Identification of phenolic constituents in Lonicera caerulea L. by HPLC with diode array detection electrospray ionisation tandem mass spectrometry

The purpose of this work was a comparative metabolomic study of extracts of Blueberried honeysuckle Lonicera caerulea L.: No1043-11 (St. Petersburg); No1043-08 (St. Petersburg) No863; (Japan); No860 (Wild Lonicera from Amur river) from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources. To identify target analytes in extracts HPLC was used in combination with a BRUKER DALTONIKS ion trap. The results showed the presence of 82 target analytes corresponding to family Caprifoliaceae. In addition to the reported metabolites, a number of metabolites were newly annotated in Lonicera caerulea L. There were flavonols: Dihydrokaempferol, Rhamnetin I, Rhamnetin II, Taxifolin-3O-glucoside, Mearnsetin-hexoside, Horridin; flavones: Chrysoeriol, Apigenin-O-pentoside, Chrysoeriol-7-Oglucoside; flavanone Naringenin; flavan-3-ols: Catechin, Epicatechin, Biochanin A-7-O-glucoside; essential amino acids: L-Pyroglutamic acid, Tyrosine; polypeptide 5-Oxo-L-propyl-L-isoleucine; sterols: Ergosterol, Fucosterol, Beta-Sitosterin; triterpenoids: Betunolic acid, Oleanoic acid; anabolic steroid Vebonol, indole sesquiterpene alkaloid Sespendole; iridoids: Monotropein, p-Coumaroyl monotropein, p-Coumaroyl monotropein hexoside; Myristoleic acid, etc.


Introduction
Blue-berried honeysuckle Lonicera caerulea L., family Caprifoliaceae is known as a natural source of food, beverages and nutraceuticals due to its rich chemical composition, enriched with nutrient and biologically active compounds. The increased focus on these berries is due to their phenolic composition, antioxidant activity, and potential health benefits. The high content of phenols in Lonicera caerulea L. is directly related to their biological activity. Popularity of phenolic compounds has grown in recent years as they are excellent antioxidants. Antioxidant intake has been shown to be effective in preventing cancer, cardiovascular disease, osteoporosis, obesity, diabetes, and other health problems [Dias et al., 2017]. The antioxidant properties of plant phenolic compounds are relevant in the field of nutrition (inhibition of lipid oxidation), physiology (protection against oxidative stress) and cosmetology. Phenolic compounds provide antioxidant activity through direct reduction of reactive oxygen species (ROS), inhibition of enzymes involved in oxidative stress, binding of metal ions Initial LC-MS/MS screening suggested that 82 target analytes detected in EtOH-extracts of Blue-berried honeysuckle. Therefore, tandem mass spectrometry was used in this study for comparative small molecule profiling of four Lonicera varieties cultivated in the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources.

Materials
The object of the study was the four varieties of Blue-berried honeysuckle Lonicera caerulea L. of breeding varieties obtained as a result of many years of research from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources. There were a varieties: №1043-11 (St. Petersburg); №1043-08 (St. Petersburg); №863 (Japan); №860 (Wild Lonicera from Amur river). The berries were harvested at the end of July 2020. All samples morphologically corresponded to the pharmacopoeial standards of the State Pharmacopoeia of the Russian Federation [SPh XIV, Russia, 2018].

Chemicals and Reagents
HPLC-grade acetonitrile was purchased from Fisher Scientific (Southborough, UK), MS-grade formic acid was from Sigma-Aldrich (Steinheim, Germany). Ultra-pure water was prepared from a SIEMENS ULTRA clear (SIEMENS water technologies, Germany), and all other chemicals were analytical grade.

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

Mass spectrometry
MS analysis was performed on an ion trap amaZon SL (BRUKER DALTONIKS, Germany) equipped with an ESI source in negative ion mode. The optimized parameters were obtained as follows: ionization source temperature: 70 ° C, gas flow: 4 l / min, nebulizer gas (atomizer): 7.3 psi, capillary voltage: 4500 V, end plate bend voltage: 1500V, fragmentary: 280 V, collision energy: 60 eV. An ion trap was used in the scan range m / z 100 -1.700 for MS and MS/MS. The capture rate was one spectrum/s for MS and two spectrum/s for MS/MS. Data collection was controlled by Windows software for BRUKER DALTONIKS. All experiments were repeated three times. A four-stage ion separation mode (MS/MS mode) was implemented.

Results and discussion
Four of the most consumed extracts of Lonicera caerulea L. have been selected. All of them have a rich bioactive composition. There were four extracts from a varieties: №1043-11 (St. Petersburg); №1043-08 (St. Petersburg); №863 (Japan); №860 (Wild Lonicera, Amur river) from the collection of N.I. Vavilov All-Russian Institute of Plant Genetic Resources.