vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ20.680vavilov-2842Research ArticleГЕНОМИКА И ТРАНСКРИПТОМИКАPLANT GENETICS AND BREEDINGВыбор оптимального метода скрининга генофонда люпина узколистного из коллекции ВИР по качественному и количественному составам алкалоидов семянSelection of an optimal method for screening the collection of narrow-leaved lupine held by the Vavilov Institute for the qualitative and quantitative composition of seed alkaloidshttps://orcid.org/0000-0002-5709-7961КушнареваА. В.KushnarevaA. V.https://orcid.org/0000-0003-3992-5353ШеленгаТ. В.ShelengаТ. V.https://orcid.org/0000-0001-6568-5248ПерчукИ. Н.PerchukI. N.https://orcid.org/0000-0002-8645-3072ЕгороваГ. П.EgorovaG. P.https://orcid.org/0000-0002-8595-1336МалышевЛ. Л.MalyshevL. L.https://orcid.org/0000-0002-3728-6968КервЮ. А.KervYu. A.ШавардаА. Л.ShavardaA. L.https://orcid.org/0000-0003-2808-7745ВишняковаМ. А.VishnyakovaM. A.m.vishnyakova.vir@gmail.comФедеральный исследовательский центр Всероссийский институт генетических ресурсов растений им. Н.И. Вавилова (ВИР)РоссияFederal Research Center the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR)Russian FederationФедеральный исследовательский центр Всероссийский институт генетических ресурсов растений им. Н.И. Вавилова (ВИР); Ботанический институт им. В.Л. Комарова Российской академии наук; Санкт-Петербургский государственный университетРоссияFederal Research Center the N.I. Vavilov All-Russian Institute of Plant Genetic Resources (VIR); V.L. Komarov Botanical Institute of the Russian Academy of Sciences; Saint-Petersburg State UniversityRussian Federation202031122020248829835Copyright © Кушнарева А.В., Шеленга Т.В., Перчук И.Н., Егорова Г.П., Малышев Л.Л., Керв Ю.А., Шаварда А.Л., Вишнякова М.А., 20202020Кушнарева А.В., Шеленга Т.В., Перчук И.Н., Егорова Г.П., Малышев Л.Л., Керв Ю.А., Шаварда А.Л., Вишнякова М.А.Kushnareva A.V., Shelengа Т.V., Perchuk I.N., Egorova G.P., Malyshev L.L., Kerv Y.A., Shavarda A.L., Vishnyakova M.A.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/2842

Люпин узколистный (Lupinus аngustifolius L.) – широко возделываемая кормовая и сидеральная зернобобовая культура с продовольственным потенциалом. Однако наличие в семенах люпина вторичных метаболитов – алкалоидов – значительно влияет на качество получаемого сырья, снижая его питательную ценность. Кроме того, высокие концентрации алкалоидов токсичны для человека и животных. Поэтому для селекции люпина актуальны сведения об изменчивости содержания алкалоидов в семенах у разных генотипов и поиск в генофонде источников их низкой концентрации. Коллекция генетических ресурсов люпина узколистного Всероссийского института генетических ресурсов растений им. Н.И. Вавилова предоставляет широкие возможности для такого поиска на основе массового скрининга. В свою очередь масштабный скрининг генофонда предполагает выбор оптимального метода оценки содержания алкалоидов в семенах: легко воспроизводимого, наиболее доступного с финансовой точки зрения и наименее трудо- и времязатратного. Представлены результаты поиска такого метода. Сравнивали качественные и количественные показатели при извлечении целевых веществ многокомпонентными смесями и отдельными реагентами (хлороформом, метанолом) и анализе экстрактов посредством газовой хроматографии, сопряженной с масс-спектрометрией. Также использовали высокоэффективную жидкостную хроматографию с масс-спектрометрией. Обнаружены пять алкалоидов, присутствовавших в экстрактах всех типов: люпанин и 13-гидроксилюпанин (доминирующие), ангустифолин, спартеин и изолюпанин. Наиболее полное извлечение алкалоидов отмечено при использовании экстрагента с добавлением щелочного агента – 425 мг/100 г. Минимальная экстракция зарегистрирована при извлечении хлороформом – 216 мг/100 г. Достоверность отличий подтверждена статистически.

Narrow-leaved lupine (Lupinus аngustifolius L.) is a widely cultivated leguminous forage and green manure crop with a potential for human nutrition. However, the presence of secondary metabolites – alkaloids – in lupine seeds considerably affects the quality of raw produce, reducing its nutritive value; in addition, high concentrations of alkaloids are toxic to humans and animals. Therefore, plant breeders working with lupine need to gain knowledge about the variability of alkaloid content in seeds of different genotypes and search for the sources of their low concentrations in the crop’s gene pool. The collection of narrow-leaved lupine genetic resources held by the N.I. Vavilov Institute of Plant Genetic Resources (VIR) offers wide opportunities for such search by means of mass screening. For its part, largescale gene pool screening requires the selection of an optimal technique to measure alkaloid content in seeds, so that it would be easily reproducible and as little labor-, time- and fund-consuming as possible. The results of the search for such method are presented. Qualitative and quantitative indices were compared when target compounds had been extracted with multicomponent mixtures and individual reagents (chloroform, methanol, etc.) and the extracts analyzed using gas chromatography-mass spectrometry. High-performance liquid chromatography combined with mass spectrometry was also employed. Five major alkaloids were found to be present in all types of extracts: lupanine, 13-hydroxylupanine (dominant ones), angustifoline, sparteine, and isolupanine. The fullest extraction of alkaloids was observed when the extractant with an added alkaline agent was used (425 mg/100 g). The lowest level of extraction was registered with chloroform (216 mg/100 g). The significance of the differences was confirmed statistically.

Lupinus angustifolius L.алкалоидыэкстракциялюпанин13-гидроксилюпанинангустифолинспартеинизолюпанинLupinus angustifolius L.alkaloidsextraction techniqueslupanine13-hydroxylupanineangustifolinesparteineisolupanineThe work was supported by the Russian Foundation for Basic Research, project No. 20-016-00072-A, and budgetary project No. 0662-2019-0002ReferencesAdejoke H.T., Louis H., Amusan O.O., Apebende G. A Review on Classes, Extraction, Purification and Pharmaceutical Importance of Plants Alkaloids. J. Med. Chem. Sci. 2019;2(4):130-139. DOI 0.26655/JMCHEMSCI.2019.8.2.Adejoke H.T., Louis H., Amusan O.O., Apebende G. A Review on Classes, Extraction, Purification and Pharmaceutical Importance of Plants Alkaloids. J. Med. Chem. Sci. 2019;2(4):130-139. DOI 0.26655/JMCHEMSCI.2019.8.2.Ageeva P.A., Pochutina N.А., Pigareva S.A. Comparative characteristics of grain and green mass quality of fodder narrow-leafed lupin varieties. Adaptivnoe Kormoproizvodstvo = Adaptive Fodder Production. 2018;1:42-48. (in Russian)Ageeva P.A., Pochutina N.А., Pigareva S.A. Comparative characteristics of grain and green mass quality of fodder narrow-leafed lupin varieties. Adaptivnoe Kormoproizvodstvo = Adaptive Fodder Production. 2018;1:42-48. (in Russian)Аrtyukhov А.I. Lupin species adaptation to agrolandscape of Russia. Zernobobovye i Krupyanye Kultury = Legumes and Groat crops. 2015;1(13):60-67. (in Russian)Аrtyukhov А.I. Lupin species adaptation to agrolandscape of Russia. Zernobobovye i Krupyanye Kultury = Legumes and Groat crops. 2015;1(13):60-67. (in Russian)Cheeke P.R., Kelly J.D. Metabolism, toxicity and nutritional implications of quinolizidine (lupin) alkaloids. In: Recent advances of research in antinutritional factors in legume seeds. Material of 1st International Workshop on Antinutritional Factors (ANF) in Legume Seeds. Netherlands, 1989;189-201.Cheeke P.R., Kelly J.D. Metabolism, toxicity and nutritional implications of quinolizidine (lupin) alkaloids. In: Recent advances of research in antinutritional factors in legume seeds. Material of 1st International Workshop on Antinutritional Factors (ANF) in Legume Seeds. Netherlands, 1989;189-201.Couch J. Relative Toxicity of the Lupine Alkaloids. J. Agric. Res. 1926; XXXII:51-67.Couch J. Relative Toxicity of the Lupine Alkaloids. J. Agric. Res. 1926; XXXII:51-67.Erdemoglu N., Ozkan S., Tosun F. Alkaloid profile and antimicrobial activity of Lupinus angustifolius L. alkaloid extract. Phytochem Rev. 2007;6(1):197-201. DOI 10.1007/s11101-006-9055-8.Erdemoglu N., Ozkan S., Tosun F. Alkaloid profile and antimicrobial activity of Lupinus angustifolius L. alkaloid extract. Phytochem Rev. 2007;6(1):197-201. DOI 10.1007/s11101-006-9055-8.Ermakov A.I., Arasimovich V.V., Yarosh N.P., Peruanski Y.V., Lukovnikova G.A., Ikonnikova M.I. Methods of biochemical study of plants. Leningrad, 1987. (in Russian)Ermakov A.I., Arasimovich V.V., Yarosh N.P., Peruanski Y.V., Lukovnikova G.A., Ikonnikova M.I. Methods of biochemical study of plants. Leningrad, 1987. (in Russian)Frick K.M., Kamphuis L.G., Siddique K.H., Singh K.B., Foley R.C. Quinolizidine alkaloid biosynthesis in lupins and prospects for grain quality improvement. Front. Plant Sci. 2017;8(87):1-12. DOI 10.3389/fpls.2017.00087.Frick K.M., Kamphuis L.G., Siddique K.H., Singh K.B., Foley R.C. Quinolizidine alkaloid biosynthesis in lupins and prospects for grain quality improvement. Front. Plant Sci. 2017;8(87):1-12. DOI 10.3389/fpls.2017.00087.Hatzold T., Elmadfa I., Gross R., Wink M., Hartmann T., Witte L. Quinolizidine alkaloids in seeds of Lupinus mutabilis. J. Agric. Food Chemistry. 1983;31(5):934-938. DOI 10.1021/jf00119a003.Hatzold T., Elmadfa I., Gross R., Wink M., Hartmann T., Witte L. Quinolizidine alkaloids in seeds of Lupinus mutabilis. J. Agric. Food Chemistry. 1983;31(5):934-938. DOI 10.1021/jf00119a003.Islam S., Ma W., Ma J., Buirchell B.J., Appels R., Yan G. Diversity of seed protein among the Australian narrow-leafed lupin (Lupinus angustifolius L.) cultivars. Crop Past. Sci. 2011;62(9):765-775. DOI 10.1071/CP11046.Islam S., Ma W., Ma J., Buirchell B.J., Appels R., Yan G. Diversity of seed protein among the Australian narrow-leafed lupin (Lupinus angustifolius L.) cultivars. Crop Past. Sci. 2011;62(9):765-775. DOI 10.1071/CP11046.Korol’ V.F., Lahmotkina G.N. The use of ultrasound in the isolation of antialimentary substances from lupine grain. Yuzhno-Sibirskiy Nauchnyy Vestnik = South Siberian Scientific Bulletin. 2018;1(21): 27-34. (in Russian)Korol’ V.F., Lahmotkina G.N. The use of ultrasound in the isolation of antialimentary substances from lupine grain. Yuzhno-Sibirskiy Nauchnyy Vestnik = South Siberian Scientific Bulletin. 2018;1(21): 27-34. (in Russian)Krasilnikov V.N., Pankina I.A. Study of the chemical composition and technological properties of narrow-leafed lupine seeds with the aim of creating combined food products. Proceedings of the international conference “The problem of vegetable protein deficiency and ways to overcome it.” Minsk, 2006;119-122. (in Russian)Krasilnikov V.N., Pankina I.A. Study of the chemical composition and technological properties of narrow-leafed lupine seeds with the aim of creating combined food products. Proceedings of the international conference “The problem of vegetable protein deficiency and ways to overcome it.” Minsk, 2006;119-122. (in Russian)Kuptsov N.S., Takunov I.P. Lupin (Genetic, selection, heterogeneous sowing). Bryansk, 2006. (in Russian)Kuptsov N.S., Takunov I.P. Lupin (Genetic, selection, heterogeneous sowing). Bryansk, 2006. (in Russian)Kurlovich B.S., Repiev S.I., Shchelko L.G., Budanova V.I., Petrova M.V., Buravtseva T.V., Stankevich A.K., Leokenе L.V., Benken I.I., Rybnikova V.A., Kartuzova L.T., Zolotov S.V., Alexandrova T.G., Debely G.A., Taranuho G.I., Teplyakova T.E., Malysh L.K. The gene pool and breeding of legumes (lupins, vetch, soy, beans). St. Petersburg, 1995;9-116. (in Russian)Kurlovich B.S., Repiev S.I., Shchelko L.G., Budanova V.I., Petrova M.V., Buravtseva T.V., Stankevich A.K., Leokenе L.V., Benken I.I., Rybnikova V.A., Kartuzova L.T., Zolotov S.V., Alexandrova T.G., Debely G.A., Taranuho G.I., Teplyakova T.E., Malysh L.K. The gene pool and breeding of legumes (lupins, vetch, soy, beans). St. Petersburg, 1995;9-116. (in Russian)Markova O.M., Karpenko V.A., Sayshkina A.S., Likhota T.T. Use of physicochemical methods in the analysis of pharmaceuticals of a vegetative origin. Bulletin of Voronezh State University. Series: Chemistry. Biology. Pharmacy. 2003;1:99-100. (in Russian)Markova O.M., Karpenko V.A., Sayshkina A.S., Likhota T.T. Use of physicochemical methods in the analysis of pharmaceuticals of a vegetative origin. Bulletin of Voronezh State University. Series: Chemistry. Biology. Pharmacy. 2003;1:99-100. (in Russian)Mironenko A.V. Methods for the determination of alkaloids. Minsk, 1966;182 p.Mironenko A.V. Methods for the determination of alkaloids. Minsk, 1966;182 p.Orekhov A.P. Chemistry of alkaloids. Moscow, 1955. (in Russian)Orekhov A.P. Chemistry of alkaloids. Moscow, 1955. (in Russian)Pankina I.A., Borisova L.M. Аlkaloidness investigation of Lupin seeds. Scientific journal ITMO University. Series “Processes and Food Production Equipment.” 2015;4:80-87. (in Russian)Pankina I.A., Borisova L.M. Аlkaloidness investigation of Lupin seeds. Scientific journal ITMO University. Series “Processes and Food Production Equipment.” 2015;4:80-87. (in Russian)Popova N.V., Potoroko I.Yu. Increase of efficiency of biologically active substance extraction from vegetable raw material by ultrasonic treatment. Bulletin of the South Ural State University. Series: Food and Biotechnology. 2018;6(1):1-9. (in Russian)Popova N.V., Potoroko I.Yu. Increase of efficiency of biologically active substance extraction from vegetable raw material by ultrasonic treatment. Bulletin of the South Ural State University. Series: Food and Biotechnology. 2018;6(1):1-9. (in Russian)Resta D., Boschin G., D’agostina A., Arnoldi A. Evaluation of total quinolizidine alkaloids content in lupin flours, lupin-based ingredients, and foods. Mol. Nutr. Food Res. 2008;52(4):490-495. DOI 10.1002/mnfr.200700206.Resta D., Boschin G., D’agostina A., Arnoldi A. Evaluation of total quinolizidine alkaloids content in lupin flours, lupin-based ingredients, and foods. Mol. Nutr. Food Res. 2008;52(4):490-495. DOI 10.1002/mnfr.200700206.Roberts M.F., Wink M. Alkaloids: Biochemistry, Ecology, and Medicinal Applications. 2013.Roberts M.F., Wink M. Alkaloids: Biochemistry, Ecology, and Medicinal Applications. 2013.Sengbusch R. Bitterstoffarme Lupinen. Zuchter. 1931;H.4:93-109.Sengbusch R. Bitterstoffarme Lupinen. Zuchter. 1931;H.4:93-109.Smirnova M.I. Biochemistry of lupine. In: Biochemistry of cultivated plants. Moscow-Leningrad: Selhozgis Publ., 1938;2:270-327. (in Russian)Smirnova M.I. Biochemistry of lupine. In: Biochemistry of cultivated plants. Moscow-Leningrad: Selhozgis Publ., 1938;2:270-327. (in Russian)Vilkhu K., Mawson R., Simons L., Bates D. Applications and opportunities for ultrasound assisted extraction in the food industry. A review. Innov. Food Sci. Emerg. Technol. 2008;9:161-169. DOI 10.1016/j.ifset.2007.04.014.Vilkhu K., Mawson R., Simons L., Bates D. Applications and opportunities for ultrasound assisted extraction in the food industry. A review. Innov. Food Sci. Emerg. Technol. 2008;9:161-169. DOI 10.1016/j.ifset.2007.04.014.Vishnyakova M.A., Seferova I.V., Buravtseva T.V., Burlyaeva M.O., Semenova E.V., Filipenko G.I., Alexandrova T.G., Egorova G.P., Yankov I.I., Bulyntsev S.V., Gerasimova T.V., Drugova E.V. Guidelines “VIR Global Collection of Grain Legumes Crop Resources: Replenishment, Preservation and Study.” St. Petersburg, 2018. (in Russian)Vishnyakova M.A., Seferova I.V., Buravtseva T.V., Burlyaeva M.O., Semenova E.V., Filipenko G.I., Alexandrova T.G., Egorova G.P., Yankov I.I., Bulyntsev S.V., Gerasimova T.V., Drugova E.V. Guidelines “VIR Global Collection of Grain Legumes Crop Resources: Replenishment, Preservation and Study.” St. Petersburg, 2018. (in Russian)Williams W., Harrison J.E.M., Jayasekera S. Genetical control of alkaloid production in Lupinus mutabilis and the effect of a mutant allele Mutal isolated following chemical mutagenesis. Euphytica. 1984; 33(3):811-817. DOI 10.1007/BF00021907.Williams W., Harrison J.E.M., Jayasekera S. Genetical control of alkaloid production in Lupinus mutabilis and the effect of a mutant allele Mutal isolated following chemical mutagenesis. Euphytica. 1984; 33(3):811-817. DOI 10.1007/BF00021907.Wink M. Quinolizidine alkaloids: biochemistry, metabolism and function in plants and cell suspension cultures. Planta Med. 1987;53(6): 509-514. DOI 10.1055/s-2006-962797.Wink M. Quinolizidine alkaloids: biochemistry, metabolism and function in plants and cell suspension cultures. Planta Med. 1987;53(6): 509-514. DOI 10.1055/s-2006-962797.Zharylgasina G.T., Nurmaganbetov Zh.S., Turmukhambetov A.Zh., Adekenov S.M. Modern methods for extraction of alkaloids from plant materials. Sci. Pract. J. 2014;3-4:105-122. (in Russian)Zharylgasina G.T., Nurmaganbetov Zh.S., Turmukhambetov A.Zh., Adekenov S.M. Modern methods for extraction of alkaloids from plant materials. Sci. Pract. J. 2014;3-4:105-122. (in Russian)

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