vuzbiochemiИзвестия вузов. Прикладная химия и биотехнологияProceedings of Universities. Applied Chemistry and Biotechnology2227-29252500-1558ИРНИТУ10.21285/2227-2925-2020-10-3-470-478vuzbiochemi-420Research ArticleФИЗИКО-ХИМИЧЕСКАЯ БИОЛОГИЯPHYSICOCHEMICAL BIOLOGYОпределениe фенольных соединений в водно-этанольных экстрактах листьев Populus tremula L. c помощью высокоэффективной жидкостной хроматографииDetermination of phenolic compounds in water-ethanol extracts of Populus tremula L. leaves using high-performance liquid chromatographyКоцупийО. В.KotsupiyO. V.

Коцупий Ольга Викторовна, к.б.н., научный сотрудник

630090, г. Новосибирск, ул. Золотодолинская, 101

Olga V. Kotsupiy, Cand. Sci. (Biology), Researcher

101, Zolotodolinskaya St., Novosibirsk, 630090

olnevaster@gmail.comЗагурскаяЮ. В.ZagurskayaYu. V.

Загурская Юлия Васильевна, к.б.н., научный сотрудник

650000, г. Кемерово, пр-т Советский, 18

Yulia V. Zagurskaya, Cand. Sci. (Biology), Researcher

18, Sovetskii Ave., Kemerovo, 650000

syjil@mail.ruУфимцевВ. И.UfimtsevV. I.

Уфимцев Владимир Иванович, к.б.н., ведущий научный сотрудник

650000, г. Кемерово, пр-т Советский, 18

Vladimir I. Ufimtsev, Cand. Sci. (Biology), Leading Researcher

18, Sovetskii Ave., Kemerovo, 650000

uwy2079@gmail.comЦентральный сибирский ботанический сад СО РАНРоссияСentral Siberian Botanical Garden SB RASRussian FederationФедеральный исследовательский центр угля и углехимии СО РАН (Институт экологии человека)РоссияFederal Research Center of Coal and Coal Chemistry SB RAS (Institute of Human Ecology)Russian Federation202008102020103470478Copyright © Коцупий О.В., Загурская Ю.В., Уфимцев В.И., 20202020Коцупий О.В., Загурская Ю.В., Уфимцев В.И.Kotsupiy O.V., Zagurskaya Y.V., Ufimtsev V.I.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://vuzbiochemi.elpub.ru/jour/article/view/420

Водно-этанольный экстракт листьев Populus tremula L. (осина обыкновенная) включает различные группы фенольных соединений с разной полярностью и различной концентрацией веществ, что осложняет аналитическую задачу при определении их содержания. Цель работы – изучить условия твердофазной экстракции и высокоэффективной жидкостной хроматографии при исследовании содержания разных групп фенольных соединений в водно-этанольных экстрактах листьев P. tremula. Для извлечения фенольных соединений проводили исчерпывающую экстракцию этанолом. Твердофазную экстракцию осуществляли с помощью патрона Диапак С16, после чего элюаты пропускали через мембранный фильтр с диаметром пор 0,45 мкм. Методом высокоэффективной жидкостной хроматографии определяли содержание фенолокислот и гликозидов флавоноидов, а также салицина и индивидуальных компонентов гликозидов флавоноидов: гиперозида, рутина, астрагалина и двух неиндентифицированных гликозидов флавоноидов в водной (аналит 1) и водно-спиртовой фракциях (аналит 2) в двух системах, различающихся по градиенту элюирования. Доказана необходимость анализа первичного водного элюата совместно или параллельно с основной водно-спиртовой фракцией при подготовке экстрактов листьев P. tremula для высокоэффективной жидкостной хроматографии с помощью картриджей твердофазной экстракции. Для разделения экстракта с целью определения содержания компонентов гидроксикоричных и гидроксибензойных кислот предпочтительнее использовать систему 2, для определения содержания фенологликозидов (салицина) – систему 1. Гликозиды флавоноидов (гиперозид, рутин, астрагалин и два неидентифицированных флавоноида) вносят наиболее весомый вклад в различие двух фракций – водной и водно-спиртовой.

The analytical task of determining the phenolic compound content of water-ethanol extracts of Populus tremula L. (common aspen) leaves is complicated by the heterogeneity of compound groups having different polarities and appearing in varying concentrations. The purpose of the present work is to study the conditions of solid-phase extraction and high-performance liquid chromatography used to analyse the content of different groups of phenolic compounds in water-ethanol extracts of leaves from the P. tremula plant. In order to facilitate the derivation of phenolic compounds, an exhaustive extraction process was carried out using ethanol. Solid-phase extraction was carried out using a Diapak C16 cartridge, after which the eluates were passed through a membrane filter having a pore diameter of 0.45 μm. The high-performance liquid chromatography method was used to determine the content of phenolic acids and flavonoid glycosides, as well as salicin and individual flavonoid glycoside components: hyperoside, rutin, astragalin and two unidentified flavonoid glycosides in aqueous (analyte 1) and aqueous-alcoholic fractions (analyte 2) in two systems along the gradient elution. The requirement of analysing the primary aqueous eluate together or in parallel with the main aqueous-alcoholic fraction in the preparation of P. tremula leaf extracts for high-performance liquid chromatography using solid-phase extraction cartridges was substantiated. For separating the extract to determine the hydroxycinnamic and hydroxybenzoic acid content, it is preferable to use system 2; for determining the phenologlycoside (salicin) content, system 1 is more effective. Flavonoid glycosides (hyperoside, rutin, astragalin and two unidentified flavonoids) make the most significant contribution to the difference between the aqueous and aqueous-alcoholic fractions.

фенольные соединенияPopulus tremulaтвердофазная экстракциявысокоэффективная жидкостная хроматографияпробоподготовкаphenolic compoundsPopulus tremulasolid-phase extractionhigh-performance liquid chromatography (HPLC)sample preparationРабота выполнена в рамках проектов Центрального сибирского ботанического сада СО РАН № АААА-А17-117012610051-5 и Федерального исследовательского центра угля и углехимии СО РАН № АААА-А17-117041410053-1.The work was carried out within the framework of the projects of Central Siberian Botanic Garden SB RAS no. AAAA-A17-117012610051-5 and Federal Research Center of Coal and Coal Chemistry SB RAS no. AAAA-A17-117041410053-1.ReferencesТемердашев З.А., Милевская В.В., Киселева Н.В., Верниковская Н.А., Коробков В.А. Сорбционно-хроматографическое определение галловой, кофейной кислот, рутина и эпикатехина в лекарственных растениях // Аналитика и контроль. 2013. Т. 17. N 2. С. 211–218.Temerdashev ZA, Milevskaya VV, Kiseleva NV, Vernikovskaya NA, Korobkov VA. Sorption-chromatographic determination of gallic, caffeic acids, rutin and (-)-epicatechin in herbs. Analitika i control = Analytics and Control. 2013;2:211–218. (In Russian)Сапрыкин Л.В., Сапрыкина Л.В. Методология аналитического применения твердофазной экстракции // Сорбционные и хроматографические процессы. 2007. Т. 7. Вып. 3. С. 397–409.Saprykin LV, Saprykina LV. Methodology of analytical application of solid-phase extraction. Sorbtsionnye i khromatograficheskie protsessy. 2007;7(3):397–409. (In Russian)Kudrinskaya V.A., Dmitrienko S.G., Zolotov Yu.A. Synthesis and study of sorption properties of molecularly imprinted polymers for quercetin // Moscow University Chemistry Bulletin. 2009. Vol. 64. Issue 3. P. 124–129. https://doi.org/10.3103/S0027131409030031Kudrinskaya VA, Dmitrienko SG, Zolotov YuA. Synthesis and study of sorption properties of molecularly imprinted polymers for quercetin. Moscow University Chemistry Bulletin. 2009;64(3):124–129. https://doi.org/10.3103/S0027131409030031Zanutto F.V., Boldrin P.K., Varanda E.A., Fernandes de Souza S., Sano P.T., Vilegas W., et al. Characterization of flavonoids and naphthopyranones in methanol extracts of Paepalanthus chiquitensis Herzog HPLC-ESI-IT-MSn and their mutagenic activity // Molecules. 2012. Vol. 18. Issue 1. P. 244–262. https://doi.org/10.3390/molecules18010244Zanutto FV, Boldrin PK, Varanda EA, Fernandes de Souza S, Sano PT, Vilegas W, et al. Characterization of flavonoids and naphthopyranones in methanol extracts of Paepalanthus chiquitensis Herzog HPLC-ESI-IT-MSn and their mutagenic activity. Molecules. 2012;18(1):244–262. https://doi.org/10.3390/molecules18010244Simirgiotis M.J., Schmeda-Hirschmann G., Bórquez J., Kennelly E.J. The Passiflora tripartita (Banana Passion) fruit: a source of bioactive flavonoid C-glycosides isolated by HSCCC and characterized by HPLC–DAD–ESI/MS/MS // Molecules. 2013. Vol. 18. Issue 2. P. 1672–1692. https://doi.org/10.3390/molecules18021672Simirgiotis MJ, Schmeda-Hirschmann G, Bórquez J, Kennelly EJ. The Passiflora tripartita (Banana Passion) fruit: a source of bioactive flavonoid C-glycosides isolated by HSCCC and characterized by HPLC–DAD–ESI/MS/MS. Molecules. 2013;18(2):1672–1692. https://doi.org/10.3390/molecules18021672Asghari A., Barfi B., Barfi A., Saeidi I., Ghollasi Moud F., Peyrovi M., et al. Comparison between conventional solid phase extraction and its simplified method for HPLC determination of five flavonoids in orange, tangerine, and lime juice samples // Acta Chromatographica. 2014. Vol. 26. Issue 1. P. 157–175. https://doi.org/10.1556/AChrom.26.2014.1.12Asghari A, Barfi B, Barfi A, Saeidi I, Ghollasi Moud F, Peyrovi M, et al. Comparison between conventional solid phase extraction and its simplified method for HPLC determination of five flavonoids in orange, tangerine, and lime juice samples. Acta Chromatographica. 2014;26(1):157–175. https://doi.org/10.1556/AChrom.26.2014.1.12Costa D.L.M.G., Rinaldo D., Varanda E.A., de Sousa J.F., Nasser A.L.M., Silva A.C.Z., et al. Flavonoid detection in hydroethanolic extract of Pouteria torta (Sapotaceae) Leaves by HPLC-DAD and the determination of Its mutagenic activity // Journal of Medicinal Food. 2014. Vol. 17. Issue 10. P. 1103–1112. https://doi.org/10.1089/jmf.2013.0116Costa DLMG, Rinaldo D, Varanda EA, de Sousa JF, Nasser ALM, Silva ACZ, et al. Flavonoid detection in hydroethanolic extract of Pouteria torta (Sapotaceae) Leaves by HPLC-DAD and the determination of Its mutagenic activity. Journal of Medicinal Food. 2014;17(10):1103–1112. https://doi.org/10.1089/jmf.2013.0116Mehta J.P., Fultariya C.R., Parmar P.H., Vadia S.H., Golakiya B.A. Determination of phenolic acids and a flavonoid in Eleusine coracana (L.) by semi-preparative HPLC photo diode array detector // Journal of Analytical Chemistry. 2015. Vol. 70, Issue 3. P. 369–373. https://doi.org/10.1134/S1061934815030090Mehta JP, Fultariya CR, Parmar PH, Vadia SH, Golakiya BA. Determination of phenolic acids and a flavonoid in Eleusine coracana (L.) by semipreparative HPLC photo diode array detector. Journal of Analytical Chemistry. 2015;70(3):369–373. https://doi.org/10.1134/S1061934815030090Hsu B.Y., Lin S.W., Stephen Inbaraj B., Chen B.H. Simultaneous determination of phenolic acids and flavonoids in Chenopodium formosanum Koidz. (djulis) by HPLC-DAD-ESI–MS/MS // Journal of Pharmaceutical and Biomedical Analysis. 2017. Vol. 132. P. 109–116. https://doi.org/10.1016/j.jpba.2016.09.027Hsu BY, Lin SW, Stephen Inbaraj B, Chen BH. Simultaneous determination of phenolic acids and flavonoids in Chenopodium formosanum Koidz. (djulis) by HPLC-DAD-ESI–MS/MS. Journal of Pharmaceutical and Biomedical Analysis. 2017; 132:109–116. https://doi.org/10.1016/j.jpba.2016.09.027Gomes S.V.F., Portugal L.A., dos Anjos J.P., de Jesus O.N., de Oliveira E.J., David J.P., Davida J.M. Accelerated solvent extraction of phenolic compounds exploiting a Box-Behnken design and quantification of five flavonoids by HPLC-DAD in Passiflora species // Microchemical Journal. 2017. Vol. 132. P. 28–35. https://doi.org/10.1016/j.microc.2016.12.021Gomes SVF, Portugal LA, dos Anjos JP, de Jesus ON, de Oliveira EJ, David JP, Davida JM. Accelerated solvent extraction of phenolic compounds exploiting a Box-Behnken design and quantification of five flavonoids by HPLC-DAD in Passiflora species. Microchemical Journal. 2017;132:28–35. https://doi.org/10.1016/j.microc.2016.12.021Tewari R., Gupta M., Ahmad F., Rout P.K., Misra L., Patwardhan A., et al. Extraction, quantification and antioxidant activities of flavonoids, polyphenols and pinitol from wild and cultivated Saraca asoca bark using RP-HPLC-PDA-RI method // Industrial Crops and Products. 2017. Vol. 103. P. 73–80. https://doi.org/10.1016/j.indcrop.2017.03.036Tewari R, Gupta M, Ahmad F, Rout PK, Misra L, Patwardhan A, et al. Extraction, quantification and antioxidant activities of flavonoids, polyphenols and pinitol from wild and cultivated Saraca asoca bark using RP-HPLC-PDA-RI method. Industrial Crops and Products. 2017;103:73–80. https://doi.org/10.1016/j.indcrop.2017.03.036Ciric A., Jelikic-Stankov M., Cvijovic M., Djurdjevic P. Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities // Biomedical Chromatography. 2018. Vol. 32. Issue 4. Art. no. e4150. https://doi.org/10.1002/bmc.4150Ciric A, Jelikic-Stankov M, Cvijovic M, Djurdjevic P. Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities. Biomedical Chromatography. 2018;32(4). Art. no. e4150. https://doi.org/10.1002/bmc.4150Kuppusamy P., Lee K.D., Song C.E., Ilavenil S., Srigopalram S., Arasu M.V., et al. Quantification of major phenolic and flavonoid markers in forage crop Lolium multiflorum using HPLC-DAD // Revista Brasileira de Farmacognosia. 2018. Vol. 28. Issue 3. P. 282–288. https://doi.org/10.1016/j.bjp.2018.03.006Kuppusamy P, Lee KD, Song CE, Ilavenil S, Srigopalram S, Arasu MV, et al. Quantification of major phenolic and flavonoid markers in forage crop Lolium multiflorum using HPLC-DAD. Revista Brasileira de Farmacognosia. 2018;28(3):282–288. https://doi.org/10.1016/j.bjp.2018.03.006Häikiö E., Makkonen M., Julkunen-Tiitto R., Sitte J., Freiwald V., Silfver T., et al. Performance and secondary chemistry of two hybrid Aspen (Populus tremula L. x Populus tremuloides Michx.) clones in long-term elevated ozone ex-posure // Journal of Chemical Ecology. 2009. Vol. 35. Issue 6. P. 664–678. https://doi.org/10.1007/s10886-009-9644-2Häikiö E, Makkonen M, Julkunen-Tiitto R, Sitte J, Freiwald V, Silfver T, et al. Performance and secondary chemistry of two hybrid Aspen (Populus tremula L. x Populus tremuloides Michx.) clones in long-term elevated ozone ex-posure. Journal of Chemical Ecology. 2009;35(6):664–678. https://doi.org/10.1007/s10886-009-9644-2Лобанова И.Ю. Фенольные соединения листьев осины обыкновенной как основа создания препарата противовоспалительного действия // Вестник уральской медицинской академической науки. 2011. N 3/1. С. 67–68.Lobanova IYu. Phenolic compounds of leaves of Populus tremula L. as the basis for creation of preparation of anti-inflammatory action. Vestnik ural'skoi meditsinskoi akademicheskoi nauki. 2011;3/1:67–68. (In Russian)Лобанова И.Ю., Турецкова В.Ф. Выделение и изучение состава флавоноидов листьев осины обыкновенной // Химия растительного сырья. 2011. N 2. С. 117–122.Lobanova IYu, Turetskova VF. Isolation and study of the composition of flavonoids of aspen leaves. Khimija rastitel’nogo syr’ja = Chemistry of plant raw material. 2011;2:117–122. (In Russian)Банаев Е.В., Шишкин С.В., Воронкова М.С., Беланова А.П., Томошевич М.А. Морфологические и биохимические особенности Populus × canescens в природных популяциях Алтайского Края // Вестник Алтайского государственного аграрного университета. 2017. N 8 (154). С. 90–97.Banayev YeV, Shishkin SV, Voronkova MS, Belanova AP, Tomoshevich MA. Morphological and biochemical features of Populus × canescens in natural populations of the Altai region. Vestnik Altaiskogo gosudarstvennogo agrarnogo universiteta = Bulletin of Altai State Agricultural University. 2017; 8:90–97. (In Russian)Сычев К.С., Даванков В.А. Материалы и методы пробоподготовки в хроматографии: твердофазное концентрирование и адсорбционная очистка // Сорбционные и хроматографические процессы. 2004. Т. 4. Вып.1. С. 5–28.Sychev KS, Davankov VA. Materials and methods for sample preparation in chromatography: solid-phase concentration and adsorption purification. Sorbtsionnye i khromatograficheskie protsessy. 2004;4(1):5–28. (In Russian)Van Beek T.A. Chemical analysis of Ginkgo biloba leaves and extracts // Journal of Chromatograhpy. A. 2002. Vol. 967. Issue 1. P. 21–55. https://doi.org/10.1016/S0021-9673(02)00172-3Van Beek TA. Chemical analysis of Ginkgo biloba leaves and extracts. Journal of Chromatograhpy. A. 2002;967(1):21–55. https://doi.org/10.1016/S0021-9673(02)00172-3Юрьев Д.В., Эллер К.И., Арзамасцев А.П. Анализ флавонолгликозидов в препаратах и БАД на основе экстракта Ginkgo biloba // Фармация. 2003. N 2. С. 7–9.Yur'ev DV., Ehller KI, Arzamascev AP. Analysis flavonolglycosides in preparations and dietary supplements based on Ginkgo biloba extract. Farmatsiya = Pharmacy. 2003;2:7–9. (In Russian)Zagurskaya Yu.V., Siromlya T.I., Kotsupiy O.V., Babaeva E.Yu., Ufimtsev V.I. Physiological and biochemical characteristics of Populus tremula leaves in anthropogenic disturbed habitats // BIO Web of Conferences. 2018. Vol. 11. Art. no. 00050. 4 p. https://doi.org/10.1051/bioconf/20181100050Zagurskaya YuV, Siromlya TI, Kotsupiy OV, Babaeva EYu, Ufimtsev VI. Physiological and biochemical characteristics of Populus tremula leaves in anthropogenic disturbed habitats. BIO Web of Conferences. 2018;11. Art. no. 00050. 4 p. https://doi.org/10.1051/bioconf/20181100050Shkarina E.I., Maksimova T.V., Nikulina I.N., Lozovskaya E.L., Chumakova Z.V., Pakhomov V.P., et al. Effect of biologically active substances on the antioxidant activity of phytopreparations // Pharmaceutical Chemistry Journal. 2001. Vol. 35. Issue 6. P. 333–340. https://doi.org/10.1023/A:1012349806071Shkarina EI, Maksimova TV, Nikulina IN, Lozovskaya EL, Chumakova ZV, Pakhomov VP, et al. Effect of biologically active substances on the antioxidant activity of phytopreparations. Pharmaceutical Chemistry Journal. 2001;35(6):333–340. https://doi.org/10.1023/A:1012349806071

The authors declare that there are no conflicts of interest present.