LC-MS/MS Screening of Phenolic Compounds in Wild and Cultivated Grapes Vitis amurensis Rupr.

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 grapes obtained from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources (St. Petersburg). The metabolome analysis was performed by liquid chromatography in combination with ion trap mass spectrometry. The results showed the presence of 118 compounds in ethanolic extracts of V. amurensis grapes. In addition, several metabolites were newly annotated in V. amurensis. The highest diversity of phenolic compounds was identified in the samples of the V. amurensis grape collected in the vicinity of Vyazemsky (Khabarovsk Territory) and the floodplain of the Arsenyevka River (Primorsky Territory), compared to the other wild samples and cultural grapes obtained in the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources.

1. Introduction

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 been found in different parts of North America [2]. In the Eocene, representatives of the genus Vitis were widespread in Eurasia and the Far North [2]. In the Paleogene, one of the best-preserved species of fossil grapes Vitis sachalinensis Krysht. was found and described 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.

Grape berries contain 65–85% water; 10–33% sugar (glucose and fructose); flofaben; gallic acid; quercetin; oenin; the glycosides monodelphinidin and delphinidin; the acids malic, hydrosilicic, ortho-hydroxybenzoic, phosphoric, tartaric, citric, succinic, formic, pectin, and tannins; salts of potassium; magnesium; calcium; manganese; cobalt; iron vitamins B1, B2, B6, B12, A, C, P, and PP; folic acid; and enzymes. The dominant class of biologically active compounds of fruits and especially grape ridges are flavonoids, in particular complexes of oligomeric proanthocyanidins (condensed tannins), which are 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 membranes [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, transferase, 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 [11,12,13].

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 extracts 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 compounds shown in Table A1 belong to different phenolic families, namely anthocyanidins, flavones, flavonols, flavan-3-ols, flavanones, hydroxycinnamic acids, hydroxybenzoic acids, stilbenes, and tannins.

Table A1

The list of compounds identified in ethanolic extracts of V. amurensis.

No.	Identified Compound	Molecular Formula	Calculated Mass	Precursor Ion, m/z		Fragment Ions, m/z	References
				[M–H]–	[M+H]+
	Anthocyanins
1.	Cyanidin 3,5-O-diglucoside	C27H31O16	611.5335		611	287; 449; 269; 231; 199; 161; 231; 213; 189; 175; 147	[35,36]
2.	Cyanidin-3-O-glucoside	C21H21O11	449.3848		449	287; 206; 143	[19,20,35,37,38]
3.	Delphinidin 3-O-glucoside	C21H21O12+	465.3905		465	303; 257; 229; 201; 165; 239; 213; 173; 145; 117	[19,20,21,39]
4.	Delphinidin-3,5-O-diglucoside	C27H30O17	626.5169		627	465; 303; 257; 153; 229; 155	[18,40]
5.	Malvidin 3,5-O-diglucoside	C32H31O15	655.5795		655	493; 331; 315; 179; 313	[17,20,21]
6.	Malvidin 3-(6-O-acetyl) glucoside	C25H27O13	535.478		537	331; 299; 261; 243; 211; 154; 111	[20,39]
7.	Malvidin 3-(6-O-coumaroyl) glucoside	C32H31O14	639.5801		639	331; 315; 299; 270; 242; 179; 150; 287; 213	[20,39,40]
8.	Malvidin 3-coumaroylglucoside-5-O-glucoside	C35H45O21	801.7192		801	639; 493; 331; 315; 287; 270; 242; 300	[39]
9.	Malvidin 3-O-acetyl hexoside	C25H27O14	535.479		537	331; 305; 261; 207; 185; 255; 229; 211	[17]
10.	Malvidin 3-O-glucoside	C23H25O12	493.4374		493	331; 315; 179	[20,39,40]
11.	Pelargonidin-3-O-glucoside (callistephin)	C21H21O10	433.3854		433	414; 271; 172; 226; 116	[35,39,41]
12.	Peonidin-3,5-О-diglucoside [Peonin; Peonidin 3-glucoside-5-glucoside]	C28H33O16	625.5520		625	301; 463; 286; 258	[21,39,40]
13.	Peonidin-3-O-glucoside	C22H23O11 +	463.4114		463	301; 286; 268; 258; 230; 202; 174; 121	[20,39,41]
14.	Petunidin 3-(6-O-coumaroyl) glucoside	C31H29O14	625.553		625	317; 302; 274; 218	[20,39,40]
15.	Petunidin 3-coumaroylglucoside-5-O-glucoside	C34H43O21	787.6926		787	625; 479; 317; 301; 246; 302; 274; 228	[39,40]
16.	Petunidin 3-galactoside	C22H23O12+	479.4108		479	317; 302; 273	[19,20,21,39]
17.	Petunidin 3,5-diglucoside	C28H33O17	641.5514		641	317; 479; 420; 257; 302; 274; 228	[39,40]
	Flavonols
18.	Dihydrokaempferol	C15H12O6	288.2522		289	271; 199; 127; 243; 189; 118	[22,42]
19.	Dihydrokaempferol glucoside	C21H22O11	450.3928	449		287; 227; 269; 225; 149	[27]
20.	Dihydroquercetin (taxifolin; taxifoliol)	C15H12O7	304.2516		305	259; 149; 199; 241; 159; 171	[20,43,44]
21.	Herbacetin [3,5,7,8-tetrahydroxy-2-(4-hydro- xyphenyl)-4H-chromen-4-one]	C15H10O7	302.2357	301		179; 273; 121; 151	[24,45]
22.	Hyperoside (quercetin 3-O-galactoside; hyperin)	C21H20O12	464.3763	463		301; 179; 257; 255; 147	[43,46,47,48]
23.	Isorhamnetin [isorhamnetol; quercetin 3’-methyl ether; 3-methylquercetin]	C16H12O7	316.2623		317	299; 270; 230; 207;177; 165;147; 123; 147; 123; 119	[49,50]
24.	Isorhamnetin 3-O-glucoside	C22H22O12	478.4029		479	317; 301; 257; 274; 228; 150	[20,47,51]
25.	Isorhamnetin 3-O-rhamonoside	C22H22O11	462.4035	461		315; 152; 219	[28,49]
26.	Kaempferide	C16H12O6	300.2629		301	283; 265; 239; 211; 185; 133; 151	[20,24,26]
27.	Kaempferol	C15H10O6	286.2363		287	269; 227; 153	[20,24,50]
28.	Kaempferol diglycoside	C27H30O16	610.5175		611	449; 287; 229; 165; 213; 111	[52,53]
29.	Kaempferol glycoside	C21H20O11	448.3769		449	287; 269; 217	[20,47]
30.	Mearnsetin	C16H12O8	332.2617		333	318; 301; 273; 245; 193; 165; 139; 289; 271; 219; 153; 136	[49]
31.	Myricetin	C15H10O8	318.2351	317		273; 191; 255; 229; 205; 187; 163; 125; 227	[20,28,54]
32.	Myricetin-3-O-galactoside	C21H20O13	480.3757	479		299; 153; 271; 243; 171	[47,48,55]
33.	Quercetin	C15H10O7	302.2357		303	285; 163; 267; 159; 239	[20,24,37,43]
34.	Quercetin 3-O-glucoside [Isoquercitrin; Hirsutrin]	C21H20O12	464.3763		465	303; 285; 257; 229; 201; 150; 155	[20,27,47,56]
35.	Quercetin-3-O-glucuronide	C21H18O13	478.3598	477		301; 179; 273; 151	[39,47,57]
36.	Quercetin-O-dihexoside	C27H30O17	626.5179		627	303; 257; 150; 229	[51,58]
37.	Rutin (quercetin 3-O-rutinoside)	C27H30O16	610.5175		611	303; 229; 257	[27,35,37,56]
38.	Taxifolin-3-O-glucoside	C21H22O12	466.3922		467	449; 303; 188; 287; 132; 260	[20]
	Flavones
39.	Apigenin [5,7-dixydroxy-2-(40hydroxyphenyl)-4H-chromen-4-one]	C15H10O5	270.2369		271	253; 181; 137	[56,59,60]
40.	Luteolin	C15H10O6	286.2363		287	271; 225; 175; 158	[43,56,59,60]
41.	Diosmetin [luteolin 4’-methyl ether; salinigricoflavonol]	C16H12O6	300.2629		301	286; 258; 229; 184; 153; 124	[61,62,63]
42.	Cirsimaritin [scrophulein; 4’,5-dihydroxy-6,7-dimethoxyflavone; 7-methylcapillarisin]	C17H14O6	314.2895	313		298; 247; 151; 270	[24]
43.	Nevadensin	C18H16O7	344.3154	343		328; 259; 313; 269	[24,63]
44.	Syringetin	C17H14O8	346.2883	345		330; 315; 246; 151; 287; 271; 203; 183; 163	[28]
45.	Pentahydroxy trimethoxy flavone	C18H16O10	392.3136		393	378; 347; 317; 284; 246; 206; 349; 321; 284; 193; 322; 304;282; 196; 154	[28]
46.	Apigenin diglycoside	C21H20O10	432.3775		433	414; 287; 186; 241; 158	[20,56,64,65]
47.	Vitexin [apigenin 8-C-glucoside]	C21H20O10	432.3775	431		249; 221; 192	[57,66,67]
48.	Luteolin diglycoside	C21H20O11	448.3769		449	287; 213; 137; 185	[20,55,56,66,68]
49.	Isovitexin 6”-O-deoxyhexoside [apigenin 6-C-glucoside 6”-O-deoxyhexoside]	C27H30O14	578.5187		579	415; 297; 177; 397; 344; 362	[66]
50.	Vitexin glucoside	C27H30O15	594.5181		595	415; 353; 283; 265; 176	[66]
51.	Apigenin glucoside	C29H32O15	620.5554		621	561; 547; 461; 533; 461; 433	[66]
	Flavan-3-ols
52.	Catechin [D-catechol]	C15H14O6	290.2681	289		245; 205; 203; 188	[43,49,55,57]
53.	Epicatechin	C15H14O6	290.2681		291	272; 175; 130; 157; 140	[20,49,55]
54.	Gallocatechin [+(-)gallocatechin]	C15H14O7	306.2675	305		179; 125	[20,28,43,44]
55.	Catechin gallate	C22H18O10	442.3723	441		289; 169; 245; 205; 203	[20,56]
	Flavanones
56.	Naringenin [Naringetol; Naringenine]	C15H12O5	272.5228		273	227; 155; 209; 139	[20,43,49]
57.	Hesperitin [Hesperetin]	C16H14O6	302.2788	301		257; 151; 228; 189	[20,43,68]
58.	Eriodictyol-7-O-glucoside [Pyracanthoside; miscanthoside]	C21H22O11	450.3928	449		269; 207; 251; 165	[48,65,68]
59.	Hexahydroxyflavanone hexoside	C21H22O13	482.3916		483	437; 359; 263; 231; 298; 255; 225; 155	[28]
	Hydroxybenzoic acids
60.	4-hydroxybenzoic acid	C7H6O3	138.1207		139	121	[20,69,70]
61.	Protocatechuic acid	C7H6O4	154.1201		155	127	[20,28,55]
62.	Gallic acid	C7H6O5	170.1195		171	126	[20,54,55]
63.	Syringic acid [benzoic acid; cedar acid]	C9H10O5	198.1727		199	154; 140; 111; 140; 123; 125	[20,55,71]
64.	Ellagic acid [benzoaric acid; elagostasine]	C14H6O8	302.1926		303	172; 158; 144; 127; 116	[27,41,44]
65.	Salvianolic acid F	C17H14O6	314.2895		315	269; 243; 213;185; 144; 207; 181; 153; 179; 161; 133	[69]
66.	Dihydroxybenzoyl-hexoside	C13H16O9	316.2607	315		153; 253; 151; 184	[66]
67.	Salvianolic acid G	C18H12O7	340.2837		341	323; 295; 255; 195; 159; 305	[63,72]
68.	Salvianolic acid D	C20H18O10	418.3509	417		373; 329; 287; 209	[69,73]
	Hydroxycinnamic acids
69.	p-Coumaric acid	C9H8O3	164.16		165	146; 119	[20,46,55,73]
70.	Sinapic acid [trans-sinapic acid]	C11H12O5	224.2100		225	179; 153; 115; 133; 115	[20,37,55,74]
71.	Caffeoylmalic acid	C13H12O8	296.2296	295		133; 179; 148; 119; 115	[28]
72.	Coutaric acid [trans-p-Coumaroyltartaric acid]	C13H12O8	296.2296	295		163; 119	[20]
73.	Caftaric acid [cis-caftaric acid; 2-caffeoyl-L-tartaric acid; caffeoyl tartaric acid}	C13H12O9	312.23	311		149; 221; 131	[20,38,64,69]
74.	Fertaric acid [fertarate]	C14H14O9	326.2556	325		193; 149; 134	[20]
75.	p-Coumaric acid-O-hexoside [trans-p-coumaric acid 4-glucoside]	C15H18O8	326.2986	325		193; 163; 119	[28,57,75]
76.	1-caffeoyl-beta-D-glucose [caffeic acid-glucoside]	C15H18O9	342.298	341		179; 161; 135	[20,66]
77.	5-O-(4’-O-p-coumaroyl glucosyl) quinic acid	C22H28O13	500.4499		501	339; 277; 203	[56]
78.	3-p-coumaroyl-4-caffeoylquinic acid	C25H24O11	500.4515		501	355; 483; 181; 225; 281; 193; 120; 133	[76]
79.	Coumaric acid derivative	C30H30O7	502.5550		503	457; 411; 382; 339; 293; 409; 391; 367; 323; 293; 233; 205	[57]
80.	Di-O-caffeoylquinic acid	C25H24O12	516.4509		517	355; 339; 202	[58,66,76]
81.	Caffeic acid-O-(sinapoyl-O-hexoside)	C26H30O14	566.5080		567	405; 520; 249; 234	[57,77]
	Other compounds
82.	Malic acid	C4H6O5	134.0874	133		115	[57,69,78]
83.	Tartaric acid	C4H6O6	150.0900	149		131	[78,79]
84.	Umbelliferone	C9H6O3	162.1421	161		115	[20,28,54]
85.	Shikimic acid	C7H10O5	174.1513		175	112	[28,78]
86.	Indole-3-carboxylic acid	C10H9NO2	175.1840		176	130	[75]
87.	Esculetin [Cichorigenin; Aesculetin]	C9H6O4	178.1415		179	133; 115	[20]
88.	Citric acid	C6H8O7	192.1235	191		111; 173; 143; 127	[57,59,79]
89.	Quinic acid	C7H12O6	192.1666	191		111; 173	[20,28,57,59]
90.	Dihydroferulic acid	C10H12O4	196.1999	195		159; 129; 113; 122	[28,80,81]
91.	Ethyl gallate	C9H10O5	198.1727	197		169; 125	[45]
92.	L-Tryptophan [tryptophan; (S)-tryptophan]	C11H12N2O2	204.2252		205	188; 146; 170; 118	[41,66]
93.	Myristoleic acid [cis-9-tetradecanoic acid]	C14H26O2	226.3550		227	209; 181; 155; 199; 181; 127	[28]
94.	Resveratrol [trans-resveratrol; stilbentriol]	C14H12O3	228.2433		229	142; 184; 114	[28,43]
95.	Linolenic acid (alpha-linolenic acid; linolenate)	C18H30O2	278.4296		279	260; 176; 120	[62,74]
96.	9-oxo-10E,12Z-octadecanoic acid [9-oxo-ODE]	C18H30O3	294.4290		295	249; 165; 220; 125	[62,82]
97.	Nonadecadienoic acid	C19H34O2	294.4721		295	278; 250; 211; 172; 204; 181; 176	[28]
98.	Protocatechuic acid-O-hexoside	C13H16O9	316.2607	315		153; 298; 151	[57,69,75]
99.	Bilobalide [(-)-Bilobalide]	C15H18O8	326.2986	325		183; 261; 119; 183	[46,50,75]
100.	3,7-dimethylquercetin	C17H14O7	330.2889		331	314; 297; 255; 228; 203; 146; 267; 227; 203; 186; 164; 134	[75]
101.	Galloyl glucose [beta-glucogallin; 1-O-galloyl-beta-D-glucose]	C13H16O10	332.2601	331		313; 195; 166	[41]
102.	Gallic acid hexoside	C13H16O10	332.2601	331		271; 169; 125	[83]
103.	Erucic acid (cis-13-docosenoic acid)	C22H42O2	338.5677		339	132; 293	[65]
104.	Esculin [aesculin; esculoside; polichrome]	C15H16O9	340.2821	339		177; 293; 131	[20,28,56]
105.	Palmatine [berbericinine; Burasaine]	C21H22NO4	352.4037		353	335; 235; 317; 235; 137	[84]
106.	Hexose-hexose-N-acetyl	C14H25NO10	367.3490	366		186; 142	[85]
107.	Fraxin (fraxetin-8-O-glucoside)	C16H18O10	370.3081		371	208; 352; 135	[20]
108.	1-O-sinapoyl-beta-D-glucose	C17H22O10	386.3576		387	205; 130	[20]
109.	Polydatin [piceid; trans-piceid]	C20H22O8	390.3839	389		227; 343; 184; 143	[27,43]
110.	Fucosterol [fucostein; trans-24-ethylidenecholesterol]	C29H48O	412.6908		413	395; 355; 271; 194; 119; 297; 199; 268; 187	[28]
111.	Stigmasterol [stigmasterin; beta-stigmasterol]	C29H48O	412.6908		413	301; 259; 189; 171	[28,86,87]
112.	Phlorizin [phloridzin; phlorizoside; floridzin: phlorrhizin; phloretin 2’-glucoside; phloretin-O-hexoside]	C21H24O10	436.4093		437	397; 217; 377	[20,27,46,49,57]
113.	Oleanoic acid	C30H48O3	456.7003		457	439; 411; 365; 337; 293; 248; 205; 364; 309; 219; 319; 301; 279; 247; 232	[24,76]
114.	Ursolic acid	C30H48O3	456.7003		457	411; 393; 365; 337; 279; 247; 292; 247; 219; 205	[63,76,86]
115.	Anmurcoic acid	C30H46O5	486.6922		487	469; 427; 397; 367; 325; 307; 304; 261; 279	[76]
116.	Dimethylellagic acid hexose	C22H20O13	492.3864		493	331; 299; 270; 242; 179; 150; 225	[41]
117.	Procyanidin A-type dimer	C30H24O12	576.501		577	425; 397; 373; 287; 245; 181; 245; 218; 189; 123	[20,55,57]
118.	Cyclopassifloic acid glucoside	C37H62O12	698.8810		699	537; 347; 271; 259; 185	[66]

2.1. Anthocyanidins and Anthocyanins

A total of 18 anthocyanin compounds have been identified in the analyzed samples of V. amurensis (Table 1). 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, petunidin 3-coumaroylglucoside-5-O-glucoside, and malvidin 3-coumaroylglucoside-5-O-glucoside were only found in the extracts of cultivated V. amurensis (St. Petersburg).

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
1.	Cyanidin 3,5-O-diglucoside			+			+	+	+	+
2.	Cyanidin-3-O-glucoside [Cyanidin 3-O-beta-D-glucoside]			+
3.	Delphinidin 3-O-glucoside		+						+
4.	Delphinidin-3,5-O-diglucoside							+
5.	Malvidin 3-(6-O-acetyl) glucoside	+	+								+
6.	Malvidin 3-(6-O-coumaroyl) glucoside		+								+
7.	Malvidin 3-(6’-p-caffeoylglucoside)	+	+	+			+		+	+
8.	Malvidin 3,5-diglucoside		+		+	+	+	+	+	+	+
9.	Malvidin 3-coumaroylglucoside-5-O-glucoside										+
10.	Malvidin 3-O-acetyl hexoside							+
11.	Malvidin 3-O-glucoside		+	+		+	+	+	+	+	+
12.	Pelargonidin-3-O-glucoside (callistephin)						+
13.	Peonidin-3,5-О-diglucoside [peonin; peonidin 3-glucoside-5-glucoside]		+			+	+	+	+	+
14.	Peonidin-3-O-glucoside						+	+	+
15.	Petunidin 3-(6-O-coumaroyl) glucoside		+
16.	Petunidin 3-coumaroylglucoside-5-O-glucoside										+
17.	Petunidin 3-O-glucoside-5-O-glucoside [Petunidin 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 (Table 2). The flavonols dihydrokaempferol, kaempferide, mearnsetin, kaempferol-3-O-glucoside, dihydrokaempferol glucoside, isorhamnetin 3-O-rhamnoside, hyperoside, taxifolin-3-O-glucoside, kaempferol 3,7-di-O-glucoside, and quercetin-O-dihexoside have been already characterized as components of Far East V. amurensis.

Table 2

Other flavonoid compounds 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
	Flavonols
1.	Quercetin-3-O-glucuronide	+	+	+	+		+		+						+		+		+
2.	Kaempferol	+	+	+			+		+						+
3.	Quercetin			+			+		+			+							+
4.	Isorhamnetin [Isorhamnetol; Quercetin 3’-Methyl ether]		+									+			+				+
5.	Isorhamnetin 3-O-glucoside								+			+			+		+
6.	Myricetin-3-O-galactoside		+						+						+		+
7.	Quercetin 3-O-glucoside [Isoquercitrin; Hirsutrin]		+						+						+				+
8.	Myricetin		+	+											+
9.	Dihydrokaempferol		+						+
10.	Dihydroquercetin (Taxifolin; Taxifoliol)						+												+
11.	Hyperoside (Quercetin 3-O-galactoside; Hyperin)	+							+
12.	Kaempferol diglycoside				+		+
13.	Kaempferol glycoside	+							+
14.	Dihydrokaempferol glucoside	+
15.	Herbacetin														+
16.	Isorhamnetin 3-O-rhamonoside								+
17.	Kaempferide		+
18.	Mearnsetin		+
19.	Quercetin-O-dihexoside		+
20.	Rutin (Quercetin 3-O-rutinoside)		+
21.	Taxifolin-3-O-glucoside						+
	Total number:	3	9	2	1		4		8			3			6		2		4
	Flavones
22.	Apigenin	+	+	+	+				+						+
23.	Syringetin			+								+			+		+
24.	Luteolin diglycoside											+			+		+
25.	Nevadensin		+				+
26.	Vitexin 2”-O-glucoside [Apigenin 8-C-glucoside 2”-O-glucoside]		+																+
27.	Luteolin				+
28.	Diosmetin [Luteolin 4’-Methyl Ether; Salinigricoflavonol]																		+
29.	Pentahydroxy trimethoxy flavone																		+
30.	Apigenin diglycoside								+
31.	Vitexin [ Apigenin 8-C-Glucoside]														+
32.	Vitexin glucoside	+
33.	Apigenin glucoside								+
	Total number:	2	3	2	2		1		3			2			4		2		3
	Dimethoxyflavone
34.	Cirsimaritin [Scrophulein; 4’,5-dihydroxy-6,7-dimethoxyflavone; 7-methylcapillarisin]		+
	Flavan-3-ols
35.	Catechin [D-Catechol]		+		+		+		+			+			+		+		+
36.	Epicatechin		+		+
37.	Gallocatechin [+(-)Gallocatechin]														+
38.	Catechin gallate								+
	Total number:	0	2	0	2		1		2			1			2		1		1
	Flavanones
39.	Naringenin [Naringetol; Naringenine]				+		+		+
40.	Eriodictyol-7-O-glucoside [Pyracanthoside; Miscanthoside]				+														+
41.	Hesperitin [Hesperetin]								+
42.	Hexahydroxyflavanone hexoside		+
	Total number:	0	1	0	2		1		2			0			0		0		1

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).

2.3. Phenolic Acids and Other Compounds

In addition, 22 phenolic acids and 37 other compounds were identified in analyzed V. amurensis samples (Table 3). It should be noted that the coumarins umbelliferone and fraxin; the sterol fucosterol; and the flavanols taxifolin-3-O-glucoside, kaempferol-3,7-di-O-glucoside; hydroxycinnamic acids 3-p-coumaroyl-4-caffeoylquinic acid, and 5-O-(4’-O-p-coumaroyl glucosyl) quinic acid were identified by mass spectrometry only in samples of wild V. amurensis grapes collected from the Pakhtusov Islands and Rikord Island, Peter the Great Bay, Sea of Japan.

Table 3

Phenolic acids and other compounds 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
	Hydroxybenzoic acids
1.	Salvianolic acid D		+		+		+		+		+
2.	Salvianolic acid G	+					+		+
3.	Ellagic acid [Benzoaric acid; Elagostasine]						+			+
4.	4-Hydroxybenzoic acid							+
5.	Protocatechuic acid							+
6.	Gallic acid						+
7.	Syringic acid [Benzoic acid; Cedar acid]								+
8.	Salvianolic acid F						+
9.	Dihydroxybenzoyl-hexoside						+
	Total number:	1	1	0	1	0	6	2	3	1	1
	Hydroxycinnamic acids
10.	Caftaric acid [cis-caftaric acid; 2-caffeoyl-L-tartaric acid; caffeoyl tartaric acid}	+		+	+	+	+		+	+	+
11.	Di-O-caffeoylquinic acid		+					+	+
12.	Sinapic acid [trans-Sinapic acid]			+			+
13.	Coutaric acid [Trans-p-Coumaroyltartaric acid]					+					+
14.	Fertaric acid [Fertarate]				+						+
15.	p-Coumaric acid-O-hexoside [Trans-p-Coumaric acid 4-glucoside]						+				+
16.	Caffeic acid-O-(sinapoyl-O-hexoside)							+	+
17.	p-Coumaric acid				+
18.	Caffeoylmalic acid		+
19.	1-Caffeoyl-beta-D-glucose [Caffeic acid-glucoside]										+
20.	5-O-(4’-O-p-coumaroyl glucosyl) quinic acid				+
21.	3-p-coumaroyl-4-caffeoylquinic acid					+
22.	Coumaric acid derivative						+
	Total number:	0	1	0	3	2	2	1	1	0	4
	Other compounds
23.	Ethyl gallate	+	+	+	+		+	+	+	+	+
24.	Malic acid	+	+		+	+	+	+	+
25.	Hexose-hexose-N-acetyl	+	+				+	+	+	+
26.	Citric acid		+	+			+	+	+
27.	Quinic acid		+		+	+	+	+
28.	Galloyl glucose [Beta-Glucogallin; 1-O-Galloyl-Beta-D-Glucose]		+	+		+			+		+
29.	L-Tryptophan [Tryptophan; (S)-Tryptophan]		+	+				+	+
30.	Cyclopassifloic acid glucoside	+	+			+	+
31.	Indole-3-carboxylic acid		+		+	+
32.	Myristoleic acid [Cis-9-Tetradecanoic acid]		+				+		+
33.	Resveratrol [trans-Resveratrol; Stilbentriol]	+	+	+
34.	Protocatechuic acid-O-hexoside				+		+			+
35.	Palmatine [Berbericinine; Burasaine]						+	+			+
36.	Polydatin [Piceid; trans-Piceid]				+		+		+
37.	Procyanidin A-type dimer		+					+	+
38.	Shikimic acid		+					+
39.	Esculetin [Cichorigenin; Aesculetin]				+		+
40.	9-oxo-10E,12Z-octadecanoic acid [9-Oxo-ODE]				+					+
41.	Gallic acid hexoside				+				+
42.	Esculin [Aesculin; Esculoside; Polichrome]			+		+
43.	1-O-Sinapoyl-beta-D-glucose			+						+
44.	Stigmasterol [Stigmasterin; Beta-Stigmasterol]	+							+
45.	Oleanoic acid		+				+
46.	Tartaric acid						+
47.	Umbelliferone				+
48.	Dihydroferulic acid						+
49.	Linolenic acid (Alpha-Linolenic acid; Linolenate)						+
50.	Nonadecadienoic acid							+
51.	Bilobalide [ (-)-Bilobalide]		+
52.	3,7 -Dimethylquercetin										+
53.	Erucic acid (Cis-13-Docosenoic acid)							+
54.	Fraxin (Fraxetin-8-O-glucoside)					+
55.	Fucosterol [Fucostein; Trans-24-Ethylidenecholesterol]				+
56.	Phlorizin [Phloridzin; Phlorizoside; Floridzin: phlorrhizin; Phloretin 2’-Glucoside; Phloretin-O-hexoside]	+
57.	Ursolic acid						+
58.	Anmurcoic acid										+
59.	Dimethylellagic acid hexose						+
	Total number	7	15	7	11	7	17	11	11	5	5

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).

3. Discussion

In general, the diversity of phytochemicals identified in wild and cultural grape V. amurensis resulted in the following descending order (number of metabolites in parenthesis): VZK (52) > ART (46) > SPB-2 (39) > SPB-1 (28) > SPB-4 (27) > PAK (25) > RIK (22) > KAL (20) > SPB-3 (19) > ARS (18). The most diverse metabolome was identified in the grapes collected in the vicinity of Vyazemsky, Khabarovsk Territory, which was rich in flavanols and phenolic acids.

The anthocyanins identified in V. amurensis in this study were previously identified and annotated in the vines [17] Solanium nigrum [18], Gaultheria Antarctica [19], and Vitis vinifera [20] and wheat [21]. Our identification of flavonoid compounds agrees with bibliographic data for Echinops [22], Rhodiola rosea [23], Ocimum [24], Alpinia officinarum [25], Brazilian propolis [26], Vitis vinifera [20], Rubus occidentalis [27], C. edulis [28], and Vaccinium macrocarpon [29].

Although wild grapes tend to be more diverse than cultivated varieties [30], this number of anthocyanins in one form is quite rare and more likely to occur in other berries, such as blueberries [31]. We hypothesize that many different anthocyanins are associated with rather low temperatures in summer and monsoon climates. To respond to adverse conditions, various anthocyanins are produced [32]. In addition, V. amurensis have an increased acidity of the fruit, which is also associated with unfavorable growing conditions [33]. As it is known, anthocyanins and many other phenolic compounds participating in the protective processes of plants are more stable in an acidic environment [34].

4. Materials and Methods

4.1. V. amurensis Samples

Ten samples of wild and cultivated grape V. amurensis were selected for the performance of metabolomic study. Six samples of wild V. amurensis were collected from different places in the Primorsky and Khabarovsk territories, Far Eastern Russia (Table 4, Figure 1). Four samples of cultivated V. amurensis, namely SPB-1, SPB-2, SPB-3, and SPB-4, were obtained from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources, St. Petersburg. The grapes were harvested at the end of August and September 2020. Each sample included 100 g of grape berries.

Table 4

Locations of wild V. amurensis grape collection.

Code Name of the Sample	Location	Geographical Values	Soil Type
ARS	Floodplain of the Arsenyevka River, Primorsky Territory	N. 44°52′18″, E 133°35′12″	brown grey bleached soils
ART	The vicinity of Artem, Primorsky Territory	N 43°21′34″, E 132°11′19″	yellow-brown soil
KAL	The vicinity of Kalinovka, Primorsky Territory	N 43°07′27″, E 133°12′30″	layered floodplains
PAK	The Pakhtusov Islands, Peter the Great Bay, Sea of Japan	N 42°53′57″, E 131°38′45″	yellow-brown soil
RIK	Rikord Island, Peter the Great Bay, Sea of Japan	N 42°52′54″, E 131°40′06″	yellow-brown earth soils
VZK	The vicinity of Vyazemsky, Khabarovsk Territory	N 47°32′15″, E 134°45′20″	podzolic brown forest heavy loamy soils

Figure 1

Region of wild V. amurensis grape collection.

4.2. Chemicals and Reagents

HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), and MS-grade formic acid was purchased from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was obtained with Siemens Ultra-Clear TWF EDI UV UF TM Water Purification System (Siemens, Munich, Germany). All the other chemicals were of analytical grade.

4.3. Fractional Maceration

Fractional maceration with ethyl alcohol was applied to obtain highly concentrated extracts of V. amurensis. Each sample of V. amurensis was divided into three parts and consistently infused. The infusion time of each part of the extractant was seven days.

4.4. Liquid Chromatography

The separation of multicomponent mixtures was performed by a Shimadzu LC-20 Prominence HPLC (Shimadzu, Kyoto, Japan) equipped with a UV detector and a Shodex ODP-40 4E reverse-phase column (4.6 × 250 mm, particle size 4 µm). The gradient elution program with two mobile phases (A, deionized water; B, acetonitrile with formic acid 0.1% v/v) was as follows: 0.01–2 min, 100% B; 2–50 min, 100–0% B; control washing 50–60 min, 0% B. The entire HPLC analysis was done with an SPD-20A detector at wavelengths of 230 and 330 nm; the temperature corresponded to 40 °C. The injection volume was 10 µL.

4.5. Mass Spectrometry

MS analysis was performed on an ion trap amaZon SL (Bruker Daltonics, Bremen, Germany). Four-stage ion separation (MS/MS mode) was implemented. All the chemical profiles of the samples were obtained by the HPLC–ESI–MS/MS method. The working parameters were as follows: ionization source temperature 50 °C, gas flow 4 L/min, nebulizer gas (atomizer) 7.3 psi, capillary voltage 4500 V, endplate bend voltage 1500 V, fragmentary voltage 280 V, and collision energy 60 eV. The ion trap was used in the scan range of 100–1.700 m/z for MS and MS/MS. The capture rate was one spectrum/s for MS and two spectrum/s for MS/MS. The mass spectra were recorded in negative and positive ion mode. Data collection was controlled by Hystar DataAnalisys 4.1 software (Bruker Daltonics, Bremen, Germany). All the measurements were performed in triplicate.