IntroductionLife expectancy in the first-world countries hassignificantly increased due to proper hygiene andnutrition. According to the World Health Organization,there are 125 million people aged 80+ in the world [1].This explains a growing need for developing drugs toprevent age-related diseases.Old age is a major risk factor for common chronic,neurodegenerative, and oncological diseases such ascancer, cardiovascular diseases, multiple sclerosis,Parkinson’s disease, etc. [2]. Aging is often accompaniedby genomic damage, mitochondrial dysfunction, telomereshortening, epigenetic changes, proteostasis dysregulation,disruption of intercellular communication, and otherprocesses. Cellular aging is defined as a response to stressthat gives cells an irreversible proliferative capacity,thus causing the body to age [3].Creating anti-aging drugs is a long process withmany variables and it is difficult to assess their effectin clinical trials [4, 5]. Metformin is one of the drugswith an anti-aging effect that is used to treat diabetesmellitus [6]. However, we should distinguish betweendrugs that are aimed at reversing the aging process andgeroprotective drugs that can prevent premature agingand increase life expectancy [2, 7, 8].Polyphenols, whose molecules contain one ormore phenolic hydroxyl groups, exhibit antioxidant,antitumorous, cardioprotective, anticancerous, andantimicrobial properties [9]. There is growing evidencethat phenolic acids, especially hydroxycinnamic acids,have an effect on the regulation of lipid metabolism. Forexample, caffeic, ferulic, and coumaric acids significantlyreduce hepatic lipids in rats with high cholesterol [10].Trans-cinnamic acid (3-phenylpropenoic acid) isthe main phenolic compound in plants [11]. Manystudies have reported its geroprotective activity due toantibacterial, antidiabetic, anticancerous, and antiagingproperties [12, 13].Many medicinal plants growing in the SiberianFederal Okrug are sources of geroprotective compounds[14]. For example, the Baikal skullcap (Scutellariabaicalensis) contains flavonoids (quercetin, rutin,catechin, luteonin, etc.), phenolic acids (caffeic, ferulic,p-coumaric, p-hydroxybenzoic, and cinnamic), vitamins,carotenoids, and terpenes [15–19]. In order to preservethe diversity of its species, trans-cinnamic acid shouldbe isolated from this plant’s cell cultures in vitro [20].Previous studies have found a significant amount oftrans-cinnamic acid in the in vitro root culture extractof S. baicalensis [21].A number of model organisms are used to study theeffect of bioactive substances of plant origin on theaging process. They include nematodes (Caenorhabditiselegans), fruit flies (Drosophila melanogaster),yeasts (Saccharomyces cerevisiae), short-lived fish(Nothobranchius furzeri), and rodents (mice and rats) [1].In this study, we used nematodes C. elegans as apreclinical experimental model mainly due to their shortlifespan under normal growth conditions [22]. Thisfeature of nematodes allows scientists to study processesthat affect aging and life expectancy. In addition, usingC. elegans is cost-effective since they feed on inexpensivemicroorganisms such as Escherichia coli bacteria.Also, growing worms can be fully automated using aflow cytometry apparatus, where they are distributedin analytical plates, as well as robots that place theexperimental samples in the wells [23]. Nematodeshave a transparent body and do not need to be stainedat all growth stages, so their internal organs are easilyvisible under a microscope. At the subcellular andtissue levels, fluorescent label reporters are used tostudy the distribution of expressing genes and theirprotein products. Powerful phase-contrast microscopesenable scientists to observe the division and death ofindividual worm cells [24, 25].C. elegans has a lifespan of only three weeks, whichmakes it a convenient model to use. With sufficientfood, optimal temperatures, and population control,nematodes can reach the adult growth stage in threedays. Their embryogenesis is faster compared to othermodel organisms. At 20–25°C, the development of eachcell can be traced in just 10–12 h. After embryonicdevelopment, nematode larvae go through several stages(L1–L4) before becoming adults [26]. The studies intothe lifespan of nematodes are usually carried out inPetri dishes using liquid and solid nutrient media [27].Thus, C. elegans can be used as model organisms tostudy the geroprotective properties of various bioactivesubstances.In this study, we aimed to isolate trans-cinnamicacid from the Baikal skullcap (S. baicalensis) and studyits geroprotective activity in the C. elegans nematode.Study objects and methodsOur study objects were:– in vitro root culture of the Baikal skullcap (Scutellariabaicalensis);– in vitro root culture extract of the Baikal skullcap(S. baicalensis);– trans-cinnamic acid obtained from the in vitro rootculture extract of the Baikal skullcap (S. baicalensis); and– soil nematodes Caenorhabditis elegans (strain N2Bristol).Germinated sterile seeds of S. baicalensis were usedto obtain the root culture (Botanical Garden of theImmanuel Kant Baltic Federal University, Kaliningrad).The seeds were sterilized in several stages: they werewashed with detergent, placed in 95% ethanol for 30 s,and transferred to a 6% NaOCl solution for 30 min.After sterilization, the seeds were rinsed with steriledistilled water and then washed three times with itfor 20 min. The seedlings grew for 14–28 days on anutrient medium containing 50.00 mg of B5 macrosalts,585Федорова А. М. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 582–59110.00 mg of B5 microsalts, 5.00 mL of Fe-EDTA,10.00 mg of thiamine, 1.00 mg of pyridoxine, 1.00 mgof nicotinic acid, 30.00 g of sucrose, 100.00 mg ofinositol, 0.05 mg of 6-benzylaminopurine, 1.00 mg ofindoleacetic acid, and 20.00 g of agar. The seedlings weretransformed with the soil agrobacteria Agrobacteriumrhizogenes 15834 Swiss (Moscow, Russia). The invitro root cultures of S. baicalensis were cultivated for5 weeks. They were first grown in 100 mL flasks (40mL of medium) and then transplanted into 300 mLflasks (100 mL of medium). The initial weight of theroot culture ranged from 0.5 to 1.0 g [21].The extract of the S. baicalensis root culture wasobtained by water-alcohol extraction. For this, the driedand crushed plant roots were treated with 30.0 ± 0.2%ethyl alcohol (1:86) at 70.0 ± 0.1°C for 6.0 ± 0.1 h. Theextraction was performed in an EKROS PE-4310 waterbath (Ekroskhim, Russia) with a reflux condenser [28].Then, trans-cinnamic acid was isolated from theobtained water-alcohol extract of the S. baicalensisroot culture by high-performance liquid chromatography(HPLC) on a liquid chromatograph (ShimadzuLC-20 Prominence, Japan). The process of isolationand purification consisted of several stages, namely:1. The extract of the S. baicalensis root culture wasevaporated under vacuum at 50°C max;2. Diethyl ether was added to the evaporated residuein three repetitions;3. The ether fraction obtained was chromatographedon PF silica gel in an n-hexane-acetone gradient(1:0 → 0:1) to isolate hydroxycinnamic acids; and4. Trans-cinnamic acid was isolated by subsequentrechromatography on PF silica gel in n-hexanechloroform(1:0 → 0:1).The trans-cinnamic acid isolated from theS. baicalensis root culture extract was at least 95% pure.Infra-red (IR) spectroscopy was performed to analyzethe chemical composition of trans-cinnamic acid on anFSM-1202 apparatus (Infraspek, St. Petersburg, Russia).IR spectra were recorded in potassium bromide disks(Fluka, Germany) in the range of 4000–400 cm–1 witha resolution of 4 cm–1 and the number of scans being 30.Air was used as a reference sample and it was recordedbefore the analysis of each sample. The Fspec (4.0.0.2)and Aspec (1.1) software was used to control the apparatusand process spectral data.Next, we assessed the effect of trans-cinnamicacid on the lifespan, stress (oxidative and thermal)resistance, and reproductive abilities of C. elegansnematodes. The N2 Bristol strain was provided by theLaboratory for the Development of Innovative Medicinesand Agrobiotechnologies (Moscow Institute of Physicsand Technology, Dolgoprudny, Russia). The nematodeswere fed on Escherichia coli OP50 provided by theV.A. Engelhardt Institute of Molecular Biology (Moscow,Russia). We used a total of 100 nematodes for all thestages of the study. The control nematodes were nottreated with solutions of trans-cinnamic acid. However,they were used in the lifespan and reproductivity testsand were subjected to oxidative and thermal stress.To assess the effect of trans-cinnamic acid onnematodes, we used a stock solution of this acid in10 mmol/L of dimethyl sulfoxide. Then, the acid wastitrated by diluting stock solutions in sterile distilledwater to concentrations of 2000, 1000, 500, and 100 μM.Each well with nematodes was filled with 15 μL offreshly prepared stock solutions, thus obtaining workingconcentrations of trans-cinnamic acid of 2000, 1000,500, and 100 μmol/L, respectively. The stocks werestored at 4°C.At the first stage, nematodes were cultivated onsolid agar. For this, a daily culture of E. coli OP50 wasobtained by inoculating one bacterial colony, which waspreviously grown on L-broth (15 g bacteriological agar,10 g tryptone, 5 g yeast extract, 5 g NaCl, 1000 mLdistilled water), in 5–10 mL of L-broth (10 g tryptone,5 g yeast extract, 10 g NaCl, 800 mL distilled water).The bacteria were cultivated at 37°C for 24 h withintensive stirring. After the incubation of E. coli OP50,50 μL of the overnight culture was inoculated intoPetri dishes with an NGM medium (3 g NaCl, 17 gbacteriological agar, 2.5 g peptone, 975 mL distilledwater) and incubated at 37°C for 24 h. Then, an NGMmedium was prepared for cultivating nematodes. Forthis, the autoclaved NGM agar medium was cooled to55°C in an EKROS PE-4310 water bath (Ekroskhim,Russia) for 15 min. Then, 1 mL of 1 M CaCl2, 1 mL of5 mg/mL cholesterol in alcohol, 1 mL of 1 M MgSO4,and 25 mL of 1 M KPO4 buffer were added to the cooledagar. The nematodes were transferred to new NGM agardishes in two ways: by a loop and by a piece of agar.The first method involved hooking a nematode with acalcined and cooled bacteriological loop and plantingit on a bacterial lawn in the center of a new Petri dishwith NGM agar. In the second method, a 5×0.5 cmpiece of agar containing a nematode was cut out witha sterile scalpel from an NGM dish and transferred tothe center of the new dish surface down. The disheswere incubated at 20°C.At the second stage, the nematodes were synchronized.For this, 5–10 mL of sterile water was added to thePetri dish with a nematode and pipetted until its eggswere completely attached to the agar. The liquid fromthe dish was placed in a 50 mL centrifuge tube andcentrifuged for 2 min (1200 rpm). Then, the supernatantwas removed and the precipitate was washed with 10mL of distilled water and centrifuged as describedabove. After repeated centrifugation, the supernatantwas removed, and 5 mL of a freshly prepared mixtureof 1 mL of 10 N NaOH, 2.5 mL of household bleach,and 6.5 mL of H2O was added to the precipitate. Themixture was thoroughly mixed on a vortex (Biosan,Latvia) for 10 min with a break every 2 min to observethe hydrolysis of nematodes under an Axio Observer Z1586Fedorova A.M. et al. Food Processing: Techniques and Technology. 2022;52(3):582–591microscope (Karl Zeiss, Germany). After that, 5 mL ofM9 medium was added to neutralize the reaction. Theresulting mixture was centrifuged for 2 min (2500 rpm).The supernatant was removed, and 10 mL of sterilewater was added to the precipitate, with washing andcentrifugation repeated 3 times. In the fourth repetition,the precipitate was washed with 10 mL of S-mediumand the test tube with nematode eggs was placed on aslow shaker for a day at room temperature so that thenematodes could enter the L1 stage.When the nematodes reached the L1 stage, anovernight bacterial culture of E. coli OP50 was addedto the S-medium. The culture had been previously washedfrom the L-broth and resuspended in the S-medium toa concentration of 0.5 mg/mL. Then, 120 μL of thesuspension containing bacteria and nematodes was addedto each well of a 96-well plate (TPP, Switzerland). Theplate was sealed with a film and left for 48 h at 20°C.After that, 15 μL of 1.2 mM 5-fluoro-2-deoxyuredin(FUDR) was poured into each well and left for a day at20°C to prevent the nematodes from reproduction. Atthe end of incubation, the worms entered the L4 stage.Then, 15 μL of a solution with trans-cinnamic acid indifferent concentrations was added to the wells and theplates were cultivated at 20°C on day 5.Next, we analyzed the effects of trans-cinnamic acidon the lifespan of C. elegans nematodes, their resistanceto oxidative and thermal stress, as well as reproductiveability.To assess the effect of trans-cinnamic acid on thelifespan of C. elegans, we used the acid at concentrationsof 0 (control), 10, 50, 100, 200 μmol/L. The experimentwas carried out in 96-well plates in the liquid S-mediumfor the cultivation of nematodes in 6 repetitions. Thenumbers of live and dead nematodes were countedevery 4–7 days during 61 days of the experiment. Theexperiment was considered completed when all thecontrol nematodes died.To determine the resistance of C. elegans to oxidativestress, we added 15 μL of 1 M paraquat to each welland continued incubation in the thermostat at 20°C.The numbers of live and dead nematodes were countedtwice: after 24 and 48 h of incubation.The resistance of C. elegans to thermal stress wasstudied by increasing the temperature to 33°C. Liveand dead nematodes were counted after 24 and 48 hof incubation.The effect of trans-cinnamic acid on the reproductiveability of C. elegans was analyzed as follows. Thesynchronized nematodes at stage L1 in the S-mediumwith E. coli OP50 were placed in 48-well 270 μLplates and 30 μL of trans-cinnamic acid at the requiredconcentration was immediately added to them. Thus,L1 larvae developed to the sexually mature stage ofL4 in the presence of trans-cinnamic acid throughoutthe experiment for 72 h. Trans-cinnamic acid at eachconcentration was added in triplicate. When thenematodes reached stage L4, each well was filledwith 300 μL of a lysis solution prepared in a 2-foldconcentration and containing 2 mL of 10 M NaOHand 5.0 mL of bleach in 3.0 mL ddH2O. The plate wascovered with an adhesive film, placed in a MaxMateplate shaker (USA), and vortexed for 5 min at 1800 rpm.Then, the wells were filled with repeat nematodes foreach test condition of each sample. The lysed nematodeswere transferred into a 2 mL Eppendorf-type tube andcentrifuged for 2 min at 1100 g at room temperature inan Eppendorf 5424 centrifuge (Eppendorf, USA). Thesupernatants were collected and transferred into a newEppendorf tube, with 1 mL of M9 buffer added to theegg precipitate. The mixture was vigorously stirred andcentrifuged for 2 min at 1100 g at room temperature.Then the procedure was repeated. Namely, the M9washing medium was transferred into a new Eppendorftube, and 1 mL of distilled water was added to the eggprecipitate. The mixture was intensively stirred andused later to count the number of eggs formed in theL4 stage nematodes under the action of trans-cinnamicacid. For the count, 100 μL was taken from 1 mL of theFigure 1. Trans-cinnamic acid obtained from the water-alcohol extract of the Scutellaria baicalensis root culture in vitro:a) structure; b) IR-spectrumРисунок 1. Транс-коричная кислота, полученная из водно-спиртово го экстракта корневой культуры in vitro Scutellaria baicalensis:a) структура; b) ИК-спектрa b3500 3000 2500 2000 1500 1000 500204060801000Wavelength, cm-1Transmission, %OOH587Федорова А. М. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 582–591aqueous suspension of eggs previously obtained afterthe nematode lysis stage and transferred to a 96-wellplate. The samples were placed in duplicate for eachconcentration of trans-cinnamic acid. The countwas performed on an Axio Observer Z1 microscope(Karl Zeiss, Germany). If the well contained morethan 100 eggs, they were additionally diluted andrecounted.Results and discussionThe results of IR spectroscopy of trans-cinnamicacid obtained from the in vitro root culture extract ofScutellaria baicalensis are shown in Fig. 1 and Table 1.As we can see in the IR spectrum of trans-cinnamicacid (phenylpropenoic acid) isolated from S. baicalensis,the 3064 cm–1 band is due to the stretching vibrationsof the acid’s diene fragment =C-H, while the 3026 cm–1band is determined by the C-H stretching vibrationsin the benzene ring. These bands can be considered ascharacteristic for trans-cinnamic acid.In this case, the 1680 cm–1 band can also be consideredas the C=C of the diene fragment. The 1631 cm–1 band isdue to the stretching vibrations of the carboxyl fragment.The 1576, 1451, 1420, and 1176 cm–1 bands correlatewith the stretching vibrations of the aromatic fragment’sС-Н bonds. The absorption band at 1451 cm–1 is due tothe deformation vibrations of the carboxyl fragment’sC-O-H. The bands at 1332, 1313, and 1221 cm–1 resultfrom the О-Н deformation vibrations and С-О stretchingvibrations, including those of the carboxyl fragment.The 1285 cm–1 band is associated with the stretchingvibrations of the C-O bond.The band at 979 cm–1 correlates with the dienefragment in the trans-form. The monosubstituted ringTable 1. Vibrational frequencies and their correlation with the structural fragments of trans-cinnamic acid obtained from the wateralcoholextract of the Scutellaria baicalensis root culture in vitroТаблица 1. Характеристичные колебательные частоты и их соотнесение с основными структурными фрагментами образца транс-коричнойкислоты, полученной из водно-спиртового экстракта корневой культуры in vitro Scutellaria baicalensisReference Wavelength, cm–1 Type of vibrations/bonds of structural fragmentsOOHOOHOOHOOHOOHOOHOOH3064 Stretching vibrations of the diene fragment = С-НOOHOOHOOHOOHOOHOOHOOH3026 Stretching vibrations of the С-Н bondOOHOOHOOHOOHOOHOOHOOH1680 С=С of the diene fragmentOOHOOHOOHOOH1631 Stretching vibrations C=O of the carboxyl groupOOHOOHOOHOOHOOHOOHOOH1576, 1451, 1420, 1176 Planar stretching vibrations of the С-С bonds of aromaticfragmentsOOHOOHOOHOOHOOHOOHOOH1332, 1313, 1221 Planar deformation vibrations of the О-Н bondOOHOOHOOHOOHOOHOOHOOH1285 Stretching vibrations of the C-O bondOOHOOHOOHOOHOOH979 Planar deformation vibrations of the ring hydroxyl groupsOOHOOHOOHOOH766 Out-of-plane deformation vibration of the C-C bond588Fedorova A.M. et al. Food Processing: Techniques and Technology. 2022;52(3):582–591is characterized by an out-of-planedeformation vibrationof the C-C bond at 766 and 711 cm–1.Thus, the spectral activity corresponded to thestructural features of trans-cinnamic acid.Figure 2 shows the effect of trans-cinnamic acid(0 (control),10, 50, 100, and 200 μmol/L) isolated fromthe water-alcohol extract of the S. baicalensis in vitroroot culture on the lifespan of Caenorhabditiselegans nematodes.As we can see in Fig. 2, on day 8 of the experiment,trans-cinnamic acid at all the concentrations under study(10, 50, 100, and 200 μmol/L) increased the lifespan ofworms (by 18.1, 26.3, 24.1, and 36.6%, respectively).From day 13 to 34, 200 μmol/L of trans-cinnamicacid did not have a positive effect on the lifespan ofnematodes, unlike the other concentrations. From day34 to 61, all the concentrations increased the percentageof surviving nematodes. The highest increase in lifespan(9.8%) was observed in the nematodes treated with50 μmol/L of trans-cinnamic acid.Figures 3 and 4 show the effect of trans-cinnamicacid isolated from the water-alcohol extract of theS. baicalensis in vitro root culture on stress resistance.As can be seen in Fig. 3, all the concentrations oftrans-cinnamic acid (10–200 μmol/L) had a positiveeffect on the resistance of nematodes to oxidative stressduring 24 and 48 h, i.e. increased their survival, comparedto the control. We also found a gradual decrease inthe percentage of surviving nematodes during 24-hoxidative stress with increased concentrations of transcinnamicacid.After 5 days of nematode incubation in the presenceof trans-cinnamic acid, the experimental plate wastransferred to a 33°C incubator. Dead worms werecounted after 24 h of nematode incubation at elevatedtemperature and after 48 h of incubation under prolongedthermal stress.According to Fig. 4, trans-cinnamic acid significantlyreduced the percentage of surviving nematodes underFigure 2. The effect of trans-cinnamic acid isolated fromthe water-alcohol extract of the Scutellaria baicalensisin vitro root culture on the lifespanof Caenorhabditis elegans nematodesРисунок 2. Влияние транс-коричной кислоты, выделеннойиз водно-спиртового экстракта корневой культуры in vitroScutellaria baicalensis, на продолжительность жизни нематодCaenorhabditis elegans0204060801001200 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, daysControl 10 μmol/L 50 μmol/L100 μmol/L 200 μmol/L85.990.786.894.392.970.769.773.481.880.7Survival, %Control 1 0 μ m o l / L 5 0 μ m ol/L 100 μmol/L 200 μmol/LCultivation time, h24 h 48 h85.990.786.894.392.970.769.773.481.880.7Survival, %Control 1 0 μ m o l / L 5 0 μ m ol/L 100 μmol/L 200 μmol/LCultivation time, h24 h 48 hFigure 3. The effect of trans-cinnamic acid isolated fromthe water-alcohol extract of the Scutellaria baicalensisin vitro root culture on the resistance of nematodesto oxidative stressFigure 3. Рисунок 3. Влияние транс-коричной кислоты,выделенной из водно-спиртового экстракта корневойкультуры in vitro Scutellaria baicalensis, на устойчивостьнематод при окислительном стрессе96.395.196.094.993.980.674.678.971.865.2Control 10 μmol/L 50 μmol/L 100μmol/L 200μmol/LSurvival, %Cultivation time, h24 ч 48 ч85.990.786.894.392.970.769.773.481.880.7Survival, %Control 1 0 μ m o l / L 5 0 μ m ol/L 100 μmol/L 200 μmol/LCultivation time, h24 h 48 hFigure 4. The effect of trans-cinnamic acid isolated fromthe water-alcohol extract of the Scutellaria baicalensisin vitro root culture on the resistance of nematodesto thermal stressРисунок 4. Влияние транс-коричной кислоты, выделеннойиз водно-спиртового экстракта корневой культуры invitro Scutellaria baicalensis, на устойчивость нематод ктемпературному стрессу589Федорова А. М. [и др.] Техника и технология пищевых производств. 2022. Т. 52. № 3. С. 582–591thermal stress with increased concentrations from10 to 200 μmol/L.Figure 5 shows the effect of trans-cinnamicacid isolated from the water-alcohol extract of theS. baicalensis in vitro root culture on the reproductiveabilities of C. elegans nematodes.As we can see in Fig. 5, trans-cinnamic acid atthe concentrations of 10, 50, and 200 μmol/L did notsignificantly affect the reproductive performance ofnematodes, compared to the control. The concentrationof 100 μmol/L was the most effective since it produced1.48 times more eggs, compared to the control.ConclusionModern medical gerontology is looking for waysto increase life expectancy with a focus on geroprotectors– special compounds that reduce the rate ofaging. Plants are the main source of geroprotectors,including Scutellaria baicalensis. Since this is a rareplant included in the Russian Red Data Book, we usedits root culture as a source of trans-cinnamic acid.In particular, we isolated trans-cinnamic acid fromthe water-alcohol extract of the S. baicalensis in vitroroot culture b HPLC. The isolated bioactive compoundwas at least 95% pure. According to IR spectroscopy, thespectral activity corresponded to the structural features oftrans-cinnamic acid.To study the geroprotective activity of trans-cinnamicacid, we evaluated its effect in various concentrations onthe lifespan, oxidative and thermal stress resistance, aswell as reproductivity of Caenorhabditis elegans usedas a model organism. As a result, we drew the followingconclusions:– all the studied concentrations of trans-cinnamic acidincreased the lifespan of C. elegans worms, with thehighest increase achieved by 50 μmol/L;– all the concentrations of trans-cinnamic acid (10–200 μmol/L) had a positive effect on the resistance ofnematodes to oxidative stress increasing their survival,compared to the control;– under thermal stress, increased concentrations of transcinnamicacid significantly reduced the percentage ofsurviving nematodes; and– trans-cinnamic acid at a concentration of 100 μmol/Lincreased the reproductive capacity of nematodes,producing 1.48 times more eggs, compared to thecontrol. The remaining concentrations did not havesuch an effect.Based on our data, trans-cinnamic acid can be usedas a bioactive substance with geroprotective properties.However, further research is needed on other modelorganisms with detailed toxicity studies to determinethe full potential of trans-cinnamic acid as an anti-agingagent capable of slowing down the aging process andextending life.ContributionAll the authors equally contributed to the studyconcept, data processing and analysis, as well as writingthe manuscript.Conflict of interestThe authors declare that there is no conflict of interestregarding the publication of this article.Критерии авторстваВсе авторы внесли равный вклад в созданиеисследования, обработку и анализ полученныхрезультатов, а также в оформлении статьи.Конфликт интересовАвторы заявляют об отсутствии конфликтаинтересов.