东方梨生理活性成分的HPLC分析研究
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摘要
中国不仅是东方梨的发源中心，同时也是世界第一产梨大国，在漫长的栽培和食用过程中，古人已经发现了梨的许多食疗和保健作用，然而到目前为止，关于梨果实中生理活性成分方面有价值的研究报道十分罕见。在前期研究中，本研究组曾首次发现东方梨果实中有高含量的熊果苷、绿原酸等酚类活性物质，本实验研究表明熊果苷和绿原酸应该是梨果实最具代表性的活性成分。
本文利用国产高效液相色谱仪建立了测定梨果实中主要活性成分的等度高效液相色谱法和梯度淋洗高效液相色谱法，等度淋洗高效液相色谱分析方法使用HYPERSIL C_(18) BDS色谱柱(4.6mm×200mm，5μm)，流动相为甲醇：水(25∶75，v／v)，磷酸调节pH值为3.20，流速0.6 ml／min，室温条件，紫外检测器波长280nm，分析时间13分。熊果苷在144-5000ng，绿原酸在200-5000ng的范围线性相关系数均为0.99998，最低检出限分别为0.153ng和0.150ng，加标回收率分别为101％和97％。对梨果实中熊果苷、绿原酸、儿茶素、表儿茶素、咖啡酸、香豆酸、槲皮素等多酚类活性成分建立了双泵高压梯度淋洗的高效液相色谱分析方法。梯度淋洗程序(min／B％)为：0／2，3／2，4／25，11／50，16／80，26／80，33／2，45／2，流动相用磷酸调pH3.20，280nm检测波长下，30分钟内样品组分全部沈出，45分钟时色谱柱重新稳定，可以分析下一个样品。采用建立的高效液相色谱法对鸭梨的花期和果实生长期活性成分含量进行了动态分析，花期的不同器官中两种成分含量均处于较高的水平，尤其熊果苷，在花芽和叶芽中含量高达1.3％左右，绿原酸为3000μg／g左右。在果实生长期，幼果中两种酚类成分含量最高，分别为1.0％和0.38％，之后快速下降，果实成熟时这些酚类成分含量降低到400μg／g以下。对东方梨系统的白梨、砂梨、秋子梨和新疆梨以及西洋梨5个种的22个品种进行了分析，东方梨果实中熊果苷的含量(17个品种平均164μg／g)高于西洋梨(5个品种平均83μg／g)，其中主栽品种白梨中的鸭梨含量最高，达到400μg／g。西洋梨中的绿原酸含量更为突出，平均309μg／g，高于东方梨的含量(平均158μg／g)。这些成分在果实中的分布差别很大，熊果苷在果皮中含量最高，分别为果心和果肉的3-5倍和10-40倍。东方梨中绿原酸的分布果心含量高于果皮，而西洋梨则相反。
China is not only the cradle center of eastern pear but also the biggest county in pear's output of the world. During the lengthy process of growth and eating, some therapy function through biting and supping and many hygienically the ancients founded function. But the infrequent investigations and reports about the constituents with physiologic activity in pear fruit were founded by far. Our research team founded the upper level content of the constituents with physiologic activity for example arbutin and chlorogenic acid in eastern pear in initial stages. It was founded that Arbutin and chlorogenic acid should be the representative components with physiologic activity in eastern pear in fact by our test.
Isocratic elution reversed-phase high-performance liquid chromatographic (HPLC) method and gradient elution reversed-phase high-performance liquid chromatographic (HPLC) method for the analysis of the main constituents with physiologic activity in pear fruit were developed with high-performance liquid chromatographic instrument which was made in china. Isocratic elution method make up of a reversed-phase column (4.6mmx200mm) filled with 5um HYPERSIL BDS as the stationary phase .The mobile phase was made of CH3OH-H2O (25: 75, v/v) dualistic system at pH3.20, which adjusted, by phosphoric acid. The flow rate of mobile phase was 0.6 ml / min. During 13 minutes the analysis was ended at 280nm wavelength with ultraviolet detector in room temperature. The linearity correlative coefficient are 0.99998 both arbutin and chlorogenic acid. The linear range is at 144-5000ng to arbutin and is at 200-5000ng to chlorogenic acid. The two components' lowest detected limit was 0.153 ng and 0.150 ng respectively. Gradient elut
ion reversed-phase HPLC method using two pumps established to the main polyphenolic compounds for example arbutin, chlorogenic acid, catechin, epicatechin, caffeic acid, p-coumaric acid, quercetin, et, al in pear fruit. Gradient elution program (min/B%): 0/2, 3/2, 4/25, 11/50, 16/80, 26/80, 33/2, 45/2 .The mobile phase at pH3.20 adjusted by phosphoric acid and detected at 280nm wavelength. In this HPLC system, the main active components were separated within 30 minutes and chromatography column condition was renewably stabilized within 45 minutes and the next sample can be run. Dynamic changes in the main constituents with physiologic activity during the florescence and the fruits development stage of "Ya" Pear were monitored by the developed HPLC methods.The contents of the two constituents in different organs during the florescence are
on upper level especially the content of arbutin .The contents of arbutin reached about 1.3% in floral bud and foliar bud while the contents of chlorogenic acid obtained about 3000u.g/g .The contents of the two polyphenolic compounds in fruit let stage during the process of fruit growing are the tiptop that reached 1.0% and 0.38% respectively . Then the content of the polyphenolic compounds decline swiftly and the content of the polyphenolic compounds reduce below 400ug/g in the maturation period of pear fruit. Twenty-two varieties include P. bretschnrideri Reh., P. pyrifolia Burm Nakai, P. ussuriensis Maxim., P. sinkiangensis Yu. of eastern pear system and five varieties of Pyrus communis L. was compared and analyzed. The content of arbutin in eastern pear fruit (the average content is 164ug/g of 17 varieties) were upper than the Pyrus communis L. (the average content is 83ug/g). Ya pear is one variety of P.bretschnrideri Reh. Which mostly planting variety in eastern pear system and its content of arbutin w
ere uppest reaching 400(4ug/g. The content of chlorogenic acid was given prominence to Pyrus communis L. with 309(ug/g average and upper than the content of eastern pear (158ug/g). The content distribution of the components was prodigiously different in pear fruit. The tiptop content of arbutin was distributing in the fruit peel and obtained 3-5 times to fruit core and 10-40 times to fruit pulp. The content distribution of chlorogenic acid fruit core was upper than frui
本文利用国产高效液相色谱仪建立了测定梨果实中主要活性成分的等度高效液相色谱法和梯度淋洗高效液相色谱法，等度淋洗高效液相色谱分析方法使用HYPERSIL C_(18) BDS色谱柱(4.6mm×200mm，5μm)，流动相为甲醇：水(25∶75，v／v)，磷酸调节pH值为3.20，流速0.6 ml／min，室温条件，紫外检测器波长280nm，分析时间13分。熊果苷在144-5000ng，绿原酸在200-5000ng的范围线性相关系数均为0.99998，最低检出限分别为0.153ng和0.150ng，加标回收率分别为101％和97％。对梨果实中熊果苷、绿原酸、儿茶素、表儿茶素、咖啡酸、香豆酸、槲皮素等多酚类活性成分建立了双泵高压梯度淋洗的高效液相色谱分析方法。梯度淋洗程序(min／B％)为：0／2，3／2，4／25，11／50，16／80，26／80，33／2，45／2，流动相用磷酸调pH3.20，280nm检测波长下，30分钟内样品组分全部沈出，45分钟时色谱柱重新稳定，可以分析下一个样品。采用建立的高效液相色谱法对鸭梨的花期和果实生长期活性成分含量进行了动态分析，花期的不同器官中两种成分含量均处于较高的水平，尤其熊果苷，在花芽和叶芽中含量高达1.3％左右，绿原酸为3000μg／g左右。在果实生长期，幼果中两种酚类成分含量最高，分别为1.0％和0.38％，之后快速下降，果实成熟时这些酚类成分含量降低到400μg／g以下。对东方梨系统的白梨、砂梨、秋子梨和新疆梨以及西洋梨5个种的22个品种进行了分析，东方梨果实中熊果苷的含量(17个品种平均164μg／g)高于西洋梨(5个品种平均83μg／g)，其中主栽品种白梨中的鸭梨含量最高，达到400μg／g。西洋梨中的绿原酸含量更为突出，平均309μg／g，高于东方梨的含量(平均158μg／g)。这些成分在果实中的分布差别很大，熊果苷在果皮中含量最高，分别为果心和果肉的3-5倍和10-40倍。东方梨中绿原酸的分布果心含量高于果皮，而西洋梨则相反。
China is not only the cradle center of eastern pear but also the biggest county in pear's output of the world. During the lengthy process of growth and eating, some therapy function through biting and supping and many hygienically the ancients founded function. But the infrequent investigations and reports about the constituents with physiologic activity in pear fruit were founded by far. Our research team founded the upper level content of the constituents with physiologic activity for example arbutin and chlorogenic acid in eastern pear in initial stages. It was founded that Arbutin and chlorogenic acid should be the representative components with physiologic activity in eastern pear in fact by our test.
Isocratic elution reversed-phase high-performance liquid chromatographic (HPLC) method and gradient elution reversed-phase high-performance liquid chromatographic (HPLC) method for the analysis of the main constituents with physiologic activity in pear fruit were developed with high-performance liquid chromatographic instrument which was made in china. Isocratic elution method make up of a reversed-phase column (4.6mmx200mm) filled with 5um HYPERSIL BDS as the stationary phase .The mobile phase was made of CH3OH-H2O (25: 75, v/v) dualistic system at pH3.20, which adjusted, by phosphoric acid. The flow rate of mobile phase was 0.6 ml / min. During 13 minutes the analysis was ended at 280nm wavelength with ultraviolet detector in room temperature. The linearity correlative coefficient are 0.99998 both arbutin and chlorogenic acid. The linear range is at 144-5000ng to arbutin and is at 200-5000ng to chlorogenic acid. The two components' lowest detected limit was 0.153 ng and 0.150 ng respectively. Gradient elut
ion reversed-phase HPLC method using two pumps established to the main polyphenolic compounds for example arbutin, chlorogenic acid, catechin, epicatechin, caffeic acid, p-coumaric acid, quercetin, et, al in pear fruit. Gradient elution program (min/B%): 0/2, 3/2, 4/25, 11/50, 16/80, 26/80, 33/2, 45/2 .The mobile phase at pH3.20 adjusted by phosphoric acid and detected at 280nm wavelength. In this HPLC system, the main active components were separated within 30 minutes and chromatography column condition was renewably stabilized within 45 minutes and the next sample can be run. Dynamic changes in the main constituents with physiologic activity during the florescence and the fruits development stage of "Ya" Pear were monitored by the developed HPLC methods.The contents of the two constituents in different organs during the florescence are
on upper level especially the content of arbutin .The contents of arbutin reached about 1.3% in floral bud and foliar bud while the contents of chlorogenic acid obtained about 3000u.g/g .The contents of the two polyphenolic compounds in fruit let stage during the process of fruit growing are the tiptop that reached 1.0% and 0.38% respectively . Then the content of the polyphenolic compounds decline swiftly and the content of the polyphenolic compounds reduce below 400ug/g in the maturation period of pear fruit. Twenty-two varieties include P. bretschnrideri Reh., P. pyrifolia Burm Nakai, P. ussuriensis Maxim., P. sinkiangensis Yu. of eastern pear system and five varieties of Pyrus communis L. was compared and analyzed. The content of arbutin in eastern pear fruit (the average content is 164ug/g of 17 varieties) were upper than the Pyrus communis L. (the average content is 83ug/g). Ya pear is one variety of P.bretschnrideri Reh. Which mostly planting variety in eastern pear system and its content of arbutin w
ere uppest reaching 400(4ug/g. The content of chlorogenic acid was given prominence to Pyrus communis L. with 309(ug/g average and upper than the content of eastern pear (158ug/g). The content distribution of the components was prodigiously different in pear fruit. The tiptop content of arbutin was distributing in the fruit peel and obtained 3-5 times to fruit core and 10-40 times to fruit pulp. The content distribution of chlorogenic acid fruit core was upper than frui
引文
[1] 蒲富慎．梨品种[M]．北京：中国农业出版社，1989．
[2] (日)林真二，吴耕民译．梨[M]．北京：农业出版社，1981．
[3] 郗荣庭．中国鸭梨[M]．北京：中国林业出版社，1999．
[4] 张鹏．我国梨属植物种和品种分类的进展[J]．山西果树，1991(1)：2～5
[5] 滕葳．我国梨品种资源分布与梨果品质现状[J]．农牧产品开发，1996(12)：37～38．
[6] 林伯年．世界及中国的梨生产与贸易[J]．中国南方果树，2001，30(6)：64～67．
[7] 傅玉瑚，陈敬谊，张喜焕．我国梨科研五十年来的主要进展和成就[J]．邯郸农业高等专科学校学报，1999，16(3)：1～5．
[8] 王伟东等．我国及世界梨的产销形势[J]．落叶果树，2002，34(5)：56．
[9] 萧梁·陶宏景，名医别录[M]，北京：人民卫生出版社．
[10] 元·李杲，食物本草[M]，北京：中国医药科技出版社．
[11] 明·李时珍，本草纲目[M]，北京：人民卫生出版社，1982．1756．
[12] 清·赵学敏．本草纲目拾遗[M]．北京：人民卫生出版社，1963．
[13] 清·吴仪洛，本草从新[M]，北京：北京市中国书店，1985．12．
[14] I. Aesculus Hippocastaneum L., Arctostaphyllos Uva-Ursi Spreng, Fraxinus Excelsior. L. Quantitative determination of biologically active constituents in medicinal plant crude extracts by thin-layer chromatography-densitometry [J]. Journal of Chromatography, 1983, 281: 263～271.
[15] Friedrich H. Studies on the phenolic constituents of Pyrus communis. Ⅳ. Content of arbutin in germinating pear seeds & distribution in young plants[J]. Pharmazie, 1958, 13(3): 153～155.
[16] Friedrich H. Studies on phenolic components of Pyrus communis. Ⅱ. Arbutin content of pear leaves[J]. Pharmazie, 1957, 12(12): 831～834.
[17] Durkee A B, Johnston F B, Thivierge P A. Arbutin and a related glucoside in immature pear fruit[J]. J Food Sci, 1968, 33: 461～463.
[18] Suau R, Cuevas A, Valpuesta V. Arbutin and sucrose in the leaves of the resurrection plant Myrothamnus flabellifolia[J]. Phytochemistry, 1991, 30(8): 2555～2556.
[19] Amiot M J, Tacchini M, Aubert S Y. Phenolic composition and browning suscerptibility of various apple cultivars at maturity[J]. J Food Sci, 1992, 57: 958～962.
[20] Veltman R H, Kho R M, Van Schaik A C R, Sanders M G. Ascorbic acid and tissue browning in pears(Pyrus communis L. cvs. Rocha and Conference)under controlled atmosphere conditions[J]. Postharv BiolTechnol, 2000, 19: 129～137.
[21] Pinto E, Lentheric I, Vendrell M. Role of fermentative and antioxidant metabolism in the induction of core browning in controlled-atmosphere stored pears[J]. J Hort
Sci Food Agric, 2001, 74: 791～795.
[22] Lentheric I, Pinto E, Vendrell M. Harvest data affects the antioxidant system in pear fruits[J]. J Hort Sci Biotechnol, 1999, 74: 791～795.
[23] Cornwell C J, Wrolstad R E. Cause of browning in pear juice concentrate during storage[J]. J. Food Sci, 1981, 46: 515～518.
[24] Amiot M J, Aubert S, Nicolas, J. Phenolic composition and browning susceptibility of various apple and pear cultivars at maturity[J]. Bull. Liaison-Groupe Polyphenols, 1992, 16, 48～51.
[25] Eskin N A M. Biochemistry of Food Spoilage: Enzymatic Browning[J]. Biochemistry of Foods, 1990, 29: 401～432.
[26] Amiot M J, Tacchini M, Auber S Y. Influence of cultivar, maturity stage, and storage conditions on phenolic composition and enzymatic browning of pear fruits[J]. J Agric Food Chem, 1995, 43: 1132～1137.
[27] Tate J N, Luh B S, York G K. Polyphenol oxidase in Bartlett pears. J. Food Sci, 1964, 29: 829～836.
[28] HalimD H, Montgomery M W. Polyphenol oxidase of d'Anjou pears(Pyrus communisL.)[J]. J. Food Sci, 1978, 43: 603～605.
[29] Veltman R H, Larrigaudiere C, Wicher H J. PPO activity and polyphenol content are net limiting factors during brown core development in pears (Pyrus communis L. cv. Conference) [J]. J. Plant Physiol, 1999, 154: 697～702.
[30] Sanchez A C G, Gil～lzquierdo A, Gil MI1. Comparative study of six pear cultivars in terms of their phenolic and vitamin C contents and antioxidant capacity[J]. J Sci Food Agric, 2003, 83: 995～1003.
[31] Veltman R H, Sanders M G, Persijn S T. Decreased ascorbic acid levels and brown core development in pears(Pyrus communis L. cv. Conference) [J]. Physiol Plant, 1999, 107: 39～45.
[32] 鞠志国，朱广廉，曹宗巽，等．莱阳茌梨果实褐变与多酚氧化酶几分类物质区域划分布的关系[J]．植物生理学报，1988，14(4)：356～361．
[33] 韩雅珊．食品化学[M]．北京：中国农业大学出版社，1998．262～268．
[34] Spanos G A, Wrolstad R E. Influence of variety, maturity, processing, and storage on the phenolic cimposition of pear juice[J]. J Agric Food Chem, 1990, 38: 817～824.
[35] Ferreira D, Barros A, Coimbra M A. Use of FT-IR spectroscopy to follow the effect of processing in cell wall polysaccharide extracts of a sun-dried pear[J]. Carbohydr. Polym, 2001, 45: 175～182.
[36] Silva B M, Andrade P B, Mendes G C. Analysis of Phenolic Compounds in the Evaluation of Commercial Quince Jam Authenticity[J]. J. Agric. Food Chem, 2000, 48: 2853～2857.
[37] Branca M. Silva, Paula B. Andrade. Phenolic profile in the evaluation of commercial quince jellies authenticity[J]. Food Chemistry, 2000, 71: 281～285.
[38] Shigeki Jin, Sato N. Benzoquinone, the substance essential for antibacterial activity in aqueous extracts from succulent young shoots of the pear Pyrus spp[J]. Phytochemistry, 2003, 62: 101～107.
[39] Rychlinska I, Gudej J. Qualitative and quantitative chromatographic investigation of hydroquinone derivatives in Pyrus communis L. flowers[J]. Acta Pol Pharm. 2003, Jul-Aug;60(4): 309～312.
[40] Hildebracd, D. C., Schroth, M. N. Relation of arbutin-hydroquinone in pear blossoms to invasion by E. amylovora [J]. Nature, 1963, 197: 513.
[41] Hildebracd, D. C., Schroth, M. N. Antibiotic activity of pear leaves against Erwinia amylovora and its relation to β-glucosidase[J]. Phytopathology, 1964a, 54: 59～63.
[42] Hildebracd, D. C. , Schroth, M. N. Arbutin-hydroquione complex in pearas a factor in fire blight development [J] Phytopathology, 1964b, 54: 640～645.
[43] Hildebracd, D.C. Fire blight resistance in Pyrus, hydroquinone formation as related to antibiotic activity[J]. Can. J. Bot, 1970, 48: 177～181.
[44] 李安良，杨淑琴，郭秀茹，等．熊果苷的进展[J]．日用化学工业，2000，4(2)：62～65．
[45] 王建新．天然活性化妆品[M]．北京：中国轻工业出版社，1997．245～247．
[46] Matsuda H, Nakata T. et al Combined effects of arbutin and prednisolone or dexamethazon[J]. Yakugaku Zasshi, 1990, 110(1): 68～76.
[47] Petkou D, Diamantidis G, Vasilakakis M. Arbutin osidation by pear(Pyrus communis L.)peroxidases Plant Science, 2002, 162: 115～119.
[48] Kimura Y. Okuda T et al. Studies on the activities of tannis and related compounds. V. Inhibitory effects on lipid peroxidation in mitochondria and microsomes of liver [J]. Planta Med, 1984, 50: 473～477.
[49] Ioku K, Terao N, Nakatani N. Antioxidant properties of arbutin, Biosci. Biotechnol. Biochem, 1992, 52: 1658～1659.
[50] Oliver AE, Hincha D K, Crowe L M. Interactions of arbutin with dry and hydrated bilayers, Biochim. Biophys. Acta, 1998, 1370: 89～97.
[51] C. Couteau. L. J. M. Coiffard. Photostability determination of arbutin, a vegetable whitening agent. Farmaco, 2000, 55: 410～413.
[52] 李咏悦，陈雅英．熊果苷在化妆品中的用途[J]．日用化学工业，1995，6：31～33．
[53] 陆晔，周名权，翁康生，等．三种美白剂对酪氨酸酶活性的抑制[J]．香料香精化妆品，2001，5：15～17．
[54] 高锦明等．绿原酸分布、提取与生物活性研究综述[J]．西北林学院学报，1999，14(2)：73～82．
[55] 武雪芬，郑超萍，李桂兰．金银花液提取物的抗氧化作用研究[J]．河南化工，1999，10：10～11．
[56] 贾宪生，吴家其，茅青．吊子银花的品质研究[J]．贵阳中医学院学报，1997，19(3)：58～59．
[57] 娄红祥．金银花中水溶性化合物的分离与结构鉴定[J]．中草药，1996，371：203～214．
[58] 张英，李世敏，林志达，等．绿茶中要用绿原酸成分分析[J]．食品科学，2001，22(11)：84～86．
[59] 张军民，高振川，张琪，等．杜仲叶及提取物营养价值和药用成分研究[J]．氨基酸和生物资源，2002，24(1)：1～2．
[60] 宰学明，吴国荣，龚祝南，等．槲寄生抗氧化物质的研究测定[J]．中草药，2001，32(12)：1081～1083．
[61] 秦文娟等．苦丁茶化学成分的研究(Ⅱ)[J]．中草药，1988，19(11)：6～8．
[62] 房志仲，杨金荣，张璇，等．解热抗炎1号口服液中绿原酸定量方法的研究[J]．天津医科大学学报，2000，6(2)：153～155．
[63] 牛志多，王威，闫喜英，等．咳喘平口服液质量控制方法研究[J]．时珍国医国药，2001，12(3)：209～210．
[64] 武雪芬，景小琦，李国茹．金银花叶药用成分的提取及抑菌试验[J]．天然产物研究与开发，2001，13(3)：43～44．
[65] 郑建仙．功能性食品[M]．北京：中国轻工业出版社，1995．
[66] San R H C. Chan R I M. Inibitory effect of phenolic compounds on aflatox in B1 metabolism and induced mutagenesis [J]. Mutation Res, 1987, 177: 229～239.
[67] 林启寿．中草药成分化学[M]．北京：科学出版社，1977．146．
[68] 柯铭清．中草药有效成分理化与药理特性[M]．长沙：湖南科学技术出版社，1982．225．
[69] 柳云溪．欧洲植物药会议介绍[J]．国外医药，植物药分册，1997，12(6)：257～260．
[70] 罗思齐．中草药活性成分含量测定概述[J]．中草药，1989，20(9)：19～26．
[71] Andrade P B, Carvalho A R F. A previous study of phenolic profiles of quince, pear and apple purees by HPLC diode-array detection for the evaluation of quince puree genuineness. [J]. Agric. Food. Chem, 1998, 46: 968～972.
[72] Oleszek W O, Amiot MJ, Aubert, S. Identification of some phenolics in pear fruit[J]. J. Agric. Food Chem, 1994, 42: 1261～1265.
[73] 崔同，王燕桓，傅承光，王继英，赵海．HPLC—二极管阵列检测法分析梨果实酚类化合物[A]．第十一次全国色谱学术报告会文集[C]，大连：1997．101～103．
[74] Chang Q, Zhu M, Zuo Z. High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma[J]. Journal of Chromatographic B, 2001, 760: 227～235.
[75] Escarpa A, Gonzalez M C. Fast separation of(poly)phenolic compounds from apples and pears by high-performance liquid chromatographic with diode-array detection[J]. Journal of Chromatographic A, 1999, 830: 301～309.
[76] Schieber A, Keller P, Carle R. Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography[J]. Journal of Chromatographic A, 2001, 910: 265～273. [71]
[77] 高原军，王素梅，孔谨．控制梨果发生酶褐变的研究[J]．食品科学，1997，6：15～17．
[78] Iwagawa T, Takahashi H, Munesada K. A phenol alloside from viburnum wrightii[J]. Phytochemistry, 1984, 23(2): 468～469.
[79] Takido M, Fukuhiro K, Yamanouchi S. Phlebotrichin, A phenolic compound from the fresh leaves of viburnum phlebotrichum[J]. Phytochemistry, 1983, 22(1): 223～225.
[80] 张国海，史国安等．野生砂梨果实的某些品质性状及抗氧化活性的比较分析[J]．河南农业大学学报，2000，9，34(3)：302～304．
[81] Tomas-Barberan FA, Ferreres F, Gil M I. Antioxidant phenolic metabolites from fruit and vegetables and changes during postharvest storage and processing[J]. Natural Products Chemistry, 2000, 23: 739～795.
[82] Blankenship S M, Richardson DG. Changs in phenolic acids and internal ethylene during long-term cold storage of pears[J]. J Am Soc Hort Sci, 1985, 110: 336～339.
[83] Escarpa A, Gonzaleze M C. Total extractable phenolic chromatographic index: an overview of the phenolic class content from different sources of foods[J]. Eur Food Res Technol, 2001, 212: 439～444.
[84] 朱健辉，崔同，锁然．薄层扫描测定果蔬中绿原酸含量[J]．中国畜产与食品，1999，6(5)：218～219．
[85] Tong Cui, Hiroshi Kayahara. Abilities of Superoxide Scavenging and Activities of Tyrosinase Inhibiting of Fruit Extracts[P]．商英凡．中国专利发明成果大典[M]．北京：国家知识产权局知识产权出版社，2002．797～801
[86] 鞠志国．酚类物质与梨果实品质的研究进展[J]．莱阳农学院学报，1988，5：59～65．
[87] 鞠志国．采期对莱阳茌梨酚类物质代谢和组织褐变的影响[J]．中国农业科学，1991，24(2)：63～68．
[88] 鞠志国，朱广廉，曹宗巽．莱阳茌梨果实褐变与多酚氧化酶几分类物质区域划分布的关系[J]．植物生理学报，1988，14(4)：356～361．
[89] 鞠志国，朱广廉，曹宗巽．气调贮藏条件下CO_2对莱阳茌梨果肉褐变的影响[J]．园艺学报，1988，15(4)：229～232．
[90] 鞠志国，刘成连，原永兵，等．莱阳茌梨酚类物质合成的调节及其对果实品质
的影响[J]．中国农业科学，1993，26(4)：44～48．
[91] 鞠志国．莱阳茌梨细胞中多份氧化酶的分布及其性质[J]．植物生理学通报，1989，3：33～35．
[92] 鞠志国，刘成连，原永兵，等．苹果果皮中酚类物质合成规律的研究[J]．莱阳农学院学报，1992，9(3)：222～225．
[93] 鞠志国，刘成连，原永兵，等．PP_(333)对梨果实生长和酚类物质合成的影响[J]．园艺学报，1993，20(3)：216～220．
[94] 张玉星，田志喜，郗荣庭，等．水杨酸对鸭梨幼果酚类物质代谢调节作用的研究[J]．河北农业大学学报，2002，25(3)：33～36．
[95] Hase T, Iwagawa T, Munesada K. A bitter monoterpene glucoside from viburnum phlebotrichum[J]. Phytochemistry, 1982, 21(6): 1435～1437.
[96] Wald B, Wray V, Galensa R. Malonated flavonol glycosides and 3, 5～dicaggeoylquinic acid from pears[J]. Phytochemistry, 28: 663～664.
[97] Petkou D, Diamantidis G, Vasilakakis M. Arbutin oxidation by pear(Pyrus communis L.) peroxidases[J]. Plant Science, 2002, 162: 115～119.
[98] Wrolstad R E, Lombard P B, Richardson D G. The pear[J]. In Quality and Preservation of Fruits, 1991: 67～95.
[99] (日)富山宏平，尹福祥译．植物的感染生理[M]．北京：农业出版社，1986．
[100] Escarpa A, Gonzalez M C. High-performance liquid chromatography with diode～array detection for the determination of phenolic compounds in peel and pulp from different apple varieties[J]. J. Chromatogr. A, 1998, 823: 331～337.
[101] Spanos G A, Wrolstad R E. Phenolics of apple, pear, and white grape juices and their changes with processing and storage-A review[J]. J. Agric. Food Chem, 1992, 40: 1478～1487.
[102] Mosel H D, Herrmann K. Changes in catechins and hydroxycinnamic acid derivatives during development of apples and pears [J]. J. Sci. Food Agric, 1974, 25: 251～256.
[103] Oszmianski J O, Lee C Y. Enzymatic oxidation reaction of catechin and chlorogenic acid in amodel system[J]. J. Agric. Food Chem, 1990, 38: 1202-1204.
[104] 宋立江．植物多酚研究与利用的意义及发展趋势[J]．化学进展，2000年，12(2)：161～170．
[105] 凌关庭．有“第七类营养素”之称的多酚类物质[J]．中国食品添加剂，2000(1)：28～37．
[106] Timberlake C F, Bridle P. The anthocyanins if apples and pears: the occurrence of acyl derivatives[J]. J Sci Food Agic, 1971, 22: 509～513.
[107] Francis F J. Anthocyanins in pears[J]. Hort Sci, 1970, 5: 42.
[108] Ranadive A S, Harrd N F. Changes in polyphenolics on ripening of selected pear
varieties[J]. J Sci Food Chem, 1998, 46: 968～972.
[109] 马良，崔同，张宇昊，等．梨果中绿原酸和熊果苷的HPLC测定方法[J]．吉林农业大学学报，2003，10(25)：115～117，120．
[110] 刈米达夫．植物化学[M]．杨本文译．北京：科学出版社，1985．
[111] T．W．古德温．E．I默塞尔．植物生物化学导论[M]．西北农业大学译．陕西杨陵：天则出版社，1988：88～89，233，531，560．
[2] (日)林真二，吴耕民译．梨[M]．北京：农业出版社，1981．
[3] 郗荣庭．中国鸭梨[M]．北京：中国林业出版社，1999．
[4] 张鹏．我国梨属植物种和品种分类的进展[J]．山西果树，1991(1)：2～5
[5] 滕葳．我国梨品种资源分布与梨果品质现状[J]．农牧产品开发，1996(12)：37～38．
[6] 林伯年．世界及中国的梨生产与贸易[J]．中国南方果树，2001，30(6)：64～67．
[7] 傅玉瑚，陈敬谊，张喜焕．我国梨科研五十年来的主要进展和成就[J]．邯郸农业高等专科学校学报，1999，16(3)：1～5．
[8] 王伟东等．我国及世界梨的产销形势[J]．落叶果树，2002，34(5)：56．
[9] 萧梁·陶宏景，名医别录[M]，北京：人民卫生出版社．
[10] 元·李杲，食物本草[M]，北京：中国医药科技出版社．
[11] 明·李时珍，本草纲目[M]，北京：人民卫生出版社，1982．1756．
[12] 清·赵学敏．本草纲目拾遗[M]．北京：人民卫生出版社，1963．
[13] 清·吴仪洛，本草从新[M]，北京：北京市中国书店，1985．12．
[14] I. Aesculus Hippocastaneum L., Arctostaphyllos Uva-Ursi Spreng, Fraxinus Excelsior. L. Quantitative determination of biologically active constituents in medicinal plant crude extracts by thin-layer chromatography-densitometry [J]. Journal of Chromatography, 1983, 281: 263～271.
[15] Friedrich H. Studies on the phenolic constituents of Pyrus communis. Ⅳ. Content of arbutin in germinating pear seeds & distribution in young plants[J]. Pharmazie, 1958, 13(3): 153～155.
[16] Friedrich H. Studies on phenolic components of Pyrus communis. Ⅱ. Arbutin content of pear leaves[J]. Pharmazie, 1957, 12(12): 831～834.
[17] Durkee A B, Johnston F B, Thivierge P A. Arbutin and a related glucoside in immature pear fruit[J]. J Food Sci, 1968, 33: 461～463.
[18] Suau R, Cuevas A, Valpuesta V. Arbutin and sucrose in the leaves of the resurrection plant Myrothamnus flabellifolia[J]. Phytochemistry, 1991, 30(8): 2555～2556.
[19] Amiot M J, Tacchini M, Aubert S Y. Phenolic composition and browning suscerptibility of various apple cultivars at maturity[J]. J Food Sci, 1992, 57: 958～962.
[20] Veltman R H, Kho R M, Van Schaik A C R, Sanders M G. Ascorbic acid and tissue browning in pears(Pyrus communis L. cvs. Rocha and Conference)under controlled atmosphere conditions[J]. Postharv BiolTechnol, 2000, 19: 129～137.
[21] Pinto E, Lentheric I, Vendrell M. Role of fermentative and antioxidant metabolism in the induction of core browning in controlled-atmosphere stored pears[J]. J Hort
Sci Food Agric, 2001, 74: 791～795.
[22] Lentheric I, Pinto E, Vendrell M. Harvest data affects the antioxidant system in pear fruits[J]. J Hort Sci Biotechnol, 1999, 74: 791～795.
[23] Cornwell C J, Wrolstad R E. Cause of browning in pear juice concentrate during storage[J]. J. Food Sci, 1981, 46: 515～518.
[24] Amiot M J, Aubert S, Nicolas, J. Phenolic composition and browning susceptibility of various apple and pear cultivars at maturity[J]. Bull. Liaison-Groupe Polyphenols, 1992, 16, 48～51.
[25] Eskin N A M. Biochemistry of Food Spoilage: Enzymatic Browning[J]. Biochemistry of Foods, 1990, 29: 401～432.
[26] Amiot M J, Tacchini M, Auber S Y. Influence of cultivar, maturity stage, and storage conditions on phenolic composition and enzymatic browning of pear fruits[J]. J Agric Food Chem, 1995, 43: 1132～1137.
[27] Tate J N, Luh B S, York G K. Polyphenol oxidase in Bartlett pears. J. Food Sci, 1964, 29: 829～836.
[28] HalimD H, Montgomery M W. Polyphenol oxidase of d'Anjou pears(Pyrus communisL.)[J]. J. Food Sci, 1978, 43: 603～605.
[29] Veltman R H, Larrigaudiere C, Wicher H J. PPO activity and polyphenol content are net limiting factors during brown core development in pears (Pyrus communis L. cv. Conference) [J]. J. Plant Physiol, 1999, 154: 697～702.
[30] Sanchez A C G, Gil～lzquierdo A, Gil MI1. Comparative study of six pear cultivars in terms of their phenolic and vitamin C contents and antioxidant capacity[J]. J Sci Food Agric, 2003, 83: 995～1003.
[31] Veltman R H, Sanders M G, Persijn S T. Decreased ascorbic acid levels and brown core development in pears(Pyrus communis L. cv. Conference) [J]. Physiol Plant, 1999, 107: 39～45.
[32] 鞠志国，朱广廉，曹宗巽，等．莱阳茌梨果实褐变与多酚氧化酶几分类物质区域划分布的关系[J]．植物生理学报，1988，14(4)：356～361．
[33] 韩雅珊．食品化学[M]．北京：中国农业大学出版社，1998．262～268．
[34] Spanos G A, Wrolstad R E. Influence of variety, maturity, processing, and storage on the phenolic cimposition of pear juice[J]. J Agric Food Chem, 1990, 38: 817～824.
[35] Ferreira D, Barros A, Coimbra M A. Use of FT-IR spectroscopy to follow the effect of processing in cell wall polysaccharide extracts of a sun-dried pear[J]. Carbohydr. Polym, 2001, 45: 175～182.
[36] Silva B M, Andrade P B, Mendes G C. Analysis of Phenolic Compounds in the Evaluation of Commercial Quince Jam Authenticity[J]. J. Agric. Food Chem, 2000, 48: 2853～2857.
[37] Branca M. Silva, Paula B. Andrade. Phenolic profile in the evaluation of commercial quince jellies authenticity[J]. Food Chemistry, 2000, 71: 281～285.
[38] Shigeki Jin, Sato N. Benzoquinone, the substance essential for antibacterial activity in aqueous extracts from succulent young shoots of the pear Pyrus spp[J]. Phytochemistry, 2003, 62: 101～107.
[39] Rychlinska I, Gudej J. Qualitative and quantitative chromatographic investigation of hydroquinone derivatives in Pyrus communis L. flowers[J]. Acta Pol Pharm. 2003, Jul-Aug;60(4): 309～312.
[40] Hildebracd, D. C., Schroth, M. N. Relation of arbutin-hydroquinone in pear blossoms to invasion by E. amylovora [J]. Nature, 1963, 197: 513.
[41] Hildebracd, D. C., Schroth, M. N. Antibiotic activity of pear leaves against Erwinia amylovora and its relation to β-glucosidase[J]. Phytopathology, 1964a, 54: 59～63.
[42] Hildebracd, D. C. , Schroth, M. N. Arbutin-hydroquione complex in pearas a factor in fire blight development [J] Phytopathology, 1964b, 54: 640～645.
[43] Hildebracd, D.C. Fire blight resistance in Pyrus, hydroquinone formation as related to antibiotic activity[J]. Can. J. Bot, 1970, 48: 177～181.
[44] 李安良，杨淑琴，郭秀茹，等．熊果苷的进展[J]．日用化学工业，2000，4(2)：62～65．
[45] 王建新．天然活性化妆品[M]．北京：中国轻工业出版社，1997．245～247．
[46] Matsuda H, Nakata T. et al Combined effects of arbutin and prednisolone or dexamethazon[J]. Yakugaku Zasshi, 1990, 110(1): 68～76.
[47] Petkou D, Diamantidis G, Vasilakakis M. Arbutin osidation by pear(Pyrus communis L.)peroxidases Plant Science, 2002, 162: 115～119.
[48] Kimura Y. Okuda T et al. Studies on the activities of tannis and related compounds. V. Inhibitory effects on lipid peroxidation in mitochondria and microsomes of liver [J]. Planta Med, 1984, 50: 473～477.
[49] Ioku K, Terao N, Nakatani N. Antioxidant properties of arbutin, Biosci. Biotechnol. Biochem, 1992, 52: 1658～1659.
[50] Oliver AE, Hincha D K, Crowe L M. Interactions of arbutin with dry and hydrated bilayers, Biochim. Biophys. Acta, 1998, 1370: 89～97.
[51] C. Couteau. L. J. M. Coiffard. Photostability determination of arbutin, a vegetable whitening agent. Farmaco, 2000, 55: 410～413.
[52] 李咏悦，陈雅英．熊果苷在化妆品中的用途[J]．日用化学工业，1995，6：31～33．
[53] 陆晔，周名权，翁康生，等．三种美白剂对酪氨酸酶活性的抑制[J]．香料香精化妆品，2001，5：15～17．
[54] 高锦明等．绿原酸分布、提取与生物活性研究综述[J]．西北林学院学报，1999，14(2)：73～82．
[55] 武雪芬，郑超萍，李桂兰．金银花液提取物的抗氧化作用研究[J]．河南化工，1999，10：10～11．
[56] 贾宪生，吴家其，茅青．吊子银花的品质研究[J]．贵阳中医学院学报，1997，19(3)：58～59．
[57] 娄红祥．金银花中水溶性化合物的分离与结构鉴定[J]．中草药，1996，371：203～214．
[58] 张英，李世敏，林志达，等．绿茶中要用绿原酸成分分析[J]．食品科学，2001，22(11)：84～86．
[59] 张军民，高振川，张琪，等．杜仲叶及提取物营养价值和药用成分研究[J]．氨基酸和生物资源，2002，24(1)：1～2．
[60] 宰学明，吴国荣，龚祝南，等．槲寄生抗氧化物质的研究测定[J]．中草药，2001，32(12)：1081～1083．
[61] 秦文娟等．苦丁茶化学成分的研究(Ⅱ)[J]．中草药，1988，19(11)：6～8．
[62] 房志仲，杨金荣，张璇，等．解热抗炎1号口服液中绿原酸定量方法的研究[J]．天津医科大学学报，2000，6(2)：153～155．
[63] 牛志多，王威，闫喜英，等．咳喘平口服液质量控制方法研究[J]．时珍国医国药，2001，12(3)：209～210．
[64] 武雪芬，景小琦，李国茹．金银花叶药用成分的提取及抑菌试验[J]．天然产物研究与开发，2001，13(3)：43～44．
[65] 郑建仙．功能性食品[M]．北京：中国轻工业出版社，1995．
[66] San R H C. Chan R I M. Inibitory effect of phenolic compounds on aflatox in B1 metabolism and induced mutagenesis [J]. Mutation Res, 1987, 177: 229～239.
[67] 林启寿．中草药成分化学[M]．北京：科学出版社，1977．146．
[68] 柯铭清．中草药有效成分理化与药理特性[M]．长沙：湖南科学技术出版社，1982．225．
[69] 柳云溪．欧洲植物药会议介绍[J]．国外医药，植物药分册，1997，12(6)：257～260．
[70] 罗思齐．中草药活性成分含量测定概述[J]．中草药，1989，20(9)：19～26．
[71] Andrade P B, Carvalho A R F. A previous study of phenolic profiles of quince, pear and apple purees by HPLC diode-array detection for the evaluation of quince puree genuineness. [J]. Agric. Food. Chem, 1998, 46: 968～972.
[72] Oleszek W O, Amiot MJ, Aubert, S. Identification of some phenolics in pear fruit[J]. J. Agric. Food Chem, 1994, 42: 1261～1265.
[73] 崔同，王燕桓，傅承光，王继英，赵海．HPLC—二极管阵列检测法分析梨果实酚类化合物[A]．第十一次全国色谱学术报告会文集[C]，大连：1997．101～103．
[74] Chang Q, Zhu M, Zuo Z. High-performance liquid chromatographic method for simultaneous determination of hawthorn active components in rat plasma[J]. Journal of Chromatographic B, 2001, 760: 227～235.
[75] Escarpa A, Gonzalez M C. Fast separation of(poly)phenolic compounds from apples and pears by high-performance liquid chromatographic with diode-array detection[J]. Journal of Chromatographic A, 1999, 830: 301～309.
[76] Schieber A, Keller P, Carle R. Determination of phenolic acids and flavonoids of apple and pear by high-performance liquid chromatography[J]. Journal of Chromatographic A, 2001, 910: 265～273. [71]
[77] 高原军，王素梅，孔谨．控制梨果发生酶褐变的研究[J]．食品科学，1997，6：15～17．
[78] Iwagawa T, Takahashi H, Munesada K. A phenol alloside from viburnum wrightii[J]. Phytochemistry, 1984, 23(2): 468～469.
[79] Takido M, Fukuhiro K, Yamanouchi S. Phlebotrichin, A phenolic compound from the fresh leaves of viburnum phlebotrichum[J]. Phytochemistry, 1983, 22(1): 223～225.
[80] 张国海，史国安等．野生砂梨果实的某些品质性状及抗氧化活性的比较分析[J]．河南农业大学学报，2000，9，34(3)：302～304．
[81] Tomas-Barberan FA, Ferreres F, Gil M I. Antioxidant phenolic metabolites from fruit and vegetables and changes during postharvest storage and processing[J]. Natural Products Chemistry, 2000, 23: 739～795.
[82] Blankenship S M, Richardson DG. Changs in phenolic acids and internal ethylene during long-term cold storage of pears[J]. J Am Soc Hort Sci, 1985, 110: 336～339.
[83] Escarpa A, Gonzaleze M C. Total extractable phenolic chromatographic index: an overview of the phenolic class content from different sources of foods[J]. Eur Food Res Technol, 2001, 212: 439～444.
[84] 朱健辉，崔同，锁然．薄层扫描测定果蔬中绿原酸含量[J]．中国畜产与食品，1999，6(5)：218～219．
[85] Tong Cui, Hiroshi Kayahara. Abilities of Superoxide Scavenging and Activities of Tyrosinase Inhibiting of Fruit Extracts[P]．商英凡．中国专利发明成果大典[M]．北京：国家知识产权局知识产权出版社，2002．797～801
[86] 鞠志国．酚类物质与梨果实品质的研究进展[J]．莱阳农学院学报，1988，5：59～65．
[87] 鞠志国．采期对莱阳茌梨酚类物质代谢和组织褐变的影响[J]．中国农业科学，1991，24(2)：63～68．
[88] 鞠志国，朱广廉，曹宗巽．莱阳茌梨果实褐变与多酚氧化酶几分类物质区域划分布的关系[J]．植物生理学报，1988，14(4)：356～361．
[89] 鞠志国，朱广廉，曹宗巽．气调贮藏条件下CO_2对莱阳茌梨果肉褐变的影响[J]．园艺学报，1988，15(4)：229～232．
[90] 鞠志国，刘成连，原永兵，等．莱阳茌梨酚类物质合成的调节及其对果实品质
的影响[J]．中国农业科学，1993，26(4)：44～48．
[91] 鞠志国．莱阳茌梨细胞中多份氧化酶的分布及其性质[J]．植物生理学通报，1989，3：33～35．
[92] 鞠志国，刘成连，原永兵，等．苹果果皮中酚类物质合成规律的研究[J]．莱阳农学院学报，1992，9(3)：222～225．
[93] 鞠志国，刘成连，原永兵，等．PP_(333)对梨果实生长和酚类物质合成的影响[J]．园艺学报，1993，20(3)：216～220．
[94] 张玉星，田志喜，郗荣庭，等．水杨酸对鸭梨幼果酚类物质代谢调节作用的研究[J]．河北农业大学学报，2002，25(3)：33～36．
[95] Hase T, Iwagawa T, Munesada K. A bitter monoterpene glucoside from viburnum phlebotrichum[J]. Phytochemistry, 1982, 21(6): 1435～1437.
[96] Wald B, Wray V, Galensa R. Malonated flavonol glycosides and 3, 5～dicaggeoylquinic acid from pears[J]. Phytochemistry, 28: 663～664.
[97] Petkou D, Diamantidis G, Vasilakakis M. Arbutin oxidation by pear(Pyrus communis L.) peroxidases[J]. Plant Science, 2002, 162: 115～119.
[98] Wrolstad R E, Lombard P B, Richardson D G. The pear[J]. In Quality and Preservation of Fruits, 1991: 67～95.
[99] (日)富山宏平，尹福祥译．植物的感染生理[M]．北京：农业出版社，1986．
[100] Escarpa A, Gonzalez M C. High-performance liquid chromatography with diode～array detection for the determination of phenolic compounds in peel and pulp from different apple varieties[J]. J. Chromatogr. A, 1998, 823: 331～337.
[101] Spanos G A, Wrolstad R E. Phenolics of apple, pear, and white grape juices and their changes with processing and storage-A review[J]. J. Agric. Food Chem, 1992, 40: 1478～1487.
[102] Mosel H D, Herrmann K. Changes in catechins and hydroxycinnamic acid derivatives during development of apples and pears [J]. J. Sci. Food Agric, 1974, 25: 251～256.
[103] Oszmianski J O, Lee C Y. Enzymatic oxidation reaction of catechin and chlorogenic acid in amodel system[J]. J. Agric. Food Chem, 1990, 38: 1202-1204.
[104] 宋立江．植物多酚研究与利用的意义及发展趋势[J]．化学进展，2000年，12(2)：161～170．
[105] 凌关庭．有“第七类营养素”之称的多酚类物质[J]．中国食品添加剂，2000(1)：28～37．
[106] Timberlake C F, Bridle P. The anthocyanins if apples and pears: the occurrence of acyl derivatives[J]. J Sci Food Agic, 1971, 22: 509～513.
[107] Francis F J. Anthocyanins in pears[J]. Hort Sci, 1970, 5: 42.
[108] Ranadive A S, Harrd N F. Changes in polyphenolics on ripening of selected pear
varieties[J]. J Sci Food Chem, 1998, 46: 968～972.
[109] 马良，崔同，张宇昊，等．梨果中绿原酸和熊果苷的HPLC测定方法[J]．吉林农业大学学报，2003，10(25)：115～117，120．
[110] 刈米达夫．植物化学[M]．杨本文译．北京：科学出版社，1985．
[111] T．W．古德温．E．I默塞尔．植物生物化学导论[M]．西北农业大学译．陕西杨陵：天则出版社，1988：88～89，233，531，560．