摘要
咖啡作为世界消费量最大的饮品,其营养价值一直备受瞩目。早在19世纪国外就对咖啡豆中的功能成分展开了研究,随着科技的进步,研究方法和技术的不断革新,咖啡豆中的营养素成分及非营养素成分已逐渐明了,相应的保健功能及药理学作用也得到了进一步证实。尤其是,咖啡豆中所含的大量非营养素成分——多酚类化合物己成为目前国际天然药物学领域的关注重点,这些物质在先导物发现和创新药物研制中扮演了越来越重要的角色。云南、海南、广西及广东是我国咖啡豆种植的主产区,且资源十分丰富。因此,建立标准化和规范化的咖啡多酚提制体系,有效开发和合理利用我国咖啡豆资源,改变仅靠原料出口的贸易模式,具有非常重要的经济价值和现实意义。
天然产物的开发利用关键在于先进的提取分离技术与手段以及准确、高效、快速的分析方法。本研究建立了咖啡豆中多酚类物质的高效液相色谱(HPLC)分析方法；确定了咖啡多酚的最优提取工艺参数,进一步研究了聚酰胺树脂初步分离纯化咖啡多酚的最优条件,并利用HPLC技术对咖啡多酚的具体组成进行了分析；评价了咖啡豆多酚体外抗氧化活性,结果如下：
建立了同时测定咖啡豆中6种多酚类化合物(咖啡酸、3-咖啡酰奎尼酸、4-咖啡酰奎尼酸、5-咖啡酰奎尼酸、3,5-二咖啡酰奎尼酸、4,5-二咖啡酰奎尼酸)的反相高效液相色谱测定方法。采用Kromasil C18柱(4.6 mm×200 mm,5μm),以乙腈和0.1%甲酸水溶液为流动相进行梯度洗脱,二极管阵列检测器检测,45 min内可对6种目标物进行同时检测,且各化合物都能达到基线分离。经测定,该方法的平均回收率为90.76%-104.73%,相对标准偏差为0.7%-3.9%。该法简便、快速、灵敏度高,适用于咖啡豆中6种酚酸类化合物的同时分析。此外,试验还利用HPLC法比较了云南四个产区咖啡豆原料中多酚含量,结果显示保山产区咖啡豆的总多酚含量最高。
通过单因素试验研究了提取溶剂、提取方法(温度)、提取时间及次数对咖啡多酚提取效果的影响,得到咖啡多酚的最佳提取工艺：用70%乙醇水溶液,按液料比1：7,80℃下回流提取两次,每次提取2h。
从7种大孔树脂中筛选出最佳树脂—聚酰胺,研究其初步纯化咖啡多酚的适宜条件。结果表明：聚酰胺树脂纯化咖啡多酚的最佳吸附和解吸条件为上样液浓度4.44 mg/ml左右,重复过柱1次,最佳洗脱剂为80%乙醇,洗脱剂用量约为3倍的柱体积,上样及洗脱流速均为2 BV/h。多酚含量由14.43%提高到53.32%,得率达到78.21%。分别采用紫外分光光度计和HPLC对咖啡多酚初步纯化物进行分析,前者测得总多酚含量为53.32%,后者检测6种多酚类物质的总含量为47.55%,表明咖啡多酚主要为绿原酸及其衍生物。
通过5种不同的体系(清除过氧化氢、羟基自由基、超氧阴离子自由基、DPPH自由基能力及还原能力的测定)研究了咖啡多酚的抗氧化性能。结果表明,咖啡多酚具有一定的自由基清除能力和还原Fe3+的能力,且自由基清除率和还原Fe3+的能力与样品浓度之间存在显著相关性。
As the largest consumption of drinks in the world, coffee's nutritional value has much more attracted people'eyes. Early in the 19th century, the abroad began to study the function of components of coffee beans. With the advances in technology, innovation in research methods and technology, the nutrient composition of coffee beans and non-nutrient components has been gradually clear, corresponding health function and pharmacological effect has been further confirmed. In particular, a large number of non-nutrient components—polyphenols in the coffee beans has become the focus in international natural medicine field, these substances play an increasingly important role in the leading compound discovery and the innovation of drugs development. Yunnan, Hainan, Guangxi and Guangdong is China's main coffee growing areas, and very rich in resources. Therefore, the establishment of the standard and regular distilling system of coffee polyphenols, in the effective development and rational utilization of the coffee resources, change the export trade mode of raw materials alone, has the very important economic value and practical significance.
The key of the exploitation and use of natural products is the advanced extraction and separation technologies, and the accurate, efficient and fast method of analysis. This study established the high performance liquid chromatography (HPLC) analytical methods of the polyphenols in the coffee beans, identified the optimal extraction parameters of the polyphenols from the coffee, and further studyed the optimal conditions that the polyamide resin separated and purified the polyphenol of the coffee, analyzed the specific composition of polyphenols of the coffee by using the HPLC techniques, evaluated the in vitro antioxidant activity of polyphenols in coffee beans, the results were as follows:
A method of reversed-phase high performance liquid chromatography coupled with diode array detector (HPLC-DAD) was established for the simultaneous determination of 6 phenolic acids (Caffeic acid,3-O-Caffeoylquinic acid,4-O-Caffeoylquinic acid,5-O-Caffeoylquinic acid, 3,5-O-Dicaffeoylquinic acid,4,5-O-Dicaffeoylquinic acid) in green coffee beans. Separation was achieved using a Kromasil C18 column (4.6 mm×200 mm,5μm), under the gradient elution with the mobile phase of acetonitrile and 0.1%(v/v) formic acid/water. The 6 phenolic acids were well separated within 45 min. The recoveries from 90.76% to 104.73% with the relative standard deviation between 0.7%-3.9%.The method is simple, rapid and highly sensitive, suitable for the simultaneous determination of 6 phenolic acids and quality control of coffee beans. In addition, using the HPLC method the test also compared the content of coffee polyphenol in four areas of Yunnan, the result showed that the highest content of total polyphenols is in Baoshan coffee.
The effects on yield of extraction solution, extraction method (temperature), extraction time, and extraction times were researched. The optimization technics parameters of the extraction of coffee polyphenols were as follows:coffee was extracted at 80℃in 70% ethanol twice, with 1:7 as ratio of solid/liquid,extraction 2h every time.
7 kinds of macroporous resins were compared for the separation of polyphenol of coffee, Polyamide resin exhibited superior adsorption and regeneration properties. The optimum conditions for separating and purifying polyphenol of coffee were obtained. The results showed that the separation optimization technics parameters with Polyamide resin were as follows:the sample concentration is 4.44mg/ml,the moving speed is 2BV/h, adsorbing twice,and eluting volume is 3BV with 80% ethanol as eluting solvent. The purity of polyphenol of coffee can be improved from 14.43% up to 53.32% and the recovery rate is 78.21%. UV spectrophotometer and HPLC were used to analyze Preliminary purified coffee polyphenols. The former measured the total polyphenol content was 53.32%, the latter measured total content of 6 kinds of the polyphenols was 47.55%, the results indicated that the main Phenolic substances in coffee are caffeyolquinic acid (CQA) and its derivatives.
Through five kinds of detection system (scavenging hydrogen peroxide, hydroxyl radical, superoxide anion radical, DPPH radical and reducing power determination), the experiment studied the antioxidant properties of coffee polyphenols. The result showed that coffee polyphenols have certain abilities of scavenging free radieals and reducing Fe3+, and Significant correlation exists between the clearance rate of the free radieals, the abilities of reducing Fe3+ and the Sample concentration.
引文
[1]咖啡豆百度百科http://baike.baidu.com/view/8402.htm
[2]田口护(日本).咖啡豆品鉴大全[M].辽宁：辽宁科学技术出版社,2009,1：5
[3]李贵平,杨世贵,黄健,等.云南咖啡种质资源调查与收集[J].热带农业科技,2007,30(4)：17
[4]李恂.朱古拉存有中国最古老的咖啡林[J].中国经贸.2008,9(1)：80
[5]云南咖啡种植面积达50万亩http://news.163.com/10/1221/20/6OF1HAS500014JB5.html
[6]胡大一,马力奥·马拉奥(巴西),达西·阿·里马(美).咖啡无罪的101个理由[M].江苏：江苏文艺出版社.2009,1：181-197
[7]凌关庭,等.抗氧化食品与健康[M].北京：化学工业出版社,2004,1：112
[8]Clifford MN,Ramirez-Martinez JR.Tannins in wet processed coffee beans and coffee pulp[J].Food. Chem.1991,40:191-200
[9]Adriana Farah, Carmen Marino Donangelo. Phenolic compounds in coffee[J]. Braz.J.Plant Physiol 2006,18(1):23-36
[10]张东明.酚酸化学[M].北京：化学工业出版社,2009,1：214
[11]Clifford MN. Hierarchical Scheme for LC-MSn Identification of Chlorogenic acids[J]. J.Agric. Food.Chem.2003,51:2900-2911
[12]Clifford MN, Knight S, Sururu B, et al. Characterization by LC-MSn of four new classes of chlorogenic acids in coffee beans:dimetoxycinnamoyilquinic acids,diferuloylquinic acids,caffe-oyldimethoxycinnamoylquinic acids and feruloyl-dimethoxycinnamoylquinic acids[J]. J.Agric. Food.Chem.2006,54:1957-1969
[13]Murata M, Okada H, Homma S. Hydroxycinnamic acid-derivatives and p-coumaroyil-(L)-try-ptophan,a novel hydroxycinnamic acid-derivative,from coffee beans[J]. Biosc.Biotech.Biochem. 1995,59:1887-1890
[14]Correia AMNG, Leitao MCA, Clifford MN. Caffeoyil-tyrosine and Angola II as characteristic markers for Angolan robusta coffees[J]. Food Chem.1995,53:309-313
[15]Schrader K, Liehne A, Engelhardt UH, et al. Determination of chlorogenic aicd with lactones in roasted coffee.J.Sci.Food Agric.1996,71:392-398
[16]Farah A, De Paulis T, Trugo LC, et al. Effect of roasting on the formation of chlorogenic aicd lactones[J]. J.Agric.Food.Chem.2005,53:1505-1513
[17]Ky C-L,Louarn, Dussert S, et al. Caffeine,trigonelline,chlorogenic acids and sucrose diversity in wild Coffea arabica L.and C.canephora ccessions[J]. Food Chem.2001,75:223-230
[18]Farah A,Monteiro MC,Calado V,et al.Correlation between cup quality and chemical attributes of Brazilian coffee[J].Food Chem.2006,98:373-380
[19]Montavon P,Duruz E,Rumo G,et al.Evolution of green coffee protein profiles with maturation and relationship to coffee cup quality[J].J.Agric.Food Chem.2003,51:1328-2334
[20]Menezes HC.The relationship between the state of maturity of raw coffee beans and the isomers of caffeoyilquinic acid[J].Food Chem.1994,50:293-296
[21]Mazza G,Miniati E.Anthocyanins in fruits,vegetables and grain[M].CRC Press,Boca Raton,1993, 1:245
[22]Milderm IEJ,Feskens EJM,Arts ICW,et al.Intake of the plant lignans secoisolariciresinol,mataire-sinol,lariciesinol and pinoresinol in dutch men and wemen[J].J.Nutr.2005,135:1201-1207
[23]Trugo LC.HPLC in coffe analysis.University of Reading,England.Doctorate thesis(1984)
[24]Clifford MN.Chlorogenic acids and other cinnamates.Nature,occurrence and dietary burden[J]. J.Sci.Food Agric.1999,79:362-372
[25]Clifford MN.Chlorogenic acids and other cinnamates-nature,occurrence,dietary burden,absorption and metabolism[J].J.Sci.Food Agric.2000,80:1033-1043
[26]Trugo LC,Macrae R.A study of the effect of roasting on the chlorogenic acid composition of coffee using HPLC[J].Food Chem.1984,15:219-227
[27]Leloup V,Louvrier A,Liardon R.Degradation mechanisms of chlorogenic acids during roasting[J]. Sci.Coll.Coffee(Kyoto)ASIC,1995,16:192-198
[28]Scholz BM,Maier HGIsomers of quinic acid and quinide in roasted coffee[J].Lebensm.Unters. Forsch.1990,190:132-134
[29]Scholz-Bottcher BM,Ludges E,Maier HGNew stereoisomers of quinic acid and their lactones[J]. Liebigs Ann.Chem.1991,25:1029-1036
[30]Tono T,Fujita S,Kawabe M.Determination of chlorogenic acid in coffee samples by difference spectral method and DEAE-Toyopearl column chromatography[J].Nippon Shokuhin Kogyo Gakkaish.1989,36:587-591
[31]Steinhart H,Luger A.An analytical Distinction between untreated and steam-treated roasted coffee[J].Sci.Coll.Coffee(Nairobi)ASIC,1997,17:150-160
[32]Mazzafera P.Trigonelline in coffee[J].Phytochemistry.1991,30:2309-2310
[33]Ashihara H,Crozier A.Caffeine:a well known but little mentioned compound in plant science[J]. Trends Plant Sci.2001,6:407-413
[34]Clifford MN.Coffee:botany,biochemistry and production of beans and beverage[M].Croom Helm,London.1985,1:305-374
[35]R.J.Clarke, R.Macrae.Coffee:Physiology[M].Springer.1988
[36]Gallus S,Tavani A,Negri E,et al.Does coffee protect against liver cirrhosis?[J].Ann Epidemiol, 2002,12:202-205.
[37]Corrao G,Zambon A,Bagnardi V,et al.Coffee,caffeine,and the risk of liver cirrhosis[J].Ann Epidemiol,2001,11:458-465
[38]Namba T,Mat suse T.A historical study of coffee in Japanese and Asian count ries:focusing the medicinal uses in Asian traditional medicines[J].Yakushigaku Zasshi.2002,37:65-75
[39]Phan TT,Sun L,Bay BH,et al.Dietary compounds inhibit proliferation and contraction of keloid and hypertrophic scar-derived fibroblasts in vitro:therapeutic implication for excessive Scarring[J]J Trauma,2003,54:1212-1224.
[40]Watanabe T,Arai Y,Mit sui Y,et al.The blood pressure-lowering effect and safety of chlorogenic acid from green coffee bean extract in essential hypertension[J].Clin Exp Hypertens.2006, 28:439-449
[41]Odegaard AO,Pereira MA,Koh WP,et al.Coffee,tea,and incident type 2 diabetes:the Singapore Chinese health study[J].Am J Clin Nutr.2008,88:979-985.
[42]Kurata R,Adachi M,Yamakawa O,et al.Growth suppression of human cancer cells by polyphen-olics from sweetpotato (ipomoea batatas L.)leaves[J].J Agric Food Chem.2007,55:185-190.
[43]Padol IT,Hunt RH.Effect of The cytokines on acid secretion in pharmacologically characte-rised mouse gastric glands[J]. Gut,2004,53:1075-1081.
[44]贾桂燕,鲁魏藐,郑毅男.咖啡中咖啡因和绿原酸的含量测定[J].黑龙江八一农垦大学学报,2008,20(3)：64-67
[45]张宇平来,黄可龙.高效液相色谱法同时测定金银花中5种有机酸[J].分析试验室.2007,26(7)：67-70
[46]Joon-Kwan Moon, Hyui Sun Yoo, Takayuki Shibamoto. Role of roasting conditions in the level of chlorogenic acid content in coffee beans:correlation with coffee acidity[J].J Agric Food Chem,2009, 57:5367
[47]魏云,赵平平.薄层扫描法测定金银花两种不同加工法绿原酸含量[J].中国中药杂 志,1999,24(6)：340-341
[48]朱玉,张书胜,张西林,陈洁.薄层色谱法分析葵花仁粕中的绿原酸[J].色谱,2001,19(1)：82-84
[49]易昌华,贺建华.紫外分光光度法测定中草药提取物中绿原酸的含量[J].兽药与饲料添加剂,2004,9(1)：24-25
[50]梅林,付雪,刘凌云,程正学,胡承波,田光珍.紫外分光光度法分析金银花及其制剂银黄口服液中绿原酸[J].激光杂志,2007,28(5)：94
[51]刘秀凤,阮美娟,蔡金星,贾文沦.咖啡浸提条件优化研究[J].工艺技术,2006,27(2)：123-126
[52]吴红,李稳宏,吴道澄.杜仲叶水提醇沉工艺对绿原酸含量的影响[J].第四军医大学学报,2002,23(10)：931.
[53]王茜,李智,何琦,侯太平.杜仲叶中绿原酸提取分离工艺条件的研究[J].离子交换与吸附,2008,24(1)：73-80
[54]尹波,王科军.超声波法提取金银花中的绿原酸[J].林产化工通讯,2005,39(1)：13-15.
[55]周军,黄琼,李志光,谢磊,黄可龙.超声波法提取金银花中的绿原酸[J].化学与生物互程2008,25(1)：31-33
[56]韦藤幼,赵群莉,阮莉姣.微波预处理法提取金银花中的绿原酸[J].中成药,2003,25(7)：534-573.
[57]谭伟,丘泰球,阳元娥.超临界C02萃取葵粕绿原酸影响因素分析[J].食品工业,2008(4)：17-19
[58]高锦明.植物化学[M].北京：科学出版社,2003,1
[59]戚向阳,张声华.杜仲叶中绿原酸的提取分离研究[J].中草药,1998,29(11)：741-742.
[60]戚向阳,张声华.杜仲叶中绿原酸的提取、分离和鉴植物资源与环境[J],中草药,1998,7(1)：61-62.
[61]钱骅,赵伯涛,张卫明.杜仲叶绿原酸的提取分离[J].中国野生植物资源,2001,20(4)：15-16.
[62]成军,白焱晶,赵玉英.杜仲叶苯并素类成分的研究[J].中国中药杂志,2001,27(1)：38-39.
[63]李进飞,黄可龙,李春华.NKA-9型树脂对绿原酸吸附分离性能的研究[J].华西药学杂志,2004,19(1)：1-4.
[64]Trichopoulou A.Vasilopoulou E, Hollman P. Nutritionalcomposition and flavonoid content of edible Wild greens and green pies:A potential rich source of antioxidant nutrients in the Mediterranean diet[J] Food Chemistry,2000,(70):319-323
[65]Tate R M. Oxgen metabolites stimulate thromboxane productionand vasoconstriction in isolated saline-perfused rabbitlungs[J].Jclin Inves,1984,74:68-72
[66]Burton K. P. Superoxide dismutase enhances recovery following myocardial is chemia[J]. American Physiol.1985,248:637-641
[67]Fogliano V,Verde V,Ralldazzo G,et al.Method for measuring antioxidant activity and its application to montioring the antioxidant capacity of wines[J],J Agric Food Chem,l999,47(3): 1035-1040.
[68]郝晓丽,苏延友.用两种方法评价四种食品抗氧化剂的抗氧化活性[J].食品科学,2003,24(2)：127-129.
[69]Miller N J,Rice-Evans C,Davies M J,et al. A novel method for meansuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates[J],Clin Sci,1993, 84(4):407-412
[70]Bartschwinkler S.Cycle of earthquake-induced aggradation and related tidal channel shifting, Upper Turnagain Arm,Alaska,USA[J].Sedimentology,2000,35(4):621
[71]Adriana Farah, Carmen Marino Donangelo.Phenolic compounds in coffee[J]. Braz J Plant Physiol,2006,18(1):26
[72]Rosa M. Alonso-Salces, Francesca Serra, Fabiano Reniero, et al. Botanical and Geographical Characterization of Green Coffee (Coffea arabica and Coffea canephora):Chemometric Evaluation of Phenolic and Methylxanthine Contents[J]. J Agric Food Chem,2009,57:4225
[73]Chin-Long Ky, Michel Noirot, Serge Hamon. Comparison of Five Purification Methods for Chlorogenic acids in Green Coffee Beans(Coffea sp.)[J]. J Agric Food Chem,1997,45:786
[74]吴卫华,康桢,欧阳冬生,等.绿原酸的药理学研究进展[J].天然产物研究与开发,2006,18：692
[75]Micheal N Clifford.Chlorogenic acids-their complex nature and rotine determination in coffee beans[J], J Agric Food Chem,1979,4:63
[76]董娟娥,马希汉.反相高效液相色谱法同时分离测定杜仲雄花及其产品中的京尼平苷酸和绿原酸[J].色谱,2007,25(2)：217
[77]杨敏丽,郝凤霞.金银花中绿原酸的分离纯化工艺研究[J].食品科学,2007,28(7)：255
[78]王茜,李智,何琦,等.杜仲叶中绿原酸提取分离工艺条件的研究[J].离子交换与吸附,2008,24(1)：73
[79]兰小艳,张学俊,龚桂珍.杜仲叶中绿原酸的研究进展[J].中国农学通报,2009,25(21)：86-89
[80]茶叶中茶多酚和儿茶素类含量的检测方法.中华人民共和国国家标准[GB/T 8313-2008]
