INTRODUCTIONAccording to the WHO, average life expectancy issteadily increasing worldwide [1]. Over the last 20 years,it has grown by 6 years as a result of advances inscience and medicine. However, behind these advancesis an increase in diseases associated with aging, whichhas become a serious problem of public health in the21st century. Aging is a process that affects the entirehuman body, in particular its cardiovascular, nervous,digestive, and immune systems.The aging process is directly related to oxidativestress. Age-related diseases cause structural changesin mitochondria, as well as changes in the functionsof the electron transport chain, which ultimately leadsto oxidative stress. The cardiovascular system isparticularly susceptible to this effect, which explains theincrease in cardiovascular diseases in the elderly [2–5].Copyright © 2022, Faskhutdinova et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 InternationalLicense (https://creativecommons.org/licenses/by/4.0/), allowing third parties to copy and redistribute the material in any medium or format and toremix, transform, and build upon the material for any purpose, even commercially, provided the original work is properly cited and states its license.Research Article Available online at http://jfrm.ru/enOpen Access https://doi.org/10.21603/2308-4057-2022-2-544https://elibrary.ru/ZVCUUWEffects of bioactive substances isolated from Siberianmedicinal plants on the lifespan of Caenorhabditis elegansElizaveta R. Faskhutdinova1,* , Andrey S. Sukhikh1 , Violeta M. Le1 ,Varvara I. Minina1 , Mohammed El Amine Khelef2 , Anna I. Loseva11 Kemerovo State University , Kemerovo, Russia2 Moscow State University of Food Production , Moscow, Russia* e-mail: faskhutdinovae.98@mail.ruReceived 10.02.2022; Revised 10.03.2022; Accepted 05.04.2022; Published online X.X.2022Abstract:Medicinal plants are sources of natural antioxidants. Acting as reducing agents, these substances protect the human body againstoxidative stress and slow down the aging process. We aimed to study the effects of bioactive substances isolated from medicinalplants on the lifespan of Caenorhabditis elegans L. used as a model organism.High-performance liquid chromatography was applied to isolate bioactive substances from the extracts of callus, suspension, androot cultures of meadowsweet (Filipendula ulmaria L.), ginkgo (Ginkgo biloba L.), Baikal skullcap (Scutellaria baicalensis L.),red clover (Trifolium pretense L.), alfalfa (Medicágo sativa L.), and thyme (Thymus vulgaris L.). Their effect on the lifespan ofC. elegans nematodes was determined by counting live nematodes treated with their concentrations of 10, 50, 100, and200 μmol/L after 61 days of the experiment. The results were recorded using IR spectrometry.The isolated bioactive substances were at least 95% pure. We found that the studied concentrations of trans-cinnamic acid,baicalin, rutin, ursolic acid, and magniferin did not significantly increase the lifespan of the nematodes. Naringenin increasedtheir lifespan by an average of 27.3% during days 8–26. Chlorogenic acid at a concentration of 100 μmol/L increased the lifespanof C. elegans by 27.7%. Ginkgo-based kaempferol and quercetin, as well as red clover-based biochanin A at the concentrations of200, 10, and 100 μmol/L, respectively, increased the lifespan of the nematodes by 30.6, 41.9, and 45.2%, respectively.The bioactive substances produced from callus, root, and suspension cultures of the above medicinal plants had a positive effecton the lifespan of C. elegans nematodes. This confirms their geroprotective properties and allows them to be used as anti-agingagents.Keywords: Plants, antioxidants, callus culture, suspension culture, root culture, nematodes, agingFunding: The study was financed by the Ministry of Science and Higher Education of the Russian Federation (Minobrnauka)(project FZSR-2020-0006 “Screening bioactive plant-based substances with geroprotective properties and developing technologyfor producing anti-aging nutraceuticals”).Please cite this article in press as: Faskhutdinova ER, Sukhikh AS, Le VM, Minina VI, Khelef MEA, Loseva AI. Effectsof bioactive substances isolated from Siberian medicinal plants on the lifespan of Caenorhabditis elegans. Foods and RawMaterials. 2022;10(2):340–352. https://doi.org/10.21603/2308-4057-2022-2-534Foods and Raw Materials. 2022;10(2)ISSN 2310-9599 (Print)ISSN 2308-4057 (Online)341Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352The problem of aging is widely covered by thefree-radical theory of aging developed by DenhamHarman in the 1950s [6]. According to this theory,the body’s defense mechanisms stop responding todamage caused by reactive oxygen species, resultingin the deterioration of cellular homeostasis, energyimbalance, and mitochondrial insufficiency [7].The molecules of reactive oxygen species includenitric oxide, hydrogen peroxide, monoxide radicals,superoxide anions, tocopherols, ascorbic acid, andpolyphenols [8]. Reactive oxygen species alter cellularactivities such as cell survival, stress responses, andinflammation. They are also involved in musclecontractions, regulate vascular tone, as well asdetermine bactericidal and bacteriostatic activity [9].However, their increase leads to oxidative stress,disrupting the balance of antioxidants and prooxidants[10]. This causes damage to macromolecules(lipids, DNA, and proteins) and subsequently to wholecells, tissues, and organs [4]. Higher concentrationsof reactive oxygen species in the body promoteinflammation, which, in turn, can accelerate theformation of their molecules [11]. Therefore, it is veryimportant to maintain a balance between antioxidantsand pro-oxidants.Antioxidants can react with free radicals andneutralize them by causing them to decay. The humanbody has three lines of defense against oxidativestress. The first line consists of body enzymes such assuperoxide dismutase, catalase, glutathione peroxidase,glutathione S-transferase, and glutathione reductase [12].They prevent cell damage by scavenging free radicalsthat cause premature aging and age-related disorders.The first line also includes non-enzymatic moleculesin the blood plasma such as transferrin, ferritin,ceruloplasmin, and albumin [13]. These preventativeantioxidants inhibit the formation of new reactiveoxygen species by binding transition metal ions (forexample, copper and iron). The second line of defense isrepresented by non-enzymatic antioxidants that provideintermediate protection against oxidative radicals. Thethird line of defense serves to regenerate biomoleculesdamaged by oxidative stress [14].However, protection against oxidative stress shouldnot be limited to the protective function of the biologicalsystem itself. Noteworthily, one antioxidant moleculeis capable of reacting with only one oxidizing radical.Therefore, there is a need to replenish antioxidantmolecules, including the use of supplements.There are three groups of exogenous antioxidants:mineral elements, nutritional antioxidants (carotenoids,vitamins E and C), and natural antioxidantsderived from natural sources commonly known asphytochemicals or phytonutrients [15].Synthetic antioxidants have been widely used untilrecently, but there is some doubt as to their usefulnessand safety. According to some studies, syntheticantioxidants are ineffective against oxidative stress.Moreover, their long-term use can lead to diseases suchas skin allergies, gastrointestinal and cardiovasculardiseases, and even increase the risk of cancer [16].Therefore, there is a need for thorough research into thesafety of synthetic antioxidants.The main sources of exogenous antioxidants arefruits, vegetables, cereals, etc. However, modernresearch is focused on traditional medicinal plants as asource of natural antioxidants [17, 18].Since ancient times, plants have been a source ofmany useful substances, including exogenous antioxidants.These substances act as reducing agentsthat scavenge free radicals, protect the body againstoxidative stress, and, as a result, maintain a balancebetween oxidants and antioxidants [19]. This is achieveddue to the presence of polyphenols, tocopherols,carotenoids, ascorbic acid, and macromolecules(including polysaccharides and peptides), as well asessential oils [20].Polyphenols are substances that contain a multiplenumber of structural units of phenol [21]. Theirnumber affects the chemical, biological, and physicalproperties of polyphenolic compounds. Polyphenolsare represented by flavonoids, phenolic acids, andnonflavonoids [22, 23]. Depending on the chemicalstructure, flavonoids are classified into flavonols,flavanones, isoflavones, anthocyanins, and flavan-3-ols.They are the most abundant class of polyphenols, withabout 8000 compounds identified to date [24]. A coupleof decades ago, researchers significantly increasedtheir interest in polyphenols due to their beneficialproperties for humans [25]. In particular, polyphenolscurb oxidative stress and related conditions through theirreductive ability to protect cellular components fromoxidative damage caused by free radicals [26].Model organisms have become an indispensablepart of biological studies that would be impossible toconduct on humans for ethical or economic reasons [27].Studies of aging and age-related diseases require anorganism with a relatively short lifespan and clearlyidentified genetic factors to ensure reproducibility andreliability [28]. Nematodes (Caenorhabditis elegans L.),drosophila (Drosophila melanogaster L.), and yeasts(Saccharomyces cerevisiae L.) are used to test the effectof bioactive substances on lifespan [29, 30].C. elegans is a non-parasitic, free-living nematodethat feeds on various bacteria, primarily Escherichiacoli [31]. This simple multicellular organism up to 1 mmlong is a hermaphrodite capable of self-fertilization [32].Its hermaphroditic structure contributes to low geneticvariability [33]. C. elegans has a short life cycle (2–4 days) and remains viable for 20–25 days at 20°C as anadult [34]. The nematode can be stored in liquid nitrogenfor an almost unlimited amount of time [35].C. elegans was first used as a model organism forbiological research by Sydney Brenner in the 1965s [36].Since then, it has been widely used to study aging andassociated oxidative stress, as well as neurodegenerationand inflammation processes [37].342Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352C. elegans has a number of advantages that explainits common uses as a model organism. Firstly, thenematode is easily cultivated and has a transparentbody, which makes it easy to track the changesmicroscopically [33, 38–40]. Secondly, C. elegans hasfour organ systems that are the same as in vertebrates(nervous, digestive, immune, and reproductive), whichallows for reliable and valuable conclusions [41]. Thirdly,its short lifespan (20–25 days) enables scientists toconduct rapid experiments aimed to study the effectof various substances on the lifespan [42]. Finally, thenematode’s genome is completely deciphered andeasily modified, which facilitates studies of the agingprocess [43–46].Plants of the Siberian Federal Okrug (Russia) arepotential sources of geroprotectors – substances that canslow down the aging process [47].Meadowsweet (Filipendula ulmaria L.) is a herbaceousperennial plant common in Russia and manyEuropean countries [48]. Extracts of this planthave anticancerous, antioxidant, and anti-inflammatoryactivity [49]. This activity is associated withtannins, phenolic compounds, phenolcarboxylic acids,catechins, flavonoids, essential oils, and other bioactivesubstances contained in the roots and flowers of theplant [50]. Many previous studies confirm its medicinalproperties [51, 52]. Rutin, one of its phytochemicals,belongs to the class of natural flavonoids and is knownas quercetin-3-O-rutinoside or vitamin P [53]. It hasantitumorous, anticarcinogenic, and antimicrobialproperties [54, 55].Ginkgo (Ginkgo biloba L.) is a special medicinalplant that contains a variety of compounds with aunique structure due to its phylogenetic divergencefrom other plants. Its extract obtained by dryingthe leaves is used to treat many neurodegenerativediseases (memory impairment, dementia, Alzheimer’sdisease) [56]. It is also widely applied as an antiinflammatory,cardioprotective, and antioxidantagent [57]. The plant’s active components areflavonoids, terpenoids, polyphenols, and organicacids [58]. Quercetin and kaempferol are two of thesebeneficial compounds. Quercetin is a flavonoid thathas a positive effect on cardiovascular diseases, breastcancer, and ischemia [59–62]. Kaempferol is a valuablecomponent with anticancerous, antitumorous, and antiinflammatoryproperties [62].Baikal skullcap (Scutellaria baicalensis L.) belongsto the genus Lamiaceae and is still widely used inChinese traditional medicine [63]. This plant growsin China, Russia, Mongolia, Japan, and North Korea.To date, scientists have identified 126 low-molecularweight compounds in it, mostly in its root. Thesecompounds include flavonoids, flavonoid glycosides, andphenylethanoid glycosides [64]. The most widespreadand studied of them is baicalin, which is used for variousmedical purposes [66].Red clover (Trifolium pretense L.) is one of themost important representatives of the Leguminosaefamily, numbering over 240 species [66]. It hasnumerous medicinal properties and therapeuticeffects on respiratory diseases, bacterial and fungalinfections, tumors, and diabetes [67]. The plant isrich in isoflavones (biochanin A, genistein, trifoside),flavonoids (quercetin, kaempferol), as well as cinnamic,caffeic, and chlorogenic acids [68].Alfalfa (Medicágo sativa L.) is a flowering plant inthe Fabaceae family, which is the largest and mostwidespread family in the world. The genus Medicágoincludes 83 species rich in alkaloids, flavonoids,naphthoquinones, and saponins [69]. Naringenin, oneof its components, is a water-soluble flavonoid of greatvalue due to its anticancerous, antioxidant, and antiinflammatoryeffects [70, 71].Thyme (Thymus vulgaris L.) is an aromatic perennialflowering plant belonging to the Lamiaceae family. Itstherapeutic properties are mainly associated with itsessential oil that has antitussive, expectorant, antiseptic,antimicrobial, and anthelmintic effects [72]. The plantis traditionally used to treat oral, gastrointestinal, andurinary tract infections, as well as respiratory diseases(cough, bronchitis, asthma) [73]. Ursolic acid, one of itsbioactive substances, is a promising agent against cancer,cardiovascular disease, brain and liver diseases, obesity,and diabetes [74, 75].We aimed to study the effect of individual bioactivesubstances on the lifespan of the model organism C.elegans.STUDY OBJECTS AND METHODSWe used individual bioactive substances isolatedfrom the extracts of suspension, callus, and root culturesof Siberian medicinal plants. The extraction parametersare presented in Table 1.Table 1 Parameters for obtaining extracts from Siberian medicinal plantsSample Extraction time, h Temperature, °С Ethanol, %Callus cultures of meadowsweet (Filipendula ulmaria L.) 5 35 70Suspension cultures of ginkgo (Ginkgo biloba L.) 6 55 70Root cultures of Baikal skullcap (Scutellaria baicalensis L.) 5 35 70Callus cultures of red clover (Trifolium pretense L.) 5 70 60Callus cultures of alfalfa (Medicágo sativa L.) 3 50 70Callus cultures of thyme (Thymus vulgaris L.) 4 50 70Root cultures of sweetvetch (Hedysarum neglectum L.) 6 30 70343Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352High-performance liquid chromatography (HPLC)was applied (Shimadzu LC-20 Prominence liquidchromatograph, Japan) to isolate the following bioactivesubstances from the extracts of the above cultures:1. Rutin from the callus culture extract of meadowsweet(Filipendula ulmaria) [76];2. Quercetin from the suspension culture extract ofginkgo (Ginkgo biloba) [77];3. Kaempferol from the suspension culture extract ofginkgo (G. biloba) [77];4. Baicalin from the root culture extract of Baikalskullcap (Scutellaria baicalensis) [78];5. Trans-cinnamic acid from the root culture extract ofBaikal skullcap (S. baicalensis) [79];6. Chlorogenic acid from the callus culture extract of redclover (Trifolium pratense) [80];7. Biochanin A from the callus culture extract of redclover (T. pratense) [80];8. Naringenin from the callus culture extract of alfalfa(Medicágo satíva) [81];9. Ursolic acid from the callus culture extract of thyme(Thymus vulgaris) [82]; and10. Magniferin from the root culture extract ofsweetvetch (Hedysarum neglectum) [83].To isolate rutin from the callus culture extractof meadowsweet, the plant’s ethanol extract wasevaporated under vacuum at a temperature under 40°Con an IKA RV 8 rotary evaporator (IKA, Germany).After adding deionized water to 1/4 of the concentrate’sinitial volume, evaporation continued until a thickprecipitate was formed. The precipitate was treatedwith a chloroform:ethylacetate mixture for 5 minwith vigorous stirring in triplicate. The extracts werecombined and mixed with anhydrous sodium sulfate(2.0 g per 100 mL of extract). The mixture was kept for3 h at +4°C and then filtered. The residue containing aflavonoid fraction was dissolved in 50% ethanol. Then,50.0 g of activated carbon was added to the mixtureand evaporated until a dry residue was formed. Theadsorbent with the extract residue was transferred to ashot filter and successively eluted with methanol, water,7% aqueous phenol, and 15% phenol in methyl alcohol.The fraction extracted with 7% aqueous phenol wastreated with 100 mL of diethyl ether in triplicate. Theresulting extract was evaporated under vacuum to athick precipitate, which was then mixed with 40.0 gof silica gel (column chromatography grade, Sigma),dried completely, and transferred to a column (5×6 cmBioRad) as a suspension in chloroform. The substanceswere eluted with a mixture of chloroform:ethanol (80:20)and evaporated to isolate rutin.To isolate quercetin and kaempferol from thesuspension culture extract of ginkgo, the extract wasfiltered through cellulose filters, diluted with water, andkept at +4°C for 48 h to filter lipid precipitates. Theextract was then concentrated in a vacuum evaporatorin the presence of sodium chloride (up to 10% by saltcontent in solution). Resinous substances were removedby decantation. Lipophilic substances were purifiedby liquid-liquid extraction with n-heptane to isolateterpenolactones. The aqueous phase was extracted withn-butanol in triplicate.The three phases were combined and concentratedunder vacuum until a dry precipitate was formed. Theprecipitate was dissolved in a water-alcohol solutionand purified by liquid-liquid extraction with ethylacetate. The resulting phase was washed with a sodiumchloride solution and evaporated. The dry residuewas dissolved in acetone containing 40 wt.% of water,cooled to 10°C, and filtered. Flavonogicosides werechromatographed on polyamide (Sigma) packed in a5.3×250 mm chromatographic column on a BioLogiclow-pressure chromatograph (BioRad) using gradientelution mixtures: chloroform-methanol (100:0 → 60:40)and then water-ethanol (100:0 → 0:100). The componentswere separated and purified by silica gelrechromatography (Lachema) using an eluent mixtureof chloroform:petroleum ether (30:70), followed byrecrystallization to isolate quercetin and kaempferol.Baicalin and trans-cinnamic acid were isolatedfrom the root culture extract of Baikal skullcap byevaporating the extract under vacuum at a temperatureunder 50°C. The evaporated residue was treated withdiethyl ether in triplicate. The resulting ether fractionwas chromatographed on silica gel (mobile phase) ina n-hexane-acetone gradient (1:0 → 0:1) to isolateflavonoids and hydroxycinnamic acids. Baicalin andtrans-cinnamic acid were isolated by subsequentrechromatography on silica gel (mobile phase) withn-hexane-chloroform (1:0 → 0:1).To isolate biochanin A from the callus cultureextract of red clover, the ethanol extract was vacuumevaporatedon a rotary evaporator at under 50°C.Deionized water was added to the precipitate up to 1/4 ofthe concentrate’s initial volume to continue evaporationto a thick precipitate. The precipitate was treated withn-hexane for 5 min in triplicate and the suspension wastreated ultrasonically. The extracts were filtered throughfilter paper and combined. Then, they were evaporatedunder vacuum to a thick precipitate. The precipitate wasmixed with 50.0 g of silica gel, dried, and transferredto a column (5×6 cm BioRad). Then, it was eluted witha petroleum ether-ethanol mixture (99:1, 98:2, 97:3,95:5, 93:7, 80:20). Biochanin A was isolated from theevaporated eluates.To isolate chlorogenic acid from the callus cultureextract of red clover, the thick precipitate obtained asdescribed above was treated with diethyl ether to isolatehydroxycinnamic and coumaric acids. The mixturewas then evaporated to a dry residue and separated onsilica gel (column chromatography grade, Sigma) on acolumn (0.65×10 cm BioRad). Then, it was eluted withisopropyl alcohol:acetic acid:hexane (65:12:23) to isolatechlorogenic acid.Naringenin was isolated from the alfalfa extractas follows. The ethanol extract was evaporated undervacuum at under 55°C on a rotary evaporator. Theresidue was mixed with deionized water added to 1/4 of344Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352the concentrate’s initial volume to continue vacuumevaporation to a thick precipitate. The precipitate wasplaced on a 5×6 cm BioRad chromatographic columnand eluted with n-hexane to collect 1-mL fractions. Theresulting extracts were evaporated to a thick precipitate,which was then dissolved in ethanol and fractionatedon LH-20 Sephadex (Aldrich) in toluene. Silica gel waseluted with isopropyl alcohol:water (40:60) and thenevaporated under vacuum to a thick residue. The residuewas dissolved in ethanol and fractionated on LH-20Sephadex (Aldrich) in a methanol gradient of 10 → 90%to isolate naringenin.Ursolic acid was isolated from the callus cultureextract of thyme. For this, the ethanolic extract wasevaporated under vacuum at under 40°C on a rotaryevaporator. The residue was mixed with deionizedwater added to 1/4 of the concentrate’s initial volumeto continue vacuum evaporation to a thick precipitate.The resulting precipitate was treated three times withdichloromethane for 5 min with vigorous stirring. Theextracts were combined and mixed with anhydroussodium sulfate (20.0 g per liter of extract). The mixturewas kept for 3 h and filtered through a paper filter. Thefiltered precipitate was dissolved in ethanol. The ethanolfraction was passed through an AN-1 anion exchanger(State Standard 20301-74) and washed with waterethanoleluents (up to 50% of ethanol). Then, it wasdesorbed with 0.1 M hydrochloric acid to isolate ursolicacid.Magniferin was isolated from the root culture extractof sweetvetch. The ethanol extract was evaporatedin a vacuum evaporator at 45°C. The residue wasfractionated on a BioLogic low-pressure chromatograph(BioRad) using silica gel (column chromatographygrade, Sigma). Petroleum ether-ethyl acetate was used asan eluent (100:0; 50:1; 20:1; 10:1; 5:1; 2:1; 1:1; and 0:1).Methanol was fed to the column to desorb gallic acid,resulting in nine 300-mL fractions collected. A crudecrystal of magniferin was obtained from fraction 3.Then, it was recrystallized from a mixture of petroleumether:acetone (20:1) and purified by rechromatographyon CL6B Sepharose (Sigma – Aldrich) using a BioLogiclow-pressure chromatograph (BioRad) to isolate puremangiferin.All the isolated bioactive substances were at least95% pure. Their IR spectra were registered on anSF-2000 instrument (OKB Spektr, Russia).Further, we analyzed the effect of bioactivesubstance concentrations on the lifespan of wild-typeCaenorhabditis elegans nematodes (strain N2 Bristol,www.wormbook.org). Our study consisted of five stagesdescribed below.Cultivation of nematodes on solid agar. Obtainingan Escherichia coli bacterial culture. E. coli OP50was seeded on Petri dishes with a Lysogeny broth(L-broth) solid medium (HiMedia Laboratories, India).Then, under sterile conditions, one bacterial colony wasselected and placed in 5–10 mL of L-broth (HiMediaLaboratories, India) to incubate at 37°C overnight withvigorous stirring. After that, the culture was transferredto a refrigerator and stored at +4°C.Inoculating E. coli OP50 on NGM agar plates. 50μL of the E. coli OP50 overnight culture was placed inthe center of a 100-mm Petri dish. Using a sterile glassrod, the drop was distributed over the center of the dishin the shape of a square, without touching the walls, andincubated at 37°C for a day. After incubation, the disheswere wrapped in parafilm and stored in the refrigeratorfor several weeks.Preparing NGM agar plates. After autoclaving, thesterile NGM agar was cooled to 55°C in a water bath.Then, the cooled nutrient medium was mixed with 1 mLof 1 M CaCl2, 1 mL of 5 mg/mL cholesterol in alcohol,1 mL of 1 M MgSO4, and 25 mL of 1 M K3PO4 buffer.After thorough mixing, it was poured into sterile Petridishes, 20 mL each. To ensure the absence of bacterialcontamination, the dishes were left for 2–3 days at roomtemperature.Transferring nematodes to new NGM agardishes. The nematodes were transferred in two ways:with a loop and with a piece of agar. The first methodinvolved hooking a nematode with a calcined and cooledbacteriological loop and planting it on a bacterial lawnin the center of a new NGM Petri dish with agar. Thesecond method involved cutting a 0.5×0.5 cm piece ofagar containing a nematode with a sterile scalpel andtransferring it to the center of the dish surface down.The dishes were incubated at 20°C.Nematode synchronization. 5–10 mL of sterilewater was pipetted on the surface of the dish containinga nematode until its eggs were completely attachedto the agar. The liquid from the Petri dish was placedin a 50 mL tube and centrifuged for 2 min (1200 rpm).Then, the supernatant was removed and the precipitatewas washed with 10 mL of distilled water to repeatcentrifugation under the above conditions.After repeated centrifugation, the supernatant wasremoved and the precipitate was mixed with 5 mLof a freshly prepared mixture of 1 mL of 10 N NaOH,2.5 mL of household bleach, and 6.5 mL of H2O. Themixture was thoroughly vortexed (Biosan, Latvia) for10 min with 2 min intervals to observe the hydrolysisof nematodes under an Axio Observer Z1 microscope(Karl Zeiss, Germany). At the end of the process, 5 mLof M9 medium was added to neutralize the reaction. Theresulting mixture was centrifuged for 2 min (2500 rpm).After that, the supernatant was removed and theprecipitate was mixed with 10 mL of sterile water torepeat the washing and centrifugation three times. Then,the precipitate was washed with 10 mL of S-mediumand the supernatant was removed. Finally, 10 mL ofS-medium was added and the test tube with nematodeeggs was placed on a slow shaker for a day at roomtemperature for the complete transfer of the nematodesto the L1 stage.345Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352Cultivation of nematodes in a liquid medium.After the nematodes passed to the L1 stage, an overnightbacterial culture of E. coli OP50 was added to theS-medium. The culture had previously been washed andresuspended in the S-medium to a bacterial concentrationof 0.5 mg/mL. Then, 120 μL amounts of the suspensioncontaining the bacteria and nematodes were poured intoeach well of a 96-well plate (TPP, Switzerland). The platewas sealed with a film and left for 48 h at 20°C.After that, 15 μL of 1.2 mM 5-fluoro-2-deoxyuredinwas poured into each well of the plate to prevent thenematodes from reproduction and left for a day at 20°C.At the end of incubation, the nematodes entered the L4stage. Then, 15-μL amounts of the bioactive substancesunder study were added to the wells in accordance withthe experiment plan.Preparation of bioactive substances. Stocksolutions of bioactive substances were prepared indimethyl sulfoxide at a concentration of 10 mmol/L.The substances were tested by diluting stock solutionsin sterile distilled water to concentrations of 2000,1000, 500, and 100 μM. Each well was filled with 15 μLof freshly prepared stock solutions so that workingconcentrations of each bioactive substance reached 2000,1000, 500, and 100 μmol/L, respectively. The stockswere stored at 4°C.Effects of bioactive substances on nematodelifespan. The effect of bioactive substances atconcentrations of 0, 10, 50, 100, and 200 μmol/L on thelifespan of C. elegans was determined by the numberof nematodes surviving in the presence of the testedsubstances. The experiment was carried out in 6-foldrepetitions using 96-well plates and a liquid S-mediumfor nematode cultivation. The numbers of live and deadnematodes were counted every 4–7 days during the61-day experiment. The experiment was consideredcompleted when there were no live nematodes left in thecontrol group.Each concentration of bioactive substances wasstudied in 6-fold repetitions. Statistical data wereanalyzed in the Microsoft Office Excel 2007. Statisticalanalysis was performed using a paired Student’s t-testfor each pair of interests. Differences were consideredstatistically significant at P < 0.05.RESULTS AND DISCUSSIONThe effects of the bioactive substances obtainedfrom the extracts of suspension, callus, and rootcultures of Siberian medicinal plants on the lifespanof Caenorhabditis elegans nematodes are graphicallypresented in Fig. 1.As can be seen in Fig. 1a, rutin, which was isolatedfrom the extract of meadowsweet callus culture, didnot significantly increase the lifespan of C. elegansnematodes. At concentrations of 50 and 100 μmol/L, ithad a positive effect from day 8 to day 34, but then thenumber of surviving nematodes approached the control.Its greatest effect was observed at a concentration of50 μmol/L on day 13, with the survival rate of 32.3%(15.3% higher than in the control group).Quercetin, which was obtained from the suspensionculture extract of ginkgo, had a significant effect onthe lifespan of C. elegans nematodes at concentrationsof 10 and 100 μmol/L (Fig. 1b). The proportion ofsurviving nematodes was 32.6–4.6% from day 8 to day45 of the experiment. On day 8, all the concentrationsof quercetin had a positive effect on the lifespan. Theproportions of surviving nematodes treated with 10, 50,100 and 200 μmol/L of this bioactive substance were72.9, 74.0, 67.5, and 63.6%, respectively (higher thanin the control nematodes by 41.9, 43.0, 36.5, and 32.6%,respectively).Of special interest was kaempferol obtained fromthe suspension culture extract of ginkgo (Fig. 1c). At aconcentration of 50 μmol/l, this substance increasedthe lifespan of nematodes throughout the experiment(except for 3 days), compared to the control. Thenematode population was active, reaching 10.3% onday 61. We also observed kaempferol’s positive effectat a concentration of 10 μmol/L in the period of 8 to 61days. The maximum proportion of surviving nematodestreated with this concentration was registered on day 8at 48.6%, which was 17.6% higher than in the controlgroup. However, the greatest increase in the nematodelifespan was provided by a concentration of 200 μmol/Lon day 8, with the survival rate of 61.6% (by 30.6%higher than in the control nematodes).Baicalin was produced from the root culture ofBaikal skullcap (Fig. 1d). At concentrations of 10, 100,and 200 μmol/L, it increased the lifespan of nematodesfrom day 8 to day 13. After that period, the number ofsurviving nematodes exposed to 200 μmol/L baicalinbecame lower than in the control group. Duringdays 13–17, their lifespan increased only at baicalin’sconcentrations of 10 and 100 μmol/L. From day 17 untilthe end of the experiment, the number of survivingnematodes treated with 10, 50, and 200 μmol/L baicalinwas greater than in the control group. Noteworthily,the end of the experiment saw greater proportionsof surviving nematodes treated with baicalin at allconcentrations (10, 50, 100, and 200 μmol/L) than that ofthe control (by 4.3, 6.7, 2.3, and 2.1%, respectively).Trans-cinnamic acid was isolated from the rootculture extract of Baikal skullcap (Fig. 1e). As we cansee, on day 8, its concentrations of 10, 50, 100, and200 μmol/L increased the lifespan of nematodes by 18.1,26.3, 24.1, and 36.6%, respectively. During days 13–34,the concentration of 200 μmol/L had no positiveeffect on the lifespan of nematodes, unlike the otherconcentrations. However, from day 34 to the end of theexperiment, trans-cinnamic acid at all concentrationsincreased the percentage of surviving nematodes. Thegreatest increase in the lifespan was observed in thenematodes treated with 50 μmol/L of this bioactivesubstance (9.8%).Chlorogenic acid obtained from the callus cultureextract of red clover showed a generally positive effect346Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352Figure 1 Beginning. Effects of bioactive substances isolated from the extracts of suspension, callus, and root culturesof medicinal plants on the lifespan of Caenorhabditis elegans nematodes: (a) rutin from the suspension culture extract ofmeadowsweet; (b) quercetin from the suspension culture extract of ginkgo; (c) kaempferol from the suspension culture extract ofginkgo; (d) baicalin from the root culture extract of Baikal skullcap; (e) trans-cinnamic acid from the root culture extract of Baikalskullcap; (f) chlorogenic acid from the callus culture extract of red clover; (g) biochanin A from the callus culture extract redclover; (h) naringenin from the callus culture extract of alfalfa; (i) ursolic acid from the callus culture extract of thyme;(j) magniferin from the root culture extract of sweetvetcha ba bc d0 3 8 13 17 20 26 30 34 45 55 61Lifespan, days10 мкмоль/л 50 мкмоль/л 200 мкмоль/л100 мкмоль/л Контроль20.070,020 26 30 34 45 55 61Surviving nematodes , %Lifespan, days10 мкмоль/50 мкмоль/200 100 Контроль0 3 8 13 17 20 26 30 34 45 55 61Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,030 34 45 55 61Surviving nematodes , %10 50 200 Контроль100 c da bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль10,020,030,040,050,060,070,080,090,0100,08 13 17 20 26 30 34 Surviving nematodes , %Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,090,0100,00 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , % Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days%a c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L controlSurviving nematodes , %0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольSurviving nematodes , %a bc d0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л 50 мкмоль/л 200 мкмоль/л100 мкмоль/л Контроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 Surviving nematodes , %Lifespan, days0.010.020.030.040.050.060.070.080,090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 Surviving nematodes , % Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,00 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , % Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , %Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, daysa b0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control10,020,030,040,050,060,070,080,090,0100,013 17 20 26 30 34 45 55 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,0Surviving nematodes, %10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,0Surviving nematodes , %a b0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control10,020,030,040,050,060,070,080,090,0100,013 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,0Surviving nematodes, %10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,0Surviving nematodes , %a b0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control10,020,030,040,050,060,070,080,090,0100,013 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61 Surviving nematodes, %10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,0Surviving nematodes , %e f60.070.080.090.0100.0a c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 100 Контрольa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,0100,00 3 8 13 17 20 26 30 34 Surviving nematodes , %Lifespan, daysa bc d0 3 8 13 17 20 26 30 34 45 55 61Lifespan, days10 мкмоль/л 50 мкмоль/л 200 мкмоль/л100 мкмоль/л Контроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days10 50 200 100 Контроль0 3 8 13 17 20 26 30 34 45 55 61Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль60.070.080.090.0100.030 34 45 55 61Surviving nematodes , Lifespan, days10 50 200 Контроль100 a c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 100 Контрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L controlSurviving nematodes , %0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольSurviving nematodes , %a c 0.010.020,030.040.050.00 3 8 13 17 20 26 30 34 45 55 61nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 100 Контрольe f0.010.020.030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 Surviving nematodes, %Lifespan, daysa b0.010.020,030.040.050.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,00 3 8 13 17 20 26 30 34 45 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 Surviving nematodes , %e f0 3 8 13 17 20 26 30 34 45 55 61Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0.010.020.030.040.050.060.070.080.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, Lifespan, days10 мкмоль/50 мкмоль/200 Контроль100 a 20,090.0100.026 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %10 мкмоль/50 мкмоль/200 100 Контрольa 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %10 мкмоль/50 мкмоль/200 100 Контрольa 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,0Surviving nematodes, %10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контрольa 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,0Surviving nematodes, %10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольa 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L controlSurviving nematodes , %0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольSurviving nematodes , %347Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352Figure 1g shows the effect of biochanin A isolatedfrom red clover callus culture. As we can see, thebest survival rate was provided by this substanceat 100 μmol/L. Unlike the other concentrations, thisconcentration had a positive effect on C. elegansthroughout the entire experiment. Days 8–20 saw thehighest survival rates, with the greatest increase inlifespan occurring on day 13 (by 45.2% compared tothe control). Noteworthily, 200 μmol/L of biochaninA had a negative effect on the survival and lifespanof C. elegans almost throughout the experiment,except for the very end. On day 61, the proportion ofsurviving nematodes increased by 1.8% and amountedto 2.5% (compared to 0.7% in the control group). Theconcentrations of 10 and 50 μmol/L increased theon the lifespan of C. elegans (Fig. 1f). As can be seen,100 μmol/L of this substance increased the survival ofnematodes throughout the experiment, with the greatestincrease (by 40.1%) on day 8. The other concentrationsshowed varying survival rates. Days 8–13 saw greaterlifespans in the nematodes exposed to chlorogenicacid at all four concentrations. During days 13–26,increased lifespan was provided by the concentrationsof 10, 50, and 100 μmol/L. The maximum survivalrate was observed on day 20 (27.7%) in the nematodestreated with 100 μmol/L of chlorogenic acid. From day26 to day 34, the concentration of 200 μmol/L had noeffect on the lifespan of C. elegans. However, from day45 to the end of the experiment, chlorogenic acid had apositive effect again at all its concentrations.Figure 1 Ending. Effects of bioactive substances isolated from the extracts of suspension, callus, and root cultures ofmedicinal plants on the lifespan of Caenorhabditis elegans nematodes: (a) rutin from the suspension culture extract ofmeadowsweet; (b) quercetin from the suspension culture extract of ginkgo; (c) kaempferol from the suspension culture extract ofginkgo; (d) baicalin from the root culture extract of Baikal skullcap; (e) trans-cinnamic acid from the root culture extract of Baikalskullcap; (f) chlorogenic acid from the callus culture extract of red clover; (g) biochanin A from the callus culture extract redclover; (h) naringenin from the callus culture extract of alfalfa; (i) ursolic acid from the callus culture extract of thyme;(j) magniferin from the root culture extract of sweetvetchg hi j0.010.00 3 Surviving nematodes, a c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L controlSurviving nematodes , %0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольSurviving nematodes , %a c 20,030.040.050.060.070.080.090.0100.08 13 17 20 26 30 34 45 55 61%Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 100 Контрольg h20.010 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 Surviving nematodes, %Lifespan, daysg h0 3 8 13 17 20 26 30 34 45 55 61Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0.010.020.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, Lifespan, days10 50 200 100 Контрольa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 Surviving nematodes , % Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, daysi jFigure 1 Effects of bioactive substances isolated from the extracts of suspension, callus, and root cultures of medicinal plants on th e nematodes: a – rutin from the suspension culture extract of meadowsweet; b – quercetin from the suspension culture extract of ginkgo; c – kaempferol extract of ginkgo; d – baicalin from the root culture extract of Baikal skullcap; e – trans-cinnamic acid from the root culture extract of Baikal callus culture extract of red clover; g – biochanin A from the callus culture extract red clover; h – naringenin from the callus culture extract of culture extract of thyme; j – magniferin from the root culture extract of sweetvetch0.010.020.030.040.050.060.0Surviving nematodes, %Lifespan, days10 мкмоль/л 200 мкмоль/л 100 мкмоль/л Контроль0.010.020.030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 Surviving nematodes, %Lifespan, daysa bc d0.020,070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control10,020,030,040,050,060,070,080,090,0100,017 20 26 30 34 45 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 Surviving nematodes , %Lifespan, daysi j1 Effects of bioactive substances isolated from the extracts of suspension, callus, and root cultures of medicinal plants on th e lifespan of Caenorhabditis nematodes: a – rutin from the suspension culture extract of meadowsweet; b – quercetin from the suspension culture extract of ginkgo; c – kaempferol from the suspension ginkgo; d – baicalin from the root culture extract of Baikal skullcap; e – trans-cinnamic acid from the root culture extract of Baikal skullcap; f – chlorogenic culture extract of red clover; g – biochanin A from the callus culture extract red clover; h – naringenin from the callus culture extract of alfalfa; i – ursolic acid culture extract of thyme; j – magniferin from the root culture extract of sweetvetch0 3 8 13 17 20 26 30 34 45 55 61Lifespan, days10 мкмоль/л 200 мкмоль/л 100 мкмоль/л Контроль0.010.020.030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %10 50 200 100 Контрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 100 Контрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 мкмоль/100 мкмоль/Контрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L controlSurviving nematodes , %0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтрольSurviving nematodes , %a c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/50 мкмоль/200 100 Контроль0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 Surviving nematodes , %Lifespan, daysa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 Surviving nematodes , %Lifespan, daysa c 0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 Surviving nematodes , %Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes , %Lifespan, daysa bc d0.010.020,030.040.050.060.070.080.090.0100.00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 μmol/L 50 μmol/L 200 μmol/L100 μmol/L control0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 Surviving nematodes , %Lifespan, days0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 45 55 61Surviving nematodes, %Lifespan, days10 мкмоль/л50 мкмоль/л200 мкмоль/л100 мкмоль/лКонтроль0,010,020,030,040,050,060,070,080,090,0100,00 3 8 13 17 20 26 30 34 Surviving nematodes , %Lifespan, days348Faskhutdinova E.R. et al. Foods and Raw Materials. 2022;10(2):340–352survival of nematodes during days 13–45 by an averageof 8.4 and 9.1%, respectively. Both concentrationsprovided maximum lifespan increases during days13–20 and had a weaker effect towards the end of theexperiment. From day 26 to day 61, biochanin A at 10,50, and 200 μmol/L had no significant effect on thelifespan of nematodes compared to the control group.Naringenin was isolated from the extract of alfalfacallus culture. As shown by Fig. 1h, its concentrationof 100 μmol/L had the greatest effect on the lifespanof nematodes compared to the other concentrations,especially during days 8–26. During that period,the survival of nematodes increased by an averageof 27.3% compared to the control group, withmaximum survival on day 13 (by 35.4%). The otherconcentrations of naringenin (10, 50, and 200 μmol/L)did not have a significant effect on the survival orlifespan of nematodes.Ursolic acid was isolated from thyme callusculture. According to Fig. 1i, it had no significanteffect on the lifespan of nematodes at all itsconcentrations. The greatest increase in survival (by14.1%) was observed on day 8 in the nematodes treatedwith 100 μmol/L of ursolic acid.Similarly, we found no positive effect in magniferinobtained from the root culture extract of sweetvetch (Fig.1j). Moreover, its concentrations of 100 and 200 μmol/Lreduced the proportion of surviving nematodes fromday 13 to day 55 of the experiment. The greatestincreases in the lifespan of nematodes were observedat magniferin concentrations of 10 and 50 μmol/L onday 8, amounting to 19.3 and 24.2%, respectively. Atthe end of the experiment, the longest lifespan wasdemonstrated by the nematodes exposed to 100 μmol/Lof magniferin (1.4%).CONCLUSIONHaving applied HPLC methods, we isolated thefollowing bioactive substances from the extracts ofcallus, suspension, and root cultures of medicinal plantsgrowing in the Siberian Federal Okrug: rutin – fromthe callus culture extract of meadowsweet (Filipendulaulmaria L.); quercetin – from the suspension cultureextract of ginkgo (Ginkgo biloba L.); kaempferol – fromthe suspension culture extract of ginkgo (G. biloba);baicalin – from the root culture extract of Baikalskullcap (Scutellaria baicalensis L.); trans-cinnamicacid – from the root culture extract of Baikal skullcap(S. baicalensis); chlorogenic acid – from the callusculture extract of red clover (Trifolium pretense L.);biochanin A – from the callus culture extract of redclover (T. pratense); naringenin – from the callusculture extract of alfalfa (Medicágo sativa L.); ursolicacid – from the callus culture extract of thyme (Thymusvulgaris L.); and magniferin – from the root cultureextract of sweetvetch (Hedysarum neglectum L.). Allthe bioactive substances were at least 95% pure andwere registered using IR spectroscopy on an SF-2000instrument (OKB Spektr, Russia).We determined the effect of the above bioactivesubstances at concentrations of 10, 50, 100, and200 μmol/L on the lifespan of Caenorhabditis elegansnematodes, which are widely used as model organismsto study the aging process. We used 96-well plates forthe experiment that lasted 61 days. Surviving nematodeswere counted every 4–7 days and the experimentwas considered completed when there were no livenematodes left in the control group. Stock solutions ofthe following bioactive substances were prepared forthe experiment: rutin, quercetin, kaempferol, baicalin,trans-cinnamic acid, chlorogenic acid, biochanin A,naringenin, ursolic acid, and magniferin.Trans-cinnamic acid, baicalin, rutin, ursolic acid, andmagniferin did not significantly increase the lifespan of thenematodes.Chlorogenic acid and naringenin had a littleeffect on the lifespan of nematodes, while quercetin,kaempferol, and biochanin A demonstrated their highsurvival.Noteworthily, the greatest proportions of survivingnematodes treated with various concentrations ofbioactive substances were recorded on days 8 to 13for all the experimental samples. Then, the lifespan ofC. elegans decreased and their survival ratesapproached those of the control group.Thus, 200 μmol/L of kaempferol, 10 μmol/L ofquercetin (both obtained from ginkgo suspensionculture extract), and 100 μmol/L of biochanin A(obtained from red clover callus culture extract)increased the lifespan of C. elegans nematodes by30.6, 41.9, and 45.2%, respectively, compared to thecontrol (days 8 and 13). These results suggest thatthe mentioned bioactive substances can be effectivelyused as anti-aging agents.CONTRIBUTIONAll the authors are equally responsible for the researchresults and the manuscript.CONFLICT OF INTERESTThe authors declare no conflict of interest.