Zostera marina L . : Supercritical CO 2 ‐ Extraction and Mass Spectrometric Characterization of Chemical Constituents Recovered from Seagrass

: Three types of Zostera marina L. collection were extracted using the supercritical CO 2 ‐ ex ‐ traction method. For the purposes of supercritical СО 2 ‐ extraction, old seagrass ejection on the surf edge, fresh seagrass ejection on the surf edge and seagrass collected in water were used. Several experimental conditions were investigated in the pressure range 50–350 bar, with the used volume of co ‐ solvent ethanol in the amount of 1 % in the liquid phase at a temperature in the range of 31– 70 °C. The most effective extraction conditions are: pressure 250 Bar and temperature 60 °C for Z. marina collected in sea water. Z. marina contain various phenolic compounds and sulfated polyphe ‐ nols with valuable biological activity. Tandem mass ‐ spectrometry (HPLC ‐ ESI–ion trap) was applied to detect target analytes. 77 different biologically active components have been identified in Z. ma ‐ rina supercritical CO 2 ‐ extracts. 38 polyphenols were identified for the first time in Z. marina. Conceptualization, L.A.T. M.P.R.; methodology, L.A.T., A.M.Z., M.P.R.; M.P.R.; investigation, K.G. and L.A.T.; data curation, V.D.S.;


Introduction
Zostera marina L. is a perennial marine herbaceous plant, genus Zostera, family Zosteraceae. Zostera lives mainly in the coastal waters of the northern hemisphere, it grows in the Azov, Black, Caspian, White and Far Eastern seas ( Figure 1). For the most part, the plant lives in shallow water or at a depth of 1-4 m (sometimes 10 m), mainly on soft sandy or muddy bottoms in the calm waters of bays and bays. In the 30s of the last century, Zostera began to die, the reason for this was a special type of animal-the labyrinthula [1]. During the epidemic, Zostera disappeared from the coasts of North America, the Atlantic and Southern Europe and still does not grow in these places.
Zostera has a branched root system, forms underwater meadows, sometimes with a very high herbage up to 100 cm high. Plants bloom and pollinate under water, pollen is carried by streams of water. In order to survive in harsh conditions that are not intended for the life of higher plants, that is, in salty sea water, the plant has acquired a number of biochemical features that determines its adaptation to a specific habitat. The plant produces a special pectin, which has no analogues in other plants. Firstly, it was isolated in 1940 by the Russian scientist V.I. Miroshnikov, who named it zosterin. Zosterin from a chemical point of view is a polysaccharide of pectin nature. It is a highly active polyanionic adsorbent, which, passing through the gastrointestinal tract, binds and removes heavy metal ions, bile acids, pathogenic microorganisms, etc. from the body [2]. Interested in the unique nature of zosterin, Yu. S. Ovodov engaged in serious research, the result of which showed that these pectins are among the most complex in structure of objects of natural origin, and this unique feature gives them a high adsorption capacity. Because of this, a pectin called zosterin has found extensive use in medicine [3].
The pectin from Zostera marina has unique features that distinguish it from the glycans of other land plants. Numerous studies have shown that zosterin has a more complex structure than land plant pectins. Although it, like other pectins, has a linear backbone of rhamnogalacturonan and a branched region, however, the latter is a much more complex configuration. Another "block" is attached to it-xylogalacturonan (chains consisting of rings of galacturonic acid and xylose). Xylogalacturonans were found earlier in pectins of some terrestrial plants (for example, in mountain pine pollen). However, in zosterin, this fragment has additional branches that increase the volume of macromolecules.
The use of zosterin as a dietary supplement has an antiulcer effect, normalizes the function of the gastrointestinal tract, enhances the feeling of satiety, thereby facilitating the tolerance of low-calorie diets. An important property of pectin is its ability to reduce blood cholesterol, which provides an anti-sclerotic effect. Features of the metabolism of pectins allow the use of zosterin in diabetes mellitus as an auxiliary antidiabetic agent. Of exceptional interest are experimental data on the antitumor properties of zosterin and its ability to prolong life, i.e., act as a potential geroprotector. The observed effects indicate the multifunctional nature of the impact of this pectin on the body [4,5]. The therapeutic effect of rosmarinic acid, luteolin and its sulfated derivatives-these are one of the most active components of Z. marina-are considered in detail in experimental studies in diseases associated with impaired carbohydrate and lipid metabolism [6].
In this research, supercritical CO2-extraction of three samples of Z. marina was used to obtain an effective amount of polyphenolic substances: old storm seagrass waste, fresh storm seagrass waste, and seagrass collected in water. We used a tandem mass spectrometry to carry out a phytochemical study involving a detailed metabolomic analysis of Z. marina. Eelgrass was collected during expedition work near Vityaz Bay, Primorsky Krai, Russia (N 42°36′10″ E 131°10′55″), during the period from 10 to 20 August 2021.

Results and Discussion
Three samples of Z. marina were subjected to a detailed research: 1. old Z. marina ejection on the surf edge, 2. fresh Z. marina ejection on the surf edge, 3. Z. marina collected in the water. All three Z. marina samples were subjected to supercritical CO2-extraction under different extraction conditions. The applied supercritical pressures ranged from 50 to 350 bar, and the extraction temperature ranged from 31 to 70 °C. The co-solvent EtOH was used in an amount of 1 % of the total amount of solvent. Used different extraction conditions for different seagrass samples showed the best result for bagging in water (Extraction conditions: pressure 250 bar and temperature 60 °C). The total yield of biologically active substances under these extraction conditions was 4.2 mg per 100 mg of supercritical CO2-extract. The quantitative ratio of the extract of biologically active substances obtained by the method of supercritical extraction was achieved by evaporating the CO2 -extract and calculating the ratio of the mass of the extracted plant matrix to the dry mass of the obtained extract. Below are 3D graphs of supercritical extraction of an old Z. marina release (     There were identified 77 compounds (53 compounds from polyphenol group and 24 compounds from other chemical groups). All the identified polyphenols and other compounds along with molecular formulas, and MS/MS data for Z. marina are summarized in Table A1   [M+H] + ion produced two fragment ions with m/z 245.02 and m/z 175.03 ( Figure 5). The fragment ion with m/z 245.02 produced one characteristic daughter ion with m/z 175.01. It was identified in the references in extract from Cassia granidis [7]; Cassia abbreviata [8]; A. cordifolia; F. glaucescens; F. herrerae [9]. It should be noted separately that the presence of many sulfated polyphenols was found in the supercritical extracts of Z. marina. For example, these are the following chemical compounds: Luteolin 7-sulfate; Diosmetin 7-sulfate, Kaempferol 7-sulfate, Isorhamnetin 3-sulfate, Apigenin7-sulfate, Chrysoeriol 7sulfate, Luteolin 7,3′-disulfate, (2S)-Naringenin 4'-O-sulfate. The CID spectrum in positive ion modes of flavone Luteolin 7-sulfate from Z. marina is shown in Figure 6.   Intens. [M+H] + ion produced one fragment ion with m/z 286.89 ( Figure 6). The fragment ion with m/z 286.89 produced two characteristic daughter ions with m/z 152.96, and m/z 286. 85. It was identified in the bibliography in extracts from Z. marina [10,11]. The CID spectrum in negative ion modes of flavone Apigenin 7-sulfate from Z. marina is shown in Figure 7. The fragment ion with m/z 225.01 formed one daughter ion with m/z 197.01. It was identified in the bibliography in extracts from G. linguiforme [9]; Z. marina [11]; sulphates [12]. We also want to separately note the first identification of the presence of a large class of anthocyanins in Z. marina: Pelargonidin 3-O-glucoside; Cyanidin 3-O-glucoside; Pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside); Cyanidin 3-(6"-malonylglucoside). All these anthocyanins were identified firstly in Z. marina. The CID spectrum in positive ion modes of anthocyanin Pelargonidin 3-O-glucoside from Z. marina is shown in Figure 8.  [13]; Strawberry [14]; Vigna unguiculata [15].
The CID spectrum in positive ion modes of anthocyanin Pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside) from Z. marina is shown in Figure 9.
[M+H] + ion produced two fragment ions with m/z 270.91, and m/z 432.81 ( Figure 9). The fragment ion with m/z 270.91 formed two daughter ions with m/z 153.00, m/z 224.93. It was identified in the bibliography in extract from Strawberry [14], Wheat [16]. Intens.  The CID spectrum in negative ion modes of phenolic acid Sagerinic acid from Z. marina is shown in Figure 11. [M-H] -ion produced two fragment ions with m/z 358.92 and m/z 197.02 ( Figure 11). The fragment ion with m/z 358.92 formed two daughter ions with m/z 179.08, m/z 161.03. It was identified in the bibliography in extract from Mentha [17]; Lamiaceae spp. [18]; Lepechinia [19]. Intens. The CID spectrum in positive ion mode of hydroxycoumarin Umbelliferone from Z. marina is shown in Figure 12. [M+H] + ion produced one fragment ion with m/z 144.99 (Figure 12). The fragment ion with m/z 144.99 produced one characteristic daughter ion with m/z 117.08 It was identified in the bibliography in extracts from F. glaucescens [9]; Sanguisorba officinalis [20]; Actinidia chinensis [21]. Separately, it should be noted that a detailed analysis of the presence of polyphenols and biologically active substances from other chemical groups showed the highest number of compounds in the seagrass collected in water and fresh release on the shore than in the old release on the shore. The ratio was 31 and 30 versus 26 for polyphenols, respectively (Table 1). Thus, it can be stated that as a result of the most detailed study by tandem mass spectrometry, new data on the content of biologically active substances in Z. marina have been obtained.

Materials
Phytomass of Z. marina was collected during expedition work near Vityaz Bay, Primorsky Krai, Russia (N 42°36′10″ E 131°10′55″), during the period from 10 to 20 August 2021. All samples were morphologically authenticated according to the current standard of Pharmacopoeia of the Eurasian Economic Union [22].

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, Munich, Germany), and all other chemicals were analytical grade.

SC-CO2 Extraction
SC-CO2 extraction was performed using the SFE-500 system (Thar SCF Waters, Milford, CT, USA) supercritical pressure extraction apparatus. System options include: Co-solvent pump (Thar Waters P-50 High Pressure Pump), for extracting polar samples. CO2 flow meter (Siemens, Germany), to measure the amount of CO2 being supplied to the system, multiple extraction vessels, to extract different sample sizes or to increase the throughput of the system. Flow rate was 10-25 mL/min for liquid CO2 and 1.00 mL/min for EtOH. Extraction samples of 20 g Z. marina were used. The extraction time was counted after reaching the working pressure and equilibrium flow, and it was 60-90 min for each sample.

Conclusions
Three types of Zostera marina L. collection were extracted using the supercritical CO2extraction method. For the purposes of supercritical СО2-extraction, old seagrass ejection on the surf edge, fresh seagrass ejection on the surf edge and seagrass collected in water were used. Several experimental conditions were investigated in the pressure range 50-350 bar, with the used volume of co-solvent ethanol in the amount of 1 % in the liquid phase at a temperature in the range of 31-70 °C. The most effective extraction conditions are: pressure 250 Bar and temperature 60 °C for Z. marina collected in sea water. Z. marina contain various phenolic compounds and sulfated polyphenols with valuable biological activity. Tandem mass-spectrometry (HPLC-ESI-ion trap) was applied to detect target analytes. High-accuracy mass spectrometric data were recorded on an ion trap amaZon SL BRUKER DALTONIKS equipped with an ESI source in the mode of negative and positive ions. The four-stage ion separation mode was implemented. 77 different biologically active components have been identified in Z. marina supercritical CO2-extracts. 38 polyphenols were identified for the first time in Z. marina.
These data could support future research for the production of a variety of pharmaceutical products containing extracts of Z. marina. The richness of various biologically active compounds, including compounds of polyphenol group, amino acids, carotenoids, Omegafatty acids, sterols, triterpenoids, iridoids, etc., provides great opportunities for the design of new nutritional and dietary supplements based on extracts from this genus Zostera.