INTRODUCTIONModern medicine and biology are actively searchingfor new drugs with geroprotective effects [1–8]. Highlyuseful in this regard are extracts of medicinal plants [8, 9].A common extraction method involves using a refluxcondenser, a Soxhlet extractor, mechanical stirring,and ultrasound. Soxhlet extraction takes place at80–90°C and lasts from 20 to 24 h. Such parametersmake it possible to efficiently extract biologically activecompounds, such as saponins [10–13].Modern extraction methods include ultra-highpressure extraction (UHPE), ultra-temperatureextraction (UTE), pressurized liquid extraction (PLE),microwave-assisted extraction (MAE), pressurizedhot-water extraction (PHWE), and supercritical fluidextraction (SFE) [9–12, 14–17].Compared to traditional techniques, modern methodsuse smaller amounts of solvents, are easily automated,and take little time. However, they are hardly moreeffective than, for example, Soxhlet extraction or mechanical mixing [13, 18]. Moreover, pressurized hotwaterextraction and supercritical fluid extraction aretechnically quite difficult to perform [10, 14, 19, 20].Among medicinal plants with geroprotective propertiesare Schisandra chinensis (L.), Scutellariabaicalensis (L.), Rhodíola rósea (L.), Ginkgo biloba (L.),and others [21–24]. The most highly valued geroprotectivemedicinal plants include Panax ginseng (L.),Aralia mandshurica (L.), and Eleutherococcussenticosus (L.) [25]. Since the 1980s, scientists haveknown of their antitumor effects [25–27].Ginseng (Panax ginseng L.) is a slowly growingperennial plant that is often used as a functionalcomponent and a phytotherapeutic agent toprevent and treat various diseases, such as cancer,allergies, inflammatory diseases, and diabetesmellitus [26–31].According to scientific literature, ginseng extract isused as an adaptogen to increase physical performance,vitality, immunity, as well as resistance to stress andaging [26, 32–34]. It also lowers total cholesterol andlow-density lipoproteins, thereby improving a bloodlipid profile [31, 32].However, this plant is included in the Red Book ofthe Russian Federation and the collection of young rootsis prohibited due to its depletion. In addition to lowseed productivity and relatively slow growth, ginsengpopulation is irreparably damaged by forest fires andhuman activities in its endemic areas [35, 36].Therefore, a justified solution would be to use theplant’s cell and organ cultures as an alternative sourceof renewable medicinal material [30, 32, 35–37]. In ourstudy, we used ginseng callus, suspension, and rootcultures – obtained in the early stages of research – as asource of biologically active substances.We aimed to select optimal extraction parametersand perform a qualitative analysis of organic compoundsisolated from ginseng callus, suspension, and rootcultures.STUDY OBJECTS AND METHODSOur study objects included callus, suspension, androot cultures of ginseng (Panax ginseng L.) obtained invitro, as well as their extracts.To determine the optimal parameters for extractingbiologically active substances from ginseng by refluxextraction, we analyzed several extraction systemsfor their effectiveness. A water-ethanol mixture wasselected as an extractant due to its safety (GRAS),economic efficiency, and the ability to extract a widerange of biologically active substances from plantmaterials [38, 39]. We screened the solvents andperformed a qualitative analysis of organic compounds(Fig. 1). The percentage of ethanol in the solvents isindicated in mass fractions. The yield of extracts (%) isexpressed in terms of 100 g of dry raw material.To extract biologically active substances fromginseng callus cultures, we placed 3 ± 0.001 g ofdry powdered callus culture in a 50 mL plastic tubeand added 40 mL of 30, 40, 50, 60, or 70% solventaccording to the screening scheme (Fig. 1). The tubewas connected to a reflux condenser. After 60 minof extraction, we separated the dry mass from thesolution by filtration. To remove suspended particles,we centrifuged the filtrate at 3900 rpm. Ethanol wasevaporated from a 100 mL pre-weighed flask underreduced pressure. After evaporation, we weighed theflask and measured the extract yield.Then, we dissolved the residue in a minimumamount of the solvent and determined the qualitativecomposition of organic compounds in the extract bythin-layer chromatography.The chromatograms for each plant were archived andanalyzed for the presence of quercetin (Sigma-Aldrich,USA, ≥ 95%), mangiferin (Sigma-Aldrich, USA, ≥ 98%),luteolin (Sigma-Aldrich, USA, ≥ 98%), rutin (Sigma-Aldrich, USA, ≥ 94%), quercetin-2-D-glucoside (Sigma-Aldrich, USA, ≥ 95%), caffeic acid (Sigma-Aldrich,USA, ≥ 98%), cinnamic acid (Acros Organics, Belgium,Figure 1 Solvents efficiency in extracting biologically active substances from ginsengSample,3 g30% ethanol extraction40% ethanol extraction50% ethanol extractionConcentration,yield measurement,thin-layerchromatographyIs the yieldhigh?Analysisof efficiencyin refluxextractionHigh-performanceliquidcFigure 1 Solvents efficiency in extracting biologically active substances from ginsengSample,3 g30% ethanol extraction40% ethanol extraction50% ethanol extractionConcentration,yield measurement,thin-layerchromatographyIs the yieldhigh?Analysisof efficiencyin refluxextractionHigh-performanceliquidchromatography60% ethanol extraction70% ethanol extraction012345630 40 Total extract yield, %468extract yield, %91215extract yield, %NOYES371Dyshlyuk L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 369–376≥ 98%), ferulic acid (Sigma-Aldrich, USA, ≥ 99%),sinapinic acid (Honeywell, USA, ≥ 95%), and malvidin(Sigma-Aldrich, USA, ≥ 90%).To prepare ginseng suspension and root cultures forthe experiment, they were pre-dried to constant weight.Then, 0.5–2.0 g samples of dried cultures were extractedwith solvents for callus cultures.Thin-layer chromatography was performed asdescribed in Pharmacopeia Article 1.2.1.2.0003.15. Afterevaporation of the solvent from the total extract, wedissolved the dry residue in 1 ml of a suitable extractant(methanol, methylene chloride or acetone) and appliedit to the plate with a glass capillary for thin-layerchromatography.Then, we placed the plate in a chamber and addeda suitable eluent. When we used silica gel withoutmodification, chromatography was performed inthe CH2Cl2:MeOH system with a 0–10% methanolgradient, in increments of 1%. For reversed-phasechromatography, we used the H2O:MeCN eluent systemwith a 0–20% acetonitrile gradient, in increments of 2%,and 0.1% trifluoroacetic acid as a modifier.We separated the fractions with high-performanceliquid chromatography (HPLC), using a ProminenceLC-20 chromatograph with diode-array detection(Shimadzu, Japan) and a 250×4.6 mm Kromasil C18chromatographic column with 5 μm sorbent particles.A mixture of water with o-phosphoric acid, pH = 4.6 (A)and acetonitrile (B) were used as a mobile phase.The gradient elution modes (% B) were 0–20 and20–60 min with a gradient change of 10–20% and20–50%, respectively. The eluent flow rate was1.0 mL/min; the temperature of the column thermostatwas 35°C. In the preparative accumulation mode, theeluent was used without the acid.The instrument was calibrated with caffeine (Sigma-Aldrich, USA, ≥ 90%).Nuclear magnetic resonance (NMR) spectra wererecorded on a Varian NMR System 400 spectrometerwith a silent Ceccato OFCS 5/8 SD compressor(Varian, USA), with DMSO-D6 used as a solvent andtetramethylsipane as the internal standard.RESULTS AND DISCUSSIONTo analyze the efficiency of various extractionsystems, we obtained average yields of solids in totalextracts. Total extract yields depending on the solvent’sconcentration are presented in Fig. 2.Based on the results, we selected 50% ethanol as asolvent to extract biologically active substances fromthe dried biomass of ginseng callus cultures by refluxextraction. Further selection parameters are shown inTables 1–2.According to Table 1, the maximum yield ofbiologically active substances extracted from driedginseng callus cultures (5.88 ± 0.59%) at 45°C wasprovided by a 1:5 ratio of solvent to biomass and theFigure 2 Solvents efficiency in extracting biologically activesubstances from ginseng callus culturesTable 1 Dry extract yield of biologically active substances from dried ginseng callus culture biomass dependingon extraction time (at 45°C)Solvent:culture Extract yield depending on duration, %10 min 30 min 60 min 120 min 180 min 360 min1:1 0.50 ± 0.05 0.81 ± 0.08 1.22 ± 0.12 1.29 ± 0.13 1.38 ± 0.14 1.38 ± 0.141:2 0.80 ± 0.08 0.94 ± 0.09 1.35 ± 0.14 1.58 ± 0.16 1.67 ± 0.17 1.71 ± 0.171:5 1.20 ± 0.12 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.581:10 1.40 ± 0.14 1.98 ± 0.20 2.98 ± 0.30 5.94 ± 0.59 5.97 ± 0.60 6.04 ± 0.601:20 1.40 ± 0.14 2.01 ± 0.20 3.01 ± 0.30 5.95 ± 0.60 6.01 ± 0.60 6.07 ± 0.61Table 2 Temperature selection for extracting biologically active substances from ginseng callus culturesTemperature, °С Extract yield depending on duration, %10 min 30 min 60 min 120 min 180 min 360 min25 1.20 ± 0.12 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.5840 1.55 ± 0.16 1.98 ± 0.20 3.92 ± 0.39 6.21 ± 0.62 6.18 ± 0.62 6.24 ± 0.6250 1.79 ± 0.18 2.35 ± 0.24 6.98 ± 0.70 7.05 ± 0.71 7.01 ± 0.70 7.12 ± 0.7180 1.62 ± 0.16 2.14 ± 0.21 6.04 ± 0.60 6.12 ± 0.61 6.14 ± 0.61 6.17 ± 0.62Analysisefficiencyin refluxextractionHigh-performanceliquidchromatography012345630 40 50 60 70Total extract yield, %Ethanol concentration, %50 60 70Ethanol concentration, %372Dyshlyuk L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 369–376extraction time of at least 120 minutes. Noteworthily, afurther increase in duration had no effect on the yield ofbiologically active substances.Next, we optimized the temperature and time ofextraction (Table 2).We found that the duration of 60 min and atemperature of 50°C produced the optimal yield ofbiologically active substances from the dried biomass ofginseng callus cultures (6.98 ± 0.70%).Next, we determined the optimal parameters toobtain total extracts from the dried biomass of ginsengsuspension cultures with various solvent concentrations(Fig. 3).Based on the results, we selected 60% ethanol asthe most optimal solvent to obtain total extracts ofbiologically active substances from the dried biomass ofginseng suspension cultures using the reflux extractionmethod. Further selection parameters are shown inTables 3–4.We found that the maximum yield of biologicallyactive substances extracted from dried ginsengsuspension cultures (8.78 ± 0.88%) at 45°C was providedby a 1:5 ratio of solvent to biomass and the extractiontime of at least 120 minutes.According to the results, the optimal parameters forextracting biologically active substances from ginsengwith 60% ethanol (extract yield of 8.95 ± 0.90%)were 50°C, 30 min extraction, and a 1:5 solvent-tobiomassratio.At the next stage, we optimized the parametersfor obtaining total extracts from in vitro ginseng rootcultures. The total extract yield depending on the solventis shown in Fig. 4.According to Fig. 4, 70% ethanol produced thehighest yield of biologically active substances fromthe dried biomass of ginseng root cultures by refluxextraction. Further selection parameters are shown inTables 5–6.According to the results, the duration of 30 to180 min and the solvent-to-biomass ratio of 1:5 and1:10 provided the maximum yield of biologically activesubstances from the dried biomass of ginseng rootcultures. In particular, the yield of 11.98% was producedat a ratio of 1:10 at 45°C during 30–60 min.Table 3 Dry extract yield of biologically active substances from dried ginseng suspension culture biomass dependingon extraction time (at 45°C)Solvent:culture Extract yield depending on duration, %10 min 30 min 60 min 120 min 180 min 360 min1:1 2.50 ± 0.25 2.51 ± 0.25 2.62 ± 0.26 2.92 ± 0.29 2.98 ± 0.30 2.83 ± 0.281:2 2.80 ± 0.28 2.94 ± 0.29 2.75 ± 0.28 2.85 ± 0.29 2.76 ± 0.28 2.91 ± 0.291:5 2.93 ± 0.29 8.78 ± 0.88 8.80 ± 0.88 8.68 ± 0.87 8.95 ± 0.90 8.21 ± 0.821:10 5.40 ± 0.54 8.98 ± 0.90 8.98 ± 0.90 8.94 ± 0.89 8.97 ± 0.90 8.84 ± 0.881:20 6.34 ± 0.63 8.61 ± 0.86 8.51 ± 0.85 8.95 ± 0.90 8.71 ± 0.87 8.77 ± 0.88Figure 3 Solvents efficiency in extracting biologically activesubstances from ginseng suspension culturesHigh-performanceliquidchromatography70% ethanol extraction01230 40 50 Total extract Ethanol concentration, 0246840 50 60 70Total extract yield, %Ethanol concentration, %0369121530 40 50 60 70Total extract yield, %Ethanol concentration, %Table 4 Temperature selection for extracting biologically active substances from ginseng suspension culturesTemperature, °С Extract yield depending on duration, %10 min 30 min 60 min 120 min 180 min 360 min25 2.20 ± 0.22 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.5840 2.55 ± 0.26 1.98 ± 0.20 3.92 ± 0.39 6.21 ± 0.62 6.18 ± 0.62 6.24 ± 0.6250 2.79 ± 0.28 8.95 ± 0.90 8.40 ± 0.84 8.75 ± 0.88 8.21 ± 0.82 8.32 ± 0.8380 3.62 ± 0.36 7.56 ± 0.76 7.34 ± 0.73 7.12 ± 0.71 7.14 ± 0.71 7.17 ± 0.72Figure 4 Solvents efficiency in extracting biologically activesubstances from ginseng root culturesliquidchromatography70% ethanol extraction0130 40 50 Total Ethanol concentration, 0246830 40 50 60 70Total extract yield, %Ethanol concentration, %0369121530 40 50 60 70Total extract yield, %Ethanol concentration, %373Dyshlyuk L.S. et al. Foods and Raw Materials, 2020, vol. 8, no. 2, pp. 369–376CONCLUSIONWe developed a solvent screening procedure andperformed a qualitative analysis of biologically activesubstances extracted from ginseng (Panax ginseng L.).The optimal parameters for extracting biologicallyactive substances (organic solvent, ratio of solvent tobiomass, time, and temperature) were 50% ethanol, 1:5ratio, 60 min, 50°C for ginseng callus cultures; 60%ethanol, 1:5 ratio, 30 min, 50°C for suspension cultures;and 70% ethanol, 1:5 ratio, 60 min, 50°C for rootcultures, respectively.Table 5 Dry extract yield of biologically active substances from dried ginseng root culture biomass dependingon extraction time (at 45°C)Solvent:culture Extract yield depending on duration, %10 min 30 min 60 min 120 min 180 min 360 min1:1 2.35 ± 0.24 2.51 ± 0.25 2.62 ± 0.26 2.92 ± 0.29 2.98 ± 0.30 2.83 ± 0.281:2 2.38 ± 0.24 2.94 ± 0.29 2.75 ± 0.28 2.85 ± 0.29 2.76 ± 0.28 2.91 ± 0.291:5 2.30 ± 0.23 11.78 ± 1.18 11.80 ± 1.18 11.68 ± 1.17 11.95 ± 1.20 11.21 ± 1.121:10 5.34 ± 0.53 11.98 ± 1.200 11.98 ± 1.20 11.94 ± 1.19 11.97 ± 1.20 11.84 ± 1.181:20 6.54 ± 0.65 11.61 ± 1.16 11.51 ± 1.15 11.95 ± 1.20 11.71 ± 1.17 11.77 ± 1.18Table 6 Temperature selection for extracting biologically active substances from ginseng root culturesTemperature, °С Extract yield depending on duration, %10 min 30 min 60 min 120 min 180 min 360 min25 3.20 ± 0.32 1.80 ± 0.18 2.78 ± 0.28 5.88 ± 0.59 5.95 ± 0.60 5.81 ± 0.5840 8.55 ± 0.86 1.98 ± 0.20 3.92 ± 0.39 6.21 ± 0.62 6.18 ± 0.62 6.24 ± 0.6250 7.79 ± 0.78 11.95 ± 1.20 12.40 ± 1.24 11.75 ± 1.18 11.21 ± 1.12 11.32 ± 1.1380 6.62 ± 0.66 11.56 ± 1.16 11.34 ± 1.13 11.12 ± 1.11 11.14 ± 1.11 10.17 ± 1.02Table 7 Optimal parameters for extracting biologicallyactive substances from the dried biomass of ginseng callus,suspension, and root culturesType ofginsengcultureOrganicsolventRatio ofsolvent tobiomassTime,minTemperature,°СCallus 50% ethanol 1:5 60 50Suspension 60% ethanol 1:5 30 50Root 70% ethanol 1:5 60 50Figure 5 Qualitative analysis of ginseng flavonoidsmAUFigure 6 NMR spectrum of mangiferin isolated from ginsengextractsglucopyranosylBased on the results in Table 6, we selected thefollowing parameters for extracting biologically activesubstances from the dried biomass of in vitro ginsengroot cultures: extraction at 50°C during 30 min at a1:5 ratio of solvent to dried biomass. These parametersproduced a yield of 11.95 ± 1.20%. We recommend touse 70% ethanol as a solvent.Thus, we determined the optimal parameters (time,temperature, organic solvent, ratio of solvent to biomass)for extracting biologically active substances fromginseng callus, suspension, and root cultures (Table 7).The qualitative analysis of standard compoundsand biologically active substances extracted from driedginseng callus, suspension, and root cultures showedthe presence of rutin, quercetin, quercetin-glycoside,mangiferin, luteolin, apigenin, and caffeic acid.The fractions were separated by preparativeHPLC (Fig. 5).As a result, we isolated a basic substance witha retention time of 24 min, which was identified asmangiferin (Fig. 6).Thus, rutin (0.25), quercetin (0.75), and mangiferin(0.57) were major biologically active substances found inthe extracts of in vitro ginseng callus, suspension, androot cultures. We also identified caffeic and sinapinicacids in the extracts.The qualitative analysis of the extracts of ginsengcallus, suspension, and root cultures showed thepresence of rutin (0.25), quercetin (0.75), and mangiferin(0.57) as predominant components. The extracts alsocontained caffeic and sinapinic acids.Thus, the extracts obtained by water-ethanolextraction from ginseng callus, suspension, and rootcultures can be used as biologically active ingredients inthe production of functional geroprotective foods.CONTRIBUTIONThe authors were equally involved in writingthe manuscript and are equally responsible forplagiarism.CONFLICT OF INTERESTThe authors declare that there is no conflict of interest.