HPLC测定番荔枝果实中酚类成分及抗氧化相关性
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摘要
目的:建立高效液相色谱(high performance liquid chromatography,HPLC)法同时测定番荔枝果实3个部位中13种酚类成分的种类及含量,并分析含量与抗氧化活性的相关性。方法:采用福林-酚法测定总酚含量;通过HPLC法对番荔枝果实中的单酚成分进行比较;通过1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基与2,2’-联氮双(3-乙基苯并噻唑啉-6-磺酸)(2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid),ABTS)阳离子自由基清除能力的检测方法测定番荔枝果实的抗氧化活性。结果:番荔枝果实中总酚含量为果皮>果肉>种子,果皮含量最高为59.48～127.84 mg/g;番荔枝单酚含量结果表明果皮显著高于果肉和种子部位,各产地酚类种类和含量差异显著。果皮和果肉中对羟基苯甲酸、儿茶素、咖啡酸、表儿茶素含量相对较高,表儿茶素最高为2 398.17 mg/mL。抗氧化结果表明番荔枝果实清除ABTS阳离子自由基能力高于DPPH自由基,且果皮活性最好,分别在质量浓度为0.39、1.56 mg/m L时均达到最高清除率。结论:番荔枝果实中果皮、果肉、种子3个部位中所含酚类物质种类及含量有显著性差异;果皮部位所含种类及含量均最高,且抗氧化活性最佳,可作为天然抗氧化剂。
Objective: To establish a high performance liquid chromatographic(HPLC) method for the simultaneous determination of the contents of 13 phenolic constituents in three fruit parts of Annona squamosa Linn., and to analyze the correlation between phenol content and antioxidant activity. Methods: Total phenols were evaluated by a colorimetric method, and the HPLC method was used to measure the composition and contents of phenols. The antioxidant activity was analyzed using the 1,1-diphenyl-2-picrylhydrazyl(DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radical cation scavenging activity assays. Results: The total phenol content of peel was the highest, ranging from 59.48 to127.84 mg/g, sequentially followed by pulp and seeds. The contents of some individual phenolic compounds in peel significantly higher than in any other parts. There were significant regional differences in the composition and contents of phenols in the same fruit parts. The contents of p-hydroxybenzoic acid, catechin, caffeic acid and(-)-epicatechin were relatively higher in peel and pulp and the highest(-)-epicatechin level was 2 398.17 mg/mL. All of the fruit parts were had more potent scavenging activity against DPPH radical than against ABTS radical cation and peel had the strongest radical scavenging with the highest percentage inhibition of DPPH radical and ABTS radical cation being observed at concentrations of 0.39 and 1.56 mg/mL, respectively. Conclusion: The composition and contents of phenols in the peel, pulp and seeds of A. squamosa fruit are significantly different. The peel contains the highest number and amount of phenols and possesses the highest antioxidant activity, indicating that it can be considered as a potential and valuable source of natural antioxidants.
Objective: To establish a high performance liquid chromatographic(HPLC) method for the simultaneous determination of the contents of 13 phenolic constituents in three fruit parts of Annona squamosa Linn., and to analyze the correlation between phenol content and antioxidant activity. Methods: Total phenols were evaluated by a colorimetric method, and the HPLC method was used to measure the composition and contents of phenols. The antioxidant activity was analyzed using the 1,1-diphenyl-2-picrylhydrazyl(DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) radical cation scavenging activity assays. Results: The total phenol content of peel was the highest, ranging from 59.48 to127.84 mg/g, sequentially followed by pulp and seeds. The contents of some individual phenolic compounds in peel significantly higher than in any other parts. There were significant regional differences in the composition and contents of phenols in the same fruit parts. The contents of p-hydroxybenzoic acid, catechin, caffeic acid and(-)-epicatechin were relatively higher in peel and pulp and the highest(-)-epicatechin level was 2 398.17 mg/mL. All of the fruit parts were had more potent scavenging activity against DPPH radical than against ABTS radical cation and peel had the strongest radical scavenging with the highest percentage inhibition of DPPH radical and ABTS radical cation being observed at concentrations of 0.39 and 1.56 mg/mL, respectively. Conclusion: The composition and contents of phenols in the peel, pulp and seeds of A. squamosa fruit are significantly different. The peel contains the highest number and amount of phenols and possesses the highest antioxidant activity, indicating that it can be considered as a potential and valuable source of natural antioxidants.
引文
[1]国家中医药管理局.中华本草[M].上海:上海科学技术出版社,1999:3.
[2]中国科学院华南植物研究所.广东植物志[M].长春:吉林人民出版社,1991:39-40.
[3] GUNABALAN M, GOVINDASAMY R, SELVARAJM R,et al.Acaricidal,insecticidal,and larvicidal efficacy of fruit peel aqueous extract of Annona squamosal extract of Annona squamosal and its compounds against blood-feeding parasites[J].Parasitology Research,2012,111(5):2189-2199.DOI:10.1007/s00436-011-2671-2.
[4] TAKAHASHI J A,PEREIRA C R,PIMENTA LP,et al.Antibacterial activity of eight Brazilian annonaceae plants[J]. Natural Product Research,2006,20(1):21-26.DOI:10.1080/14786410412331280087.
[5] MUKHLESUR R M, PARVIN S,EKRAMUL H M,et al.Antimicrobial and cytotoxic constituents from the seeds of Annona squamo-sal[J]. Fitoterapia, 2005, 76(5):484-489. DOI:10.1016/j.fi to te.2005.04.002.
[6]王续楠,陈勇,李祥,等.番荔枝内酯类化合物基于耐阿霉素肝癌细胞SMMC-77 21/ADR的构效关系研究[J].中国中药杂志,2017,42(13):2518-2523.DOI:10.19540/j.cnki.cjcmm.20170419.003.
[7] PADMAJA V, THANKAMANY V, HARA N, et al. Biological activities of Annona glabra[J].Journal of Ethnopharmacology, 1995,48:21-24.DOI:10.1016/0378-8741(95)01277-K.
[8] REJON-ORANTES J D C,GONZALEZ-ESQUINCA A R,DE LA MORA M P,et al.Annomontine.,an alkaloid isolated from Annona spurpurea,has anxiolytic-like effects in the elevated plus-maze[J].Planta Media,2011,77(4):322-327.DOI:10.1055/S-0030-1250406.
[9] OKOYE T C,AKAH P A,OMEJE E O,et al.Anticonvulsant effect of kaurenoic acid isolated from the root bark of Annona senegalensis[J].Pharmacology Biochemistry and Behavior, 2013, 109:38-43.DOI:10.1016/j.pbb.2013.05.001.
[10]丁利君,任乃林,周燕芳,等.番荔枝果肉营养成分分析[J].营养学报,2006,28(3):275-276.DOI:10.3321/j.issn:0512-7955.2006.03.025.
[11]孟富宣,段元杰,杨玉皎,等.中国番荔枝生产与科研现状及发展建议[J].热带农业科学,2017, 37(6):100-105. DOI:10.12008/j.issn.1009-2196.2017.06.022.
[12] WALTER N L, SAUTHIER M, CAVALCANTE D D, et al.Mineral composition, nutritional properties, total phenolics andflavonoids compounds of the atemoya fruit(Annona squamosa L.×Annona cherimola Mill.)and evaluation using multivariate analysis techniques[J].Anais da Academia Brasileira de Ciencias,2016,88(3):1243-1252.DOI:10.1590/0001-3765201620150537.
[13]付丽,卢文青,周晓敏,等.番荔枝酚类提取物的抗菌抗氧化作用[J].热带医学杂志,2016,16(4):460-463.DOI:1672-3619(2016)04-0460-04.
[14]赵文红,邓泽元,范亚苇,等.阿魏酸体外抗氧化作用研究[J].食品科学,2010,31(1):219-223.DOI:1002-6630(2010)01-0219-05.
[15]刘强,王亚强,许培仁,等.儿茶素类对h-VRS的体外抗菌活性研究[J].中国抗生素杂志,2011,36(7):557-561. DOI:10.13461/j.cnki.cja.004829.
[16]姜劲迈,张颖,叶百宽,等.棉酚抗癌作用机理研究[J].中国中医基础医学杂志,2002,8(2):35-37.DOI:1006-3250(2002)02-0035-03.
[17] GOVEA-SALAS M, RIVAS-ESTILLA A M, RODRIGUEZHERRERA R,et al.Gallic acid decreases hepatitis C virus expression through its antioxidant capacity[J].Experimental and Therapeutic Medicine,2016,11(2):619-624.DOI:10.3892/etm.2015.2923.
[18]伊娟娟.红松球果多酚类化合物分离鉴定及抗肿瘤作用机制研究[D].哈尔滨:哈尔滨工业大学,2017.
[19]王小红,张婵溪,李贵云,等.天然小分子酚的抗焦虑作用研究进展[J].中国中药杂志,2017,42(8):1557-1565. DOI:10.19540/j.cnki.cj cmm.20170222.005.
[20]李颖畅,吕艳芳,励建荣,等.Folin-Ciocalteu法测定不同品种蓝莓叶中多酚含量[J].中国食品学报,2014,14(1):273-277.DOI:10.16429/j.1009-7848.2014.01.005.
[21]李巨秀,王柏玉.福林-酚比色法测定桑椹中总多酚[J].食品科学,2009,30(18):292-295.DOI:1002-6630(2009)18-0292-04.
[22]韦献雅,殷丽琴,钟成,等.DPPH法评价抗氧化活性研究进展[J].食品科学,2014,35(9):137-322. DOI:10.7506/spkx1002-6630-201409062.
[23]丁豪,杨海燕,辛志宏.昆仑雪菊黄酮类化合物的抗氧化相互作用研究[J].食品科学,2015,36(21):26-31.DOI:10.7506/spkx1002-6630-201521006.
[24]邱金东,汤昆.DPPH和ABTS法测定核桃仁的体外抗氧化活[J].中成药,2008, 30(8):1215-1216. DOI:10.3969/j.issn.1001-1528.2008.08.039.
[25]李培源,霍丽妮,苏炜,等.总抗氧化能力检测试剂盒(ABTS)法测定江南星蕨的抗氧化活性[J].中国实验方剂学杂志,2011,17(1):162-164.DOI:10.13422/j.cnki.syfjx.2011.01.057.
[26] KINGSLEY C,OKOLIE P N.Proximate composition,phytochemical analysis, and in vitro antioxidant potentials of extracts of Annona muricata(Soursop)[J]. Food Science&Nutrition,2017, 5(5):1029-1036.DOI:10.1002/fsn3.498.
[27] HENRIQUE S A,GUSTAVO A P,DAMILA R M,et al.Determination of free,esterified,glycosylated and insoluble boud-phenolics composition in the edible part of araticum fruit(Annona crassiflora Mart.)and its by products by HPLC-ESI-MS/MS[J].Food Chemistry,2018, 245:738-749.DOI:10.1016/j.foodchem.2017.11.120.
[28] KELEBEK H,SELLI S.,GUBBUK H,et al. Comparative evaluation of volatiles,phenolics,sugars,organic acids and antioxidant properties of Sel-42 and Tainung papaya varieties[J].Food Chemistry,2015,173:912-919.DOI:10.1016/j.foodchem.2014.10.116.
[29] ZAHID M,ARIF M,RAHMAN M A,et al.Solvent Extraction and gas chromatography-mass spectrometry analysis of Annona squamosa L.seeds for determination of bioactives,fatty acid/fatty oil composition,and antioxidant activity[J].Journal of Dietary Supplements,2017, 2:1-11.DOI:10.1080/19390211.2017.1366388.
[30]张华,周志钦,席万鹏,等.15种柑橘果实主要酚类物质的体外抗氧化活性比较[J].食品科学, 2015.,36(11):64-70. DOI:10.7506/spkx1002-6630-201511013.
[31] ADEFEGHA S A,OYELEYE S I,OBOH G.Distribution of phenolic contents, antidiabetic potentials, antihypertensive properties, and antioxidative effects of soursop(Annona muricata L.)fruit parts in vitro[J]. Biochemistry Research International, 2015,2015:347673.DOI:10.1155/2015/347673.
[32] SSENYANGE C W, NAMULINDWA A, OYIK B, et al. Plants used to manage type II diabetes mellitus in selected districts of central Uganda[J]. African Health Sciences, 2015, 15(2):496-502.DOI:10.4314/ahs.v15i2.24.
[33] JUSTINO A B, PEREIRA M N, PEIXOTO L G, et al.Hepatoprotective properties of a polyphenol-enriched fraction frorm Annona crassiflora Mart.,fruit peel against diabetes-induced oxidative and nitrosative stress[J].Journal of Agricultural and Food Chemistry,2017,65(22):4428-4438.DOI:10.1021/acs.jafc.7b01355.
[2]中国科学院华南植物研究所.广东植物志[M].长春:吉林人民出版社,1991:39-40.
[3] GUNABALAN M, GOVINDASAMY R, SELVARAJM R,et al.Acaricidal,insecticidal,and larvicidal efficacy of fruit peel aqueous extract of Annona squamosal extract of Annona squamosal and its compounds against blood-feeding parasites[J].Parasitology Research,2012,111(5):2189-2199.DOI:10.1007/s00436-011-2671-2.
[4] TAKAHASHI J A,PEREIRA C R,PIMENTA LP,et al.Antibacterial activity of eight Brazilian annonaceae plants[J]. Natural Product Research,2006,20(1):21-26.DOI:10.1080/14786410412331280087.
[5] MUKHLESUR R M, PARVIN S,EKRAMUL H M,et al.Antimicrobial and cytotoxic constituents from the seeds of Annona squamo-sal[J]. Fitoterapia, 2005, 76(5):484-489. DOI:10.1016/j.fi to te.2005.04.002.
[6]王续楠,陈勇,李祥,等.番荔枝内酯类化合物基于耐阿霉素肝癌细胞SMMC-77 21/ADR的构效关系研究[J].中国中药杂志,2017,42(13):2518-2523.DOI:10.19540/j.cnki.cjcmm.20170419.003.
[7] PADMAJA V, THANKAMANY V, HARA N, et al. Biological activities of Annona glabra[J].Journal of Ethnopharmacology, 1995,48:21-24.DOI:10.1016/0378-8741(95)01277-K.
[8] REJON-ORANTES J D C,GONZALEZ-ESQUINCA A R,DE LA MORA M P,et al.Annomontine.,an alkaloid isolated from Annona spurpurea,has anxiolytic-like effects in the elevated plus-maze[J].Planta Media,2011,77(4):322-327.DOI:10.1055/S-0030-1250406.
[9] OKOYE T C,AKAH P A,OMEJE E O,et al.Anticonvulsant effect of kaurenoic acid isolated from the root bark of Annona senegalensis[J].Pharmacology Biochemistry and Behavior, 2013, 109:38-43.DOI:10.1016/j.pbb.2013.05.001.
[10]丁利君,任乃林,周燕芳,等.番荔枝果肉营养成分分析[J].营养学报,2006,28(3):275-276.DOI:10.3321/j.issn:0512-7955.2006.03.025.
[11]孟富宣,段元杰,杨玉皎,等.中国番荔枝生产与科研现状及发展建议[J].热带农业科学,2017, 37(6):100-105. DOI:10.12008/j.issn.1009-2196.2017.06.022.
[12] WALTER N L, SAUTHIER M, CAVALCANTE D D, et al.Mineral composition, nutritional properties, total phenolics andflavonoids compounds of the atemoya fruit(Annona squamosa L.×Annona cherimola Mill.)and evaluation using multivariate analysis techniques[J].Anais da Academia Brasileira de Ciencias,2016,88(3):1243-1252.DOI:10.1590/0001-3765201620150537.
[13]付丽,卢文青,周晓敏,等.番荔枝酚类提取物的抗菌抗氧化作用[J].热带医学杂志,2016,16(4):460-463.DOI:1672-3619(2016)04-0460-04.
[14]赵文红,邓泽元,范亚苇,等.阿魏酸体外抗氧化作用研究[J].食品科学,2010,31(1):219-223.DOI:1002-6630(2010)01-0219-05.
[15]刘强,王亚强,许培仁,等.儿茶素类对h-VRS的体外抗菌活性研究[J].中国抗生素杂志,2011,36(7):557-561. DOI:10.13461/j.cnki.cja.004829.
[16]姜劲迈,张颖,叶百宽,等.棉酚抗癌作用机理研究[J].中国中医基础医学杂志,2002,8(2):35-37.DOI:1006-3250(2002)02-0035-03.
[17] GOVEA-SALAS M, RIVAS-ESTILLA A M, RODRIGUEZHERRERA R,et al.Gallic acid decreases hepatitis C virus expression through its antioxidant capacity[J].Experimental and Therapeutic Medicine,2016,11(2):619-624.DOI:10.3892/etm.2015.2923.
[18]伊娟娟.红松球果多酚类化合物分离鉴定及抗肿瘤作用机制研究[D].哈尔滨:哈尔滨工业大学,2017.
[19]王小红,张婵溪,李贵云,等.天然小分子酚的抗焦虑作用研究进展[J].中国中药杂志,2017,42(8):1557-1565. DOI:10.19540/j.cnki.cj cmm.20170222.005.
[20]李颖畅,吕艳芳,励建荣,等.Folin-Ciocalteu法测定不同品种蓝莓叶中多酚含量[J].中国食品学报,2014,14(1):273-277.DOI:10.16429/j.1009-7848.2014.01.005.
[21]李巨秀,王柏玉.福林-酚比色法测定桑椹中总多酚[J].食品科学,2009,30(18):292-295.DOI:1002-6630(2009)18-0292-04.
[22]韦献雅,殷丽琴,钟成,等.DPPH法评价抗氧化活性研究进展[J].食品科学,2014,35(9):137-322. DOI:10.7506/spkx1002-6630-201409062.
[23]丁豪,杨海燕,辛志宏.昆仑雪菊黄酮类化合物的抗氧化相互作用研究[J].食品科学,2015,36(21):26-31.DOI:10.7506/spkx1002-6630-201521006.
[24]邱金东,汤昆.DPPH和ABTS法测定核桃仁的体外抗氧化活[J].中成药,2008, 30(8):1215-1216. DOI:10.3969/j.issn.1001-1528.2008.08.039.
[25]李培源,霍丽妮,苏炜,等.总抗氧化能力检测试剂盒(ABTS)法测定江南星蕨的抗氧化活性[J].中国实验方剂学杂志,2011,17(1):162-164.DOI:10.13422/j.cnki.syfjx.2011.01.057.
[26] KINGSLEY C,OKOLIE P N.Proximate composition,phytochemical analysis, and in vitro antioxidant potentials of extracts of Annona muricata(Soursop)[J]. Food Science&Nutrition,2017, 5(5):1029-1036.DOI:10.1002/fsn3.498.
[27] HENRIQUE S A,GUSTAVO A P,DAMILA R M,et al.Determination of free,esterified,glycosylated and insoluble boud-phenolics composition in the edible part of araticum fruit(Annona crassiflora Mart.)and its by products by HPLC-ESI-MS/MS[J].Food Chemistry,2018, 245:738-749.DOI:10.1016/j.foodchem.2017.11.120.
[28] KELEBEK H,SELLI S.,GUBBUK H,et al. Comparative evaluation of volatiles,phenolics,sugars,organic acids and antioxidant properties of Sel-42 and Tainung papaya varieties[J].Food Chemistry,2015,173:912-919.DOI:10.1016/j.foodchem.2014.10.116.
[29] ZAHID M,ARIF M,RAHMAN M A,et al.Solvent Extraction and gas chromatography-mass spectrometry analysis of Annona squamosa L.seeds for determination of bioactives,fatty acid/fatty oil composition,and antioxidant activity[J].Journal of Dietary Supplements,2017, 2:1-11.DOI:10.1080/19390211.2017.1366388.
[30]张华,周志钦,席万鹏,等.15种柑橘果实主要酚类物质的体外抗氧化活性比较[J].食品科学, 2015.,36(11):64-70. DOI:10.7506/spkx1002-6630-201511013.
[31] ADEFEGHA S A,OYELEYE S I,OBOH G.Distribution of phenolic contents, antidiabetic potentials, antihypertensive properties, and antioxidative effects of soursop(Annona muricata L.)fruit parts in vitro[J]. Biochemistry Research International, 2015,2015:347673.DOI:10.1155/2015/347673.
[32] SSENYANGE C W, NAMULINDWA A, OYIK B, et al. Plants used to manage type II diabetes mellitus in selected districts of central Uganda[J]. African Health Sciences, 2015, 15(2):496-502.DOI:10.4314/ahs.v15i2.24.
[33] JUSTINO A B, PEREIRA M N, PEIXOTO L G, et al.Hepatoprotective properties of a polyphenol-enriched fraction frorm Annona crassiflora Mart.,fruit peel against diabetes-induced oxidative and nitrosative stress[J].Journal of Agricultural and Food Chemistry,2017,65(22):4428-4438.DOI:10.1021/acs.jafc.7b01355.