Supercritical CO 2 -Based Extraction and Detection of Phenolic Compounds and Saponins from the Leaves of Three Medicago varia Mart. Varieties by Tandem Mass Spectrometry

: A comparative metabolomic study of three varieties of alfalfa ( Medicago varia Mart.) was performed via extraction with supercritical carbon dioxide modified with ethanol (EtOH) and the detection of bioactive compounds via tandem mass spectrometry. Several experimental conditions were investigated in the pressure range of 50–250 bar, with ethanol used as a co-solvent in an amount of 1% of the total volume in the liquid phase at a temperature in the range of 31–70 ◦ C. The most effective extraction conditions were as follows: a pressure of 250 Bar and a temperature of 60 ◦ C for M. varia . M. varia contains various phenolic compounds and sulfated polyphenols with valuable biological activity. Tandem mass spectrometry (HPLC-ESI–ion trap) was applied to detect the target analytes. A total of 103 bioactive compounds (59 polyphenols and 44 compounds belonging to other chemical groups) were tentatively identified in extracts from aerial parts of alfalfa. For the first time, twenty-one chemical constituents from the polyphenol group (flavones: Formononetin, Chrysoeriol, Cirsimaritin, Cirsiliol, Cirsilineol, tricin-O -hexoside, Apigenin C -glucose C -deoxyhexoside, Apigenin 7-O -diglucuronide, 2 ′ -Hydroxygenistein 4 ′ ,7-O -diglucoside, etc.) and six from other chemical groups (saponins: Soyasaponin II, Soyasaponin gamma g, Soyasaponin I, Soyasaponin Bd, Soyaysaponin beta g, etc.) were identified in the aerial parts of M. varia .


Introduction
One of the most versatile and cost-effective crops is alfalfa (Medicago varia Mart., family Fabaceae Lindl.), which plays an important role not only in sustainable agriculture but also in expanding the raw material base of the food, cosmetic and pharmaceutical industries.For instance, studies have shown that Medicago varieties contain bioactive compounds, identified as phenolic compounds, in particular, flavonoids.The flavonoids found include flavones (luteolin and apigenin), isoflavones (genistein), flavanones (naringenin), flavanols (catechin and epicatechin) and anthocyanidins (cyanidin and delphinidin) [1,2].The interest in flavonoid research increased because of the potential of these substances to prevent or treat factors related to metabolic disorders [3].GC-MS analysis of M. sativa seeds revealed their enrichment in crude protein (33.79%), crude oil (8.11%), squalene, hexadecanoic acid methyl ester, n-hexadecanoic acid, 9,12-octadecadienoic acid methyl ester, 9-octadecenamide and vitamin E.Moreover, Medicago sativa seed inclusion in the diet is recommended to normalize serum cholesterol levels in type II hyperlipoproteinemia patients [4][5][6].Additionally, there are several interesting scientific studies showing the presence of simple phenolic compounds in Medicago [7,8].These compounds have a range of biological activities, including anti-inflammatory, antioxidant, phytoestrogenic and anticarcinogenic abilities, as well as their interactions with intracellular signaling pathways and regulation of cell survival.Extracts and balms of M. sativa have long been used as traditional herbal medicines in many countries, such as China, India and America [9,10].Most varieties of alfalfa are autotetraploids (2n = 4x = 32), with the main number of chromosomes being eight [11,12].Alfalfa is characterized by its exceptional ability to grow under a wide range of natural conditions, its stable global yield, and its longevity and reproduction of soil fertility through fixation of atmospheric nitrogen.This crop is used as fodder in its green form or for the preparation of fodder (hay, haylage and grass meal).Lucerne hay is a quality forage containing high levels of protein, phosphorus, calcium and essential amino acids [13].Increased forage production is possible through the development of more productive and higher-quality lucerne varieties.Each soil-climatic zone requires a diverse set of complementary varieties adapted to different extreme growing conditions [14].Therefore, the study of the genetic diversity of alfalfa source material is of great theoretical and practical importance.M. varia contains omega-3 fatty acids, which are necessary to improve milk quality and increase meat production in ruminants [15].The plant complex of alfalfa, which contains substances necessary for humans (especially in unfavorable environmental conditions), is used in technology for the production of fermented milk products [16].It is also worth noting the insecticidal and fungicidal potential of the use of saponins identified in Medicago.The combined deterrent and toxic effects on insects make Medicago saponins suitable for use against insect pests in agriculture and horticulture [17].An effective way of using Medicago saponins against insect herbivores is to select varieties that accumulate high levels of saponins [17].For example, the development, survival and reproduction of pea aphids fed on high-saponin alfalfa were reduced compared to those fed on low-saponin alfalfa.In addition, it has been shown that zanhic acid tridesmoside and medicagenic acid, which accumulate in a high-saponin cultivar, are the main compounds contributing to the resistance of alfalfa to pea aphids [18,19].The nematocidal activity of saponins allows the use of Medicago biomass as a biological agent to control plant-parasitic nematodes, which are widespread in the soil.The antifungal activity of alfalfa saponins may also reduce the presence of phytopathogenic fungi in the soil.Total saponins and selected compounds from different Medicago species have been shown to prevent fusarium on tulip bulbs [20,21].Saponins against phytopathogenic fungi and nematodes in plant material make Medicago biomass particularly useful as an agent against soil-borne plant pathogens and as a biological fertilizer.Supercritical fluid extraction with the use of pressured CO 2 (SC-CO 2 ) has been used over the last 50 years in analytical methodologies to investigate the composition of food products, for the removal of undesirable substances and for the isolation of valuable molecules.The goal of the present work was to identify and select bioactive compounds from M. varia via extraction with SC-CO 2 .Also, a tandem mass spectrometry protocol was used for the detailed screening of phytochemicals present in three varieties of M. varia.

Materials
The subject of the study was the green mass of M. varia varieties (Demetra, Nakhodka and Sarga) (Figure 1A-D) collected and grown at the Sakhalin Agricultural Scientific Research Institute-Branch of N.I.Vavilov All-Russian Institute of Plant Genetic Resources.Standard agronomic practices were used for growing the accessions/varieties in their respective locations.The aerial parts of M. varia were harvested at the end of July 2023.All plant tissues used in this work conformed to the standard established by the State Pharmacopoeia of the Russian Federation [22].
Standard agronomic practices were used for growing the accessions/varieties in their respective locations.The aerial parts of M. varia were harvested at the end of July 2023.All plant tissues used in this work conformed to the standard established by the State Pharmacopoeia of the Russian Federation [22].

Chemicals and Reagents
All reagents used in the study were of analytical grade.HPLC-grade acetonitrile was purchased from Fisher Scientific (Kent, UK), and MS-grade formic acid and ethanol (EtOH) were purchased from Sigma-Aldrich (Steinheim, Germany).Ultrapure water was obtained from Siemens (SIEMENS water technologies, Munich, Germany).

Extraction
SC-CO2 extraction was performed using the SFE-500 supercritical pressure extraction system (Thar SCF Waters, Milford, CT, USA).The system options include the following: a co-solvent pump (Thar Waters P-50 High Pressure Pump) for the extraction of polar samples; a CO2-flow meter (Siemens, Munich, Germany) to measure the amount of CO2 supplied to the system; and multiple extraction vessels to extract different sample sizes or to increase the throughput of the system.The flow rate was 10-25 mL/min for liquid CO2 and 1.00 mL/min for EtOH.Extraction samples of 200 g M. varia were used.The extraction time was counted after reaching working pressure and equilibrium flow and was 60-90 min for each sample.This method of SC-CO2 extraction of plant matrices was tested by the authors on numerous plant samples, including aboveground and underground parts of the plant [23][24][25].

Chemicals and Reagents
All reagents used in the study were of analytical grade.HPLC-grade acetonitrile was purchased from Fisher Scientific (Kent, UK), and MS-grade formic acid and ethanol (EtOH) were purchased from Sigma-Aldrich (Steinheim, Germany).Ultrapure water was obtained from Siemens (SIEMENS water technologies, Munich, Germany).

Extraction
SC-CO 2 extraction was performed using the SFE-500 supercritical pressure extraction system (Thar SCF Waters, Milford, CT, USA).The system options include the following: a co-solvent pump (Thar Waters P-50 High Pressure Pump) for the extraction of polar samples; a CO 2 -flow meter (Siemens, Munich, Germany) to measure the amount of CO 2 supplied to the system; and multiple extraction vessels to extract different sample sizes or to increase the throughput of the system.The flow rate was 10-25 mL/min for liquid CO 2 and 1.00 mL/min for EtOH.Extraction samples of 200 g M. varia were used.The extraction time was counted after reaching working pressure and equilibrium flow and was 60-90 min for each sample.This method of SC-CO 2 extraction of plant matrices was tested by the authors on numerous plant samples, including aboveground and underground parts of the plant [23][24][25].

Liquid Chromatography
High-performance liquid chromatography was carried out on a Shimadzu LC-20 Prominence HPLC (Shimadzu, Kyoto, Japan) instrument equipped with a UV sensor and a C18 silica reverse phase column (4.6 × 150 mm, particle size: 2.7 µm). Mobile-phase eluent A was deionized water containing 0.1% formic acid, and eluent B was acetonitrile containing 0.1% formic acid.The gradient elution was started at 0-2 min, 0% eluent B 2-50 min, 0-100% B; control washing: 50-60 min, 100% B. The mobile-phase flow rate and column temperature were maintained at 0.3 mL/min and 30 • C, respectively.A UV-vis detector, the SPD-20A (Shimadzu, Kyoto, Japan), was used for detection and compound identification at a wavelength of 230 nm.The injection volume was 10 µL.Additionally, liquid chromatography was combined with a mass spectrometric ion trap to identify compounds.

Mass Spectrometry
MS analysis was performed on an ion trap, the amaZon SL (Bruker Daltoniks, Bremen, Germany), equipped with an ESI source in negative ion mode.MS analysis was carried out in electrospray ionization (ESI) mode using negative and positive polarity for all samples with data-independent MSE acquisition.The optimized parameters were obtained as reported earlier [23][24][25].Similarly, the data collection and compound identification were carried out as per our previous reports [23][24][25].

Statistical Analysis
To more clearly present the similarities and differences of bioactive substances identified in different variants of M. varia, the team of authors used the Jaccard index.The Jaccard index, also known as the Jaccard similarity coefficient, is a statistical measure used to evaluate the similarity and diversity of sets of samples.Nine replicate samples were analyzed.Jaccard indices were calculated using a the "Compare Lists-Multiple List Comparator" hosted on molbiotools server (https://molbiotools.com/listcompare.php(accessed on 21 March 2024)).

SC-CO 2 Extraction of Aerial Parts of M. varia
Three M. varia varieties, i.e., Sarga, Nakhodka and Demetra, were examined by SC-CO 2 extraction under different extraction conditions.The supercritical pressures applied ranged from 50 to 250 bar, and the extraction temperature ranged from 31 to 70 • C. The cosolvent, EtOH, was used in an amount of 1% of the total solvent amount.The Table 1 shows the global yield of bioactive compounds (variety Sarga) by SC-CO 2 extraction.Figure 1D shows a 3D plot of the global yield of bioactive compounds during SC-CO 2 extraction of the aerial parts (variety Sarga).

Global Metabolome Profile of M. varia
The structural identification of each compound was carried out on the basis of its accurate mass and MS/MS fragmentation by HPLC-ESI-ion trap-MS/MS.A total of 103 chemical compounds were identified from the extracts of the three M. varia varieties (Table 4).Fifty-nine and forty-four chemical compounds were classified as polyphenols and others, respectively (see the chemical structure of some of these compounds in Figure 2).The polyphenols detected in our study were categorized as flavones, flavonols, flavan-3-ols, anthocyanidins, phenolic acids, lignans, coumarins, stilbenes, etc.In total, the metabolites detected in our study belonged to 19 compound classes.The highest number of metabolites was recorded for flavones (24), followed by flavonols (20), anthocyanins (6) and flavan-3-ols (3).These numbers of compounds in respective groups indicate that M. varia extracts are rich in flavonoids.The highest numbers of chemical compounds from other groups were recorded for polysaccharides (8) and saponins (11).
). Fifty-nine and forty-four chemical compounds were classified as polyphenols and oth ers, respectively (see the chemical structure of some of these compounds in Figure 2).The polyphenols detected in our study were categorized as flavones, flavonols, flavan-3-ols anthocyanidins, phenolic acids, lignans, coumarins, stilbenes, etc.In total, the metabolite detected in our study belonged to 19 compound classes.The highest number of metabo lites was recorded for flavones (24), followed by flavonols (20), anthocyanins ( 6) and fla van-3-ols (3).These numbers of compounds in respective groups indicate that M. vari extracts are rich in flavonoids.The highest numbers of chemical compounds from othe groups were recorded for polysaccharides (8) and saponins (11).Moreover, to present the similarities and differences in bioactive substances in different variations of M. varia, we used the Jaccard index (Table 4).The Jaccard index, also known as the Jaccard similarity coefficient, is a statistic used to evaluate the similarity and diversity of sets of samples [26][27][28].It showed that the highest degree of similarity existed between the varieties Demetra and Nakhodka-0.4409.

Discussion
The biologically active compounds of aerial parts of plants are effectively extracted using organic solvents such as methanol and ethanol.But extraction products in the final phase require additional purification from trace amounts of used solvents.SC-CO 2 extraction can be used as an alternative to traditional extraction methods: maceration or Soxhlet extraction [119,120].SC-CO 2 extraction has been used in the evaluation of food products, the isolation of bioactive substances, and the determination of lipid levels in foods and levels of toxic substances.With SC-CO 2 extraction, the products do not contain organic solvent residues that occur with conventional extraction methods, and the solvents can be toxic, as in the case of methanol and n-hexane, for example.Easy solvent removal from the final product, high selectivity and moderate extraction temperatures are the main attractions of SC-CO 2 technology, leading to a significant increase in research for applications in the food and pharmaceutical industries.When comparing possible supercritical solvents, carbon dioxide has the most attractive advantages, being a non-toxic, non-flammable, environmentally friendly and renewable resource [121].Popova et al. investigated the influence of SC-CO 2 extraction parameters and the quality of Ledum palustre feedstock on the global yields of chlorophylls and carotenoids.The data obtained were significant for the pharmaceutical, food, and perfume and cosmetic industries, which require natural dyes and antioxidants [122].Baananou et al. reported the anti-inflammatory activity of two extracts from the aerial parts of Rhododendron [123].Aliev et al., in their research, have shown that SC-CO 2 extraction is an effective method for extracting a wide range of lipophilic fractions from plant materials in one experimental procedure, which provides additional opportunities for research [124].
Thus, the use of SC-CO 2 extraction is an effective approach for the extraction of bioactive compounds.Our results are consistent with these reports that SC-CO 2 extraction is a useful approach to extract and study bioactive compounds.
The extracts obtained showed both a high content of polyphenolic compounds and a high content of saponin group compounds.Earlier studies revealed the presence and detection of polyphenols in legume species.Chiriac et al. [125] used UHPLC-Q exactive hybrid quadrupole orbitrap high-resolution mass spectrometry and identified 29 compounds from the sprouts of Medicago sativa and Trifolium pratense, based on their mass, FIs, retention time and data in the literature.However, using SC-CO 2 extraction, the number of polyphenol compounds was higher in our results.This difference could be due to the extraction method or the different tissues under study.Other than Medicago, polyphenols are also abundant in other legume species, such as Phaseolus vulgaris [126], soybean [127], chickpea, grass pea, lentils [128], peanut [129], etc.These polyphenols act as dietary antioxidants in humans and impart protective effects against certain diseases [130].
Apart from polyphenols, the presence of saponins in Medicago varieties is a useful observation.These observations are consistent with earlier studies which reported the presence of saponins in Medicago truncatula [100,107,118].Saponins are active compounds present in edible legumes and impart health benefits [131,132].Bioinformatic analyses in legumes have revealed the presence of the triterpene biosynthesis pathway and conserved genes [133,134].Thus, the detection of eleven compounds classified as saponins and two triterpenoid acids (ursolic acid and oleanolic acid) in the leaves of M. varia is consistent with the above-cited studies.Though several studies on soybean and other legumes have highlighted that seeds and roots [135] are the major sinks for saponins, their presence in leaves and stems has also been reported in, e.g., Jatropha curcas [136], Acanthopanax sieboldianus [137], Quillaja lancifolia [138], etc.The detection of saponins in the leaves of M. varia together with a range of polyphenols suggests this plant as a potential raw material for use in traditional medicine as well as modern pharmacology.
It is well recognized that using some fungicides can be harmful to both the environment and human health.As a result, during the past twenty years, an increasing amount of research has been carried out on the potential use of plant-based substances that would be less harmful than chemicals produced in factories.The increasing prospect of employing saponins as natural fungicides is highlighted in a large number of international articles [139].
Fourteen triterpene saponins from the roots of Medicago hybrida have been identified and their structures have been established [145].Six fungi were tested in vitro to determine the antifungal activity of the roots' saponins, and eight main saponin glycosides were tested against Botrytis tulipae, one of the most sensitive fungi [20].
It should be noted that different concentrations of saponins equally inhibited the mycelial growth of Botrytis cinerea and B. tulipae.However, the higher concentrations inhibited the mycelial growth of B. cinerea somewhat less.It is assumed that lower concentrations of saponins are sufficient to block all active sites in the mycelial hyphae.It has also recently been shown that M. hybrida saponins have insecticidal activities as high as those of M. arabica and Medicago murex [146].On the other hand, it is known that M. arabica and M. murex saponins are also rich in highly fungicidal saponins [147].In conclusion, Medicago saponins have significant antifungal activity, and the roots of this plant can be a rich source of natural fungicides.Therefore, their detection in the aerial parts of M. varia has added to the existing list of beneficial compounds.

Conclusions
M. varia species contain many polyphenolic components and components of other chemical groups that have valuable biological activities.SC-CO 2 extraction of M. varia (three varieties) was successfully carried out by the team of authors.Certain extraction conditions were selected, and the extracts obtained showed both a high content of polyphenolic compounds and a high content of saponin group compounds.Tandem mass spectrometry (HPLC-ESI-ion trap) was used to detect target analytes.Mass spectrometric data were recorded on an ion trap equipped with an ESI source in negative and positive ion modes.A four-stage ion separation mode was implemented.One hundred and three different biologically active compounds were found in M. varia extracts.Twenty-seven phenolic compounds were tentatively identified for the first time.Also, for the first time, the following saponins were identified with reliable accuracy in M. varia: soyasaponin II, soyasaponin gamma g, soyasaponin I, soyasaponin Bd, soyasaponin beta g, steroidal alkaloids (alpha-chaconine), etc.Although our study aimed only to study the aerial parts of Medicago varia, which belongs to the legume family (Fabaceae), the same approach can be applied in the future to study factors influencing the metabolite profiles of legume seeds, including seasonal variations, cultivation and storage conditions.In addition, our study may provide new interesting details for future taxonomic studies, especially if they target larger genotypes.

Figure
Figure 1F shows a 3D graph of the global yield of biologically active substances during SC-CO 2 extraction of alfalfa aerial parts (variety Demetra).The maximum global yield of bioactive substances from alfalfa aerial parts (variety Demetra) was observed under the following extraction conditions: -Pressure: 150 Bar, extraction temperature: 50 • C, extraction time: 1 h; the global yield of biologically active substances was 3.2 mg/100 mg of plant sample; the share of the EtOH modifier was 2%.

Table 4 .Figure 2 .
Figure 2. Chemical structures of some phenolic compounds identified in M. varia.

Figure 3
Figure 3 shows the numbers of common and specific compounds.Nineteen com pounds were commonly detected from the three M. varia varieties.These nineteen com pounds belong to compound classes such as flavones, flavonols, anthocyanins and

Figure 2 .
Figure 2. Chemical structures of some phenolic compounds identified in M. varia.

Figure 3
Figure3shows the numbers of common and specific compounds.Nineteen compounds were commonly detected from the three M. varia varieties.These nineteen compounds belong to compound classes such as flavones, flavonols, anthocyanins and saponins, suggesting that flavonoids and saponins are major active compounds in M. varia leaves.The applied methods were able to detect 71 (Demetra), 63 (Nakhodka) and 38 (Sarga) compounds.

Figure 3 .
Figure 3. Venn diagram showing numbers of common and specific compounds in M. varia varieties.

Figure 3 .
Figure 3. Venn diagram showing numbers of common and specific compounds in M. varia varieties.
produced two characteristic daughter ions with m/z 227.15 and m/z 171.18.The FI with m/z 227.15 generated an ion with m/z 198.18.
. [M − H] − ions produced four FIs with m/z 285.20, m/z 401.15, m/z 327.21 and m/z 255.18 (Figure 4C).The FI with m/z 285.20 produced one characteristic daughter ion with m/z 255.19, m/z 227.15 and m/z 151.250.The FI with m/z 255.19 produced one characteristic daughter ion with m/z 227.

Table 2
showing the global yield of bioactive compounds (variety Nakhodka) by SC-CO 2 extraction is presented below.

Table 2 .
The global yield of bioactive compounds (variety Nakhodka) by SC-CO 2 extraction.

Table 3 .
The global yield of bioactive compounds (variety Demetra) by SC-CO 2 extraction.

Table 4 .
Jaccard indices for three varieties of M. varia.

Table 5 .
Chemical compounds identified from the SC-CO 2 -extracts of M. varia in positive and negative ionization modes by HPLC-ion trap-MS/MS.