壳聚糖微球的制备及其对茶多酚EGCG的微胶囊载体作用
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摘要
近年来,壳聚糖微球对理化性质不稳定的多酚类化合物的微胶囊载体作用,引起了广泛重视。本文以壳聚糖微球为研究对象,对材料合成、样品形貌表征及其对EGCG的微胶囊载体作用等方面进行了研究。
以壳聚糖醋酸溶液为水相,液体石蜡为油相,Span80为乳化剂,表没食子儿茶素没食子酸酯为芯材,通过戊二醛交联,采用乳化交联法制备了壳聚糖微球及载EGCG壳聚糖微球。通过光学显微镜及扫描电子显微镜,考察了壳聚糖微球的表面形貌及粒径大小。当油水比为9：1时、戊二醛的量占乳液总体积为1%(v/v)、反应温度为60℃、反应时间为4h时,制备的壳聚糖微球形态圆润,表面光滑,尺寸均一。
进一步研究了EGCG在壳聚糖微球中的包封率、EGCG的释放及载EGCG微球的抗氧化性。壳聚糖微球对EGCG的包封率可达80%。在pH 7.4的PBS缓冲液中,不同制备条件下的EGCG壳聚糖微球的释放性能表现出显著的差异性。EGCG壳聚糖微球对DPPH自由基的淬灭能力实验显示,作为微胶囊载体的壳聚糖微球对EGCG抗氧化性能的保持具有重要作用。
Public and scientific interest has been greatly intensified in the microcapsule carrier effects of chitosan microspheres on polyphenols with unstable physicochemical properties in recent years. In this paper, we synthesized chitosan microspheres and further investigated their microcapsule carrier effects on (-)-epigallocatechin gallate (EGCG).
Chitosan microspheres encapsulated EGCG were prepared by the emulsion-crosslinking method, using chitosan solution of ethylic acid as water phase, liquid paraffin as oil phase, Span80 as emulsifier, EGCG as core material, and glutaraldehyde as crosslinking agent. The microspheres' particle size distribution and surface morphology were characterized by optical microscope and scanning electron microscopy (SEM). When Voil:Vwater is 9:1. the volume of glutaraldehyde is 1%(v/v), reaction time is 4 h, and reaction temperature is 60℃, chitosan microspheres are mellow, smooth and homogeneous.
Further studies on encapsulation efficiency, release and antioxidant activity of EGCG in chitosan microspheres were carried out. The encapsulation efficiency of EGCG in chitosan microspheres was achieved about 80%. EGCG release in pH 7.4 from chitosan microspheres obtained with different conditions exhibited different behaviors. The free radical scavenging activity test of EGCG in chitosan microspheres indicated that chitosan microspheres used as the microcapsule carrier had important effect on the antioxidant function of EGCG.
以壳聚糖醋酸溶液为水相,液体石蜡为油相,Span80为乳化剂,表没食子儿茶素没食子酸酯为芯材,通过戊二醛交联,采用乳化交联法制备了壳聚糖微球及载EGCG壳聚糖微球。通过光学显微镜及扫描电子显微镜,考察了壳聚糖微球的表面形貌及粒径大小。当油水比为9：1时、戊二醛的量占乳液总体积为1%(v/v)、反应温度为60℃、反应时间为4h时,制备的壳聚糖微球形态圆润,表面光滑,尺寸均一。
进一步研究了EGCG在壳聚糖微球中的包封率、EGCG的释放及载EGCG微球的抗氧化性。壳聚糖微球对EGCG的包封率可达80%。在pH 7.4的PBS缓冲液中,不同制备条件下的EGCG壳聚糖微球的释放性能表现出显著的差异性。EGCG壳聚糖微球对DPPH自由基的淬灭能力实验显示,作为微胶囊载体的壳聚糖微球对EGCG抗氧化性能的保持具有重要作用。
Public and scientific interest has been greatly intensified in the microcapsule carrier effects of chitosan microspheres on polyphenols with unstable physicochemical properties in recent years. In this paper, we synthesized chitosan microspheres and further investigated their microcapsule carrier effects on (-)-epigallocatechin gallate (EGCG).
Chitosan microspheres encapsulated EGCG were prepared by the emulsion-crosslinking method, using chitosan solution of ethylic acid as water phase, liquid paraffin as oil phase, Span80 as emulsifier, EGCG as core material, and glutaraldehyde as crosslinking agent. The microspheres' particle size distribution and surface morphology were characterized by optical microscope and scanning electron microscopy (SEM). When Voil:Vwater is 9:1. the volume of glutaraldehyde is 1%(v/v), reaction time is 4 h, and reaction temperature is 60℃, chitosan microspheres are mellow, smooth and homogeneous.
Further studies on encapsulation efficiency, release and antioxidant activity of EGCG in chitosan microspheres were carried out. The encapsulation efficiency of EGCG in chitosan microspheres was achieved about 80%. EGCG release in pH 7.4 from chitosan microspheres obtained with different conditions exhibited different behaviors. The free radical scavenging activity test of EGCG in chitosan microspheres indicated that chitosan microspheres used as the microcapsule carrier had important effect on the antioxidant function of EGCG.
引文
[1]Graham H N. Green tea composition, consumption and polyphenol, chcmistry[J]. Prev Mod, 1992,21:3341
[2]He B Y, Ou G N. Study on the compared antioxidation activity of tea polyphenols,BHT and TBHQ[J]. J Jimei Univ-Nat Sci.1999, 4(3):40-41
[3]Dai F. Chen W F.Zhou B. Antioxidant synergism of green tea polyphenols with α-toco-pherol and L-ascorbic acid in SDS micelles[J]. Biochinie. 2008. 90: 1499-1505
[4]Devika P T, Slanely Mainzen Prince P. Protective effect of (-)-epigallocatechin gallate (EGCG) on lipid peroxide metabolism in isoproterenol induced myocardial infarction in male Wister rats: A histopathological study [J]. Biomedicine& Pharmacotherapy.2008. 62(10):701-7()8
[5]Sriram N, Kalayarasan S. Sudhandiran G. Fpigallocatechin-3-gallate(EGCG) augments antioxidant activiitics and inhibits inflammation during bleomycin-induced experimental pulmonary fibrosis through Nrf2-Keapl signaling[J].Pulmonary Pharmacology & fherapeutics.2009.22(3): 221-236
[6]Henning Susanne M, Niu Y T,Liu Y. et al. Bioavailability and anlioxidant effect of epigallocatechin gallate administered in purified form versus as green tea extract in health individuals[J]. Journal of Nutritional Biochemistry.2005. 16: 610-616
[7]Saffari Y, Hossein Sadrzadeh S M. Green tea metabolite EGCG protects membranes against oxidative damage in vitro[J]. Fife Sciences.2004. 74(12): 1513-1518
[8]Yin S T, Fang M L, Su L, et al. Effects of Fpigallocatechin-3-gallate on lead-induced oxidative damage[J].Foxicology.2008. 249(1): 45-54
[9]EI-Mowafy A M, AI-Gayyar M , Salem H A, et al. Novel chemotherapeutic and renal protective effects for the green tea (EGCG): Role of oxidative stress and inflammatory-cylokine signaling[J]. Phvtomedicine. 2010. 17(14):1067-1075
[10]Higuchi A, Yonemitsu K. Koreeda A. et al. Inhibitory activity of epigallocatechin gallate (EGCG) in paraquatinduced microsomal lipid peroxidation-a mechanism of protective effects of EGCG against paraquat loxicity[J].Toxicology.2003. 183(1-3): 143-149
[11]Raza H, John A. Green tea polyphenol cpigallocatechin-3-gallale differentially modulates oxidative stress inPC12 cell compartments[J]. Toxicology and Applied Pharmacology. 2005. 207(3): 212-220
[12]Brown Marishka K, Evans Joseph L,Luo Y. Beneficial effects of natural antioxidants EGCG and α-lipoic acid on life span and age-dependent behavioral declines in Caenorhabditis elegans[J]. Pharmacology, Biochemistry and Behavior.2006,85: 620-628
[13]Larsen Christine A, Dashwood Roderick H. (-)-Epigallocatechin-3-gallate inhibits Met signaling, proliferation, and invasiveness in human colon cancer cells[J]. Archives of Biochemistry and Biophysics.2010.501(1):52-5
[14]Lambert J D, Sang S, Yang C S. N-Acetylcysteine enhances the lung cancer inhibitory effect of epigallocatechin-3-gallate and forms a new adduct[J]. Free Radical Biology & Medicine.2008,44:1069-1074
[15]Rieko Y, Kaori S. Kenichi Y, et al. Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549[J]. Toxicology in Vitro.2009,23(5):834-839
[16]Albrecht D S, Clubbs E A, Mario F, et al. Epigallocatechin-3-gallate(EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation [J].Chemico-Biological Interactions.2008.171(1):89-95
[17]Masahito S, Atsuko D, Yukihiko H, et al. EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells[J]. Biochemical and Biophysical Research Communication.2005,334:947-953
[18]Hwang J T, Ha J, Park I J, et al. Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway [J]. Cancer Letters.2007.247:115-121
[19]Yu H N, Shen S R. Yin J J. Effects of interactions of EGCG and Cd2+ on the growth of PC-3 cells and their mechanisms[J]. Food and Chemical Toxicology.2007,45(2):244-249
[20]Nishikawa T, Nakajima T, Moriguchi M. et al. A green tea polyphenol, epigalocatechin-3-gallate, induces apoptosis of human hepatocellular carcinoma, possibly through inhibit-tion of Bcl-2 family proteins[J], Journal of Hepatology.2006,44(6):1074-1082
[21]Msatoshi Y, Mitsuyo N. Yoshi F N. et al. Antiproliferative effects of the major tea polyp-henol, (-)-epigallocatechin gallate and retinoic acid in cervical adenocarcinoma [J]. Gyne-cologiconcology.2008,108(2):326-331
[22]Mitsuyo N, Masatoshi Y, Sumiko W, et al. Inhibitory effect of the tea polyphenol, (-)-epi-gallocatechin gallate, on growth of cervical adenocarcinoma cell lines[J]. Cancer Letters. 2006,234(2):135-142
[23]Peng H Q, Cai W M, Xiang X, et al. The anti influenza virus A3 action in vitro of tea polyphenols EJ]. J Tea Sci.2003,23(1):79-81
[24]Koushi Y, Mitsuo H, Hajime I, et al. Inhibitory effects of (-)-Epigallocatechin-3-gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1)[J]. Antiviral Rcsear-ch.2002,53(1):19-34
[25]Nance C L, Siwak K B, Shearer W T. Precinical development of the given tea eateehin e-pigallocatechin gallate, as an HIV-1 therapy[J]. Journal of Allergy and Clinical Immunol--ogy. 2009, 123(2): 459-465
[26]彭章平,程普新,葛瑶等,茶多酚对血液有形成分影响的临床研究.中国疗养医学.2001,10(5)：43-44
[27]Raederstorff D G, Schlachter M F, Volker E, et al. Lffecl of EGCG on lipid absorption a-nd plasma lipid levels in rats[J]. Journal of Nutritional Biochemistry.2003. 14:326-332
[28]胡秀芳,杨贤强,陈留记.茶多酚对皮肤的保护与治疗作用.福建茶叶2000(2)：44-45
[29]J.Zimeri, C.H.Tong.Degradation kinetics of (-)-epigallocatechin gallate as a function of pH and dissolved oxygen in a liquid model system. Journal of Food Science.64 (5) (1999). pp. 753-758
[30]Y.L. Su. L.K. Leung. Y. Huang, et al.Stability of lea thealavins and cateehins. Food Chemistry.83 (2) (2003). pp. 189-195
[31]Y.L. Su.L..K. Leung, Y. Huang, et al. Stability of tea theaflavins and caleehins. Food Chemistry. 83 (2) (2003). pp. 189-195
[32]Y. Mizooku. M. Yoshikawa. T. Tsuneyoshi. et al.Analysis of oxidized epigallocatechin gallate by liquid chromatography/mass spectromelry Rapid Communications in Mass Spectrometry. 17 (16) (2003). pp. 1915-1918
[33]J. Hong. H. Lu. X. Meng. et al.Stability, cellular uptake, biotransformalion. and efflux of tea polyphenol (-)-epigallocatechin-3-gallate in HT-29 Human colon adenocarcinoma cells Cancer Research. 62 (24) (2002), pp. 7241-7246
[34]D. Paolino. D. Cosco. F. Cilurzo, et al. Innovative drug delivery systems for the administration of natural compounds. Curr. Bioact. Compd.3 (2007). pp. 262-277
[35]A.E. Giakoustidis.D.E.Giakoustidis. S. Hindis, et al. Attenuation of intestinal isehemia/reperfusion induced liver and lung injury by intraperitoneal administration of ( )-epigallocateehin-3-gallale. Free Radic. Res.40 (2006). pp. 103-110
[36]Chovv H-H.S., Cai Y., Alberts D. S., et al. Phase Ⅰ pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer epidemiol. Biomarkers Prev.2001, 10. 53-58
[37]Chow H-H.S., Cai Y. Alberts D. S.. et al. Pharmacokinetics and safely of green tea polyphenols after multiple-dose administration of epigallocaleehin gallale and polyphenon E in healthy individuals. Clin. Cancer Res. 2003. 9. 3312-3319
[38]赵锦花,孙先多.新型微球载体材PACA-co- PLG的性能应用化学.2003.20(5)：502-504
[39]杨远,贾文祥,杨春等.PELA作为基因载体的研究.西部医学.2004,16(1)：6-9
[40]余东升,黄洪章,李苏等.转基因载体聚乙二醇-聚谷氨酸纳米微球的制备.家用癌症杂志.2004.19(1)：5-8
[41]郑爱萍,刘海宏,李宏斌等.5-氟脲嘧啶壳聚糖微球的制备及体外释放特性.中国药科大学学报.2004,35(4)：318-323
[42]S.Santhosh, T.K.Sini, R.Anandan etc.Effect Of chitosan supplemenration on antitubercul-ar drugs-induced hepatotoxicity in rats[J].Toxicology.2006,219:53-59
[43]Hermann Ehrlich,Barbara Krajewska,Thomas Hanke etc.Chitosan membrane as a templa-te for hydroxyapatite crystal growth in a model dual membrane diffusion system[J]. Mem-brane Science.2006,273:124-128
[44]倪有娣,章志量,茹金泉等.凝胶化改性壳聚糖抑菌及抗腹腔感染作用研究[J].浙江预防医学.2005,17(7)：6-7
[45]徐风彩等.壳聚搪固定化木瓜蛋白酶的研究。生物化学杂志.1992,8(5)：608
[46]刘真,常雁红,刘仁霖等.磁性壳聚糖微球的制备及对乳酸氧化酶的固定化研究[J].现代化工.2007.27(2)：310-313
[47]刘海英,于森,杨桂琴.壳聚糖对肉鸡生产性能、脂肪代谢的影响[J].饲料博览.2003.5：1-4
[48]董学畅.杨燕兵.甲壳素和聚糖应用研究新动向[J].云南民族学院学报.2002.11(1)：566-584
[49]严瑞洝.水溶性高分子[M].北京：化学工业出版社.1999.540-565.
[50]陈新梅.壳聚糖微球研究进展[A],中国药师.2009.12(6)：734—737
[51]王珊,黄怡,叶光辉等.“类乒乓球”状壳聚糖微球的制备及其缓释性能的研究.食品与生物技术学报.2009.28(2)：214-218
[52]候东耀,葛喜珍,刘军锋等.小檗碱壳聚糖微球制备及其抗真菌活性测定.过程控制学报.2008,8(5)：963-965
[53]米晨华,沈鹤柏,徐端云等.磁性壳聚糖微球对牛血清白蛋白的吸附性能.物理化学学报.2007,23(10)：1583-1588
[54]FILIPOVIC-GRCIC J, PERISSUTTI B, MONEGHINI M.et al.Spray-dried carbamazepi-ne loaded chitosan and HPMC microspheres:preparation and characterisation[J].J Pharm Pharmacol.2003,55(7):921-931
[55]E.Yenilmez. E.Basaran. Y.Yazan. Release characteristics of vitamin E incorporated chito-san microspheres and in vitro-in vivo evaluation for topical application. Carbohydrate Po-lymers.84 (2011)807-811
[56]GIUNCHEDI P, JULIANO C, GAVINI E, et al. Formulation and in vivo evaluation of chlorhexidine buccal tablets prepared using drug-loaded chitosan microspheres[J]. Eur J Pharm Biopharm.2002 53(2):233-239
[57]MI FL,TAN YC,LIANG HF,et al.In vivo biocompatibility and Degradability of novel inj-ectable chitosan base- dimplant[J].Biomaterials.2002.23(1):181-191
[58]蔡金君,程巧鸳,赵宁等.喷雾干燥法制备川芎嗪壳聚糖微球的研究.中草药.2008,39(5)：679-681
[59]SINHA VR, SINGLA AK, WADhAWAN S, et al .Chitosan microspheres as a potential carrier for drugs[J]. Int J Pharm.2004,274(11/2): 1-33
[60]Y0SHINO T, MACHIDAY, ONISHI H, et al. Preparation and charaetor of chitosan microspheres containing doxilluridine [J]. Drug Dev Ind Pharm.2003 29(4): 417-427
[61]VANDKNBl'RG GW, DROLET C, SCOTT SL, et al . Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation[J].J Control Release.2001.77(3):297-307
[61]赵大庆,高明.pH值调节法制备壳聚糖空白微球[J].中国新药杂志.200615(17)：1461-1463
[63]余艺华,薛博,孙彦,何炳林.壳聚糖亲和磁性毫微粒的制备及其对蛋白质的吸附性能研究.高分子学报.2000(3)：340-344
[64]WANG LY, GU YH, SU ZG. Preparation and improvement of release behavior of chi-tosan microspheres containing insulin[J].Int J Pharm.2006,311(1/2): 187-195
[65[CHANDY T, DAS GS, RAO GH. 5-Fluorouracil-loaded chitosan Coated polylactic acid microspheres as biodegradable drug carriers for cerebral tumours[J].J. Microencapsul. 2000, 17(5): 625-638
[66]JACANATHIAN KS, RAO YU, SINGH P, et al. Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D,L-lactic-co-glycolic acid) versus chitosan microspheres[J].. Int J Pharm.2005, 294(1/2): 23-32
[67]董海丽,任晓燕.磁性壳聚糖微球对大豆乳清废水中蛋白质的吸附作用.食品科学.2007,28(7)1：205-207
[68]纵伟,夏文水.磁性高分子微球及其在食品工业中应用.山西企品工业.2003(4)：31-43
[69]寇灵梅,李冰,郭祀远,李琳,黄采坚.磁性固定化纤维素酶的制备及期响性明.食品科学.2006,(12):335-339
[70]洪爱真,魏燕芳,陈盛,磁性壳聚糖微球对酸性偶氮染料废水的脱色研究.福建轻纺.2003,165(2):2-5
[71]韩德艳,谢长生.贴壳聚糖磁性微球对甲基橙废水的脱色研究.湖北师范院学报.2006,26(1):19-21
[72]S.L. Kosaraju.L.. D'ath. A. Lawrence. Carbohydr. Polym. 64 (2006) 163
[73]刘利萍,李苹,吴泽志,等.5-FU壳聚糖/丝素复合磁微球的制备及体外性质研究[J].中国药学杂志.2003,38(10):774-777
[74]Yu-Ying Zhang, Yan Yang. Guo-I.in Zou, et al. Physicochemical characterization and a-ntioxidant Activity of quercetin-loaded chitosan nanoparticles. Journal of Applied Polym-er Science.2007. 892-897
[75]聂少平,谢明勇,罗珍.用清除有机自由基DPPH法评价茶叶多栅的抗氧化活性.食品科学.2006.Vol.27.No.03,34-36
[76]Admire Dube, Ken Ng, Joseph A. Nicolazzo, et al. Effective use of reducing agents and nanoparticle encapsulation in stabilizing catechins in alkaline solution. Food Chem.122 (2010)662-667
[77]Ya Lun Su. Lai Kwok Leung, Yu Huang, et al. Stability of tea theaflavins and catechins. Food Chem.83 (2003) 189-195
[78]K.C. Gupta, Fawzi Habeeb Jabrail. Glutaraldehyde cross-linked chitosan microspheres for controlled release of centchroman. Carbohydrate Research.2007,342(15):2244-2252
[79]K.C. Gupta. Fawzi Habeeb Jabrail.Effects of degree of deacetylation and cross-linking on physical characteristics, swelling and release behavior of chitosan microspheres. Carbohydrate Polymers.2006.66(1):43-54
[80]S.R.Jameela. A. Jayakrishnan. Glutaraldehyde cross-linked chitosan microspheres as a long acting biodegradable drug vehicle:studies on the in vitro release of mitoxantrone and in vivo degradation of microspheres in rat muscle.Biomaterials.1995.16(10):769-775
[81]S Jose,J F Fangueiro, J Smitha, et al. Cross-linked chitosan microspheres for oral delivery of insulin:Taguchi design and in vivo testing. Colloids and Surfaces B: Biointerfaces.2012(92):175-179
[82]Zhu N., Wang M., Wei G., el al. Identification of reaction products of (-)-epigallocatechin, (-)-epigallocatechin gallate andpyrogallol with 2,2-diphenyl-1-picrylhydrazyl radical.F-ood Chem.2001,73:345-349
[83]Yu-Ying Zhang. Yan Yang, Guo-Lin Zou. el al. Physicochemical characterization and a-ntioxidant Activity of quercetin-loaded chitosan nano- particles. Journal of Applied Poly-mer Science.2007.892-897
[84]Rangika Weerakody, Peter Fagan, Shantha L. Kosaraju. Chitosan microspheres for encap-sulation of α-lipoic acid. International Journal of Pharmaceutics.2008.357(1-2):213-218
[85]Shibao Bao. Shiying Xu. Zhang Wang. Antioxidant activity and properties of gelatin fil-ms incorporated with tea polyphenol-loaded chitosan nanoparticles. J Sci Food Agric. 2009; 89:2692-2700
[2]He B Y, Ou G N. Study on the compared antioxidation activity of tea polyphenols,BHT and TBHQ[J]. J Jimei Univ-Nat Sci.1999, 4(3):40-41
[3]Dai F. Chen W F.Zhou B. Antioxidant synergism of green tea polyphenols with α-toco-pherol and L-ascorbic acid in SDS micelles[J]. Biochinie. 2008. 90: 1499-1505
[4]Devika P T, Slanely Mainzen Prince P. Protective effect of (-)-epigallocatechin gallate (EGCG) on lipid peroxide metabolism in isoproterenol induced myocardial infarction in male Wister rats: A histopathological study [J]. Biomedicine& Pharmacotherapy.2008. 62(10):701-7()8
[5]Sriram N, Kalayarasan S. Sudhandiran G. Fpigallocatechin-3-gallate(EGCG) augments antioxidant activiitics and inhibits inflammation during bleomycin-induced experimental pulmonary fibrosis through Nrf2-Keapl signaling[J].Pulmonary Pharmacology & fherapeutics.2009.22(3): 221-236
[6]Henning Susanne M, Niu Y T,Liu Y. et al. Bioavailability and anlioxidant effect of epigallocatechin gallate administered in purified form versus as green tea extract in health individuals[J]. Journal of Nutritional Biochemistry.2005. 16: 610-616
[7]Saffari Y, Hossein Sadrzadeh S M. Green tea metabolite EGCG protects membranes against oxidative damage in vitro[J]. Fife Sciences.2004. 74(12): 1513-1518
[8]Yin S T, Fang M L, Su L, et al. Effects of Fpigallocatechin-3-gallate on lead-induced oxidative damage[J].Foxicology.2008. 249(1): 45-54
[9]EI-Mowafy A M, AI-Gayyar M , Salem H A, et al. Novel chemotherapeutic and renal protective effects for the green tea (EGCG): Role of oxidative stress and inflammatory-cylokine signaling[J]. Phvtomedicine. 2010. 17(14):1067-1075
[10]Higuchi A, Yonemitsu K. Koreeda A. et al. Inhibitory activity of epigallocatechin gallate (EGCG) in paraquatinduced microsomal lipid peroxidation-a mechanism of protective effects of EGCG against paraquat loxicity[J].Toxicology.2003. 183(1-3): 143-149
[11]Raza H, John A. Green tea polyphenol cpigallocatechin-3-gallale differentially modulates oxidative stress inPC12 cell compartments[J]. Toxicology and Applied Pharmacology. 2005. 207(3): 212-220
[12]Brown Marishka K, Evans Joseph L,Luo Y. Beneficial effects of natural antioxidants EGCG and α-lipoic acid on life span and age-dependent behavioral declines in Caenorhabditis elegans[J]. Pharmacology, Biochemistry and Behavior.2006,85: 620-628
[13]Larsen Christine A, Dashwood Roderick H. (-)-Epigallocatechin-3-gallate inhibits Met signaling, proliferation, and invasiveness in human colon cancer cells[J]. Archives of Biochemistry and Biophysics.2010.501(1):52-5
[14]Lambert J D, Sang S, Yang C S. N-Acetylcysteine enhances the lung cancer inhibitory effect of epigallocatechin-3-gallate and forms a new adduct[J]. Free Radical Biology & Medicine.2008,44:1069-1074
[15]Rieko Y, Kaori S. Kenichi Y, et al. Identification of epigallocatechin-3-gallate in green tea polyphenols as a potent inducer of p53-dependent apoptosis in the human lung cancer cell line A549[J]. Toxicology in Vitro.2009,23(5):834-839
[16]Albrecht D S, Clubbs E A, Mario F, et al. Epigallocatechin-3-gallate(EGCG) inhibits PC-3 prostate cancer cell proliferation via MEK-independent ERK1/2 activation [J].Chemico-Biological Interactions.2008.171(1):89-95
[17]Masahito S, Atsuko D, Yukihiko H, et al. EGCG inhibits activation of the insulin-like growth factor-1 receptor in human colon cancer cells[J]. Biochemical and Biophysical Research Communication.2005,334:947-953
[18]Hwang J T, Ha J, Park I J, et al. Apoptotic effect of EGCG in HT-29 colon cancer cells via AMPK signal pathway [J]. Cancer Letters.2007.247:115-121
[19]Yu H N, Shen S R. Yin J J. Effects of interactions of EGCG and Cd2+ on the growth of PC-3 cells and their mechanisms[J]. Food and Chemical Toxicology.2007,45(2):244-249
[20]Nishikawa T, Nakajima T, Moriguchi M. et al. A green tea polyphenol, epigalocatechin-3-gallate, induces apoptosis of human hepatocellular carcinoma, possibly through inhibit-tion of Bcl-2 family proteins[J], Journal of Hepatology.2006,44(6):1074-1082
[21]Msatoshi Y, Mitsuyo N. Yoshi F N. et al. Antiproliferative effects of the major tea polyp-henol, (-)-epigallocatechin gallate and retinoic acid in cervical adenocarcinoma [J]. Gyne-cologiconcology.2008,108(2):326-331
[22]Mitsuyo N, Masatoshi Y, Sumiko W, et al. Inhibitory effect of the tea polyphenol, (-)-epi-gallocatechin gallate, on growth of cervical adenocarcinoma cell lines[J]. Cancer Letters. 2006,234(2):135-142
[23]Peng H Q, Cai W M, Xiang X, et al. The anti influenza virus A3 action in vitro of tea polyphenols EJ]. J Tea Sci.2003,23(1):79-81
[24]Koushi Y, Mitsuo H, Hajime I, et al. Inhibitory effects of (-)-Epigallocatechin-3-gallate on the life cycle of human immunodeficiency virus type 1 (HIV-1)[J]. Antiviral Rcsear-ch.2002,53(1):19-34
[25]Nance C L, Siwak K B, Shearer W T. Precinical development of the given tea eateehin e-pigallocatechin gallate, as an HIV-1 therapy[J]. Journal of Allergy and Clinical Immunol--ogy. 2009, 123(2): 459-465
[26]彭章平,程普新,葛瑶等,茶多酚对血液有形成分影响的临床研究.中国疗养医学.2001,10(5)：43-44
[27]Raederstorff D G, Schlachter M F, Volker E, et al. Lffecl of EGCG on lipid absorption a-nd plasma lipid levels in rats[J]. Journal of Nutritional Biochemistry.2003. 14:326-332
[28]胡秀芳,杨贤强,陈留记.茶多酚对皮肤的保护与治疗作用.福建茶叶2000(2)：44-45
[29]J.Zimeri, C.H.Tong.Degradation kinetics of (-)-epigallocatechin gallate as a function of pH and dissolved oxygen in a liquid model system. Journal of Food Science.64 (5) (1999). pp. 753-758
[30]Y.L. Su. L.K. Leung. Y. Huang, et al.Stability of lea thealavins and cateehins. Food Chemistry.83 (2) (2003). pp. 189-195
[31]Y.L. Su.L..K. Leung, Y. Huang, et al. Stability of tea theaflavins and caleehins. Food Chemistry. 83 (2) (2003). pp. 189-195
[32]Y. Mizooku. M. Yoshikawa. T. Tsuneyoshi. et al.Analysis of oxidized epigallocatechin gallate by liquid chromatography/mass spectromelry Rapid Communications in Mass Spectrometry. 17 (16) (2003). pp. 1915-1918
[33]J. Hong. H. Lu. X. Meng. et al.Stability, cellular uptake, biotransformalion. and efflux of tea polyphenol (-)-epigallocatechin-3-gallate in HT-29 Human colon adenocarcinoma cells Cancer Research. 62 (24) (2002), pp. 7241-7246
[34]D. Paolino. D. Cosco. F. Cilurzo, et al. Innovative drug delivery systems for the administration of natural compounds. Curr. Bioact. Compd.3 (2007). pp. 262-277
[35]A.E. Giakoustidis.D.E.Giakoustidis. S. Hindis, et al. Attenuation of intestinal isehemia/reperfusion induced liver and lung injury by intraperitoneal administration of ( )-epigallocateehin-3-gallale. Free Radic. Res.40 (2006). pp. 103-110
[36]Chovv H-H.S., Cai Y., Alberts D. S., et al. Phase Ⅰ pharmacokinetic study of tea polyphenols following single-dose administration of epigallocatechin gallate and polyphenon E. Cancer epidemiol. Biomarkers Prev.2001, 10. 53-58
[37]Chow H-H.S., Cai Y. Alberts D. S.. et al. Pharmacokinetics and safely of green tea polyphenols after multiple-dose administration of epigallocaleehin gallale and polyphenon E in healthy individuals. Clin. Cancer Res. 2003. 9. 3312-3319
[38]赵锦花,孙先多.新型微球载体材PACA-co- PLG的性能应用化学.2003.20(5)：502-504
[39]杨远,贾文祥,杨春等.PELA作为基因载体的研究.西部医学.2004,16(1)：6-9
[40]余东升,黄洪章,李苏等.转基因载体聚乙二醇-聚谷氨酸纳米微球的制备.家用癌症杂志.2004.19(1)：5-8
[41]郑爱萍,刘海宏,李宏斌等.5-氟脲嘧啶壳聚糖微球的制备及体外释放特性.中国药科大学学报.2004,35(4)：318-323
[42]S.Santhosh, T.K.Sini, R.Anandan etc.Effect Of chitosan supplemenration on antitubercul-ar drugs-induced hepatotoxicity in rats[J].Toxicology.2006,219:53-59
[43]Hermann Ehrlich,Barbara Krajewska,Thomas Hanke etc.Chitosan membrane as a templa-te for hydroxyapatite crystal growth in a model dual membrane diffusion system[J]. Mem-brane Science.2006,273:124-128
[44]倪有娣,章志量,茹金泉等.凝胶化改性壳聚糖抑菌及抗腹腔感染作用研究[J].浙江预防医学.2005,17(7)：6-7
[45]徐风彩等.壳聚搪固定化木瓜蛋白酶的研究。生物化学杂志.1992,8(5)：608
[46]刘真,常雁红,刘仁霖等.磁性壳聚糖微球的制备及对乳酸氧化酶的固定化研究[J].现代化工.2007.27(2)：310-313
[47]刘海英,于森,杨桂琴.壳聚糖对肉鸡生产性能、脂肪代谢的影响[J].饲料博览.2003.5：1-4
[48]董学畅.杨燕兵.甲壳素和聚糖应用研究新动向[J].云南民族学院学报.2002.11(1)：566-584
[49]严瑞洝.水溶性高分子[M].北京：化学工业出版社.1999.540-565.
[50]陈新梅.壳聚糖微球研究进展[A],中国药师.2009.12(6)：734—737
[51]王珊,黄怡,叶光辉等.“类乒乓球”状壳聚糖微球的制备及其缓释性能的研究.食品与生物技术学报.2009.28(2)：214-218
[52]候东耀,葛喜珍,刘军锋等.小檗碱壳聚糖微球制备及其抗真菌活性测定.过程控制学报.2008,8(5)：963-965
[53]米晨华,沈鹤柏,徐端云等.磁性壳聚糖微球对牛血清白蛋白的吸附性能.物理化学学报.2007,23(10)：1583-1588
[54]FILIPOVIC-GRCIC J, PERISSUTTI B, MONEGHINI M.et al.Spray-dried carbamazepi-ne loaded chitosan and HPMC microspheres:preparation and characterisation[J].J Pharm Pharmacol.2003,55(7):921-931
[55]E.Yenilmez. E.Basaran. Y.Yazan. Release characteristics of vitamin E incorporated chito-san microspheres and in vitro-in vivo evaluation for topical application. Carbohydrate Po-lymers.84 (2011)807-811
[56]GIUNCHEDI P, JULIANO C, GAVINI E, et al. Formulation and in vivo evaluation of chlorhexidine buccal tablets prepared using drug-loaded chitosan microspheres[J]. Eur J Pharm Biopharm.2002 53(2):233-239
[57]MI FL,TAN YC,LIANG HF,et al.In vivo biocompatibility and Degradability of novel inj-ectable chitosan base- dimplant[J].Biomaterials.2002.23(1):181-191
[58]蔡金君,程巧鸳,赵宁等.喷雾干燥法制备川芎嗪壳聚糖微球的研究.中草药.2008,39(5)：679-681
[59]SINHA VR, SINGLA AK, WADhAWAN S, et al .Chitosan microspheres as a potential carrier for drugs[J]. Int J Pharm.2004,274(11/2): 1-33
[60]Y0SHINO T, MACHIDAY, ONISHI H, et al. Preparation and charaetor of chitosan microspheres containing doxilluridine [J]. Drug Dev Ind Pharm.2003 29(4): 417-427
[61]VANDKNBl'RG GW, DROLET C, SCOTT SL, et al . Factors affecting protein release from alginate-chitosan coacervate microcapsules during production and gastric/intestinal simulation[J].J Control Release.2001.77(3):297-307
[61]赵大庆,高明.pH值调节法制备壳聚糖空白微球[J].中国新药杂志.200615(17)：1461-1463
[63]余艺华,薛博,孙彦,何炳林.壳聚糖亲和磁性毫微粒的制备及其对蛋白质的吸附性能研究.高分子学报.2000(3)：340-344
[64]WANG LY, GU YH, SU ZG. Preparation and improvement of release behavior of chi-tosan microspheres containing insulin[J].Int J Pharm.2006,311(1/2): 187-195
[65[CHANDY T, DAS GS, RAO GH. 5-Fluorouracil-loaded chitosan Coated polylactic acid microspheres as biodegradable drug carriers for cerebral tumours[J].J. Microencapsul. 2000, 17(5): 625-638
[66]JACANATHIAN KS, RAO YU, SINGH P, et al. Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D,L-lactic-co-glycolic acid) versus chitosan microspheres[J].. Int J Pharm.2005, 294(1/2): 23-32
[67]董海丽,任晓燕.磁性壳聚糖微球对大豆乳清废水中蛋白质的吸附作用.食品科学.2007,28(7)1：205-207
[68]纵伟,夏文水.磁性高分子微球及其在食品工业中应用.山西企品工业.2003(4)：31-43
[69]寇灵梅,李冰,郭祀远,李琳,黄采坚.磁性固定化纤维素酶的制备及期响性明.食品科学.2006,(12):335-339
[70]洪爱真,魏燕芳,陈盛,磁性壳聚糖微球对酸性偶氮染料废水的脱色研究.福建轻纺.2003,165(2):2-5
[71]韩德艳,谢长生.贴壳聚糖磁性微球对甲基橙废水的脱色研究.湖北师范院学报.2006,26(1):19-21
[72]S.L. Kosaraju.L.. D'ath. A. Lawrence. Carbohydr. Polym. 64 (2006) 163
[73]刘利萍,李苹,吴泽志,等.5-FU壳聚糖/丝素复合磁微球的制备及体外性质研究[J].中国药学杂志.2003,38(10):774-777
[74]Yu-Ying Zhang, Yan Yang. Guo-I.in Zou, et al. Physicochemical characterization and a-ntioxidant Activity of quercetin-loaded chitosan nanoparticles. Journal of Applied Polym-er Science.2007. 892-897
[75]聂少平,谢明勇,罗珍.用清除有机自由基DPPH法评价茶叶多栅的抗氧化活性.食品科学.2006.Vol.27.No.03,34-36
[76]Admire Dube, Ken Ng, Joseph A. Nicolazzo, et al. Effective use of reducing agents and nanoparticle encapsulation in stabilizing catechins in alkaline solution. Food Chem.122 (2010)662-667
[77]Ya Lun Su. Lai Kwok Leung, Yu Huang, et al. Stability of tea theaflavins and catechins. Food Chem.83 (2003) 189-195
[78]K.C. Gupta, Fawzi Habeeb Jabrail. Glutaraldehyde cross-linked chitosan microspheres for controlled release of centchroman. Carbohydrate Research.2007,342(15):2244-2252
[79]K.C. Gupta. Fawzi Habeeb Jabrail.Effects of degree of deacetylation and cross-linking on physical characteristics, swelling and release behavior of chitosan microspheres. Carbohydrate Polymers.2006.66(1):43-54
[80]S.R.Jameela. A. Jayakrishnan. Glutaraldehyde cross-linked chitosan microspheres as a long acting biodegradable drug vehicle:studies on the in vitro release of mitoxantrone and in vivo degradation of microspheres in rat muscle.Biomaterials.1995.16(10):769-775
[81]S Jose,J F Fangueiro, J Smitha, et al. Cross-linked chitosan microspheres for oral delivery of insulin:Taguchi design and in vivo testing. Colloids and Surfaces B: Biointerfaces.2012(92):175-179
[82]Zhu N., Wang M., Wei G., el al. Identification of reaction products of (-)-epigallocatechin, (-)-epigallocatechin gallate andpyrogallol with 2,2-diphenyl-1-picrylhydrazyl radical.F-ood Chem.2001,73:345-349
[83]Yu-Ying Zhang. Yan Yang, Guo-Lin Zou. el al. Physicochemical characterization and a-ntioxidant Activity of quercetin-loaded chitosan nano- particles. Journal of Applied Poly-mer Science.2007.892-897
[84]Rangika Weerakody, Peter Fagan, Shantha L. Kosaraju. Chitosan microspheres for encap-sulation of α-lipoic acid. International Journal of Pharmaceutics.2008.357(1-2):213-218
[85]Shibao Bao. Shiying Xu. Zhang Wang. Antioxidant activity and properties of gelatin fil-ms incorporated with tea polyphenol-loaded chitosan nanoparticles. J Sci Food Agric. 2009; 89:2692-2700