Maackia amurensis Rupr. et Maxim.: Supercritical CO2 Extraction and Mass Spectrometric Characterization of Chemical Constituents

Three types of extraction were used to obtain biologically active substances from the heartwood of M. amurensis: supercritical CO2 extraction, maceration with EtOH, and maceration with MeOH. The supercritical extraction method proved to be the most effective type of extraction, giving the highest yield of biologically active substances. Several experimental conditions were investigated in the pressure range of 50–400 bar, with 2% of ethanol as co-solvent in the liquid phase at a temperature in the range of 31–70 °C. The most effective extraction conditions are: pressure of 100 bar and a temperature of 55 °C for M. amurensis heartwood. The heartwood of M. amurensis contains various polyphenolic compounds and compounds of other chemical groups 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 equipped with an ESI source in the modes of negative and positive ions. The four-stage ion separation mode was implemented. Sixty-six different biologically active components have been identified in M. amurensis extracts. Twenty-two polyphenols were identified for the first time in the genus Maackia.


Introduction
Maackia amurensis Rupr. et Maxim. is the only representative of the Fabaceae family in the flora of the Russian Far East. Probably, this species can be considered a relic of the Tertiary flora, which survived in more severe climatic conditions than the species of the genera Cladrastis and Sophora. The distribution area of Maackia amurensis is in the Amur River basin and in the south of Primorsky Krai, Russia. The natural reserves of this plant are large and actively self-renewal [1][2][3]. A close species to maackia is M. amurensis Rupr. et Maxim. var. buergeri (Maxim.) C.K. Schneeder ( Figure 1). However, its chemical composition differs sharply from that of the Far Eastern species, M. amurensis [2][3][4][5]. Until 1985, the only data on the chemical composition of Russian maackia were data on alkaloids contained in the bark and green parts of the plant. In the subsequent detailed chemical study of alcoholic extracts of heartwood, it was shown that the main components of maackia are plant polyphenols [3][4][5]. These include isoflavones: genistein, daidzein, retusin, afromosin, formononetin, orobol, tectorigenin, 3-hydroxyvestiton, pterocarpans maakiain, medicarpin [5][6][7]. The peculiarity of Maackia amurensis growing in Primorye is the high content of monomeric stilbenes resveratrol and piceatannol and isoflavonstilben maackiasin in its wood [5,6]. These polyphenols were not found in the variety Maackia amurensis (var. buergeri) growing in Japan [1].
The polyphenolic complex from M. amurensis heartwood, called Maksar ® preparation, is registered in the Russian Federation as a hepatoprotective medicine (P N003294/01). Maxar ® increases the body's antioxidant system activity and reduces the lipid peroxidation level. Its application in clinical practice showed that this drug is effective for treating liver fatty dystrophy. It prevents the increase in total serum lipid content and the development of hyperlipoproteinemia in experimental animals. Maxar ® also possesses antithrombogenic, antiplatelet, and antitumor properties [10,11]. Recently, new research has been carried out which shows that stilbenolignan maackolin may be a good candidate as a SARS-CoV-2 Mpro inhibitor in vivo studies [12].
The use of supercritical fluids in food material applications and more broadly in the food industry began in the late 1960s and probably represents the most successful application of supercritical fluids to date. The "green technology" of supercritical CO2 extraction using high pressures is an excellent technique for obtaining natural thermolabile compounds. In addition, there are no residues of organic solvents in the products, which occurs with conventional extraction methods-conventional solvents can be toxic, for example, in the case of methanol and hexane. Easy removal of the solvent from the final product, a high selectivity, and the use of moderate temperatures in the extraction process are the main attractive factors of SFE, leading to a significant increase in research for applications in the food and pharmaceutical industries [13].
The polyphenolic complex from M. amurensis heartwood, called Maksar ® preparation, is registered in the Russian Federation as a hepatoprotective medicine (P N003294/01). Maxar ® increases the body's antioxidant system activity and reduces the lipid peroxidation level. Its application in clinical practice showed that this drug is effective for treating liver fatty dystrophy. It prevents the increase in total serum lipid content and the development of hyperlipoproteinemia in experimental animals. Maxar ® also possesses antithrombogenic, antiplatelet, and antitumor properties [10,11]. Recently, new research has been carried out which shows that stilbenolignan maackolin may be a good candidate as a SARS-CoV-2 Mpro inhibitor in vivo studies [12].
The use of supercritical fluids in food material applications and more broadly in the food industry began in the late 1960s and probably represents the most successful application of supercritical fluids to date. The "green technology" of supercritical CO 2 extraction using high pressures is an excellent technique for obtaining natural thermolabile compounds. In addition, there are no residues of organic solvents in the products, which occurs with conventional extraction methods-conventional solvents can be toxic, for example, in the case of methanol and hexane. Easy removal of the solvent from the final product, a high selectivity, and the use of moderate temperatures in the extraction process are the main attractive factors of SFE, leading to a significant increase in research for applications in the food and pharmaceutical industries [13].
Chemical reactions that have made the greatest contribution to food technologies were enzyme-catalyzed reactions [14], hydrogenations designed to control particular trans isomers occurring in lipid mixtures [15], and hydrolysis conducted in the presence of enzymes or a medium such as subcritical water [16]. Considerable activity in producing fine particles for use in the pharmaceutical industry began in the 1990s. In the last three decades, focus on the development of technologies was displaced by the combination of SCF technologies in the food industry and the obtainment of bioactive agents from natural matrixes [17,18].
In this research, supercritical CO 2 extraction, MeOH maceration, and EtOH maceration of the samples of M. amurensis were used to obtain an effective amount of biologically active substances. We used a tandem mass spectrometry to carry out a phytochemical study

Results
Three samples of wood substance of M. amurensis were subjected to supercritical CO 2 extraction under different extraction conditions. The applied supercritical pressures ranged from 150 to 400 bar, and the extraction temperature ranged from 31 to 65 • C. The co-solvent EtOH was used in an amount of 2% of the total amount of solvent. A pronounced extraction extremum is shown in the 3D graph ( Figure 2). The best extraction conditions for M. amurensis (heartwood) were the following: pressure of 100 bar and temperature at 55 • C. The total yield of biologically active substances under these extraction conditions fine particles for use in the pharmaceutical industry began in the 1990s. In the last three decades, focus on the development of technologies was displaced by the combination of SCF technologies in the food industry and the obtainment of bioactive agents from natural matrixes [17,18].
In this research, supercritical CO2 extraction, MeOH maceration, and EtOH maceration of the samples of M. amurensis were used to obtain an effective amount of biologically active substances. We used a tandem mass spectrometry to carry out a phytochemical study involving a detailed metabolomic analysis of M. amurensis. The bark of M. amurensis was collected during expedition work near Ussuri River, Primorsky Krai, Russia (N 42°36′10″ E 131°10′55″), during the period from 1 to 20 August 2022.

Results
Three samples of wood substance of M. amurensis were subjected to supercritical CO2 extraction under different extraction conditions. The applied supercritical pressures ranged from 150 to 400 bar, and the extraction temperature ranged from 31 to 65 °C. The co-solvent EtOH was used in an amount of 2% of the total amount of solvent. A pronounced extraction extremum is shown in the 3D graph ( Figure 2). The best extraction conditions for M. amurensis (heartwood) were the following: pressure of 100 bar and temperature at 55 °C. The total yield of biologically active substances under these extraction conditions was 4.  The research data presented in Figure 2 show the presence of a confident maximum of supercritical CO2 extraction in the pressure range of 100 to 150 bar and temperature range from 50 °C to 55 °C. In this range, the highest yield of biologically active compounds from the plant matrix of M. amurensis is observed. It should be noted that during the experiment, the extraction time was also small-1 h; therefore, what can we say about the effectiveness of the applied method of supercritical CO2-extraction? The research data presented in Figure 2 show the presence of a confident maximum of supercritical CO 2 extraction in the pressure range of 100 to 150 bar and temperature range from 50 • C to 55 • C. In this range, the highest yield of biologically active compounds from the plant matrix of M. amurensis is observed. It should be noted that during the experiment, the extraction time was also small-1 h; therefore, what can we say about the effectiveness of the applied method of supercritical CO 2 -extraction?
It should also be noted that a detailed analysis of the presence of polyphenols and biologically active substances from other chemical groups showed the highest number of flavonoids at supercritical CO2 extraction at a pressure of 100 bar-43 compounds. Accordingly, with other types of extraction investigated in this study, such as maceration with ethanol (20 compounds), maceration with methanol (23 compounds), and supercritical CO2-extraction at a higher extraction pressure (19 compounds), the yield efficiency of biologically active substances is much lower. (Table 1).   24; they were identified in extracts from Astragali Radix [19][20][21]. The CID spectrum in the positive ion mode of calycosin-7-O-beta-D-glucoside-6"-O-malonate from M. amurense is shown in Figure 6.
It should also be noted that a detailed analysis of the presence of polyphenols and biologically active substances from other chemical groups showed the highest number of flavonoids at supercritical CO 2 extraction at a pressure of 100 bar-43 compounds. Accordingly, with other types of extraction investigated in this study, such as maceration with ethanol (20 compounds), maceration with methanol (23 compounds), and supercritical CO 2 -extraction at a higher extraction pressure (19 compounds), the yield efficiency of biologically active substances is much lower. (Table 1). Thus, it can be stated that as a result of the most detailed study using tandem mass spectrometry, new data on the content of biologically active substances in M. amurensis have been obtained.
M. amurensis extracts exhibited different DPPH scavenging effects compared to quercetin ( Table 2). CO 2 extract obtained at 100 bar possessed the most considerable activity compared to quercetin, which is mainly due to the high content of monomeric and dimeric stilbenes. EtOH extract from M. amurensis was the least active, because the main components of this extract were glycosides of isoflavones, which are rather weak antioxidants.

Materials
Wood substance of M. amurensis was collected during expedition work near Ussuri River, Primorsky Krai, Russia (N 42 • 36 10" E 131 • 10 55"), during the period from 1 to 20 August 2022. All samples were morphologically authenticated according to the current standard of Russian Pharmacopeia [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.

Fractional Maceration
The fractional maceration technique was applied to obtain highly concentrated extracts [23]. From 500 g of the wood substance, 20 g of wood were randomly selected for maceration. The total amount of the extractant (ethyl alcohol of reagent grade) was divided into 3 parts, and the parts of plant were consistently infused with the first, second, and third parts. The solid-solvent ratio was 1:20. The infusion of each part of the extractant lasted 7 days at room temperature.

Extraction
SC-CO 2 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; CO 2 flow meter (Siemens, Munich, Germany), to measure the amount of CO 2 being 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 100 g of wood substance of M. amurensis were used. The extraction time was counted after reaching the working pressure and equilibrium flow, and it was 60-90 min for each sample.

Liquid Chromatography
HPLC was performed using Shimadzu LC-20 Prominence HPLC (Shimadzu, Kyoto, Japan) equipped with a UV sensor and C18 silica reverse phase column (4.6 × 150 mm, particle size: 2.7 µm) to perform the separation of multicomponent mixtures. The gradient elution program with two mobile phases (A, deionized water; B, acetonitrile with formic acid 0.1% v/v) was as follows: 0-2 min, 0% B; 2-50 min, 0-100% B; control washing 50-60 min 100% B. The entire HPLC analysis was performed with a UV-vis detector SPD-20A (Shimadzu, Kyoto, Japan) at a wavelength of 230 nm for identification compounds; the temperature was 50 • C, and the total flow rate 0.25 mL min −1 . 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 amaZon SL (BRUKER DALTONIKS, Bremen, Germany) equipped with an ESI source in the 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: 1500 V; fragmentary: 280 V; and 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 using Windows software for BRUKER DALTONIKS. All experiments were repeated three times. A four-stage ion separation mode (MS/MS mode) was implemented.

Antiradical Activity
We determined the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging effect of extracts from M. amurensis heartwood. The extracts were added to DPPH solution in MeOH (10 −4 M) at concentrations from 1 to 85 µg/mL. We kept the reacting mixture in the dark at room temperature for 20 min. Then, we measured the absorbance at 517 nm using a Shimadzu UV-1800 spectrophotometer (Shimadzu, Canby, OR, USA). We used Equation (1) to calculate the DPPH radical-scavenging effect (%): where A 0 is the absorbance of DPPH solution without M. amurensis extracts (blank sample); A x is the absorbance of DPPH solution in the presence of different concentrations of extracts.
Quercetin was used as a reference compound. All experiments were performed in triplicate. The half maximal inhibitory concentrations (IC 50 ) for extracts were calculated by plotting the DPPH scavenging effect (%) against the concentrations of M. amurensis extracts. IC 50 values are given as the mean ± SEM.

Conclusions
Three types of extraction were used to obtain biologically active substances from the wood substance of M. amurensis: supercritical CO 2 extraction, maceration with EtOH, and maceration with MeOH. The supercritical extraction method proved to be the most effective type of extraction, giving the highest yield of biologically active substances. Several experimental conditions were investigated in the pressure range of 50-400 bar, with the used volume of co-solvent ethanol being 2% in the liquid phase at a temperature in the range of 31-70 • C. The most effective extraction conditions are: pressure of 100 bar and temperature at 55 • C for the wood substance of M. amurensis. The wood of M. amurensis contains various polyphenolic compounds and compounds of other chemical groups 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. Sixty-six different biologically active components have been identified in M. amurensis extracts. Twenty-two polyphenols were identified for the first time in the genus Maackia.
These data could support future research for the production of a variety of pharmaceutical products containing ultra-pure extracts of M. amurensis. The richness of various biologically active compounds, including compounds of the polyphenol group and compounds of other chemical groups (amino acids, Omega fatty acids, sterols, triterpenoids, etc.), provides great opportunities for the design of new nutritional and dietary supplements based on extracts from this Maackia genus.