Study of the Chemical Composition and Biologically Active Properties of Glycyrrhiza glabra Extracts

Glycyrrhiza glabra or licorice has long been known as a commonly used Ayurvedic herb. This study aims to investigate the effect of extraction methods on the chemical composition and biologically active properties of Glycyrrhiza glabra extract samples. The highest yield of the Glycyrrhiza glabra extract (21.31 ± 0.64 wt.%) was produced using the Soxhlet extraction method with methanol. The highest concentrations of biologically active substances (3,4-dihydroxybenzoic acid, n-coumaric acid, luteolin-7-glucoside, acacetin, apigenin-7-O-glucoside, chicoric acid, and hesperetin) were found in these samples of Glycyrrhiza glabra extracts. When applying the maceration method using a mixture of solvents methanol-NaOH, rosmarinic acid was identified, and catechin was found in large quantities with a mixture of methanol-trifluoroacetic acid (TFA). Growth inhibition zones were determined for Escherichia coli (13.6 ± 0.41 mm), Pseudomonas aeruginosa (10.8 ± 0.32 mm), Bacillus subtilis (16.1 ± 0.48 mm), and Candida albicans (13.2 ± 0.39 mm) when exposed to samples of Glycyrrhiza glabra extracts obtained by the Soxhlet method with methanol. The antioxidant activity of Glycyrrhiza glabra extract samples obtained by the Soxhlet method was 117.62 ± 7.91 µmol Trolox equivalent/g, using the ABTS method (highest value), and 23.91 ± 1.12 µmol Trolox equivalent/g according to the FRAP method (smallest). The antioxidant activity of the extract samples according to the DPPH method was an intermediate value of 58.16 ± 3.90 µmol Trolox equivalent/g. Antibacterial and antioxidant activities are manifested by the polyphenolic compounds and flavonoids contained in the samples of the methanol extract of Glycyrrhiza glabra produced using the Soxhlet method. These Glycyrrhiza glabra extract samples have the potential to become a natural alternative to existing therapies for the elimination of bacterial infections or the prevention of premature aging caused by free radicals and oxidative stress in the human body.


Introduction
Nature has always been a source of medicinal substances, providing man with a wide range of medicinal plants containing valuable phytochemicals. Licorice (Glycyrrhiza glabra) belongs to the bean family, and Fabaceae. G. glabra is a medicinal plant found throughout Asia, as well as in some parts of Europe [1]. It is believed that G. glabra originated in Iraq [2]. such as 30% water in choline chloride solution: NADES lactic acid, liquid/solid ratio 40:1 (v/mL/g), temperature 40 • C, stirring speed 400 rpm, and extraction time 30 min. The yield of GA under these conditions was 53.72 ± 0.57 mg/g GA. The NADES extraction method successfully provided increased yield and improved anti-inflammatory activity of the extracts in vitro, which was evaluated using the human erythrocyte membrane stabilization method. In addition, it was found to be energy efficient and economical compared to traditional extraction methods. The results provided valuable information for the introduction of NADES to develop an extraction process for biologically active compounds of G. glabra [23].
A study [24] described supercritical fluid extraction (SFE) using a modified supercritical carbon dioxide co-solvent and a two-step separation/purification method, which was used as a way to increase the purity of glabridin, one of the many bioactive components of G. glabra (licorice). The SFE parameters were optimized using an analytical scale SFE system in the temperature range of 40-80 • C and pressure range of 10-50 MPa. The extraction was then increased 100-fold using the SFE preparative system under the following set of recommended conditions: 40 • C, 30 MPa, and SCCO 2 modified with 25% (v/v) ethanol equivalent. The purity of glabridin obtained by the enhanced SFE system was 6.2%, which is much higher than the purity obtained by organic solvent extraction. The licorice extract obtained by scaling up the SFE system was isolated and purified using alcohol precipitation/filtration and adsorption chromatography with 80% aqueous ethanol to obtain a purer product. It was confirmed that the purity of glabridin in the final extract product increased to 37% without a significant loss of glabridin after two separation/purification steps [24].
The study [25] aimed to extract glycyrrhizic acid from G. glabra, which is widely used in medicine as an anti-inflammatory, antiulcer, antiallergic, and antipsoriatic agent. This study focused on extracting glycyrrhizic acid from the roots of G. glabra and assessing its anti-inflammatory activity in vitro. GA was extracted by maceration. Its physicochemical properties, biochemical tests, and phytochemical properties were evaluated. Anti-inflammatory activity in vitro was assessed by the method of albumin denaturation. The results of the studies showed that the ash content and the yield of the extract were below the limits set by the Ayurvedic Pharmacopoeia of India. Flavonoids, saponins, and triterpenoids were identified in the G. glabra extract by phytochemical parameters. Thin layer chromatography showed a retention value of 0.5 cm. Percent inhibition indicated that G. glabra extract had some potential for healing inflammation. Thus, it was demonstrated that GA was successfully extracted from licorice roots. The evaluation parameters showed the presence of fewer impurities in the extract with the potential for anti-inflammatory action [25].
This study aims to investigate the effect of the extraction method on the chemical composition and biologically active properties of samples of Glycyrrhiza glabra extracts. The novelty of the research lies in the selection of the extraction (maceration) method, in which the maximum amount of BAS is released from G. glabra, exhibiting the most pronounced antimicrobial and antioxidant activities.
A null hypothesis was proposed prior to the start of the experiment, stating that the extraction method had no significant effect on the content of biologically active substances in G. glabra extracts, as well as their antioxidant and antibacterial activities. All extraction methods were assumed to be equally effective. Table 1 presents the total yield of various G. glabra extracts.  The Soxhlet extraction method with methanol produced the highest yield of extracts; therefore, chromatograms for samples of G. glabra extracts obtained by the Soxhlet method with methanol as an extractant are provided below.  The Soxhlet extraction method with methanol produced the highest yield of extracts; therefore, chromatograms for samples of G. glabra extracts obtained by the Soxhlet method with methanol as an extractant are provided below.            Liquiritin apioside (Figure 4, peak M1), G. glabra G2 saponin ( Figure 4, peak M2), glycyrrhizin ( Figure 4 Table 3.  Figure 5, peak M10) were identified in the extract of the medicinal plant G. glabra obtained by the Soxhlet extraction method. The amount of these substances is presented in Table 3.     The fractionation of Soxhlet G. glabra extracts by HPLC identified an apigenin derivative with a yield of 1 × 10 2 mg/kg at a retention time of 15.495 min. Table 4 presents the antimicrobial activity of G. glabra extracts obtained by Soxhlet method and maceration methods with solvent mixtures. The antimicrobial activity of samples of G. glabra extracts, obtained by the Soxhlet method and maceration methods with solvent mixtures, was determined by the zone of inhibition of the growth of test microorganisms (diameter of the zone of inhibition of growth of test microorganisms) under the influence of these extracts. The diameter of the zone of inhibition of test microorganisms is presented in Table 4. Dilutions of 2 and 5 times were prepared for extracts of G. glabra obtained using the Soxhlet method that demonstrated activities against these microorganisms in order to determine the minimum inhibitory concentration. However, none of the diluted extracts of G. glabra demonstrated activity against P. aeruginosa, C. albicans, E. coli, and B. subtilis.  Table 5 presents the results of the study of the antioxidant activity of G. glabra extracts according to Soxhlet and extracts obtained by maceration with a mixture of solvents methanol-NaOH and methanol-TFA.

Discussion
A comparison of data on the content of biologically active substances in samples of methanolic G. glabra extracts obtained by the Soxhlet method and presented in Table 2 shows that, in the G. glabra extract using pure methanol as an extractant, the largest amount of biologically active substances was identified: 3,4-dihydroxybenzoic acid, n-coumaric acid, luteolin-7-glucoside, acacetin, apigenin-7-O-glucoside, chicoric acid, and hesperetin. A significant amount of rosmarinic acid was identified when the maceration method was used with a mixture of solvents methanol-NaOH. A record amount of catechin was found when using the maceration method with a methanol-TFA mixture of solvents.
When studying the antibacterial activity, it was found that samples of the G. glabra extract obtained by Soxhlet extraction with methanol showed antimicrobial activity against all test strains of microorganisms. It is assumed that the presence of antimicrobial activity of this extract is due to the presence in its composition of such BAS as polyphenolic compounds and flavonoids: 3,4-dihydroxybenzoic acid, n-coumaric acid, luteolin-7-glucoside, acacetin, apigenin-7-O-glucoside, chicoric acid, and hesperetin. Saponins, alkaloids, and flavonoids contained in G. glabra extracts provide certain antibacterial activities. G. glabra preparations inhibit the activity of the bacterial genes of pathogenic microorganisms and slow down the growth of bacteria and their production of toxins that are harmful to the human body [26]. Hesperelin and a number of flavonoids contained in G. glabra have powerful antioxidant properties and thus protect the human body from the damaging effects of free radicals [27].
Soxhlet extraction is one of the most popular, simple, and effective extraction methods [28]. It is used for a wide range of samples such as soils, sediments, and animal and plant tissues. A wide range of organic solvents can be used for this method. The use of non-polar solvents alone is not recommended. Since its discovery in 1879, the standard Soxhlet method has been used regularly in almost every analytical laboratory. The Soxhlet extraction method continues to be the standard method against which the performance of modern extraction methods is compared. Over the years, intensive research has been carried out in various modifications to overcome the main shortcomings of the traditional Soxhlet method. New approaches to this method have been implemented to reduce extraction times and extractant volumes [29].
Recently, many studies have been devoted to modifications that include automated Soxhlet extraction, focused microwave Soxhlet extraction, ultrasonic Soxhlet extraction, high pressure Soxhlet extraction, and fluidized bed extraction. However, in all modifications, the Soxhlet extraction method remains the most accurate method with a high yield of extracts of various, including medicinal, plants [28]. We obtained the highest yield of extracts and biologically active substances from G. glabra using Soxhlet extraction with an organic solvent: methanol. In pharmaceutical technology, water, organic solvents and their mixtures, as well as aqueous solutions of acids and alkalis, can be used as selective solvents (extractants) during Soxhlet extraction [30].
The efficiency of the extraction process is characterized by the ability of the extracted substance to penetrate due to diffusion into the immobile solvent medium. The efficiency of diffusion and, consequently, of extraction increases with increasing temperatures and decreases with an increase in the viscosity of the medium and the size of the diffusing BAS particles. However, literature data show [28,30] that the fulfillment of the conditions of the diffusion law in some cases leads to a deterioration in the quality of the obtained extract [29,31]. Namely, the improper selection of the extractant, elevated temperature, and too fine grinding of raw materials leads to an increase in the proportion of ballast substances in the extract. The purification of such an extract requires additional technological stages [32]. When using methanol as an extractant in our studies, BAS from G. glabra pass to the full extent into extracts, and increasing the temperature when using the Soxhlet method also leads to a more complete yield of extractable BAS from G. glabra. As a result of the study, it was discovered that using methanol as an extractant in Soxhlet extraction results in a more complete extraction of biologically active substances from G. glabra while retaining significant antioxidant and antibacterial properties [29].
Samples of methanol extracts of G. glabra obtained by the Soxhlet method had the highest antibacterial activity (the largest diameter of the zone of inhibition of growth of all test microorganisms): 13.6 mm zone of inhibition of E. coli, 10.8 mm zone of inhibition of P. aeruginosa, 16.1 mm zone of inhibition of B. subtilis, and fungicidal activity: 13.2 mm zone of inhibition of C. albicansa (Table 4). For samples of G. glabra extracts obtained by the methanol-NaOH maceration method, the growth inhibition zone for E. coli was 6.7 mm, and the growth inhibition zone for B. subtilis was 8.6 mm. These extracts had no inhibitory effect on P. aeruginosa and C. albicansa strains. The growth inhibition zones for P. aeruginosa, B. subtilis, and C. albicansa for samples of G. glabra extracts obtained by the methanol-TFA maceration method were 6.4 mm, 10.2 mm, and 6.6 mm, respectively. These extracts did not have an inhibitory effect on E. coli strain.
When studying the antioxidant activity of G. glabra extracts obtained by the Soxhlet method and the biologically active derivative of apigenin isolated from the extracts by HPLC, presented in Table 5, it can be concluded that the G. glabra extracts have significant antioxidant activities. The highest antioxidant activity was found in the Soxhlet G. glabra extract samples using the ABTS method at 117.62 ± 7.91 µmol Trolox equivalent/g, while the lowest was found using the FRAP method: 23.91 ± 1.12 µmol Trolox equivalent/g.
The study demonstrated that the null hypothesis could not be confirmed. A significant effect of the extraction method on the content of biologically active substances in samples of G. glabra extracts, as well as on their antioxidant and antibacterial activities, was established.
The study [33] described the antimicrobial activity of G. glabra. The antimicrobial activity of the aqueous and methanol extracts of G. glabra leaves was analyzed against Klebsiella pneumoniae, Candida albicans, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis. The results confirmed that the alcoholic extract of G. glabra had antimicrobial potential against C. albicans and Gram-positive bacteria in a dose-dependent manner. An alcoholic extract of G. glabra leaves has been shown to be effective against Gram-positive bacteria, suggesting that it could be a potential antimicrobial agent against a variety of bacterial strains [34].
Karahan et al. investigated the antioxidant and antimicrobial properties of methanolic extracts of G. glabra roots. MIC and disk diffusion methods were used to study antimicrobial efficacy. Antimicrobial analyses led to the conclusion that methanolic extracts of G. glabra roots are less effective against Gram-negative bacteria than against Gram-positive bacteria. In addition, methanolic extracts of G. glabra root were found to be more effective against Candida species than against other microorganisms. The results of the study showed that environmental factors affect the chemical content and biological properties of G. glabra in Life 2022, 12, 1772 10 of 17 each habitat. In addition, the results of the study support the traditional practice of using G. glabra and suggest that it may be useful in the treatment of various infections [35]. Gupta et al. demonstrated that G. glabra roots have antimicrobial potential at a concentration of 500 g/mL in their study. They proved that the glabridin contained in the roots inhibits the growth and development of the Mycobacterium tuberculosis strain at a concentration of 29.16 µg/mL [36]. Research by Shirazi et al. also proved the antimicrobial properties of G. glabra. Studies showed that G. glabra inhibits the growth and development of S. aureus and P. aeruginosa test strains [37]. Thus, G. glabra has antimicrobial activity of both Grampositive and Gram-negative bacteria [36][37][38].
G. glabra has practical applications due to its antioxidant properties, which are due to the presence of flavonoids, isoflavonoids, and other compounds [39,40]. Singh et al. [40] reported that compounds such as ispaglabridin A, glabridin, and 30-hydroxy-4-Omethylglabridin have high antioxidant activity. Later, it was proved that dihydrostilbene derivatives and lycochalcones B and D present in G. glabra can slow down oxidative processes [41,42]. In vitro studies, for example, demonstrated that licochalcones B and D can inhibit microsomal lipid peroxidation [42]. Thus, this proves the conclusion of Castangia et al. [43] who showed that G. glabra extract can be used in the production of innovative products for skin and cosmetics since it counteracts oxidative stress, maintaining skin homeostasis due to its high content of antioxidants.
Multidrug-resistant microorganisms are a serious problem in clinical medicine, and they require the search for new active substances. Various authors reported antimicrobial properties of G. glabra, especially against Gram-positive and Gram-negative bacteria such as S. aureus, E. coli, P. aeruginosa, C. albicans, and B. subtilis. [36,44]. Secondary metabolites, such as saponins, alkaloids, and flavonoids, are responsible for the observed antibacterial activity [8]. This activity is primarily attributed to glabridin, glabrol, glabren, hispaglabridin A, hispaglabridin B, 40-methylglabridin, and 3-hydroxyglabrol isolated from G. glabra [8]. The mechanism underlying this might be a decrease in bacterial gene expression, the inhibition of bacterial growth, and a reduction in bacterial toxin production [34]. In 2014, Ahn et al. [45] demonstrated that G. glabra prevents bacterial caries caused by Streptococcus mutans and Streptococcus sobrinus. Similarly, in vitro studies revealed that G. glabra aqueous and alcoholic extracts have inhibitory activity against Streptococcus pyogenes [34]. On the other hand, various authors have also reported the ability to inactivate methicillin-resistant S. aureus by downregulating SaeR and Hla, key virulence genes in S. aureus [34,45]. It is also assumed that lycochalcone E can be used for the chemical synthesis of new anti-S. aureus compounds that reduce α-toxin production in methicillin-sensitive S. aureus [34].
In addition, α-hemolysin is an important exotoxin in the pathogenesis of S. aureus infections [46]. Such infections are associated with a wide range of diseases, ranging from endocarditis to mild skin infections, toxinosis, and fatal pneumonia. Liquiritigenin, one of the most active compounds in G. glabra, was shown to prevent human lung cells (A549) from α-hemolysin-mediated damage by reducing α-hemolysin production [34,45] Similarly, glabrin and glycyrrhetinic acid demonstrated antibacterial activity against S. aureus [40].
Various authors reported the antibacterial activity of G. glabra against M. tuberculosis [34], demonstrating that glabridin rather than hispaglabridin B is responsible for this activity [14]. Antituberculous phenolic compounds have previously been identified in extracts of G. glabra, such as lycoisoflavone and lycochalcone A [47].
In a mouse lung infection model, G. glabra exhibited therapeutic activities against a multidrug-resistant strain of P. aeruginosa [48], and the hydroalcoholic extract led to a decrease in the microbial load in the blood, mainly due to the presence of stigmasterol, ergosterol, licochalcone, and glabridin [47].
G. glabra was also reported to be active against Helicobacter pylori [49]. According to Krausse et al. [49], the compounds glabridin and glabrene are responsible for this activity. Cao et al. [50] also reported that 18β-glycyrrhetinic acid significantly attenuated H. pylori gastritis infection. Asha et al. [51] found that flavonoid glabridin was active against H. pylori, while glycyrrhizin was inactive even at a concentration of 250 µg/mL. These flavonoids also showed activity against strains of H. pylori resistant to clarithromycin and amoxicillin [52]. The likely mechanism of this action is the inhibition of protein, DNA gyrase, and dihydrofolate reductase synthesis [52]. In addition, G. glabra polysaccharides also exhibit activity against the adhesion of Porphyromonas gingivalis, which is of great importance, since no specific adhesion inhibitors were described [53].
The antifungal activity of G. glabra is also described [54]. Sato et al. [54] reported that the methanol extract of G. glabra exhibits fungicidal activity against Arthrinium sacchari and Chaetomium funicola, while glabridin was found to be the active compound responsible for the observed effects [54]. In fact, isoflavones such as glabridin, glabrol, and their derivatives, are responsible for the in vivo inhibition of M. smegmatis, Shigella, Salmonella, E. coli, S. mutans, and Lactobacillus acidophilus [55]. Recently, Chandra and Gunasekaran [56] also demonstrated the antifungal activity of a crude methanol extract of G. glabra against Aspergillus niger.
Various authors reported that C. albicans is sensitive to G. glabra extracts due to their rich content of lykyritigenin, lykyritin, licochalcon A, and glabridin [57][58][59]. However, according to Karahan, Avsar, Ozyigit, and Berber [35], chemical content and biological and antimicrobial activities can be influenced by environmental conditions.

Reagents
All chemicals (ethylacetate, methanol, trifluoroacetic acid, formic acid, trichloroacetic acid, sodium hydroxide, and ultrapure water) used in the studies had at least ACS classification and were purchased from Sigma Aldrich (Sigma-Aldrich Rus, Moscow, Russia). Distilled water from a distiller (Khimlabpribor, Klin, Moscow, Russia) was used to prepare working solutions.

G. glabra Extraction
In the first stage, extraction parameters were chosen in accordance with the Soxhlet method in order to analyze the content of secondary metabolites of a phenolic nature. To achieve this, extraction was conducted using various organic extractants and pH values (methanol, methanol in an acidic medium, and methanol in an alkaline medium). Trifluoroacetic (TFA) and formic acids were used to create an acidic environment, and sodium hydroxide and ammonia were used to create an alkaline environment. Extracts were obtained by extraction and maceration (fractional extraction). The maceration process basically involved adding an extractant (methanol) to the crushed G. glabra raw material, sealing the vessel, and infusing the mixture at a temperature of 15-20 • C for 7 days while occasionally shaking or stirring. After infusion, the extract was poured out, and the leftover material was squeezed out, washed with a little extractant, and then squeezed out once more. The squeezed extract was combined with the drained extract, and the combined extract was brought back to its original volume using the extractant. This procedure was repeated three times. As a result, plant extracts of G. glabra were obtained by using 6 different methods: • Extraction with methanol according to the Soxhlet method; • Extraction with methanol by maceration; • Extraction with methanol in a medium of 0.1 N sodium hydroxide by maceration (pH = 12.5); • Extraction with methanol in a medium of 0.1 N ammonia solution by maceration (pH = 10.9); • Extraction with methanol in a medium of 0.1 N trifluoroacetic acid by maceration (pH = 1.2); • Extraction with methanol in a medium of 0.1 N formic acid by maceration (pH = 3.2).
Extraction with methanol according to the Soxhlet method was carried out for 8 h (15 cycles) at the boiling temperature of the solvent. However, according to literature data, an increase in temperature can cause the destruction of thermolabile biologically active substances (BAS) [54]; therefore, for comparison, extraction by maceration was performed without heating. Extraction by maceration was carried out for 8 h at room temperature with constant stirring and an extraction modulus of 1:40. During the experiment, the maximum yield of dry extract was evaluated.

Determination of the Qualitative Composition of G. glabra Extracts by Mass Spectrometry
To study the qualitative composition of G. glabra extracts by liquid chromatographymass spectrometry, samples of extracts obtained by maceration with methanol, methanolic ammonia solution, and methanolic formic acid solution were analyzed using reversephase quadrupole time-of-flight ultra-high-performance chromatography-mass spectrometry (RP-UHPLC-Q-TOF-MS/MS) [55]. This analysis was performed using a Waters ACQUITY UPLC I-Class UPLC System (Waters GmbH, Eschborn, Germany) ultra-highperformance chromatograph (UHPLC) connected online to an AB Sciex TripleTOF 6600 hybrid quadrupole-time-of-flight mass spectrometer (Q-TOF-MS) (AB Sciex, Darmstadt, Germany) with electrospray (ES) ionization. Before performing the analysis of semi-polar metabolites, an optimization procedure was performed using extracts of test samples during which the optimal injection volume of the analyzed extract for analysis was established, which did not cause an overload of the analytical column and the associated negative changes in the linearity of measurements and the accuracy of determining the mass-to-charge ratio (m/z). The strategy of non-targeted metabolomic analysis [56] was used, in which UHPLC-MS/MS was performed in the data-independent acquisition (DIA) mode. The PHA algorithm was based on SWATH (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) technology. Two UHPLC-MS/MS experiments were performed. At the same time, the chromato-mass-spectrometric analysis was repeated twice to ensure the detection in the mode of both positively and negatively charged ions of secondary metabolites, [M + X] + and [M-H] − , respectively, where X denotes singly charged cations. The two sets of obtained chromato-mass-spectrometric data were processed separately from each other. The quality of chromatographic-mass-spectrometric data (chromatographic and mass-spectrometric resolution, symmetry of chromatographic peaks, and accuracy of mass and retention time) was evaluated using the PeakView program (AB Sciex Pte. Ltd., PeakView 2.2 Software version, 2014, Colorado Springs, CO, USA). Calibration curves were plotted to determine the concentrations of the components. The error of the method did not exceed 3-7%.

Determination of the Antimicrobial Activity of G. glabra Extracts
Samples of G. glabra extracts obtained by maceration with 0.1 N methanolic formic acid (methanol-TFA) were tested for antimicrobial activity [60]. When obtaining extracts, 100 mL of solvent accounted for 2.5 g of plant material. Solvents were removed from the extracts using an IKA RV 8 V vacuum rotary evaporator (DV-Ekspert, Moscow, Russia). The extracts were then dried using a Triad freeze dryer (Labconco Corporation, Kansas City, Missouri, USA). Drying conditions: vacuum 0.037 mbar, coolant temperature minus 80 • C. Extracts obtained from 2.5 g of plant material were dissolved in 10.0 mL of a mixture of methanol and water (9:1, respectively). The methanol:water ratio of 9:1 was chosen based on previous research and data from the literature on the use of mixed extractants for BAS extraction from plants [61,62].
The antimicrobial activity of extract samples and their individual active components was studied using the disk diffusion method. Four strains of test microorganisms were used for the study: Gram-positive bacteria Bacillus subtilis and Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa on LB agar medium, andyeast-like fungi of the genus Candida albicans on Ringer's agar medium. Test strains of microorganisms E. coli, P. aeruginosa, B. subtilis, and C. albicans were purchased from the National Bioresource Center of the All-Russian Collection of Industrial Microorganisms of the Kurchatov Institute Research Center (Moscow, Russia). These microorganisms are standard and frequently used test strains for determining the antibacterial and fungicidal activities of various preparations, including medicinal plant extracts. These test strains of microorganisms were used in accordance with the recommendations of the National Committee for Clinical Laboratory Standards [63]. The growth inhibition of these microorganism test strains allows for the highly accurate determination of the G. glabra extract's antibiotic activities. Clinical isolates of these test strains were obtained after inoculation on culture media and were used to determine the antibacterial activity of G. glabra extracts. The concentration of the microbial suspension was 1.5 × 10 8 CFU/mL. The disc diameter was 6 mm, and the thickness of the agar layer was 4.0 ± 0.5 mm. The antibiotic kanamycin at a concentration of 50 µg/disk (for bacteria) and fluconazole 500 µg/disk (for yeast-like fungi) were used as controls. The comparison was carried out with a mixture consisting of 1% TFA (31%) and acetonitrile (69%), in which the individual active components were dissolved. The measurements were taken three times. The mean value was taken as the measurement result [64].

Determination of the Antioxidant Activity of G. glabra Extracts
When determining the antioxidant activity by the DPPH method, 20 µL of the extract sample and a solution of an individual compound or a standard solution (trolox) were mixed with 300 µL of a freshly prepared 0.1 mM solution of 2,2-diphenyl-1-picrylhydrazyl. The mixture was incubated in the dark at room temperature for 30 min. The decrease in optical density compared to the control, consisting of a 0.1 mm solution of 2,2-diphenyl-1-picrylhydrazyl and the appropriate solvent used for extraction or for the isolation of fractions and individual compounds, was recorded at 515 nm [65].
When determining the antioxidant activity by the ABTS method, a solution of the ABTS radical was preliminarily prepared. The ABTS radical was generated by mixing aliquots of 7.0 mM ABTS solution and 2.45 mM potassium persulfate solution. The solution was kept for 16 h in a dark place at room temperature. To start the reaction, 300 µL of the ABTS + radical cation solution was added to 20 µL of the G. glabra extract, a solution of a single compound, or a standard (trolox). The absorbance was measured at 734 nm after the mixture was incubated for 15 min at 37 • C in the dark. A sample with the ABTS reagent and the corresponding solvent used for extraction or for the isolation of fractions and individual compounds was used as a control [66].
To determine the restorative power of G. glabra extracts, a freshly prepared FRAP reagent was used, prepared by mixing 10 parts of 0.3 M acetate buffer (pH 3.6), one part of a 10 mM solution of 2.4.6-tripyridyl-s-triazine in 40 mM HCl, and one part of aqueous 20 mM iron chloride solutio FeCl 3 × 6H 2 O. The reaction was started by mixing 300 µL of the FRAP reagent and 20 µL of the test extract, a solution of an individual compound, or a standard solution (trolox). The reaction time was 10 min at 37 • C in the dark. The dptical density was measured at 593 nm. As a control, a sample with the FRAP reagent and the corresponding solvent used for extraction or for the isolation of fractions and individual compounds was used.
When measuring antioxidant activity using DPPH, ABTS, and FRAP methods, solutions of Trolox (6-hydroxy-2.5.7.8-tetramethylchroman-2-carboxylic acid) of known concentrations were used as standard solutions. When analyzing extracts, the results of the analyzes were expressed in µmol Trolox equivalents per gram of dry plant weight (µmol Trolox equivalent/g); when analyzing fractions and/or individual compounds, antioxidant activity was expressed in mmol Trolox equivalents per gram of individual compound (mmol trolox equivalents/g). All spectrophotometric measurements were performed using a CLARIOstar microplate reader (BMG Labtech, Ortenberg, Germany).

Statistical Analysis
Each experiment was repeated three times, and data are expressed as means ± standard deviation. Data processing was carried out via the standard methods of mathematical statistics. For each sample, three repeated preparations and three repeated measurements were performed (n = 3). The data were subjected to analysis of variance (ANOVA) using Statistica 10.0 (StatSoft Inc., 2007, Tulsa, OK, USA). Post hoc analyses (Duncan's test) were undertaken to identify samples that were significantly different from each other. The equality of the variances of the extracted samples was checked using the Levene test. Differences between means were considered significant when the confidence interval is smaller than 5% (p < 0.05).

Conclusions
The Soxhlet extraction method with methanol resulted in the highest yield of G. glabra extract. Samples of the G. glabra extract obtained using the Soxhlet method with methanol demonstrated significant antibacterial activity against all test strains. The presence of antimicrobial activity of this extract can apparently be explained by the presence in its composition of such BAS as polyphenolic compounds and flavonoids. The highest antioxidant activity was found in methanol samples of G. glabra extracts (Soxhlet method) using the ABTS method, while the lowest was found using the FRAP method.
As a result of the studies, it was found that Soxhlet extraction with methanol is much more effective for isolating BAS contained in G. glabra compared to maceration (with methanol and with a mixture of solvents methanol-TFA, methanol-NH 4 OH, methanol-NaOH, and methanol-CHOOH). G. glabra extracts obtained via Soxhlet methanol extraction may become natural alternatives to existing therapies for the elimination of bacterial infections or the prevention of early aging in the human body due to free radicals and oxidative stress. The conducted studies allow filling a gap in the studies of BAS from G. glabra, growing in the Kaliningrad region. The number of phytocomponents and their