Research of 5 extracts of wild Amur grape Vitis amurensis Rupr. and identification of its polyphenolic composition by tandem mass spectrometry (HPLC-MS / MS)

Vitis amurensis Ruprecht contains a large number of polyphenolic compounds which are biologically active components. For the most efficient and safe extraction supercritical carbon dioxide was used. In this work, for the first time, a comparative metabolomic study of biologically active substances of wild grapes collected from five different places of the Primorsky and Khabarovsk territories is carried out. To identify target analytes in ethanol extracts of grape berries, high performance liquid chromatography (HPLC) was used in combination with an amaZon SL ion trap (manufactured by BRUKER DALTONIKS, Germany) equipped with an ESI electrospray ionization source in negative and positive ion modes. The mass spectrometer was used in the scan range m / z 100 1.700 for MS and MS / MS. Used fragmentation of the 4th order. Primary mass spectrometric results showed the presence of 89 biologically active compounds corresponding to the species V. amurensis, moreover, salvianolic acids F, D and G, oleanoic, ursolic, myristoleic acids, berbericinin, mearnsetin, esculin, nevadensin, stigmasterol, fucosterol, phlorizin, L-tryptophan identified for the first time in V. amurensis.


Introduction
Researchers attribute the appearance of the first representatives of the Vitaceae family, belonging to the genus Vitis, to the Upper Cretaceous period, when there were already types of plants that were very similar in leaves to vines. The absence of seeds does not allow, however, to have complete confidence in their belonging to the genus Vitis [1,2]. To these types, researchers include the Vitis dakotana Berry vine found in the Upper Cretaceous sediments in Harding County in South Dakota, which is very similar in appearance to modern vines [3,4].
The evolution of grape plants approaching the cultivated vine, judging by the fossil finds, took place especially intensively in Central and Southern Europe during the second half of the Tertiary period and then especially in the Quaternary period. On the territory of Russia, quite a lot of finds of fossils are also known belonging to the genera Cissites, Ampelopsis, Parthenocissus and especially to the genus Vitis: V. sachalinensis Krysht. and V. crenata Heer on Sakhalin, V. teutonica A. Br. -near Taganrog and on the Irtysh River, as well as V. praevinifera Sap. -on the Krynka river. All these data show that the evolution of the vine on the territory of Russia proceeded from ancient times. And now in Russia in many areas wild grapes V. sylvestris Gmel grow [5,6,7].
Very little information is available about the culture of East Asian grape varieties. V. lanata Roxb is cultivated in eastern India. and V. tomentosa Heyne, in Japan and in Korea -V. Thunbergii Sieb. et Zucc. called V. Seiboldii hort [8]. More complete information is available regarding V. amurensis Rupr., which was first introduced into the culture by I.V. Michurin. In his work "Results of half a century of work" I.V. Michurin describes four forms of V. amurensis Rupr., which were isolated in the Far East [9,10].
The dominant class of biologically active compounds of fruits, and especially grape ridges, are bioflavonoids and, in particular, the so-called complexes of oligomeric proanthocyanidins or condensed tannins, which are polymeric forms of flavonoids from the group of catechins [11].
In European medicine, until recently, grapes were widely used as a means of therapy and rehabilitation for a wide range of diseases: chronic recurrent inflammatory processes, tuberculosis, kidney disease, arterial hypertension, etc. The aim of this work was a comparative metabolomic study of biologically active substances of wild grapes harvested in five different places in the Russian Far Eastern taiga in the Primorsky and Khabarovsk territories. High performance liquid chromatography (HPLC) in combination with a BRUKER DALTONIKS ion trap (tandem mass spectrometry) was used to identify target analytes in extracts. This paper presents a detailed study of the metabolomic composition of grape juice from fruits taken from five habitats of V. amurensis in the Far East: Pakhtusov Islands and Rikord Island (Peter the Great Bay, Sea of Japan), the vicinity of Artem (Primorsky Territory), the vicinity of the river Arsenyevka (Primorsky Territory), environs of Vyazemsky (Khabarovsk Territory).

Materials
The object of the study was the berries of the wild grape V. amurensis, collected in the floodplain of the Arsenyevka River, Primorsky Territory (N. 44

Fractional maceration
To obtain highly concentrated extracts, fractional maceration was applied. In this case, the total amount of the extractant (ethyl alcohol of reagent grade) is divided into 3 parts and is consistently infused with grapes with the first part, then with the second and third. The infusion time of each part of the extractant was 7 days.

Tandem mass spectrometry
Mass spectrometric data were obtained using an amaZon SL ion trap (manufactured by BRUKER DALTONIKS, Germany) equipped with an ESI electrospray ionization source in negative and positive ion modes. The optimized parameters are obtained as follows: ionization source temperature: 70 ° C, gas flow: 4 L / min, nebulizer gas (nebulizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500V, fragmentary: 280V, collision energy: 60 eV. The mass spectrometer was used in the scan range m / z 100 -1.700 for MS and MS / MS. Used fragmentation of the 4 th order.

Conclusions
Amur grape V. amurensis Ruprecht contains a large number of polyphenolic complexes, which are biologically active compounds. In this work, we have tried for the first time to conduct a comparative metabolic study of biologically active substances of wild grapes obtained from five different places in the Primorsky and Khabarovsk territories. HPLC in combination with a BRUKER DALTONIKS ion trap (tandem mass spectrometry) was used to identify target analytes in extracts. The results showed the presence of 89 biologically active compounds corresponding to the species V. amurensis, and Salvianolic acids F, D and G, Oleanolic, Ursolic, Myristoleic acids, Berbericinin, Mearnsetin, Esculin, Nevadensin, Stigmasterol, Fucosterol, Phlorizin, L-Tryptophan were identified for the first time in V. amurensis.
The findings may support future research into the production of various pharmaceutical and dietary supplements containing V. amurensis extracts. A wide variety of biologically active polyphenolic compounds opens up rich opportunities for the creation of new drugs and biologically active additives based on extracts from this family of grapes (Vitaceae).