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.


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.

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.
Dimethylellagic acid hexose + Total number

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 paren-  (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.
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].

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.

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.

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). Ultrapure 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.

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.

Conflicts of Interest:
The authors declare no conflict of interest.