molecules Article LC-MS/MS Screening of Phenolic Compounds in Wild and Cultivated Grapes Vitis amurensis Rupr. Mayya Razgonova 1,2,* , Alexander Zakharenko 1,3, Konstantin Pikula 1 , Yury Manakov 3, Sezai Ercisli 4 , Irina Derbush 1, Evgeniy Kislin 1, Ivan Seryodkin 5, Andrey Sabitov 1, Tatiana Kalenik 2 and Kirill Golokhvast 1,2,3,5 1 N.I. Vavilov All-Russian Institute of Plant Genetic Resources, B. Morskaya 42-44, 190000 Saint-Petersburg, Russia; [email protected] (A.Z.); [email protected] (K.P.); [email protected] (I.D.); [email protected] (E.K.); [email protected] (A.S.); [email protected] (K.G.) 2 Far Eastern Federal University, 10 Ajax Bay, Russky Island, 690922 Vladivostok, Russia; [email protected] 3 Siberian Federal Scientific Centre of Agrobiotechnology, Centralnaya, Presidium, 633501 Krasnoobsk, Russia; [email protected] 4 Department of Horticulture, Agricultural Faculty, Ataturk University, 25240 Erzurum, Turkey; [email protected] 5 Pacific Geographical Institute, Far Eastern Branch of the Russian Academy of Sciences, Radio 7, 690041 Vladivostok, Russia; [email protected] * Correspondence: [email protected] Abstract: This work represents a comparative metabolomic study of extracts of wild grapes obtained from six different places in the Primorsky and Khabarovsk territories (Far East Russia) and extracts of Citation: Razgonova, M.; grapes obtained from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources Zakharenko, A.; Pikula, K.; Manakov, Y.; Ercisli, S.; Derbush, I.; Kislin, E.; (St. Petersburg). The metabolome analysis was performed by liquid chromatography in combination Seryodkin, I.; Sabitov, A.; Kalenik, T.; with ion trap mass spectrometry. The results showed the presence of 118 compounds in ethanolic et al. LC-MS/MS Screening of extracts of V. amurensis grapes. In addition, several metabolites were newly annotated in V. amurensis. Phenolic Compounds in Wild and The highest diversity of phenolic compounds was identified in the samples of the V. amurensis grape Cultivated Grapes Vitis amurensis collected in the vicinity of Vyazemsky (Khabarovsk Territory) and the floodplain of the Arsenyevka Rupr.. Molecules 2021, 26, 3650. River (Primorsky Territory), compared to the other wild samples and cultural grapes obtained in the https://doi.org/10.3390/ collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources. molecules26123650 Keywords: Amur grape; identification; mass spectrometry; metabolites; metabolomics Academic Editor: Eulogio J. Llorent-Martínez Received: 17 May 2021 1. Introduction Accepted: 14 June 2021 Published: 15 June 2021 The appearance of the first representatives of the Vitaceae family (genus Vitis) dates from the Upper Cretaceous period [1]. Several types of fossil grapes of genus Vitis have Publisher’s Note: MDPI stays neutral been found in different parts of North America [2]. In the Eocene, representatives of the with regard to jurisdictional claims in genus Vitis were widespread in Eurasia and the Far North [2]. In the Paleogene, one of the published maps and institutional affil- best-preserved species of fossil grapes Vitis sachalinensis Krysht. was found and described iations. in the sediments of the Sakhalin Island, the Russian Far East. These data show that the evolution of the vine in the territory of Russia proceeded from ancient times. Moreover, now wild grapes of the genus Vitis grow in many Russian regions [3,4]. At the same time, there is very little information about the culture of East Asian grapes. Copyright: © 2021 by the authors. Grape berries contain 65–85% water; 10–33% sugar (glucose and fructose); flofaben; Licensee MDPI, Basel, Switzerland. gallic acid; quercetin; oenin; the glycosides monodelphinidin and delphinidin; the acids This article is an open access article malic, hydrosilicic, ortho-hydroxybenzoic, phosphoric, tartaric, citric, succinic, formic, distributed under the terms and pectin, and tannins; salts of potassium; magnesium; calcium; manganese; cobalt; iron conditions of the Creative Commons vitamins B1, B2, B6, B12, A, C, P, and PP; folic acid; and enzymes. The dominant class Attribution (CC BY) license (https:// of biologically active compounds of fruits and especially grape ridges are flavonoids, in creativecommons.org/licenses/by/ particular complexes of oligomeric proanthocyanidins (condensed tannins), which are 4.0/). Molecules 2021, 26, 3650. https://doi.org/10.3390/molecules26123650 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 3650 2 of 17 polymeric forms of flavonoids from the group of catechins, and their monomeric units, namely catechins and leuсoanthocyanidins [5]. Many studies have been devoted to the biological activity of flavonoids and complexes of oligomeric proanthocyanidins [6,7]. Complexes of oligomeric proanthocyanidins act as traps of free radicals and block the process of lipid peroxidation of biological mem- branes [8,9]. Their antioxidant activity is many times higher than that of vitamins E and C. They can inhibit the activity of many enzymes (hydrolase, oxidoreductase, kinase, trans- ferase, among others) [10]. Due to the wide spectrum of action, the active compounds of the grapes V. amurensis have a pronounced positive effect on various organs and systems of the body, such as antihypertensive and vasostrengthening effects, as well as antidiabetic, anti- inflammatory, antiallergy, anticarcinogenic, antistress, radioprotective, and antirheumatic effects. Moreover, flavonoids have an anti-Alzheimer’s activity [11–13]. This work presents a detailed comparative study of the metabolomic composition of wild V. amurensis grape berry extracts taken from six different locations of the Russian Far East and four cultural specimens of V. amurensis obtained from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources (St. Petersburg). High-performance liquid chromatography (HPLC) in combination with tandem mass spectrometry was used to identify target analytes in the extracts. Previously, the authors carried out metabolomic studies of Far Eastern plant species, such as Schizandra chinensis, Rhodiola rosea, Rhododendron adamsii, and Panax ginseng [14,15]. 2. Results The metabolome of ten samples of wild and cultural V. amurensis was analyzed and compared. A combination of both ionization modes (positive and negative) in MS full scan mode was applied for the molecular mass determination of the compounds in ethanolic ex- tracts of V. amurensis. Compound identification was performed by comparing the observed m/z values and the fragmentation patterns with the literature. The list of compounds identified in the ethanolic extract of V. amurensis are represented in Table A1. The 118 com- pounds shown in Table A1 belong to different phenolic families, namely anthocyanidins, flavones, flavonols, flavan-3-ols, flavanones, hydroxycinnamic acids, hydroxybenzoic acids, stilbenes, and tannins. 2.1. Anthocyanidins and Anthocyanins A total of 18 anthocyanin compounds have been identified in the analyzed samples of V. amurensis (Table1). The anthocyanins pelargonidin-3- O-glucoside, cyanidin-3-O- glucoside, and petunidin-3-(6-O-coumaroyl) glucoside have already been characterized as a component of Far East V. amurensis [16]. The anthocyanins malvidin-3-O-acetylhexoside, delphinid-3,5-O-diglucoside, malvidin-3-O-rutinoside, malvidin 3-acetyl-5-glucoside, petu- nidin 3-coumaroylglucoside-5-O-glucoside, and malvidin 3-coumaroylglucoside-5-O- glucoside were only found in the extracts of cultivated V. amurensis (St. Petersburg). Molecules 2021, 26, 3650 3 of 17 Table 1. Anthocyanins identified in the ethanolic extracts of V. amurensis. No. Identified Compound ARS ART KAL PAK RIK VZK SPB-1 SPB-2 SPB-3 SPB-4 Cyanidin 1. + + + + + 3,5-O-diglucoside Cyanidin-3-O-glucoside 2. [Cyanidin + 3-O-beta-D-glucoside] Delphinidin 3. + + 3-O-glucoside Delphinidin-3,5-O- 4. + diglucoside Malvidin 3-(6-O-acetyl) 5. + + + glucoside Malvidin 6. 3-(6-O-coumaroyl) + + glucoside Malvidin 7. + + + + + + 3-(6’-p-caffeoylglucoside) 8. Malvidin 3,5-diglucoside + + + + + + + + Malvidin 9. 3-coumaroylglucoside-5- + O-glucoside Malvidin 3-O-acetyl 10. + hexoside 11. Malvidin 3-O-glucoside + + + + + + + + Pelargonidin-3-O- 12. glucoside + (callistephin) Peonidin-3,5-О- diglucoside [peonin; 13. + + + + + + peonidin 3-glucoside-5-glucoside] 14. Peonidin-3-O-glucoside + + + Petunidin 15. 3-(6-O-coumaroyl) + glucoside Petunidin 16. 3-coumaroylglucoside-5- + O-glucoside Petunidin 3-O-glucoside- 5-O-glucoside [Petunidin 17. + + + + + 3,5-di-O-beta-D- glucoside] 18. Petunidin-3-O-glucoside + Total number 2 10 5 1 3 8 7 8 6 6 ARS, wild V. amurensis sample obtained from floodplain of the Arsenyevka River (Primorsky Territory); ART, wild V. amurensis sample obtained from the vicinity of Artem (Primorsky Territory); KAL, wild V. amurensis sample obtained from the vicinity of Kalinovka (Primorsky Territory); PAK, wild V. amurensis sample obtained from the Pakhtusov Islands (Sea of Japan); RIK, wild V. amurensis sample obtained from Rikord Island (Sea of Japan); VZK, wild V. amurensis sample obtained from the vicinity of Vyazemsky (Khabarovsk Territory); SPB-1, SPB-2, SPB-3, and SPB-4, samples of cultivated V. amurensis provided by N.I. Vavilov All-Russian Institute of Plant Genetic Resources (St. Petersburg). 2.2. Other Flavonoid Compounds A total of 42 flavonoid compounds were identified in analyzed V. amurensis samples (Table2). The flavonols dihydrokaempferol, kaempferide, mearnsetin, kaempferol-3- O-glucoside, dihydrokaempferol