Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(1): 83-92, 2020 Firenze University Press www.fupress.com/caryologiaCaryologia International Journal of Cytology, Cytosystematics and Cytogenetics ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-116 Citation: N.A. Şuţan, A.N. Matei, E. Oprea, V. Tecuceanu, L.D. Tătaru, S.G. Moga, D.Ş. Manolescu, C.M. Topală (2020) Chemical composition, anti- oxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts. Caryologia 73(1): 83-92. doi: 10.13128/caryologia-116 Received: January 9, 2019 Accepted: February 22, 2020 Published: May 8, 2020 Copyright: © 2020 N.A. Şuţan, A.N. Matei, E. Oprea, V. Tecuceanu, L.D. Tătaru, S.G. Moga, D.Ş. Manolescu, C.M. Topală. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts Nicoleta Anca Şuţan1,*, Andreea Natalia Matei1, Eliza Oprea2, Victorița Tecuceanu3, Lavinia Diana Tătaru1, Sorin Georgian Moga1, Denisa Ştefania Manolescu1, Carmen Mihaela Topală1 1 University of Piteşti, Faculty of Sciences, Physical Education and Informatics, Depart- ment of Natural Sciences, 1 Targu din Vale Str., 110040 Pitesti, Romania 2 University of Bucharest, Faculty of Chemistry, Department of Organic Chemistry, Bio- chemistry and Catalysis, Blvd. Regina Elisabeta, No. 4-12, 030018, Bucharest, Romania 3 Organic Chemistry Centre of the Romanian Academy “Costin D. Nenitescu”, 202B Splaiul Independentei, 78100 Bucharest, Romania *Corresponding author. E-mail addresses: ancasutan@yahoo.com, mateiandreeanata- lia@gmail., eliza_oprea2003@yahoo.com, vichi_tecu@yahoo.com, lavinia.tataru@upit.ro, sorin.g.moga@gmail.com, stefaniamanolescu@yahoo.com, carmen.topala@gmail.com. Abstract. Through time, in the traditional medicine Ligularia genus has been used as a remedy to cure several diseases and affections. The paper represents an essential step in offering more information about the antioxidant activity, chemical composi- tion and cytogenetic activity of L. sibirica (L.) Cass. rhizomes and roots extracts. The antioxidant activity of the extracts has been achieved by analyzing the total phenolic content, total flavonoids, the organic chemical compound and trolox equivalent anti- oxidant capacity and their major polyphenolic constituents were quantified by liquid chromatography electrospray ionization-tandem mass spectrometry. The extracts were obtained by the Supercritical Fluid Extraction (SFE-CO2) technique, SFE-CO2 extrac- tion with co-solvent and absolute ethanol extraction. The best results for the antiox- idant activity have been fulfilled through the last two techniques. High Performance Liquid Cromatography (HPLC) has been applied in order to identify and quantify selected phenolic acids and flavonoids in the ethanolic extracts of L. sibirica (L.) Cass. The cytogenotoxic effects of the extracts completed the present study, with a further- ance of antiproliferative potential highlighted by the statmokinetic effect and an addi- tional genoprotective effect. Keywords. antioxidant activity, phenols, SFE extraction, HPLC, cytogenotoxic effects, Ligularia sibirica. Abbreviation: TP - total phenols, TF - total flavonoids, DPPH - the organic chemical compound 2,2-diphenyl-1-picrylhydrazyl, TEAC - Trolox Equivalent Antioxidant Capacity, FTIR - Fourier Transform Infrared spectroscopy, HPLC - High-performance liquid chromatography, SFE - Supercritical fluid extraction, MIR - middle infrared region, ATR - Attenuated total reflection, GAE - gal- lic acid equivalents, ABTS - 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), MI - mitotic index. 84 Nicoleta Anca Şuţan et al. INTRODUCTION The genus Ligularia from the Asteracae family has been extensively researched from phytochemical point of view especially in the last years, having a real signifi- cance in the traditional medicines as a remedy for bron- chitis, asthma, tuberculosis, haemoptysis, soothing pain, rheumatoid arthritis, coughs, inflammations, jaundice, scarlet fever and hepatic diseases (Pinglin et al. 2008; Xie et al. 2010). Ligularia genus has many biological activities such as antibacterial activity, antifeedant and insecticidal activities, antihepatotoxicity, antioxidant, antithrombotic and anticoagulant activity (Yang et al. 2011). The pharmaceutical studies as well as the chemi- cal studies on Ligularia species demonstrate the specific presence of constituents such aseremophilane-type ses- quiterpenes (Wu et al. 2016). Over the glacial and interglacial period, a large number of species migrated from the Asian continent to Europe, including the glacial relict Ligularia sibiri- ca (L.) Cass. which is the target of the present study. L. sibirica (L.) Cass., a perennial hemicryptophyte species, has a short and thick rhizome with long lateral fascicu- lated roots, leaves with long petioles, and inflorescence stem straight up to 200 cm. The blooming period starts from July till the end of August (Pop, 1960; Šmídová et al. 2011). An overall small number of studies have been pub- lished with regards to the chemical composition and cytogenotoxic effects of L. sibirica (L.) Cass. Scientif- ic literature highlights the use of this species as a cure for treating phlegm and for reducing cough (Liao et al. 1002; Tori et al. 2008; Yuan et al. 2013). It is worth to mention here that there is not a clear situation regarding the L. sibirica (L.) Cass. populations in Romania related to its distribution in the Natura 2000 sites (Brînzan et al. 2013), and the protection of the spe- cies through the Bern Convention, Annex I - Strictly Protected Flora Species (Berne Treaty No. 104), comple- mented by the IUCN threat status, Data Deficient (Bilz et al. 2011). Due to the fact that the species of communi- ty interest is protected by the Habitats Directive through Annexes IIb and IVb, GEO 57/2007 (Law 49/2011): Annexes 3, 4 A and mentioned in the Carpathian List of Endangered Species in the category Near Threatened (Mihăilescu et al. 2015), the material used in our study was collected after a complex survey regarding the quan- titative and qualitative analysis of L. sibirica (L.) Cass. population in each site, as follows: Apa Roşie Peat Bog, Hărman Marsh and Zănoaga Gorges. The necessary material for study was harvested in minimum amounts, in order to preserve the existing vegetal communities. In the present study, we analyzed the chemical com- position, antioxidant activity and cytogenotoxic prop- erties of ethanol extracts obtained from roots and rhi- zomes of L. sibirica (L.) Cass. Our study represents a first step in estimating the possible phytotherapeutic applications of L. sibirica (L.) Cass., and to understand to what extent this species can be incorporated into farming systems as a medicinal herb. MATERIALS AND METHODS Plant material Roots and rhizomes of L. sibirica (L.) Cass were harvested in August 2017, from 3 distinct sites, both in terms of habitat, pedo-climatic conditions, but also with the same level of anthropogenic activity, in order to decide which of them hold a higher potential for further studies. The first sample was collected from Zănoagei Gorges (Lat N45°28’, Long E25°25’), which are part of Bucegi Natural Park an interesting limestone mountain system, the studied area being characterized by a cool- wet climate type, the average annual temperature being of 4.9°C where the rainfalls varies with the altitude, cov- ered by rendzina soils (Beldie, 1967). The second sam- ple of L. sibirica (L.) Cass. was collected from Hărman Marsh (Lat N45°43’, Long E25°39’), an eutrophic marsh with hydric and alluvial soils and CaCO3 deposits, locat- ed in the Braşov Depression, with an annual tempera- ture of 7°C and low rainfall. The last sample was collect- ed from Apa Roşie Peat Bog (Lat N46°10’, Long E26°15’), located in Nemira Mountains and characterized by a cool-wet climate type rich in precipitations, with an average annual temperature of 2–4°C, occupying hydric soils without CaCO3 deposits (Brînzan et al. 2013). After weighing the plant material, the fresh rhi- zomes and roots were washed in tap water to remove the soil, rinsed well in distilled water, pat dried with paper towel, and then minced at room temperature. Reagents and Chemicals The reagents used: gallic acid monohydrate ACS rea- gent ≥ 98% and Folin Ciocalteu’s phenol reagent of 2M concentration, caffeic, cinnamic, ferulic, rosmarinic and syringic, catechin, myricetin, naringenin, methanol, and ethanol were from Sigma-Aldrich (USA). Chlorogenic acid and quercetin were from Alfa Aesar (Germany) and Fluka, respectively. Ethanol (HPLC degree) was obtained from Merck Co. (Darmstadt, Germany). The used water was double-distilled. The carbon dioxide (99.5% purity) 85Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts used in SFE extraction and the rest of the reagents used in the experiments were purchased from commercial sources. The weight of the samples was measured on an analytical balance of Shimadzu Corporation with a pre- cision of 0.1 mg. Extraction Procedures Ethanolic extract was prepared by mixing 5 g of each type of plant material, roots and rhizomes, in 50 mL absolute ethanol stored for 8 h at room temperature (22°C). Using Whatman filter paper no. 1, the extracts were filtered and the resulted filtrates were used to indi- cate their cytogenotoxic potential using Allium cepa test. The SFE extracts were achieved through the specif- ic equipment, a pilot unity called SFT-110 Supercritical Fluid Extractor (Supercritical Fluid Technologies, Inc.). The temperature of the restrictor valve was adjusted to 50° or 60°C for all extraction processes. The extract was collected in a 50 mL glass vial. The output of CO2 in the system was of 6.0 g/min. The yields of extraction were around 2%. For the co-solvent extraction (sample 4), using 1% and 2% ethanol in relation to the CO2 the experiment was performed in conditions of 50°C, 250 bar and flow of CO2 at 6 g/min (Erdogan et al. 2011). In this case the extraction yield was 2.5% from the raw material. Five samples of extracts were prepared: Hărman Mash – 1, Zănoagei Gorges – 2 and Apa Roşie Peat Bog – 3 through SFE extraction with CO2, then it was added a SFE extraction with CO2 and EtOH as cosolvent from Apa Roşie Peat Bog – 4 and the last one was the abso- lute ethanol extraction from Apa Roşie Peat Bog – 5. The extracts of every single assay were individualized by studying the antioxidant activity through: TP, TF, DPPH and TEAC. The functional groups were analysed using FTIR in the region 4000–400 cm-1. The ethanolic extract (sample 5) was used to evaluate cytogenotoxic activity. Evaluation of chemical composition of the rhizome and roots extracts FT-MIR The extracts of L. sibirica (L.) Cass. were investi- gated for their chemical composition: extract with CO2 (1, 2, 3), CO2 and ethanol (4), ethanolic extract (5). For FTIR spectroscopy, was used a Jasco 6300 spectrom- eter. The FTIR spectroscopy of each rhizomes and roots extract was recorded in the MIR region. An ATR acces- sory equipped with a diamond crystal (Pike Technolo- gies) was used for sampling. Samples were carried out at 100 scans with resolution of 4 cm-1 in the regions of 4000-400 cm-1. Spectra Manager II software was used for processed the spectra data. Total phenolic content In order to assess the TP content of the material extract, it has been used the Folin-Ciocalteu method (Singleton and Rossi, 1965). The TP content was brought out by mixing 0.5 mL of sample or standard (gallic acid) with 5 mL of Folin - Ciocalteu reagent and 4 mL of 1M sodium carbonate. It was measured the absorbance at 746 nm (Shimadzu UV-1800 Spectrophotometer) and the calibration curve with standard solutions of gallic acid concentrations ranging from 0.05 to 1 mgL-1 was traced. Equation of standard cur ve was y=0.0067x+0.0105 (R2=0.9960). The results were expressed as mg of GAE in 100 g of the rhizomes and roots. Extract constituents The identification and quantification of selected phe- nolic acids and flavonoids in the plant extracts was per- formed by high performance liquid chromatography (HPLC) using a Varian 310-MS LC/MS/MS triple quad- rupole mass spectrometer (USA) fitted with an electro- spray ionization interface (ESI). A sample of each dry plant extract was dissolved in HPLC-grade methanol. The mobile phase was double distilled water/methanol, fr = 20:80. A Varian C18 column (150 mm 9 3.0 mm; 5 mm) was used at a flow-rate of 0.6 ml/min, in an isocratic elu- tion. The injection volume was 20 ml and triplicate injec- tions were used for each sample. The drying gas was air at a pressure of 131 kPa and 250 °C, and the nebulizing gas was nitrogen at 276 kPa. The capillary voltage was set at the potential -4500 V for negative ionization. The result- ing deprotonated molecular ion was selected by the first quadrupole; in the second quadrupole, the deprotonated molecular ion was fragmented by collision with an inert gas (argon) at a pressure of 0.2 Pa; the fragments were ana- lyzed in the third quadrupole. Prior to these experiments, the tuning of the mass spectrometer was performed using a polypropylene glycol standard for both positive and neg- ative modes. The determinations have been carried out in triplicate. The results are expressed as μg of selected phe- nolic acid or flavonoid per 1 g of dry extract. Determination of antioxidant activity 1. DPPH Antioxidant activity. In order to deter- mine the radical scavenging activity of the ethanol 86 Nicoleta Anca Şuţan et al. extract of L. sibirica (L.) Cass. the Brand-Williams DPPH assay has been used (Brand-Williams et al. 1995). DPPH is stable organic nitrogen radical, with a purple colour solution, which reacts with the antioxidant com- pounds. The method it stands on the measurement of the reducing ability of antioxidants toward this radical. The ability can be evaluated by measuring the decrease of DPPH absorbance at 517 nm. The percentage of the DPPH remaining was calculated using the equation: I% = [(Ablanck-Asample)/Ablanck] x100, where: Ablank is the absorbance for the blank (ethanol - DPPH. ethanolic solution) and Asample is the absorbance for the sample mixed with 0.04 mg/mL DPPH solution. The results were expressed as IC50 (the extract con- centration which inhibits the activity of DPPH by 50%). In short, to 4 mL of extract at 1 mL of DPPH (0.04 mg/ mL) ethanol solution was added. After incubating for 30 min in dark, the absorbance of the resulting solutions was measured at 517 nm using UV-VIS Jasco 730 Spec- trophotometer (Kedare and Singh 2011). 2. TEAC assay. A stock solution of ABTS•+ was obtained after reacting the ABTS chemical compound with potassium persulfate. Then the mixture has been left at dark at room temperature for 12–16 hours before use. The ABTS•+ used solution was prepared by dilu- tion of this solution with ethanol till the absorbance was around 0.70 (Pellegrini et al 2003). The absorbance was measured at 734 nm. The calibration curve was made with Trolox (with concentrations between 0.125 and 2 mM). The results were done in mmol of Trolox per 100g rhizomes and root. Equation of standard curve was y=45.432x+20.334 (R2=0.9896). Evaluation of cytogenetic effects of L. sibirica ethanol extract The two samples extracted with ethanol (sample 4 and 5) showed the best antioxidant properties, taking into account a higher volume of extraction, the sample 5 was studied in order to evaluate the cytogenotoxic effects of L. sibirica (L.) Cass. extracts. The cytogenotoxic effects were evaluated through the changes of the mitotic indices and through the fre- quency of the phases of mitosis (prophase, metaphase, anaphase, telophase), as well as through the chromo- somal aberrations frequency and the nuclear anomalies induced in root tip cells of A. cepa L. The onion bulbs (a local variety), about 4 cm diam- eter, have been checked to fit in the phytosanitary stand- ards. In order to expose root primordia, the outer scales have been gently removed and the bulbs were placed in 30 mL containers, with the discoid stem being in con- tact with distilled water. The Allium test was performed through static exposure, initially at the action of dis- tilled water for 24 or 48 hours as well as at various con- centrations of L. sibirica (L.) Cass. ethanol extracts for 24 or 48 hours. Further the samples were defined by the extracts concentration, respectively 5%, 10%, and 15%, as well as by the incubation time of onion roots, 24 hours and 48 hours (L 5% 24h, L 5% 48h, L 10% 24h, L 10% 48h, L 15% 24h, L 15% 48h). For the cytogenetic analysis roots with a length of about 10 mm were used. The roots were fixed in a mix- ture of absolute ethanol: glacial acetic acid 3:1 overnight and then transferred to 70º ethanol for long-term pres- ervation. For each experimental variant, a number of 5 roots were subjected to attenuated hydrolysis with HCl 1N for 15 minutes at 60°C. Fixed and macerated roots were stained with aceto-orcein solution 1% at 60°C for 15 minutes. The root tips were cut on a glass slide, in a drop of 45% glacial acetic acid, and used to perform microscopic slides using the squash technique. The coverslips were glued with several layers of nail polish, and the resulted microscopic slides were analyzed on an Olympus CX-31 microscope, at a 400x magnifica- tion. The microscopic analysis aimed at establishing the numbers of cells at different stages of mitosis, the fre- quency of chromosomal and nuclear aberrations related to about 3000 cells for each experimental sample. The MI was determined as the percentage ratio between the number of cells in mitosis and the total number of ana- lysed cells (Tedesco et al. 2012). Depending on the total number of cells that carry out mitosis, it was determined the percentage of the cells mitosis stages. The frequency of chromosomal aberrations and nuclear abnormalities was calculated as the percentage between the number of damaged cells and total number of cells in the appropri- ate stage of the cell cycle and mitosis. The results were statistical analysed using the IBM SPSS Statistics 20 program. Significant differences among samples were determined using the analysis of variance (one-way ANOVA), as well as the Duncan mul- tiple comparison test. The P≤0.05 values were considered statistically significant. The graphs and tables were elab- orated based on average values ± the standard error (SE) of more independent experiments. RESULTS AND DISCUSSION Evaluation of chemical composition of the rhizome and roots extracts by FT-MIR The ATR-MIR spectra (4000-400 cm-1) of each extract was registered and the specific wavenumbers 87Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts and intensities were considered in order to present the FTIR-ATR spectra of alcoholic and CO2 extracts (Figure S1 in the Supplementary Material), as well as the FT-IR absorption bands for rhizomes and roots extracts (Table S1 in the Supplementary Material). The vibrational assignments for extracts were compared with literature data (Szymanska-Chargot and Zdunek 2013). The identi- fication of the functional groups was based on the FTIR peaks attributed to stretching and bending vibrations. Eight areas were identified in the MIR domain and the fingerprint region was localized between 900 and 1760 cm-1 (areas 1-6) (Zavoi et al. 2011). Figure 1 shows the spectral regions 1-7 for sample analysis. Area 1 (< 1000 cm-1) corresponds to C-H bend- ing vibrations from isoprenoids, area 2 (997-1140 cm-1) to stretching vibrations C-O of carbohydrates, with sig- nals at 1005, 1009, 1040, 1080 and 1136 cm-1, while area 3 (1150-1270 cm-1) corresponds to stretching vibrations of carbonyl C-O or O-H bendings. The intense peaks at 1040 cm−1 and to nearly 1080 cm-1 are attributed to character- istic functional groups of polyphenols. C-O (amide) and C-C stretchings vibrations appear in the region 4 (1300- 1450 cm-1) Region 5 (1500-1600 cm-1) corresponds to aro- matic group and N-H bending vibrations. Domain 6 is a complex one (1600-1760 cm-1) and corresponds to bending vibrations N-H (amino acids), C=O stretchings (aldehydes and ketones, esters) as well to free fatty acids (1766 cm-1) and glycerides (1738 cm-1). Area 7 (2800-2900 cm-1), cor- responds to C-H stretching vibrations of CH3 and CH2 from lipids, lipid derivatives, C-H (aldehydes). Region 8 (3350-3600 cm-1) is assigned to stretching vibrations of OH (from water, alcohols, phenols, carbohydrates). The spectra show relatively more bands in the region of 400-700 cm-1. The inorganic constituents can be observed in the region between 470-480 and 530-540 cm-1. The variation may be due to the differences in the extraction and purification methods. Antioxidant activity and phenol content of plant extracts There are a few studies about compounds with anti- oxidant activity from the genus Ligularia. According to them, L. fischeri (Ledeb.) Turcz. showed high total phe- nolic contents (215.8 ± 14.2 mg gallic acid equivalent/g) with low contents of total flavonoid (86.9 ± 3.8 mg rutin equivalent/g) (Lee et al. 2013). Liu (2010) has reported the isolation and structural elucidation of the fura- noeremophilane-type sesquiterpenes and benzofuran derivatives (part of them with phenol groups) from L. veitchiana (Hemsl.) Greenm., and some results about its biologic activity (Liu et al. 2010). L. macrophylla (Ledeb.) DC. has been reported to contain at least two f lavonoids: 6-acetyl-8-methoxy-2,3-dimethylchromen- 4-one and 4-14 (2S)-3’-hydroxy-5’,7-dimethoxyflavanone in root and rhizome, while in L. duciformis’s root have been identified some derivatives of sinapyl alcohol and coniferyl alcohol, with known antioxidant activities (Yang et al. 2011). From our knowledge, phenols, fla- vonoids or other compounds with antioxidant activity from L. sibirica (L.) Cass. were not assessed. The Table 1 presents the TP and TF content of L. sibirica (L.) Cass. rhizome and root extracts and its TEAC. The highest concentrations of TP and TF were obtained for extracts of L. sibirica (L.) Cass. prepared by the co-solvent meth- od (EtOH), as expected. Our results have also shown that supercritical CO2 extractions were the least efficient, predictable since phenols are polar compounds and thus are less extract- ible by CO2. Something more effective than this was the extraction method at cold temperatures with etha- nol (method used for extraction of phenols from vegetal matrix (Santos-Buelga et al. 2012). The polyphenol constituents present in extract 5, as identified and quantified by LC–ESI–MS/MS, are listed in Table 2. Based on these results, it was concluded that the antioxidant properties of the extract originate in Figure 1. ATR-MIR spectra registered for sample 3 (SFE extraction with CO2). Table 1. TP, TF and TEAC assay for L. sibirica rhizome and root extracts. Sample TP (mg gallic acid equivalent/100g root) TF (mg quercetol equivalent/100g root) TEAC assay (mmoli Tolox equivalent/100g root) 1 2.13 n. d. a 0.03 2 3.08 n. d. 0.04 3 10.01 n. d. 0.09 4 494.26 5.25 1.42 5 49.25 n. d. 0.47 a not detected. 88 Nicoleta Anca Şuţan et al. two main classes of compounds, namely flavonoids (cat- echin, myricetin, naringenin, quercetin) and, to a greater extent, phenolic acids (caffeic, chlorogenic, cinnamic, ferulic, gallic, rosmarinic, syringic acid). Evaluation of cytogenetic effects of L. sibirica ethanol extracts Starting with Fiskejio (1988) the Allium test was considerably used for the evaluation of cytotoxic and genotoxic effects, as well as for the genoprotective poten- tial of various natural or synthetic chemical compounds. The Allium test follows some endpoints, such as the mitotic index, chromosomal aberrations and the nuclear anomalies (Bonciu et al. 2018). For each experimental sample, the results were com- pared with the control. Figure 2 shows the MI variation in onion roots exposed for 24 or 48 hours at the action of L. sibirica (L.) Cass. extracts of 5%, 10% and 25% con- centration. Statistical analyzis of the microscopic results revealed that ethanol extracts of L. sibirica (L.) Cass. have determined a significant decrease in the frequency of cells in various phases of mitosis. The highest MI was determined for the control for which the calculated per- centage was 7.64%. L. sibirica (L.) Cass. extracts had a statistically important mitoinhibitory effect, when com- pared to the control, showing an indirect correlation with their concentration. The lowest MI (0.7%) was cal- culated after the roots incubation in the extract with the concentration of 5% for 48h. This decreased frequency of cells in the mitosis was followed at a statistically signifi- cant difference by that determined in the experimental sample, defined by the 5% concentration, respectively 24h. In the root tip cells incubated in L. sibirica (L.) Cass. extracts, the highest MI with a 6.19% value was calculated for the L 15% 24h experimental sample. The overall interpretation of the microscopic results revealed that the variation of the mitotic index was independent of the exposure time. The decrease of the MI in A. cepa L. meristematic root cells demonstrates the presence of bioactive substances with antiproliferative potential. The effects of L. sibirica (L.) Cass. extracts on the distribution of the mitotic division phases in the onion meristem cells are summarized in Figure 3. The frequen- cy of prophase and metaphase has significantly varied, in a concentration dependent manner. Therefore L 5% 24h has induced the highest percentage of prophases, significantly different compared to the other tested con- centrations. When compared to the control, the ethanol extracts of L. sibirica (L.) Cass. have induced an increase and a significant increase of metaphases frequency. The Table 2. The phenolic profile of the plant extract-5, as determined by LC–ESI–MS/MS. Class compound Compound Concentration (μg compounds/g dry extract) Phenolic acids Caffeic acid 69.778±1.0229 Chlorogenic acid 189.114±1.7604 Cinnamic acid 0.438±0.0177 Ferulic acid 1.856±0.0764 Gallic acid 7.420±0.1071 Rosmarinic acid 25.359±0.1292 Syringic acid 2.532±0.0955 Flavonoids Catechin 6.901±0.0124 Myricetin 0.343±0.0158 Naringenin 0.704±0.0283 Quercetin 13.032±0.6992 Figure 2. The influence of L. sibirica extracts on the mitotic index in root meristem cells of Allium cepa L. (The data are the averages ± SE of three repetitions; a, b, c, d, e, - the interpretation of statistical significance and of significant differences through the Duncan test, p <0.05). Figure 3. The influence of L. sibirica extracts on the distribution of mitotic division phases in the radicular meristem cells of A. cepa L. (the data are ± SE averages of three repetitions; a, b, c, d, e, f, g, h, i, j, k – the interpretation of statistical significance and of significant differences through the Duncan test, p <0.05). 89Chemical composition, antioxidant and cytogenotoxic effects of Ligularia sibirica (L.) Cass. roots and rhizomes extracts highest metaphases percentage was determined in L 10% 24h and L 10% 48h variants. The calculated percentage values for anaphase and telophase were not significantly different compared to the control. On the basis of these observations it can be appreci- ated that the MI decline is due to cells arresting in the metaphase at a 5% extracts concentration. The overall interpretation of the results also reveals a global slow- down of mitotic progression at a higher concentration of the extracts. The decrease of the MI may be reflect- ing a cytogenotoxic effect of L. sibirica (L.) Cass. etha- nol extracts and could be interpreted as cellular death (Yuet Ping et al. 2012). According to Nieva-Moreno et al. (2005) cited by Stojković et al. (2013) the main mecha- nisms of cancer chemoprevention are anti-mutagenesis and anti-proliferation or mitotic anti-progression. An extensive review by Yang et al. in 2011, as well as other recent studies (Dong et al. 2015) shows that among the bioactive chemical constituents of Ligularia species, the most common phytochemical types are sesquiter- penes, which have demonstrated a strong cytotoxic or inhibitory activity on some tumor cell lines. Among the volatile compounds identified by Gas chromatography with mass spectrometry detection in the L. sibirica (L.) Cass. extracts obtained by microwave assisted hydrodistillation, there were sabinene, limonene and terpinolene (monoterpenoids), as well as alkaloids that were most likely tussilagine and isotussilagine. In Figure 4. Chromosomal aberrations and nuclear abnormalities identified in meristematic root cells of A. cepa L. (a) telophase bridge – L 5% 24h; (b) C-mitosis – L 5% 48h; (c) laggards – L 5% 24h; (d) micronucleus and nuclear buds - L 5% 48h; (e) giant cell – L 10% 48h; (f ) vagrant – L 10% 24h; (g) anaphase bridges – L 15% 24h; (h) altered nuclear shape – L 10% 48h; (h) fragments – L 5% 24h. 90 Nicoleta Anca Şuţan et al. our study, the statmokinetic effect may be attributed to these terpenoids. Many of the isolated terpenoids from natural sources have chemopreventive effects (Akihisa et al. 2003; Yang et al. 2011). Nuclear and chromosomal aberrations observed in the root tip meristem cells of A. cepa L. are shown in Figure 4. Their frequency in the various experimental samples is shown in Supplementary Material (Table S2). The average percentage value of the nuclear and chromosomal aberrations calculated for the control it was only 0.35 ± 0.07, and the percentages 1.53 ± 0.12, 3.46 ± 0.55, 3.25 ± 0.94, 1.83 ± 0.22, 1.25 ± 0.42 and 1.26 ± 0.35 for L 5% 24h, L 5% 48h, L 10% 24h, L 10% 48h, L 15 % 24h, L 15% 48h. The significant higher frequen- cy of the chromosomal and nuclear aberrations in L 5% 48h, L 10% 24h, L 10% 48h variants, may be attributed to the potentially lower antioxidant activity as a result of the dilution of the tested extracts. This antimutagenic activity may be attributed to the secondary metabolites or to synergistic action of the antioxidant compounds. Besides that, the FT-MIR analysis has revealed the exist- ence of OH group characteristic for phenols, to which may be due the genoprotective activity. Oxidative DNA damage can contribute to single double strand breaks formation or to oxidation of the purines or pyrimidines bases, inducing a genomic instability and also the devel- opment of cancer (Chobotokova 2009). The biologically active phenols are known for the antioxidant properties exerted by the absorption of the free radicals (Hidalgo and Almajano 2017; Shahidi et al 2015) as well as for the wide spectrum of biological and physiological actions (Durazzo 2017). Mitoinhibitory effect of L. sibirica (L.) Cass. extracts was supported by the C-mitosis high percentage, which indicates the spindle disturbance in metaphase (Firbas and Amon 2014; Bonciu 2018). However, the nuclear aberrations identified in the meristematic root cells were much more varied and had a higher frequency compared to chromosomal aberrations. The nuclear abnormalities defined by the nuclear morphology alterations in the interphase, such as micronuclei, nuclear buds, altered nuclei shape may be an indicator of some processes such as cell death or tumorigenesis (Pellegrini 2003; Nefic et al. 2013). In our study, except for the L 10% 24h experimen- tal sample, changes in the shape of the nucleus was observed with a very low frequency. Nuclear envelope proteins ensure the nucleus rigidity to the distortion associated forces caused by the cytoplasmic microtu- bules. These changes may be the consequence of micro- tubule generated forces in the cytoplasm, if any of the membrane nuclear proteins is inactive (Nefic 2013; King et al. 2008). Other studies have shown that the inacti- vation of the associated proteins with the endoplasmic reticulum affects the nucleus form (Higashio et al. 2000; Matynia et al. 2002; Webster et al. 2009). Moreover, the chromosomal aberrations and nucle- ar abnormalities identified by microscopic analysis are indicative of both clastogenic and aneugenic effects and of genotoxic damage. In this context, for a deeper esti- mation of the potential of this species additional in vitro and in vivo studies are required, aiming the extraction conditions, extract concentration and exposure time. CONCLUSION The paper represents an essential step in offering more information about the antioxidant activity, chemi- cal composition and cytogenotoxic activity of ethanol extracts obtained from roots and rhizomes of L. sibir- ica (L.) Cass. The best results for phenols and flavo- noids extraction from roots and rhizomes of L. sibirica (L.) Cass. were obtained in supercritical CO2 extraction with cosolvent (ethanol) followed by ethanol extraction. The ethanol extracts of L. sibirica (L.) Cass. have dem- onstrated a very strong mitoinhibitory effect on in vitro root meristem cells of A. cepa L. 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