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Natural deep eutectic solvents (NADES) are a new alternative to toxic organic solvents. Their constituents are pri- mary metabolites, non-toxic, biocompatible and sustainable. In this study four selected NADES were applied for the extraction of two medicinal plants: Sideritis scardica, and Plantago major as an alternative to water-alcohol mixtures, and the antimicrobial and genotoxic potential of the extracts were studied. The extraction efficiency was evaluated by measuring the extracted total phenolics, and total flavonoids. Best extraction results for total phenolics for the studied plants were obtained with choline chloride-glucose 5:2 plus 30% water; but surprisingly these extracts were inactive against all tested microorganisms. Extracts with citric acid-1,2-propanediol 1:4 and choline chloride-glycerol 1:2 showed good activity against S. pyogenes, E. coli, S. aureus, and C. albicans. Low genotoxicity and cytotoxicity were observed for all four NADES and the extracts with antimicrobial activity. Our results confirm the potential of NADESs for extraction of bioactive constituents of medicinal plants and further suggest that NADES can improve the effects of bioactive extracts. Further studies are needed to clarify the influence of the studied NADES on the bioactivity of dis- solved substances, and the possibility to use such extracts in the pharmaceutical and food industry. Keywords: Green extraction, Natural deep eutectic solvents, Sideritis scardica, Plantago major, Antimicrobial activity, Genotoxicity Introduction components of the mixture and are liquid at ambient One of the most important aims of green chemistry temperature. In the case of NADES, the constituents of has been to find green solvents for extraction of bioac - the eutectic mixture are natural compounds: primary tive compounds from natural sources in order to replace metabolites, which are easily available, non-toxic, bio- the currently used hazardous organic solvents. One of compatible and sustainable . NADES have low vapor the eco-friendly alternatives are deep eutectic solvents pressure, an advantage with respect to environmental and (DES) and particularly the natural deep eutectic solvents human health protection. This property, however, poses a (NADES). DES are mixtures of organic compounds that serious problem to the recovery of the active ingredients have melting points lower than those of the individual from the extract. Recently, several studies have demon- strated that NADESs retained or even improved the bio- logical activity of dissolved substances [2, 3]. Therefore, *Correspondence: firstname.lastname@example.org the NADES could function as an active ingredient, and Institute of Organic Chemistry With Centre of Phytochemistry, Bulgarian the extract could be directly used as part of cosmetic or Academy of Sciences, Acad. G. Bonchev Str., Bl. 9, 1113 Sofia, Bulgaria Full list of author information is available at the end of the article pharmaceutical formulations, bypassing the difficulties of © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Grozdanova et al. BMC Chemistry (2020) 14:73 Page 2 of 9 solute recovery. The aim of the present study was to apply absorption spectra were recorded. The λ was used to max selected NADES for extraction of two popular Bulgar- calculate the molar transition energy ENR, based on the ian medicinal plants: the mountain tea Sideritis scardica equation: ENR = hc /λ = 28,591/λ , (ENR in kcal/ NA max max Griesb., and the broadleaf plantain Plantago major L. as mol, λ in nm). max an alternative to water–alcohol mixtures, and to evaluate the antimicrobial, cytotoxic and genotoxic potential of Density measurements both NADES solvents and extracts. The NADESs density was determined as follows: 2 ml of NADES were put in a volumetric flask at 20 °C and the Materials and methods weight of the liquid was measured. The density was cal - Chemicals and reagents culated using the formula: ρ = m /V , where ρ is NADES NADES Ethanol (absolute) was obtained from Alkaloid (Skopje, density, g/ml at 20 °C, m − weight, g at 20 °C and NADES Macedonia). Glycerol and 1,2-propanediol were pur- V − volume in ml at 20 °C (2 ml). For each solvent NADES chased from Valerus (Sofia, Bulgaria); choline chloride the procedure was performed in duplicate. and Nile Red from Sigma Aldrich (Switzerland), cit- ric acid and glucose from Chem-Lab NV (Zedelgem, Extraction Belgium) and Fisher Chemical (Loughborough, UK), Air-dried plant material was ground using a coffee mill, respectively. The chemicals used in the in vitro cytotox - the average particle size was 0.75 mm. The extraction was icity assay were purchased from Sigma Life Science, performed in a 2 ml Eppendorf tube with 50 mg of plant (Steinheim, Germany): 3-(4,5-dimethylthiazolyl-2)-2,5- material and 1.5 ml solvent in an ultrasound bath (Elma- diphenyltetrazolium bromide (MTT, #M2128-1G), sonic S 30 H), without heating, for 1 h. The mixture was ethylenediaminetetraacetic acid (EDTA, #E6635), then centrifuged at 13,000 rpm for 40 min and filtered l-glutamin (#G7513) and Dulbecco’s phosphate buff - through cotton in a 1 ml volumetric flask. This extract ered saline (PBS, #D8537). Media, enzymes and sera was further used for antimicrobial tests, and analyzed to for cultivation CCL-1 cells originated from Capricorn , determine the main groups of bioactive compounds in Germany: MEM (#MEM-A), horse serum (#HOS-1A), the extracts. Each extraction procedure was performed in Pen/Strep 100x(#PS-B), Trypsin (#TRY-1B10, #TRY- triplicate. 2B10). HCOOH was delivered from Chimspektar OOD (Bulgaria). Quantitative determination of total phenolics and total flavonoids Plant material For measuring those two groups of bioactive compounds, Aerial parts of the studied plants were used. Plantago previously reported spectrophotometric methods were major was collected in March 2018 in the valley of River used . For blank: solution of respective NADES instead Struma (: 41°56′46.24″ N 23°5′54.23″ E, 212 m a.s.l.), of the test sample was used in analogous procedures. a voucher specimen (No. SOM 1390) has been depos- Total phenolics content was estimated using caffeic acid ited in the Herbarium of the Institute of Biodiversity as standard, and total flavonoid content with rutine as and Ecosystem Research, Bulgarian Academy of Sci- standard. Every assay was performed in triplicate. ences (IBER-BAS). Sideritis scardica collected in June 2017, was cultivated in the Western Rhodope Mountain Antimicrobial activity (41°57′36.34″ N 23°39′19.32″ E, 1202 m a.s.l.), a voucher Test microorganisms specimen has been deposited in the Herbarium of the For antimicrobial activities of extracts and solvents, IBER-BAS, No. SOM 1391. Plant material was collected the following test-microorganisms were used: Bacillus and identified by Assoc. Prof. Dr. Ina Aneva. cereus ATCC 9634 (American Type Cell Culture Collec- tion, USA), Escherichia coli ATCC 35218, Staphylococcus Preparation of NADES aureus ATCC 29213, Pseudomonas aeruginosa ATCC The NADES were prepared by mixing the components 27853, Listeria monocytogenes C12, Salmonella typhimu- and subsequently stirring in water bath (300 rpm) com- rium 123, Streptococcus pyogenes 10535, Yersinia entero- bined with mild heating at 50 °C until a homogeneous colitica 864 O:3 and the fungus Candida albicans 562 liquid was formed . from the SAIM-BAS collection. Polarity measurements Culture medium and growth conditions The NADESs polarity was measured by the solvatochro - Sterilized Brain Heart Infusion Broth and Agar (BHIB, mic dye Nile red . The dye was dissolved in each GM210, resp. BHIA, M1611, HiMedia, India) were used NADES in the concentration range 0.01–0.1 mM and as the cultivation media for all bacteria excepting S. Gr ozdanova et al. BMC Chemistry (2020) 14:73 Page 3 of 9 thyphimurium, S. aureus and E. coli growing on Muller flasks and controlled environment (incubator Pana - Hinton Agar and Broth (MHA, CM0337B, resp. MHB, sonic MCO-18AC, Japan) at 37 °C, 5% C O and approx. CM0405B, Thermo Scientific-Oxoid, UK), and C. albi - 95% humidity. The cultivation medium MEM was sup - cans SAIMC 562—on Sabouraud-Glucose agar supple- plemented with 2 mM l-glutamine, 10% heat-inacti - mented with gentamicin (40 μg/ml) (CM0041, Oxoid, vated horse serum, 10 Units/l penicillin G sodium and Basingstoke, UK). All microorganisms were grown at 100 mg/l streptomycin sulphate. Cells were sub-culti- 4 2 37 °C overnight except B. cereus, which was cultivated at vated at a seeding density of 1 × 10 cells/cm 1–3 times 30 °C and Y. enterocolitica—at 26 °C. All microbiological per week after reaching 80–90% confluence. Sequen - procedures were performed under sterile conditions into tially, applied solutions of 0.05% EDTA in PBS (1–2 ml, a Class II laminar box (FASTER BH-EN 2003, Ferarra, 5–10 min) and 0.25% (w/v)/0.53 mM trypsin/EDTA Italy). (1–2 ml, 5–10 min) were used for the detachment of the cell monolayer and cell separation. Minimal inhibitory (MIC) and bactericidal (MBC) concentrations MTT test—calculation of IC and statistics The antimicrobial activity was studied by the broth The MTT test was conducted according to Annex C, 5 −1 microdilution method according to ISO 20776-1:2006. ISO 10993-5 [7, 8]. Cells with a density of 1 × 10 ml Briefly, bacterial and fungal inoculums with concentra - were seeded in 96-well plates (flat bottom, 100 µl/well). tion 105 CFU/ml were added to 96-well plates contain- For cells to start exponential growth (log phase), plates ing BHIB or MHB loaded with twofold serial dilutions of were incubated for 24 h. After entering the log phase cells pure solvents or extracts differing in the concentration of were exposed to NADES and their extracts at concentra- total phenolics. Pure solvents were applied in an equiva- tions ranging between 2 and 0.004% volume fraction for lent concentration as for testing the antimicrobial activ- 24 and 72 h. PBS was used as a solvent and 4 wells were ity. Plates were incubated overnight at 37 °C, excepting used for each treatment. MTT (0.5 mg/ml final concen - the plates with B. cereus and Y. enterocolitica, which were tration) was added to each well, followed by a 2-h incuba- incubated at 30 °C, respectively 26 °C. Gentamicin, peni- tion at 37 °C. The medium above the cells was removed cillin and tetracycline were used as reference antibiotics and 100 μl/well 2-propanol supplemented with 5% formic for bacteria and amphotericin B—for C. albicans, follow- acid were used to dissolve the formed formazan crys- ing the requirements of EUCAST. Experiments were per- tals and as a blank solution. Absorption was measured formed in triplicate. MICs and MBCs were determined at 540 nm (reference filter 690) on a microplate reader as described before . ELx800 (BioTek Instruments, Inc., United States). The IC values (inhibitory concentration 50 which reduces Dehydrogenase (DEHA) activity vital cells by half) were calculated with a non-linear The DEHA activity of the test microorganisms was regression analysis (inhibition dose–response model, assessed by MTT-test (3-(4,5-dimethylthiazolyl-2)-2,5- variable slope) using the GraphPad Prizm software. diphenyltetrazolium bromide, M2128-1G, Sigma- Untreated cells were considered as negative control and Aldrich). The method is based on the reduction of the normalized for 100%. MTT dye by the membrane located bacterial enzyme NADH: ubiquinone reductase (H+-translocation) to Genotoxic activity insoluble formazan crystals. Briefly, the treated and The method of neutral Comet Assay untreated bacterial, respectively fungal cells, were incu- The method of Comet Assay was performed under neu - bated for 2 h with MTT dye in a final concentration of tral conditions. CCL1 cells—control and treated with 0.05 mg/ml. An equivalent volume of 5% HCOOH in iso- increasing concentrations of the tested NADES solvents propanol dissolved the formed crystals. Absorption was and extracts for 24 h were mixed with 1.4% low-melt- measured using ELISA reader (BioTek Elx800, USA) at ing agarose and spread onto already pre-coated with 550 nm (reference 690 nm) against a blank solution. 0.5% normal agarose microscopic slides. The microgels, covered with coverslips to assure equal distribution, In vitro cytotoxicity were incubated at 4 °C for 10 min. The coverslips were Cell line and culture conditions removed after solidification of the microgels. This was The cell line CCL-1 (mouse fibroblasts, NCTC clone followed by a 20-min incubation in a lysis buffer (146 mM 929, ATCC—American Type Culture Collection, Manas- NaCl, 30 mM EDTA, pH 8; 10 mM Tris–HCl, pH 8; 0.1% sas, Virginia, USA) recommended in Annex C of ISO N-lauroyl sarcosine). Incubation of the gels 3 × 10 min in 10993-5 (ISO 10993-5:2009 2017) for evaluation of 0.5×TBE buffer followed. Slides were electrophoresed in in vitro cytotoxicity was cultured in sterile cell culture 0.5×TBE buffer at 0.45 V/cm for 20 min. The slides were Grozdanova et al. BMC Chemistry (2020) 14:73 Page 4 of 9 then dehydrated subsequently in 75% and 96% ethanol and data of their density and polarity measured with and left to fully dry at room temperature. The results the solvatochromic dye Nile red (lower E values mean NR were visualized under an epifluorescent microscope higher polarity, . Leitz—Orthoplan, Vario Orthomat 2 (450/490 nm), after The dry aerial parts of both selected plants were staining with SYBR Green (Roche Diagnostics GmbH). extracted with the four NADES and 70% ethanol as a Treatment of CCL1 cells with 5 mM H O for 30 min at reference solvent. Ultrasound assisted extraction was 2 2 37 °C was performed as a positive control for genotoxic- applied to accelerate the process, because of the sig- ity. Results were analyzed by using the CometScore soft- nificant viscosity of the NADES. The NADES choline ware. Three repetitions of the experiment have been done chloride-glucose 5:2 was too viscous and to enable mass and data were evaluated using Excel 2016 software where transfer, water was added to make it suitable for extrac- values for the measured Olive Moment in all probes were tion. It is known that addition of water decreases the presented as MEAN values ± STDEV. viscosity of NADES and weakens the hydrogen bonding interaction between its components, but dilution under FACS analysis for probing the cytostatic activity of the tested 50% usually does not lead to a matrix disruption into solvents and extracts individual components . CCL1 cells treated for 24 h with the NADES solvents and The extraction yield was evaluated by measuring the extracts were fixed in 100% ice-cold ethanol and stored total phenolics and total flavonoids in the extracts. at − 20 °C overnight. The cell pellets were washed twice Extraction with 70% ethanol under the same conditions with 1xPBS (2.68 mM KCl, 1.47 mM K H PO , 1.37 mM was used as a reference. The results are presented in 2 4 NaCl, 8 mM Na HPO ), pH 7. Incubation with RNase A Table 2. 2 4 (100 µg/ml) for 30 min at 37 °C followed. Before FACS The results demonstrated that some of the NADES analysis the cells were stained with propidium iodide extracted more bioactive compounds than the classic (50 µg/ml) for 20 min at dark. FACS analysis was per- water-alcohol mixture. The water-containing NADES formed by a BD FACSCanto apparatus and the results XXGlH was the most effective one, it extracted more were analyzed by FlowJo software V10. phenolics and more flavonoids from the mountain tea that 70% ethanol. In the case of plantain phenolics Results and discussion XXGlH was again the most effective solvent: it extracted Extraction and evaluation of extraction efficiency 25% more phenolics, compared to the reference solvent. Four NADES were selected based on literature data of However, none of the four NADES extracted flavonoids their polarity, close or higher than 70% ethanol. They are from plantain more efficiently than the 70% ethanol. This described in Table 1, with corresponding abbreviations, difference could be explained considering the specific Table 1 Composition and characteristics of the NADES Abbreviation Component 1 Component 2 Molar ratio Water (%) E (kcal/mol) Density (g/ml) NR XXGly Choline chloride Glycerol 1:2 0 50.03 1.17 CAPD Citric acid 1,2-Propanediol 1:4 0 48.87 1.19 XXGlH Choline chloride Glucose 5:2 30 49.17 1.16 XXPD Choline chloride 1,2-Propanediol 1:3 0 50.69 1.06 EtOH 70% 50.87 0.88 Table 2 Amount of extracted plant constituents (percentage of dry plant material) Extraction solvent S. scardica P. major Total phenolics, % Total flavonoids, % Total phenolics, % Total flavonoids, % 70% EtOH 5.4 ± 0.2 1.14 ± 0.02 5.55 ± 0.03 1.11 ± 0.06 XXGly 4.6 ± 0.3 1.02 ± 0.03 3.27 ± 0.2 0.51 ± 0.06 CAPD 4.2 ± 0.1 0.87 ± 0.03 3.06 ± 0.03 0.69 ± 0.06 XXGlH 6.3 ± 0.2 1.20 ± 0.06 6.99 ± 0.5 0.22 ± 0.06 XXPD 5.6 ± 0.2 1.20 ± 0.06 4.62 ± 0.2 0.90 ± 0 Gr ozdanova et al. BMC Chemistry (2020) 14:73 Page 5 of 9 chemical structures of flavonoids in the two medicinal metabolic activity of the microorganisms after the treat- plants. In S. scardica, flavonoid diglycosides predominate ment was studied by measuring the dehydrogenase activ- , while the major flavonoids in P. major are flavone ity (DEHA) with the MTT test. The results of these tests monoglycosides , which are less polar than diglyco- are shown in Table 3. sides. As the chosen NADESs have polarities higher than The most potent antimicrobial activity was observed 70% ethanol, the better extraction of more polar flavo - for CAPD/S against S. pyogenes, E. coli, S. aureus and C. noid diglycosides can be explained by this fact, at least to albicans with MICs between 0.098 and 0.39 µg/ml total some extent. phenolics. CAPD/P was active against S. aureus, E. coli and C. albicans (MICs were in the range 1.99–3.98 µg/ml Antimicrobial activity total phenolics). The plantain extracts had MBC = MIC Recently, it became clear that NADES components can for the three test microorganisms against which they be selected not only to fine-tune solvent physicochemi - were active. In this case the DEHA activity cannot be cal characteristics but also to improve the biological determined because it is equal to zero, as there are no activity of dissolved active compounds [12, 13]. That is metabolically active cells present. The extracts XXPD/S, why we decided to check the antimicrobial activity of XXPD/P, XXGlH/S, and XXGlH/P were inactive against the extracts, and to compare the results with the activ- the tested microorganisms. Surprisingly, the extracts ity of ethanol extracts, based on total phenolics concen- with the NADES which was most effective in the extrac - tration. The minimum inhibitory concentrations (MIC) tion of phenolics and flavonoids, XXGlH, showed no and minimum bactericidal concentrations (MBC) of total antimicrobial activity. This could be due to qualitative phenolics in the extracts were measured. In addition, the differences between the extracts obtained with different Table 3 MIC, MBC and dehydrogenase activity (MTT test) of total phenolics in different solvent extracts of S. scardica and P. major a b a b Sample/indicator XXGly/S XXGly/P CAPD/S CAPD/P 70% EtOH/S 70% EtOH/P S. pyogenes SAIM 10535 MIC (µg/ml) 380 NA 0.19 NA > 67.5 > 69.3 DEHA (%) – NA 39.88 ± 9.6 NA – – MBC (µg/ml) 380 NA 1.56 NA > 67.5 > 69.3 E. coli ATCC 35218 MIC (µg/ml) 47.5 38.75 0.39 3.984 > 67.5 > 69.3 DEHA (%) – – 1.31 ± 0.87 – – – MBC (µg/ml) 47.5 38.75 0.78 3.984 > 67.5 > 69.3 S. aureus ATCC 29213 MIC (µg/ml) NA NA 0.098 1.99 > 67.5 > 69.3 DEHA (%) NA NA 9.8 ± 2.3 – – – MBC (µg/ml) NA NA 0.39 1.99 > 67.5 > 69.3 P. aeruginosa ATCC 27853 MIC (µg/ml) 23.75 19.37 NA NA > 67.5 > 69.3 DEHA (%) – – NA NA – – MBC (µg/ml) 23.75 19.37 NA NA > 67.5 > 69.3 C. albicans SAIM 562 MIC (µg/ml) 23.75 19.37 0.19 1.99 > 67.5 > 69.3 DEHA (%) – – 7.595 ± 2.1 – – – MBC (µg/ml) 23.75 19.37 0.78 1.99 > 67.5 > 69.3 B. cereus ATCC 9634 MIC (µg/ml) 23.75 38.75 NA NA > 67.5 > 69.3 DEHA (%) 50 ± 12.3 – NA NA – – MBC (µg/ml) 47.5 38.75 NA NA > 67.5 > 69.3 Extracts with XXPD and XXGlH are inactive against all tested microorganisms Extract of S. scardica Extract of P. major NA: no activity. DEHA data are expressed as the mean ± SD of three measurements Grozdanova et al. BMC Chemistry (2020) 14:73 Page 6 of 9 solvents, however, qualitative analysis of the extracts 24 h exposure time were used for performing the geno- was not performed. The present work aimed to find the toxicity assay. The calculated IC concentrations of all most effective NADES for extraction of S. scardica and tested samples are given in Table 4. P. major in terms of antimicrobial potential. Optimiza- As visible from the median inhibitory concentrations tion of the extraction conditions with the most effective in Table 4, the solvent XXGly was less cytotoxic than solvent is the subject of future work, when we intend to CAPD. The same trend was observed for the relevant apply more detailed analysis of individual constituents. extracts. Interestingly, the cytotoxicity of the solvents In most cases where antimicrobial activity was XXGly, CAPD and XXGIH diminished with time and was observed, it was much higher than the one of the tradi- less pronounced after 72 h of exposure than after 24 h. tional ethanol extracts. Such antimicrobial potentiat- All extracts showed dose and time dependent cytotoxic- ing effects of NADESs was also observed in the case of ity. The XXGly/S extract exhibited more potent antipro - propolis NADES extracts . Although the addition of liferative effect on mouse fibroblasts than XXGly/P and more than 50% of water to NADES during the antimicro- was more cytotoxic to the cells than the solvent itself bial tests breaks the NADES supramolecular complex, in both exposure periods. The cytotoxicity of XXGly/P the individual NADES constituents could contribute to was more pronounced than that of the solvent after the the overall effect of the solution  and some synergis - longer exposure period (72 h). CAPD/S showed a two- tic effects are also to be considered. Especially in the case fold higher antiproliferative effect on the cells after 24 h of the CAPD extract of S. scardica, the increased effect of exposure than CAPD. However, after 72 h the effect compared to the other extracts can be partly explained of CAPD/S diminished slightly compared to that of the by the effect of the presence of citric acid as an element solvent at the first incubation period. The cytotoxicity of of the NADES. In the literature there are indications that the CAPD/P extract was time-dependent and the IC NADES containing organic acids possess higher antimi- values were significantly lower than those of the pure sol - crobial activity, since some organic acids present many vent, especially after long exposure time. Considering the pharmacological effects . MIC values determined by the BMD assay, it can be seen that CAPD/P exhibited anti-staphylococcal and anti- Genotoxicity and cytotoxicity fungal activities at concentrations 3.9 and 1.99 µg/ml tP, Any further implication of the tested four NADES in respectively, that are significantly lower than the median the practice requires proofs for their genotoxicity safety. inhibitory concentration cytotoxic for normal mouse Many data in the literature show the genotoxicity of a fibroblasts (IC /24 h = 4.6 µg/ml tP). broad range of food, cosmetic and pharmaceutical addi- After estimation of the cytotoxicity of the studied tives regardless of their nature [17–19]. Some data point NADES solvents and extracts, CCL1 cells were sub- to the fact that these substances added to the products jected to Comet assay. CCL1 cells were treated with the consumed by or applied on people exert their genotox- four tested NADES and extracts of S. scardica and P. icity at allowed concentrations [17, 20, 21]. This unam - major with XXGly and CAPD, for 24 h at optimal con- biguously entails the application of sensitive tests for fast ditions. Both the pure NADES and the NADES extracts and accurate evaluation of the potential of any substance were applied to the monolayer cells at concentrations of that is planned to be implicated in food, pharmaceuti- IC , ½IC and ¼IC , estimated by MTT tests (Table 4). 50 50 50 cal and cosmetic practices to induce general cytotoxicity Untreated cells were used as negative control while CCL1 via inhibiting cell proliferation and/or damage in DNA cells treated for 30 min at 37 °C with 5 mM H O were 2 2 [22–24]. The method of Comet Assay is a brilliant tech - used as a positive control for genotoxicity. nology for fast and sensitive analysis of genotoxicity [25, The Comet Assay results were quantified with the 26]; it requires single cells, is fast and with high preci- CometScore software and the values for the Olive sion evaluates all kinds of DNA damages. Data quantifi - Moment are presented in Fig. 1. Results demonstrated cation allows precise estimation of genotoxicity. In this that all NADESs at a concentration of I C showed mod- study, the tested NADES solvents and the extracts which erate to subtle genotoxicity effect except XXGly that at a demonstrated antimicrobial activity: the extracts with concentration of I C demonstrated the highest genotoxic XXGly and CAPD of S. scardica and P. major, were tested potential in comparison to all other solvents applied at for their genotoxicity. The preparation for this test first IC . Dilution two and four times of the applied I C of all 50 50 required the evaluation of the in vitro cytotoxicity of the solvents showed lack of genotoxic potential on the tested tested substances. Therefore, the in vitro cytotoxicity of CCL1 cells. The less genotoxic were all tested concentra - the NADESs and extracts was determined on the normal tions of CAPD (Fig. 1a). Further, extracts of S. scardica mouse fibroblast cell line CCL-1 (Table 4) for two expo- and P. major with XXGly and CAPD were tested for gen- sure times—24 and 72 h. The IC values calculated after otoxicity with the method of Comet Assay and results are 50 Gr ozdanova et al. BMC Chemistry (2020) 14:73 Page 7 of 9 Table 4 Calculated IC values for the tested four NADES solvents and the extractss from S. scardica and P. major Tested samples Median inhibitory concentrations, parameters of the model and exposure time 24 h 72 h a b c IC (% v/v, μl)95% CI R IC (% v/v, μl) 95% CI R 50 50 Solvents XXGly 1.180 0.985–1.412 0.904 1.394 1.261–1.542 0.950 CAPD 0.089 0.075–0.105 0.940 0.141 0.126–0.158 0.971 XXGIH 0.077 0.043–0.139 0.980 0.096 0.085–0.108 0.964 XXPD 2.677 1.516–4.611 0.906 1.503 1.197–1.887 0.707 Extracts XXGly/S 0.803 0.563–1.148 0.952 0.589 0.391–0.886 0.901 XXGly/P 1.267 0.957–1.679 0.912 1.094 0.845–1.418 0.894 CAPD/S 0.046 0.040–0.054 0.955 0.148 0.136–0.161 0.971 CAPD/P 0.045 0.039–0.053 0.956 0.024 0.021–0.027 0.962 IC (µg/ml tP ) 95% CI R IC (µg/ml tP) 95% CI R 50 50 Extracts XXGly/S 12.212 8.550–17.450 0.952 8.953 5.941–13.490 0.901 XXGly/P 15.711 11.863–20.820 0.912 13.566 10.470–17.590 0.894 CAPD/S 0.046 0.040–0.054 0.955 0.148 0.136–0.161 0.971 CAPD/P 4.607 4.026–5.376 0.956 0.243 0.210–0.275 0.962 IC : inhibitory concentration that inhibits the cell growth by 50% CI: confidence interval R: coefficient of correlation tP: total phenolics shown in Fig. 1b. All extracts applied at concentration of at all tested concentrations, especially at ¼IC showed IC demonstrated genotoxicity on CCL1 cells, especially fewer cells in G0/G1. These results correspond with the CAPD/S. Specifically, XXGly/S and XXGly/P, CAPD/S detected genotoxicity in the samples. The detected gen - and CAPD/P showed a decrease in the detected genotox- otoxicity led to moderate cytostatic effect on the CCL1 icity when applied at ½ and ¼ of IC . The most harmless cells. was CAPD/P. All tested NADES proved harmless genome integrity of These NADES probes demonstrated a lack of geno - the tested CCL1 cells. The detected little changes in the toxicity at all tested concentrations. Conserning the percentage of cells in different phases of the cell cycle can detected genotoxicity the NADES solvents can be clas- be due to cell culture asynchronization. sified in a row showing an increase in genotoxicity with an increase in the tested concentrations of the com- Conclusions pounds. The NADES solvents and extracts are listed in In conclusion, our results confirm the promising poten - the direction left to right, which marks increased geno- tial of NADESs as solvents for extraction of the biologi- toxicity. The NADES solvents are arranged as follows: cally active constituents of popular medicinal plants and CAPD < XXGIH < XXPD < XXGly. The extracts can be confirm the suggestion that NADES can improve the classified like this: CAPD/P < XXGly/P < XXGly/S < CAP biological effects of bioactive extracts . Best extrac - D/S. tion results for total phenolics for the studied plants were Generally, all tested NADES revealed little changes in obtained using XXGlH, but surprisingly these extracts the distribution of cells in the different phases of the cell were inactive against all tested microorganisms and were cycle after treatment for 24 h with increasing concentra- not subjected to further studies of in vitro cytotoxic and tions. The closer look at the graph in Fig. 2a shows that genotoxic activity. The most effective were the extracts XXGIH at all tested concentrations, most explicitly at ½ with CAPD. The presence of citric acid and some syner - and ¼IC demonstrated decrease in the population of gistic effects with Sideritis constituents may play a role, cells in G0/G1, suggesting a slight cytostatic effect. CCL1 as CAPD extract of S. scardica was much more active cells treated with CAPD/S applied at I C , and CAPD/P compared to the respective P. major extract. 50 Grozdanova et al. BMC Chemistry (2020) 14:73 Page 8 of 9 Fig. 1 Genotoxicity of the tested NADES c solvents and extracts on Fig. 2 Cell cycle evaluation using FACS of CCL1 cells treated with the CCL1 cells. a Tail Olive Moment of CCL1 cells treated with the tested tested NADES solvents and extracts. a Distribution of cells through NADES. b Tail Olive Moment of CCL1 cells treated with the tested the cell cycle phases in CCL1 cells treated with NADES probed on NADES extracts CCL1 by FACS cells after 24 h of treatment. b Distribution of cells through the cell cycle phases in CCL1 cells treated with NADES extracts The use of NADESs allows avoiding organic solvents and the significant antimicrobial potential of the NADES extracts combined with the low toxicity and genotoxicity XXPD: Choline chloride-1,2-propanediol 1:3; /P: Extract of Plantago major; /S: Extract of Sideritis scardica. of the solvents and the extracts present a very promising perspective for using these extracts in food, cosmetic and Authors’ contributions pharmaceutical formulations. Of course, further studies VB conceived the study and was in charge of overall direction and planning, TG, BT, MP and KA performed chemical experiments: preparation of NADES, are needed to answer the question of the influence of the extractions, chemical analyses; LD and MMZ performed the antimicrobial NADES on the bioactivity of the dissolved substances, assays, measured the dehydrogenase activity and participated in the data and to elucidate the fine molecular mechanisms of their analysis, MMZ and YI performed cytotoxicity test, HN directed the design of microbiological tests and the analysis of the data; MG, GM, BV performed action in model cells and organisms. genotoxicity tests and data analysis; VN, HN and MG drafted the manuscript. All authors read and approved the final manuscript. Abbreviations Funding ATCC : American Type Cell Culture Collection; BHIB: Brain Heart Infusion Broth; This work was supported by the Bulgarian National Science Fund (Grant DH BHIA: Brain Heart Infusion Agar; CAPD: Citric acid-1,2-propanediol 1:4; DEHA: 19/4). Dehydrogenase activity; DES: Deep eutectic solvent(s); FACS: Fluorescence activated cell sorting; MBC: Minimum bactericidal concentration; MHA: Muller Availability of data and materials Hinton Agar; MHB: Muller Hinton Broth; MIC: Minimum inhibitory concentra- All materials used in the present study are mentioned in “Materials and meth- tion; MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ods” section and the data will be available upon request. NADES: Natural deep eutectic solvent(s); tP: Total phenolics; XXGlH: Choline chloride-glucose 5:2 and 30% water; XXGly: Choline chloride-glycerol 1:2; Gr ozdanova et al. 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Murador DC, de Souza Mesquita LM, Vannuchi N, Braga ARC, de Rosso VV (2019) Bioavailability and biological effects of bioactive compounds Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : fast, convenient online submission thorough peer review by experienced researchers in your ﬁeld rapid publication on acceptance support for research data, including large and complex data types • gold Open Access which fosters wider collaboration and increased citations maximum visibility for your research: over 100M website views per year At BMC, research is always in progress. Learn more biomedcentral.com/submissions
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