲氧基黄酮的含量，表明其多甲氧基黄酮总量与桔黄酮的比值(PMFs／TNG)以及甜橙黄酮与桔黄酮的比值(SNT／TNG)有较大差异，可为甜橙和宽皮柑桔的区分提供参考依据。
China, one of the main citrus original in the globe, has rich citrus germplasm resources, whose annual production ranks the second all over the world at present. According to the epidemiological and animal researches, citrus fruits have many active effects on humans' health due to the flavonoids, carotenoids, limonoids, and etc., such as antioxidation, anticancer, prevention of circulatory disorders and so on; therefore, flavonoids in citrus have been more and more applied to functional food. As a result, it is necessary to investigate the flavonoid contents and distribution of various citrus fruits in China.
Testing methods of 7 flavanones (narirutin, naringin, hesperidin, neohesperidin, didymin, naringenin, hesperetin) and 3 polymethoxylated flavones (sinensetin, nobiletin, and tangeretin) by high performance liquid chromatographic (HPLC) , and extracting effects of different solvents on these compounds in citrus, were investigated in this paper; then on the basis of the above analyzing technology, contents of flavonoids in various citrus fruits were tested, including 124 juice and 81 peel samples. The main study results were listed as follows:
1. The simultaneous testing method of 10 flavonoid compounds (7 flavanones & 3 polymethoxylated flavones) by HPLC was established.
After extraction by 70% methanol(v/v) for juice or 50% dimethyl formamide (DMF, v/v) for peel, pre-treated citrus fruit samples were determinated by HPLC; and the detail HPLC conditions were adopt: C_(18) reversed-phase column(250×4.6mm, 5μm), gradient elution by acetonitrile and 0.2% acetic acid (v/v) as mobile phase, diode array detector (287nm, 330nm). This method was able to separate 10 flavonoids completely in 40min; moreover, the average recoveries, the relative standard deviations, precisions and detection limits of 7 flavanones arranged from 94.20% to 104.7%, 1.48% to 5.41%, 0.54% to 4.05%, 0.005 to 0.01mg/L, respectively; the previous 3 indexes of 3 polymethoxylated flavones arranged from 97.37% to 106.58%, 1.95% to 2.48%, 0.31% to 0.78%, respectively, and the detection limits were all 0.01 mg/L. Therefore, this pre-treating and testing methods satisfied the need of quick analysis, quality control and testing, due to simpleness, facility and the higher precision.
2. According to the analyzed results of 10 flavonoids from 124 juice samples, flavonoid composition ratios in juice varied with different citrus varieties.
The most major composition of flavanones in sweet orange, mandarin, tangor and lemon juices was hesperidin, whose contents arranged from 68.39 to 233.88mg/L, and the secondary compositions were narirutin and didymin, however naringin, neohesperidin, naringenin and hesperetin were detected rarely. Naringin in pomelo juice stayed the highest, whose average content was 166.41mg/L, almost accounting for 90% of 7 flavanones, and other less compositions were narirutin and hesperidin; naringin and narirutin constitute the most major composition of flavanones in grapefruit juices, whose average levels were 753.34mg/L, 199.23mg/L, respectively, and trace hesperidin existed; but neohesperidin, naringenin and hesperetin in both pomelo and grapefruit juice, were not detected. In a word, there was little polymethoxylated flavones in citrus juice, and contents of polymethoxylated flavones in mandarin juice were higher than that in sweet oranges, of which nobiletin level in mandarin juice was the highest, reaching 4.30mg/L.
3. From the study of 10 flavonoids in 81 citrus peel samples, peel portion of the whole fruit was abundant in flavonoids, whose flavanone contents were almost 50-100 times than juice, and levels of polymethoxylated flavones outclass juice.
The flavanone and polymethoxylated flavone contents in peels varied with different citrus fruit varieties. The prior compositions were flavanones in sweet orange, but polymethoxylated flavones in mandarin, whose levels were 5.24 times higher than in sweet orange peel, such as ponkan, in whose peels, 107.24 mg/g polymethoxylated flavones reached the top in studied mandarin.
According to the testing results, the content sequences of diverse flavonoids were shown: nobiletin＞hesperidin＞tangeretin＞narirutin＞sinensetin＞didymin, in mandarin peels; hesperidin＞nobiletin＞narirutin＞sinensetin＞didymin＞tangeretin, in sweet orange peels, of which levels of hesperidin, nobiletin and narirutin were higher than others.
4. Comparing the contents of flavanones (narirutin, hesperidin, didymin, etc) and polymethoxylated flavones (sinensetin, nobiletin, etc) in sweet orange with those in mandarin, the results indicated siginificant difference between the values of PMFs/TNG and SNT/TNG; therefore, values of PMFs/TNG and SNT/TNG were considered as the indexes of classification for sweet orange and mandarin.
本文研究了利用高效液相色谱法同时检测柑桔中柚皮素-7-β-芸香糖苷、柚皮苷、橙皮苷、新橙皮苷、香风草甙、柚皮苷元、橙皮素等7种黄烷酮和甜橙黄酮、川皮苷、桔黄酮等3种多甲氧基黄酮的分析方法，测定了我国甜橙、宽皮柑桔、柚、柠檬及杂柑等主要种类的124个果汁样品(柑桔原汁)和81个果皮样品(干果皮)中的黄烷酮和多甲氧基黄酮的含量，并初步探讨了不同种类柑桔果汁和果皮中类黄酮的分布规律。主要研究结果如下：
1、在国内首次建立了同时检测柑桔中橙皮苷、柚皮苷、柚皮素-7-β-芸香糖苷、甜橙黄酮等10种类黄酮化合物的高效液相色谱法。果汁样品用70％甲醇提取，果皮样品用50％二甲基甲酰胺提取，Waters Symmetry C_(18)分析柱(250×4.6mm，5μm)，0.2％乙酸水溶液和乙腈为流动相，梯度洗脱，二极管阵列检测器(287 nm，330nm)检测，40min内能将10种类黄酮完全分离，7种黄烷酮的平均回收率在94.20％～104.7％之间，相对标准偏差为1.48％～5.41％，精密度0.54％～4.05％，最低检测限0.005 mg／L～0.01mg／L；3种多甲氧基黄酮的平均回收率在97.37％～106.58％之间，相对标准偏差为1.95％～2.48％，精密度0.31％～0.78％，最低检测限均为0.01 mg／L。结果表明该方法操作简单，方便快速，具有较高的灵敏度和精密度，可满足柑桔类水果的质量控制和品质快速检测的需要。
2、系统分析了甜橙、宽皮柑桔、杂柑和柠檬等种类的124个柑桔果汁样品。结果表明，不同种类柑桔果汁中黄烷酮化合物的组成不同。甜橙、宽皮柑桔、杂柑和柠檬汁中的黄烷酮化合物主要为橙皮苷，介于68.39mg／L～233.88mg／L之间，其次是柚皮素-7-β-芸香糖苷和香风草甙，柚皮苷、新橙皮苷、柚皮苷元和橙皮素很少检出；柚汁中的黄烷酮化合物以柚皮苷为主，平均含量为166.41mg／L，几乎为7种黄烷酮总含量的90％，少量为柚皮素-7-β-芸香糖苷和橙皮苷；葡萄柚汁中的黄烷酮化合物主要为柚皮苷和柚皮素-7-β-芸香糖苷，平均含量分别为753.34mg／L和199.23mg／L，以及微量的橙皮苷(4.75mg／L)。在所测果汁样品中，以湖南产冰糖橙果汁中的橙皮苷、柚皮素-7-β-芸香糖苷和香风草甙含量最高，分别为482.18mg／L、612.88 mg／L和186.41 mg／L。
同样，不同种类柑桔果汁中多甲氧基黄酮化合物的组成也不同。甜橙黄酮、川皮苷和桔黄酮主要存在于甜橙、宽皮柑桔和杂柑果汁中，而柚、葡萄柚和柠檬汁均未检出。主栽宽皮柑桔果汁中的多甲氧基黄酮总含量平均为6.89 mg／L，橙汁为5.64 mg／L，杂柑果汁为3.26mg／L。而国家种质资源圃中扁桔汁中的川皮苷、桔黄酮和甜橙黄酮含量最高，分别为16.98mg／L、7.89 mg／L和3.12mg／L。
3、对81个柑桔果皮样品中的10种类黄酮化合物的含量测定表明，果皮中的类黄酮含量丰富，其黄烷酮几乎是果汁中的50～100倍，多甲氧基黄酮含量远远高于果汁。
不同种类柑桔果皮中黄烷酮和多甲氧基黄酮含量不同，甜橙果皮中的类黄酮以黄烷酮为主，约为类黄酮总含量的73.99％，而宽皮柑桔中的类黄酮则以多甲氧基黄酮为主，约为类黄酮总含量的53.19％，其多甲氧基黄酮的含量是甜橙果皮中的5.24倍。
宽皮柑桔果皮中的类黄酮含量大小依次为川皮苷＞橙皮苷＞桔黄酮＞柚皮素-7-β-芸香糖苷＞甜橙黄酮＞香风草甙，而甜橙果皮为橙皮苷＞川皮苷＞柚皮素-7-β-芸香糖苷＞甜橙黄酮＞香风草甙＞桔黄酮。
椪柑果皮中多甲氧基黄酮含量远远高于其它品种，其中又以浙江产椪柑果皮中含量最高，高达1.5％(以干重计)，可作为提取多甲氧基黄酮化合物的优质原料，为柑桔皮渣综合利用开辟了新途径。
4、通过比较甜橙和宽皮柑桔中柚皮素-7-β-芸香糖苷、橙皮苷、香风草甙等黄烷酮以及甜橙黄酮、川皮苷等多甲氧基黄酮的含量，表明其多甲氧基黄酮总量与桔黄酮的比值(PMFs／TNG)以及甜橙黄酮与桔黄酮的比值(SNT／TNG)有较大差异，可为甜橙和宽皮柑桔的区分提供参考依据。
China, one of the main citrus original in the globe, has rich citrus germplasm resources, whose annual production ranks the second all over the world at present. According to the epidemiological and animal researches, citrus fruits have many active effects on humans' health due to the flavonoids, carotenoids, limonoids, and etc., such as antioxidation, anticancer, prevention of circulatory disorders and so on; therefore, flavonoids in citrus have been more and more applied to functional food. As a result, it is necessary to investigate the flavonoid contents and distribution of various citrus fruits in China.
Testing methods of 7 flavanones (narirutin, naringin, hesperidin, neohesperidin, didymin, naringenin, hesperetin) and 3 polymethoxylated flavones (sinensetin, nobiletin, and tangeretin) by high performance liquid chromatographic (HPLC) , and extracting effects of different solvents on these compounds in citrus, were investigated in this paper; then on the basis of the above analyzing technology, contents of flavonoids in various citrus fruits were tested, including 124 juice and 81 peel samples. The main study results were listed as follows:
1. The simultaneous testing method of 10 flavonoid compounds (7 flavanones & 3 polymethoxylated flavones) by HPLC was established.
After extraction by 70% methanol(v/v) for juice or 50% dimethyl formamide (DMF, v/v) for peel, pre-treated citrus fruit samples were determinated by HPLC; and the detail HPLC conditions were adopt: C_(18) reversed-phase column(250×4.6mm, 5μm), gradient elution by acetonitrile and 0.2% acetic acid (v/v) as mobile phase, diode array detector (287nm, 330nm). This method was able to separate 10 flavonoids completely in 40min; moreover, the average recoveries, the relative standard deviations, precisions and detection limits of 7 flavanones arranged from 94.20% to 104.7%, 1.48% to 5.41%, 0.54% to 4.05%, 0.005 to 0.01mg/L, respectively; the previous 3 indexes of 3 polymethoxylated flavones arranged from 97.37% to 106.58%, 1.95% to 2.48%, 0.31% to 0.78%, respectively, and the detection limits were all 0.01 mg/L. Therefore, this pre-treating and testing methods satisfied the need of quick analysis, quality control and testing, due to simpleness, facility and the higher precision.
2. According to the analyzed results of 10 flavonoids from 124 juice samples, flavonoid composition ratios in juice varied with different citrus varieties.
The most major composition of flavanones in sweet orange, mandarin, tangor and lemon juices was hesperidin, whose contents arranged from 68.39 to 233.88mg/L, and the secondary compositions were narirutin and didymin, however naringin, neohesperidin, naringenin and hesperetin were detected rarely. Naringin in pomelo juice stayed the highest, whose average content was 166.41mg/L, almost accounting for 90% of 7 flavanones, and other less compositions were narirutin and hesperidin; naringin and narirutin constitute the most major composition of flavanones in grapefruit juices, whose average levels were 753.34mg/L, 199.23mg/L, respectively, and trace hesperidin existed; but neohesperidin, naringenin and hesperetin in both pomelo and grapefruit juice, were not detected. In a word, there was little polymethoxylated flavones in citrus juice, and contents of polymethoxylated flavones in mandarin juice were higher than that in sweet oranges, of which nobiletin level in mandarin juice was the highest, reaching 4.30mg/L.
3. From the study of 10 flavonoids in 81 citrus peel samples, peel portion of the whole fruit was abundant in flavonoids, whose flavanone contents were almost 50-100 times than juice, and levels of polymethoxylated flavones outclass juice.
The flavanone and polymethoxylated flavone contents in peels varied with different citrus fruit varieties. The prior compositions were flavanones in sweet orange, but polymethoxylated flavones in mandarin, whose levels were 5.24 times higher than in sweet orange peel, such as ponkan, in whose peels, 107.24 mg/g polymethoxylated flavones reached the top in studied mandarin.
According to the testing results, the content sequences of diverse flavonoids were shown: nobiletin＞hesperidin＞tangeretin＞narirutin＞sinensetin＞didymin, in mandarin peels; hesperidin＞nobiletin＞narirutin＞sinensetin＞didymin＞tangeretin, in sweet orange peels, of which levels of hesperidin, nobiletin and narirutin were higher than others.
4. Comparing the contents of flavanones (narirutin, hesperidin, didymin, etc) and polymethoxylated flavones (sinensetin, nobiletin, etc) in sweet orange with those in mandarin, the results indicated siginificant difference between the values of PMFs/TNG and SNT/TNG; therefore, values of PMFs/TNG and SNT/TNG were considered as the indexes of classification for sweet orange and mandarin.
引文
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[13]Wang X, Li F W, Zhang H X, et al. Preparative isolation and purification of polymethoxylated flavones from Tangerine peel using high-speed counter-current chromatography[J]. J.Chromatogra. A. 2005, 1090(1-2 ): 188-192.
[14]胡燕飞，王长海．微波法提取陈皮中3，5，6，7，8，3′，4′-七甲氧基黄酮及其含量测定[J]．烟台大学学报(自然科学与工程版)．2005，18(1)：45-49．
[15]González-barrio R, Trindade L M, Manzanares P, et al. Production of bioavailable flavonoid glucosides in fruit juices and green tea by use of fungal alpha-L-rhamnosidases[J]. J. Agric. Food Chem., 2004, 52(20): 6136-6142.
[16]郝云彬，叶兴乾，徐黎．超声技术在桔皮中提取橙皮苷的应用[J]．食品与发酵工业．2005，31(11)：63-66．
[17]元晓梅，刘贵贤．比色法测定柑桔饮料及桔皮制剂中总黄酮含量[J]．食品与发酵工业．1996，3(1)：13-21．
[18]Pérez-Ruíz T, Martinez-Lozano C, Tomás V, et al. Spectrofluorometric determination of hesperidin by manual and flow-injection methods[J]. Fresenius J. Anal. Chem.1999,364(3): 279-283.
[19]邵伟，熊泽，李昕．橙皮中橙皮苷的分光光度法测定[J]．光谱实验室．2005，22(5)：1044-1046．
[20]Fisher F. Fluorometric Determination of Limonin in Grapefruit and Orange Juice[J]. J. Agric. Food Chem. 1973, 21(6):1109-1110.
[21]田庆国，丁霄霖．测定桔核中柠檬苦素类似物的分光光度法[J]．分析测试学报．1999，18(5)：45-47．
[22]Rouseff L, Ting S V. Quantitation of polymethoxylated flavones in orange juice by high-performance liquid chromatography[J]. J.Chromatogra. A. 1979,176(1): 75-87.
[23]Pupin A. M., Dennisb M. J., Toledo M. C. F. Polymethoxylated flavones in Brazilian orange juice[J]. Food Chem. 1998, 63(4):513-518.
[24]董朝青，钟世安，周春山．反相高效液相色谱法同时测定柚皮中柚皮苷和橙皮苷的含量[J]．理化检验-化学分册，2005，41(1)：44-46．
[25]Caccamese S, Manna L, Scivoli G. Chiral HPLC separation and CD spectra of the C-2 diasteromers of naringin in grapefruit during maturation[J]. Chirality, 2003, 15 (2): 661-667.
[26]Manthey J A, Grohmann K. Phenols in citrus peel byproducts concentrations of hydroxycinnamates and polymethoxylated flavones in citrus peel[J]. J. Agric. Food Chem., 2001, 49(7): 3268-3273.
[27]Rapisarda P, Fanella F, Maccarone E. Reliability of analytical methods for determining anthocyanins in blood orange juices[J]. J. Agric. Food Chem., 2000, 48(5): 2249-2252.
[28]Dugo P, Mondello L, Morabito D. et al. Characterization of the anthocyanin fraction of Sicilian blood orange juice by micro-HPLC-ESI/MS[J]. J. Agric. Food Chem., 2003,51(5): 1173-1176.
[29]惠伯棣，张西，文镜．反相C_(30)柱在HPLC分析类胡萝卜素中的应用[J]．食品科学．2005，26(1)：264-270．
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[32]Kanaze F I, Gabrieli C, Kokkalou E, et al. Simultaneous reversed-phase high-performance liquid chromatographic method for the determination of diosmin, hesperidin and naringin in different citrus fruit juices and pharmaceutical formulations[J]. Journal of Pharmaceutical and Biomedical Analysis, 2003,33(2):243-249.
[33]Pellati F, Benvenuti S, Melegari M. High-performance liquid chromatography methods for the analysis of adrenergic amines and flavanones in Citrus aurantium L. var[J]. Phytochem. Anal., 2004, 15(2):220-225.
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[37]Pupin A M, Dennis M J, Toledo M C F, HPLC analysis of carotenoids in orange juice[J]. Food Chem. 1999, 64(2):269-275.
[38]Aturki Z, Sinibaldi M. Separation of diastereomers of flavanone-7-O-glycosides by capillary electrophoresis using sulfobutyl ether-β-cyclodextrin as the selector. J. Sep. Sci. 2003,26(9-10):844-850.
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[40]赖闻玲，陈京才．液相色谱-电喷雾电离质谱法及其应用[J]．赣南师范学院学报．2001，12(6)：60-63．
[41]Aturki Z, Brandi V, Sinibaldi M. Separation of flavanone-7-O-glycoside diastereomers and analysis in citrus juices by multidimensional liquid chromatography coupled with mass spectrometry[J]. J. Agric. Food Chem., 2004,52(17):5303-5308.
[42]Anagnostopoulou M A, Kefalas P, Kokkalou E. et al. Analysis of antioxidant compounds in sweet orange peel by HPLC-diode array detection-electrospray ionization massspectrometry[J]. Biomedical chromatography, 2004,19(2):138-148.
[43]Manners G D, Breksa A P, Schoch T K, et al. Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionozation and electrospray ionization liquid chromatography-mass spectrometry[J]. J. Agric. Food Chem., 2003, 51(6):3709-3714.
[44]Manthey J A. Fourier transform infrared spectroscopic analysis of the polymethoxylated flavone content of orange oil residues[J]. J. Agric. Food Chem., 2006, 54 (9):3215-3218.
[45]肖润林，陈朝明，李玲．不同品种柑桔果实中柚皮苷的含量变化[J]．植物生物学通讯．1990，6(1)：24-26．
[46]Ooghe W. C., Ooghe S. J., Detavernier C. M., et al. Characterization of orange juice (Citrus sinensis) by flavanone glycosides[J]. J. Agric. Food Chem. 1994,42(10):2183-2190.
[47]Leuzzi U.,Caristi C.,Panzera V.,et al. Flavonoids in pigmented orange juice and secong-pressure[J]. J. Agric. Food Chem., 2000, 48(11): 5501-5506.
[48]Kawaii, S., Tomono, Y., Katase, et al. Quantitation of flavonoid constituents in Citris fruits[J]. J. Agric. Food Chem. 1999,47(9):3565-3571.
[49]Nogata Y.,Sakamoto K.,Shiratsuchi H.,et al. Flavonoids composition of fruit tissues of citrus species[J].Biosci. Biotechnol. Biochem. 2006,70(1): 178—192.
[50]Green C O., Wheatley, A O. Osagie A U.,et al. Determination of polymethoxylated flavones in peels of selected Jamaican and Mexican citrus (Citrus spp.) cultivars by high-performance liquid chromatography[J]. Biomed. Chromatogr. 2007,21 (1): 48-54.
[51]Vanamala J, Reddivari L, Yoo K S.,et al. Variation in the content of bioactive flavonoids in different brands of orange and grapefruit juices[J]. Journal of Food Composition and Analysis.2006,19(2-3): 157-166.
[52]Robards K., Li X., Antolovich M.et al. Characterisation of citrus by chromatographic analysis of flavonoids[J]. J. Sci. Food Agric. 1997,75(2):87-101.
[53]Manthey J. A.,Grohmann K. Concentrations of hesperidin and other orange peel flavonoids in citrus processing byproducts[J]. J. Agric. Food Chem. 1996,44(3):811-814.
[54]Pupin A. M.,Dennis M. J.,Toledo M.C.F. Flavanone glycosides in Brazilian orange juice[J]. Food Chem. 1998,61(3):275-280.
[55]Kahle K.,Kraus M.,Richling E. Polyphenol profiles of apple juices[J]. Mol. Nutr. Food Res.2005,49(3):797-806.
[56]Wang Y.C.,Chuang Y.C.,Ku Y.H. Quantitation of biactive compounds in citrus fruits cultivated in Taiwan. 2007,102(4):1163-1171.
[57]庞小明．用分子标记研究柑桔属及其近源属植物的亲缘关系和枳的遗传多样性：[博士学位论文]．武汉：华中农业大学，2002，11：35-41．
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[59]张淑芬，马炳伦，郑大威等．柑桔果皮中多甲氧基黄酮的LC-APCI-MS定性分析[J]．分析测试学报．2004，23(Z1)：110-112．
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[4]赵雪梅，朱大元，叶兴乾等．柑桔属中类黄酮的研究进展[J]．天然产物研究与开发．2001．14(1)：89-92．
[5]Peterson J.J. et al. Flavanones in oranges, tangerines (mandarins), tangors, and tangelos: a compilation and review of the data from the analytical literature[J]. J. Food Composition and Analysis. 2006, 19:S66-S73.
[6]Mouly P, Gaydou E M, Auffray A. Simultaneous separation of flavanone glycosides and polymethoxylated flavones in citrus juices using liquid chromatography[J]. J.Chromatogra. A. 1998, 800 (2):171-179.
[7]张虹，张艳萍．柠檬苦素类似物的研究进展[J]．食品与发酵工业．2001．28(2)：80-83．
[8]Heimhuber B, Galensa R, Herrmann K. High-performance liquid chromatographic determination of polymethoxylated flavones in orange juice after solid-phase extraction[J]. J. Chromatogr. A. 1988, 439(2): 481-483.
[9]Mouly P, Gaydou E M, Arzouyan C. Seperation and quantitation of orange juices using liquid chromatography of polymethoxylated flavones[J]. Analusis. 1999, 27(3): 284-288.
[10]Senorans F J, Ruiz-Rodriguez A, Cavero S, et al. Isolation of antioxidant compounds from orange juice by using countercurrent supercritical fluid extraction (CC-SFE)[J]. J. Agric. Food Chem.2001, 49(12): 6039-6044.
[11]Simoa C, Ibanez E, Senoraans F. J., et al. Analysis of antioxidants from orange juice obtained by countercurrent supercritical fluid extraction, using micellar electrokinetic chromatography and reverse-phase liquid chromatography[J]. J. Agric. Food Chem,, 2002, 50(23): 6648-6652.
[12]Hillebrand S., Schwarz M., Winterhalter P. Characterization of anthocyanins and pyranoanthocyanins from blood orange[citrus sinensis (1.) osbeck]juice[J]. J. Agric. Food Chem. 2004, 52(24): 7331-7338.
[13]Wang X, Li F W, Zhang H X, et al. Preparative isolation and purification of polymethoxylated flavones from Tangerine peel using high-speed counter-current chromatography[J]. J.Chromatogra. A. 2005, 1090(1-2 ): 188-192.
[14]胡燕飞，王长海．微波法提取陈皮中3，5，6，7，8，3′，4′-七甲氧基黄酮及其含量测定[J]．烟台大学学报(自然科学与工程版)．2005，18(1)：45-49．
[15]González-barrio R, Trindade L M, Manzanares P, et al. Production of bioavailable flavonoid glucosides in fruit juices and green tea by use of fungal alpha-L-rhamnosidases[J]. J. Agric. Food Chem., 2004, 52(20): 6136-6142.
[16]郝云彬，叶兴乾，徐黎．超声技术在桔皮中提取橙皮苷的应用[J]．食品与发酵工业．2005，31(11)：63-66．
[17]元晓梅，刘贵贤．比色法测定柑桔饮料及桔皮制剂中总黄酮含量[J]．食品与发酵工业．1996，3(1)：13-21．
[18]Pérez-Ruíz T, Martinez-Lozano C, Tomás V, et al. Spectrofluorometric determination of hesperidin by manual and flow-injection methods[J]. Fresenius J. Anal. Chem.1999,364(3): 279-283.
[19]邵伟，熊泽，李昕．橙皮中橙皮苷的分光光度法测定[J]．光谱实验室．2005，22(5)：1044-1046．
[20]Fisher F. Fluorometric Determination of Limonin in Grapefruit and Orange Juice[J]. J. Agric. Food Chem. 1973, 21(6):1109-1110.
[21]田庆国，丁霄霖．测定桔核中柠檬苦素类似物的分光光度法[J]．分析测试学报．1999，18(5)：45-47．
[22]Rouseff L, Ting S V. Quantitation of polymethoxylated flavones in orange juice by high-performance liquid chromatography[J]. J.Chromatogra. A. 1979,176(1): 75-87.
[23]Pupin A. M., Dennisb M. J., Toledo M. C. F. Polymethoxylated flavones in Brazilian orange juice[J]. Food Chem. 1998, 63(4):513-518.
[24]董朝青，钟世安，周春山．反相高效液相色谱法同时测定柚皮中柚皮苷和橙皮苷的含量[J]．理化检验-化学分册，2005，41(1)：44-46．
[25]Caccamese S, Manna L, Scivoli G. Chiral HPLC separation and CD spectra of the C-2 diasteromers of naringin in grapefruit during maturation[J]. Chirality, 2003, 15 (2): 661-667.
[26]Manthey J A, Grohmann K. Phenols in citrus peel byproducts concentrations of hydroxycinnamates and polymethoxylated flavones in citrus peel[J]. J. Agric. Food Chem., 2001, 49(7): 3268-3273.
[27]Rapisarda P, Fanella F, Maccarone E. Reliability of analytical methods for determining anthocyanins in blood orange juices[J]. J. Agric. Food Chem., 2000, 48(5): 2249-2252.
[28]Dugo P, Mondello L, Morabito D. et al. Characterization of the anthocyanin fraction of Sicilian blood orange juice by micro-HPLC-ESI/MS[J]. J. Agric. Food Chem., 2003,51(5): 1173-1176.
[29]惠伯棣，张西，文镜．反相C_(30)柱在HPLC分析类胡萝卜素中的应用[J]．食品科学．2005，26(1)：264-270．
[30]Rouseff R, Raley L, Hofsommer H J. Application of diode array detection with a C-30 reversed phase column for the separation and identification of saponified orange juice carotenoids[J]. J. Agric. Food Chem., 1996, 44(8), 2176-2181.
[31]Lee H S. High-performance liquid chromatography for the characterization of carotenoids in the new sweet orange (Earlygold) grown in Florida, USA[J]. J.Chromatogra. A. 2001, 913(1-2):371-377.
[32]Kanaze F I, Gabrieli C, Kokkalou E, et al. Simultaneous reversed-phase high-performance liquid chromatographic method for the determination of diosmin, hesperidin and naringin in different citrus fruit juices and pharmaceutical formulations[J]. Journal of Pharmaceutical and Biomedical Analysis, 2003,33(2):243-249.
[33]Pellati F, Benvenuti S, Melegari M. High-performance liquid chromatography methods for the analysis of adrenergic amines and flavanones in Citrus aurantium L. var[J]. Phytochem. Anal., 2004, 15(2):220-225.
[34]Peleg H, Naim M, Rouseff R L, et al. Distribution of bound and free phenolic acids in oranges (Citrus sinensis) and grapefruits (Citrus paradisi)[J]. J. Sci. Food Agric. 1991,57(2): 417-426.
[35]Cortés C, Esteve M J, Frígola A, et al. Identification and quantification of carotenoids including geometrical isomers in fruit and vegetable by liquid chromatography with ultraviolet-diode army detection[J]. J. Agric. Food Chem. 2004, 52(8):2203-2212.
[36]Meléndez-Martínez A J, Vicario I M, Heredia F J, A routine high-performance liquid chromatography method for carotenoid determination in ultrafrozen orange juices[J]. J. Agric. Food Chem. 2003, 51(7):4219-4224.
[37]Pupin A M, Dennis M J, Toledo M C F, HPLC analysis of carotenoids in orange juice[J]. Food Chem. 1999, 64(2):269-275.
[38]Aturki Z, Sinibaldi M. Separation of diastereomers of flavanone-7-O-glycosides by capillary electrophoresis using sulfobutyl ether-β-cyclodextrin as the selector. J. Sep. Sci. 2003,26(9-10):844-850.
[39]Desiderio C, Rossi A, Sinibaldi D M. Analysis of flavanone-7-O-glycoside in citrus juices by short-end capillary electrochromatography[J]. J.Chromatogra. A. 2005,1081 (2): 99-104.
[40]赖闻玲，陈京才．液相色谱-电喷雾电离质谱法及其应用[J]．赣南师范学院学报．2001，12(6)：60-63．
[41]Aturki Z, Brandi V, Sinibaldi M. Separation of flavanone-7-O-glycoside diastereomers and analysis in citrus juices by multidimensional liquid chromatography coupled with mass spectrometry[J]. J. Agric. Food Chem., 2004,52(17):5303-5308.
[42]Anagnostopoulou M A, Kefalas P, Kokkalou E. et al. Analysis of antioxidant compounds in sweet orange peel by HPLC-diode array detection-electrospray ionization massspectrometry[J]. Biomedical chromatography, 2004,19(2):138-148.
[43]Manners G D, Breksa A P, Schoch T K, et al. Analysis of bitter limonoids in citrus juices by atmospheric pressure chemical ionozation and electrospray ionization liquid chromatography-mass spectrometry[J]. J. Agric. Food Chem., 2003, 51(6):3709-3714.
[44]Manthey J A. Fourier transform infrared spectroscopic analysis of the polymethoxylated flavone content of orange oil residues[J]. J. Agric. Food Chem., 2006, 54 (9):3215-3218.
[45]肖润林，陈朝明，李玲．不同品种柑桔果实中柚皮苷的含量变化[J]．植物生物学通讯．1990，6(1)：24-26．
[46]Ooghe W. C., Ooghe S. J., Detavernier C. M., et al. Characterization of orange juice (Citrus sinensis) by flavanone glycosides[J]. J. Agric. Food Chem. 1994,42(10):2183-2190.
[47]Leuzzi U.,Caristi C.,Panzera V.,et al. Flavonoids in pigmented orange juice and secong-pressure[J]. J. Agric. Food Chem., 2000, 48(11): 5501-5506.
[48]Kawaii, S., Tomono, Y., Katase, et al. Quantitation of flavonoid constituents in Citris fruits[J]. J. Agric. Food Chem. 1999,47(9):3565-3571.
[49]Nogata Y.,Sakamoto K.,Shiratsuchi H.,et al. Flavonoids composition of fruit tissues of citrus species[J].Biosci. Biotechnol. Biochem. 2006,70(1): 178—192.
[50]Green C O., Wheatley, A O. Osagie A U.,et al. Determination of polymethoxylated flavones in peels of selected Jamaican and Mexican citrus (Citrus spp.) cultivars by high-performance liquid chromatography[J]. Biomed. Chromatogr. 2007,21 (1): 48-54.
[51]Vanamala J, Reddivari L, Yoo K S.,et al. Variation in the content of bioactive flavonoids in different brands of orange and grapefruit juices[J]. Journal of Food Composition and Analysis.2006,19(2-3): 157-166.
[52]Robards K., Li X., Antolovich M.et al. Characterisation of citrus by chromatographic analysis of flavonoids[J]. J. Sci. Food Agric. 1997,75(2):87-101.
[53]Manthey J. A.,Grohmann K. Concentrations of hesperidin and other orange peel flavonoids in citrus processing byproducts[J]. J. Agric. Food Chem. 1996,44(3):811-814.
[54]Pupin A. M.,Dennis M. J.,Toledo M.C.F. Flavanone glycosides in Brazilian orange juice[J]. Food Chem. 1998,61(3):275-280.
[55]Kahle K.,Kraus M.,Richling E. Polyphenol profiles of apple juices[J]. Mol. Nutr. Food Res.2005,49(3):797-806.
[56]Wang Y.C.,Chuang Y.C.,Ku Y.H. Quantitation of biactive compounds in citrus fruits cultivated in Taiwan. 2007,102(4):1163-1171.
[57]庞小明．用分子标记研究柑桔属及其近源属植物的亲缘关系和枳的遗传多样性：[博士学位论文]．武汉：华中农业大学，2002，11：35-41．
[58]陈竹生，万良珍．中国柑桔良种彩色图谱[M]．成都：四川科技出版社．1993，12：36-49．
[59]张淑芬，马炳伦，郑大威等．柑桔果皮中多甲氧基黄酮的LC-APCI-MS定性分析[J]．分析测试学报．2004，23(Z1)：110-112．