Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 76(2): 67-81, 2023 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-2330 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Nicoleta Anca Şuţan, Diana Ionela Popescu (Stegarus), Oana Alexandra Drăghiceanu, Carmen Topală, Claudiu Şuţan, Aurelian Den- is Negrea, Denisa Ştefania Vîlcoci, Georgiana Cîrstea, Sorin Georgian Moga, Liliana Cristina Soare (2023). In vitro cytotoxic activity of phytosynthe- sized silver nanoparticles using Clem- atis vitalba L. (Ranunculaceae) aque- ous decoction. Caryologia 76(2): 67-81. doi: 10.36253/caryologia-2330 Received: May 10, 2023 Accepted: October 29, 2023 Published: December 31, 2023 Copyright: © 2023 Nicoleta Anca Şuţan, Diana Ionela Popescu (Stegarus), Oana Alexandra Drăghiceanu, Car- men Topală, Claudiu Şuţan, Aurelian Denis Negrea, Denisa Ştefania Vîlco- ci, Georgiana Cîrstea, Sorin Georgian Moga, Liliana Cristina Soare. This is an open access, peer-reviewed arti- cle published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits 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. ORCID NAŞ: 0000-0001-7459-628X OAD: 0000-0001-8351-0919 LCS: 0000-0002-2874-3135 DIP: 0000-0002-3442-3760 CT: 0000-0002-9117-4983 CŞ: 0000-0002-1719-1814 ADN: 0000-0001-7525-1056 DŞV: 0000-0002-9777-6782 GC: 0000-0002-7442-7109 SGM: 0000-0001-9620-4283 In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba L. (Ranunculaceae) aqueous decoction Nicoleta Anca Şuţan1, Diana Ionela Popescu (Stegarus)2, Oana Alex- andra Drăghiceanu1, Carmen Topală3, Claudiu Şuţan3,*, Aurelian Denis Negrea4, Denisa Ştefania Vîlcoci4, Georgiana Cîrstea4, Sorin Georgian Moga4, Liliana Cristina Soare1 1 Department of Natural Sciences, National University of Science and Technology POLITEHNICA Bucharest, Pitesti University Centre, Romania 2 National Research and Development Institute for Cryogenics and Isotopic Technologies– ICSI Ramnicu Valcea, 4th Uzinei Street, 240050 Ramnicu Valcea, Romania 3 Department of Environmental Engineering and Applied Sciences, National University of Science and Technology POLITEHNICA Bucharest, Pitesti University Centre, Romania 4 Regional Center of Research and Development for Materials, Processes and Innovative Products Dedicated to the Automotive Industry, National University of Science and Tech- nology POLITEHNICA Bucharest, Pitesti University Centre, Romania *Corresponding author. E-mail: claudiu.sutan@upb.ro Abstract. In this study, we report a bottom-up approach for silver nanoparticles (AgNPs) synthesis using aqueous decoction of aerial parts of Clematis vitalba L. The phytosynthesized AgNPs were characterized by X-ray diffraction (XRD), UV-vis spec- troscopy, Fourier Transform-Infrared Spectroscopy (FTIR), Scanning Electron Micros- copy coupled with Energy Dispersive X-ray Spectroscopy (SEM-EDS) and Bright Field Scanning Transmission Electron Microscopy (BFSTEM). The cytogenotoxicity and phytotoxicity assays of AgNPs were assessed by using Allium test, Evans blue and 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, root and stem growth potential, and biomass evaluation. The results revealed that AgNPs were in the size range of 1-15 nm and spherical shape. The biosynthesized AgNPs augment the mitodepressive effect, disruption of cellular metabolism, impairment of root and stem growth, and biomass reduction induced by C. vitalba aqueous extracts. These results outline the toxicologi- cal profile of the C. vitalba extracts, as well as of the phytogenerated AgNPs and pro- vides scientific perspectives on the use of C. vitalba extracts as reducing and stabilizing agent for the phytosynthesis of metallic nanoparticles. Keywords: Clematis vitalba, AgNPs, biosynthesis, cytogenotoxicity, phytotoxicity. INTRODUCTION Clematis L., one of the best represented genera of the buttercup family (Ranunculaceae), includes about 300 species (He et al. 2019), most climb- 68 Nicoleta Anca Şuţan et al. ing plants and shrubs widespread in temperate zones of the northern and southern hemispheres, in mountain- ous and tropical regions. Over 600 ornamental varieties are commercially cultured worldwide (Weng-Tsai 2003; Woudenberg et al. 2009). Clematis vitalba L. is a deciduous perennial climber vine which grows in fields and woody areas of Europe, USA, Australia and New Zealand (Bungard 1996; Ogle et al. 2000; Redmond and Stout 2018). In various parts of the world the species C. vitalba is known by differ- ent common names, such as Old Man’s Beard due to its seeds with fluffy heads, Mile-A-Minute based to its high growth rate, or Traveler’s Joy, being found along the roads (O’Halloran 2019). Like most buttercups, C. vitalba is toxic and irritating, containing protoanemonin lac- tones and convulsive poisons, but most exposures have resulted in minimal to no toxicity (Duke 1985; Lewis et al. 2020). A synthesis of the compounds identified in Clematis species was reported by Da-Cheng (2019), who mentioned the presence of saponin, coumarin, f lavo- noids, anthocyanins, and alkaloids. In relation to envi- ronmental risk, alkaloids and saponins were character- ized by Günthardt et al. (2018) as some of the most toxic secondary metabolites. In fact, a large number of papers have shown that buttercups can inhibit mitosis, induce apoptosis and alter the human cell cycle (Naz et al. 2020), but also affect plant cells, being phytotoxic. Thus, the inhibition of root system growth in Triticum aestivum L. by ethanolic extracts of Anemone nemorosa L. was reported by Ancuceanu et al. (2018). Methanolic extracts from Anemone reflexa Steph. & Willd. and Clematis tri- chotoma Nakai showed strong herbicidal activity, inhib- iting the growth of barnyardgrass (Echinochloa crus-galli (L.) P. Beauv.) seedlings (Kim and Lee 2007). Phytotoxicity and cytogenotoxicity tests are recom- mended for assessing the impact of nanoparticles (NPs) on vascular plants. Among the parameters indicated are germination rate, root and stem growth rate, number of leaves, biomass, enzyme activities, photosynthetic rate, mitotic index, i.e. (Roy et al. 2019). The investigation of morphological, physiological and genetic changes in plants under the influence of NPs from natural or anthro- pogenic sources is still in its infancy (Ogle et al. 2000), from a toxicological perspective the impact proving both positive and negative (RuttkayNedecky et al. 2017). During the last decades, nanobiotechnology has become an ingenious strategy for green synthesis of NPs. Applying the principle of self-assembly and self-organi- zation through supramolecular interactions and under the action of external stimuli, the bottom-up approach offers the possibility of NPs synthesis in a simple, fast and cost-efficient manner. The use of plant extracts for extracellular synthesis of NPs is currently an eco-friend- ly alternative to improve their potential impact, as well as to reduce their side effects (Shende et al. 2022; Şuţan et al. 2016). Along with other metallic nanoparticles, biosynthesized silver nanoparticles (AgNPs) have found their utility in medical, ecological, textile, agriculture, food security and other applications (Eswaran et al. 2021; Hassan and El-latif 2018; Prakash 2013; Roy et al. 2017; Shende et al. 2022). Considered an invasive species (Hill et al. 2001) or potentially invasive (Filippin et al. 2009) in some parts of the world, being so common and having a rapid growth rate, and also being rich in novel secondary metabolites such as flavonoids and alkaloids responsible for the reduction of ionic into nanoparticles, C. vitalba could become a valuable resource for the bio- synthesis of NPs. Moreover, the biomolecules from the C. vitalba could act as capping agents, thus increasing the stability and monodispersity of biosynthesized NPs. Thus, the aim of this study is to emphasize the potential applicability of the extracts of C. vitalba in nanobiotechnology, and their impact, prior to and after AgNPs phytosynthesis on cell viability and metabolic activity, in view of highlighting their cytogenotoxic and phytotoxic properties. MATERIALS AND METHODS Collection of plant materials Overground parts of C. vitalba plants were collect- ed from 44°51’23.3”N 24°53’17.7”E (GPSMAP® 60CSx), Piteşti, Romania. The voucher specimen no. 2510 was deposited in the Herbarium of Pitesti University Cen- tre, National University of Science and Technology POLITEHNICA Bucharest, Romania. Decoction method and synthesis of silver nanoparticles Authenticated plant material was washed insistently with tap water, rinsed with distilled water and dry until constant weight, at room temperature. The dried plants were ground in pulses for 3 min at 4000 RPM and con- tinuously for 10 sec at 10 000 RPM through a laboratory knife mill (Retsch Knife Mill Grindomix GM 200). The aqueous extract of C. vitalba was obtained according to the protocol proposed by Muala et al. (2021) with slight modifications. The powdered raw material was extracted in distilled water (1:10 w/v), in a water bath at 95 oC for 15 min and kept 24 hours at room temperature, in the dark conditions. The slurry was filtered through Watt- man No.1 paper to obtain crude extracts. 69In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba aqueous decoction Aqueous extract of C. vitalba (Cv) were treated with equal volume of 10-3 M AgNO3 and left at room tem- perature. In order to demonstrate the biosynthesis of AgNPs, as well as to correlate the involvement of second- ary metabolites from the extract with the biosynthesis process, the extracts were subjected to Fourier Trans- form-Infrared Spectroscopy (FTIR) analysis and nano- formulations were subjected to X-ray diffraction (XRD), UV-vis spectroscopy, Scanning Electron Microscopy cou- pled with Energy Dispersive X-ray Spectroscopy (SEM- EDS) and Bright Field Scanning Transmission Electron Microscopy (BFSTEM). The cytogenotoxicity and phy- totoxicity assays of extracts and their nanoformulations were assessed by using Allium test, Evans blue and 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining. Physicochemical characterization of C. vitalba aqueous extract and metallic nanoparticles Aqueous extract was evaluated for its chemical composition. Fourier transform infrared spectroscopy (FTIR) was made with a FTIR Jasco 6300 spectrometer with an ATR accessory equipped with a diamond crys- tal (Pike Technologies). The spectra were recorded in the region of 4000-400 cm-1, detector TGS, apodization Cosine. The spectral data were processed with JASCO Spectra Manager II software. AgNPs biosynthesis (CvAg) was evaluated through color change after 4 h and confirmed by UV-Vis spec- troscopy. Qualitative analysis of compounds in Cv (dilution factor 20x) and CvAg was carried out in the wavelength ranging from 300 to 500 nm and the resolution of 2 nm by using UV-Vis Ocean Optics HR2000+. CvAg sample was centrifuged at 6000 rpm for 15 min and the resus- pended sediment in a 10 mg/mL solution was scanned for the characteristic peaks (Kaur et al. 2017). In order to perform X-ray Diffraction Analysis (XRD), the supernatant was removed, and the sediment was centrifugated twice with distilled water at the same speed and time. The XRD pattern of as-separated NPs was recorded with a Rigaku Ultima IV diffractometer. The experimental conditions were: CuKα radiation, BB geometry, D/teX Ultra detector with graphite monochro- mator, continuously mode, 2theta range [350-900], step 0.050, and speed 20/min. Scanning Electron Microscopy coupled with Ener- gy Dispersive X-ray Spectroscopy (SEM-EDS) and Bright Field Scanning Transmission Electron Micros- copy (BFSTEM) analyses were performed using the FESEM - HITACHI SU8230 microscope. SEM-EDS was used to confirm the presence of Ag, while STEM was used to investigate particles shape and size distribution. For SEM-EDS, samples of Cv and CvAg were homog- enized in the ultrasonic bath (Guyson’s Kerry Pulsa- tron KC2) for 1 min, dropped on the conductive carbon tape (C) and dried for 24 h in the desiccators. In order to enhance the fluorescence data, a sediment sample obtained by CvAg centrifugation was spread on the con- ductive carbon tape. EDS data have been collected from the conductive carbon tape (in order to extract sample’s support contribution from the EDS spectra), from the Cv, CvAg and CvAg sediment. For BFSTEM analysis, CvAg sample was homogenized in the ultrasonic bath for 1 min, dropped on carbon coated nickel STEM grid (Ni-C) and dried for 24 h in the desiccator. Size distribu- tion of biosynthesized AgNPs was analyzed with ImageJ software (Rasband 1997-2018). Evaluation of cytogenotoxic effects by Allium test Healthy bulbs of Allium cepa L. (2n=16) were pro- vided by a private organic farm. Bulbs with a diameter of about 3 cm were used for cytogenotoxic assessment. The bulbs were incubated with the discoidal stem in contact with distilled water for 48 hours, in the dark, at room temperature (20-22 oC). The bulbs with new roots were transferred for 12, 24 and 48 h in aqueous extracts of C. vitalba (Cv12, Cv24 and Cv24) and nanostructured mixture with AgNPs, respectively (CvAg12, CvAg24, CvAg48). Distilled water (C) was used as a negative control and the cytogenotoxic effects were evaluated at appropriate times of 12, 24 and 48 h (C12, C24, C48) of incubation. Microscopic slides were prepared following the protocol exhaustively previous presented (Sutan et al. 2019). About 3000 cells for each sample were evalu- ated. Mitotic index (MI), distribution of mitosis phases, chromosomal aberrations and nuclear abnormalities in the analyzed cell population were the endpoints of the cytogenotoxic assessment (Şuţan et al. 2020). Evaluation of cell viability Cell viability was assessed by performing Evan’s blue test, following the protocol proposed by Vijayara- ghavareddy et al. (2017) with slight modifications. After the completion of the experimental treatments, 10 fresh- ly harvested roots corresponding to each experimen- tal variant were incubated for 15 min in 2 ml of 0.25% Evan’s blue. The roots were rinsed insistently with dis- tilled water and kept in fresh distilled water overnight. Root tips of 5 mm in length were transferred to 2 ml of 1% aqueous SDS solution and kept for 1 h at 50 oC in 70 Nicoleta Anca Şuţan et al. water bath. The roots were qualitatively analyzed and the Evan’s blue uptake was macro-imaged. For a quantitative estimation of cell viability, the optical density (OD) of the released pigment was read by the spectrophotometer (UV-Vis, T70+) at 600 nm. Evaluation of mitochondrial activity The 2,3,5-triphenyl tetrazolium chloride (TTC) staining assay was applied for assessing the metabolic activity of root tip cells. The onion roots were immersed in 0.5% aqueous TTC solution overnight and then extracted into 3 ml of 95% ethanol for 5-15 min, with- out heating. Due to the instability of TTC to light, the assay was performed in the dark. The absorbance of the extracts was read at 485 nm with a T70 + UV-Vis spec- trophotometer (Towill and Mazur 1974; Prajitha and Thoppil 2017). Assessment of the phytotoxic effect Phytotoxicity of aqueous extracts and nanostruc- tured mixture were assessed using seeds of Triticum aestivum L., Miranda variety. The seeds were soaked in 150 ml distilled water for 2 h. For each experimental variant 10 imbibed seeds were transferred in C. vitalba extract with and without phytosynthesized AgNPs for one hour, in the dark, and watered periodically with distilled water. Distilled water was used as negative control. After 4 days, fresh and dry biomass, the length of the roots and stems were evaluated. In order to establish dry biomass, the fresh materials were kept in the oven, at 80 oC, until constant weight was obtained (Azooz et al. 2012). Statistical interpretation Three replicates were used to quantify the cytog- enotoxic and phytotoxic effects of the extracts and nanostructured mixtures. Statistical analysis of the results was performed using IBM SPSS Statistics 20.0 (2011). Statistical significance and significant differences between variables were determined using variance anal- ysis (One Way ANOVA) and the Ducan test for multi- ple comparisons, respectively. The values of P≤0.05 were considered statistically significant. Graphs and tables were compiled based on mean values ± standard error (SE) of several independent experiments. For linear asso- ciation between variables, Pearson’s correlation was sig- nificant at the 0.01 level (2-tailed). RESULTS Physicochemical characterization of C. vitalba extract prior to and after AgNPs phytosynthesis The FTIR analysis revealed the band at 3347 cm-1 which intensity has almost reduced and shifted to 3359 cm-1 in IR spectra of AgNPs. In the case of AgNPs the peaks at 1267 cm-1 and at 1046 cm-1 are shifted. The strong band at 1636 cm-1 existing in the spectrum of C. vitalba aqueous extract, are shifted in the FTIR spec- trum of the AgNPs as it is shown in Figure 1. Figure 2 shows a typical metallic Ag XRD pattern, according to ICDD PDF4+ DB04-002-1347. The crystal- lite size of AgNPs was calculated using Rigaku PDXL2 and Wagner-Halder method (Halder and Wagner, 1966). The obtained value was 10,7±0,4 nm. Furthermore, the biosynthesis of AgNPs was indicated by the visible color shift from greenish-brown to light brown after 2 hours of incubation in the dark, without stirring the mixture (Fig. 3). The UV-vis spectra of aqueous extracts of C. vitalba (Fig. 4A) showed a shoulder peak in the range of 320- 330, while the UV-vis spectra of CvAg sample showed a distribution of peaks varying between 400-500 nm, with maximum absorbance at 436 nm (Fig. 4B). EDS analysis was performed to confirm the pres- ence of Ag. The EDS spectra obtained for the conduc- tive carbon tape, Cv, CvAg and CvAg sedimented on the carbon grid are shown in figure 5. Comparison of the obtained EDS spectra revealed that the aqueous extract of C. vitalba contains as majority elements K, Ca, Cl and minority elements such as P, Br, Mg. From analysis of all the superimposed spectra, the presence of Ag can be noticed only in the samples CvAg and CvAg sedimented. Moreover, the EDS pattern did not show any evidence for nitrogen. In Figure 6A-D the dispersion of biosynthesized AgNPs is represented in BFSTEM at successive magni- fications (x20k, x100k, x200k and x500k). The size of the identified particles was below 15 nm, and their shape was relatively spherical (Fig. 6D). It should be noted that a microparticle consisting of AgNPs embedded in a complex biological matrix was identified on the edges of the Ni-C grid and an EDS mapping analysis was per- formed (Fig. 7). The analysis of the AgNPs size distribution was performed on the micrograph obtained in BFSTEM (Figure 6D). For the selected area of interest and for the inclusion criterion defined for the range 1-15 nm it was found that AgNPs with domains between 1-10 nm are prevalent. 71In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba aqueous decoction Cytogenotoxic effects of C. vitalba extract and nanostruc- tured mixture The effects of C. vitalba extracts prior to and after AgNPs biosynthesis on onion meristematic root cells are presented in Table 1. Statistical analysis of the results revealed a significant decrease in MI in the sam- ples defined by aqueous extracts and nanostructured mixtures. The highest MI (8.7%) was recorded for the negative control C12, and the lowest MI (0.6%) was determined in the CvAg48 sample. The Pearson corre- lation coefficient was -0.87 indicating a significant time dependent inhibition of MI. Nanoformulations with AgNPs determined a severe mitoinhibitory activity, the number of cells identified in the various stages of mitosis being almost zero, after 48 hours from the initiation of treatment. Suppression of mitosis resulted in a low frequen- cy of chromosomal and nuclear aberrations. However, remarkable differences were noted between the control and the samples defined by C. vitalba extracts. Thus, in Figure 1. (ATR)-FTIR spectra of C. vitalba leaves extract (black) and of AgNPs (red). 35 40 45 50 55 60 65 70 75 80 85 90 0 200 400 600 800 1000 1200 1400 1600 1800 [2 22 ][3 11 ] [2 20 ][2 00 ]In te ns ity (c ou nt s) 2� (deg.) Ag (Silver-3C, syn) PDF4+ 2021 DB 04-002-1347 [1 11 ] Figure 2. XRD spectra of the phytosynthesized AgNPs using aque- ous extract of C. vitalba. Figure 3. Color of C. vitalba extract prior (Cv) and after AgNPs phytosynthesis (CvAg). 0 72 Nicoleta Anca Şuţan et al. the control only chromosomal aberrations such as ana- phase bridges and very rarely laggards were identified, while C. vitalba extracts before and after AgNPs phyto- synthesis induced the formation of binucleate and giant cells with displaced nuclei. Additionally, concave plas- molysis was very often revealed by cytological analysis (Fig. 8). A B Figure 4. UV-vis spectra of C. vitalba prior to (A) and after AgNPs phytosynthesis (B). Figure 5. EDS spectra obtained for the conductive carbon tape, Cv, CvAg and CvAg sediment (inset zoom EDS spectra reveal prominent silver peaks). 73In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba aqueous decoction Evaluation of cell death by Evans blue staining The permeability of cell membranes in the experi- mental variants defined by C. vitalba aqueous decoction decreased compared to control (Fig. 9). In opposition, incubation of meristematic cells in the CvAg mixture led to an increased uptake of Evans blue, irrespective of treatment duration, suggesting the cytotoxic effect of biosynthesized AgNPs. Evaluation of viable cells using TTC staining In Figure 9 is presented the formation of red formazan in meristematic root cells of A. cepa and the formazan absorbance. The data suggests that the reduc- tion of TTC by electrons from the mitochondrial elec- tron transport chain (Towill and Mazur 1975) progres- sively decreased from negative control to aqueous decoc- tion of C. vitalba. The absorbance of formazan increased to 0.68 after 12 h from the start of treatment with AgNPs-supplemented extract and remained low after 48 h of incubation. Assessment of phytotoxicity on T. aestivum In the Figure 10 is presented the phytotoxicity of C. vitalba extract prior to and after AgNPs biosynthesis. The application of C. vitalba extract with and without AgNPs x20k (A) x100k (B) x200k (C) x500k (D) Figure 6. AgNPs dispersion and dimensional analysis on dispersion in BFSTEM. 74 Nicoleta Anca Şuţan et al. on T. aestivum seeds had a stimulatory effect on root and stem elongation, and fresh weight compared to control, without significant differences between these variants. The dry biomass of wheat seeds was significantly reduced by AgNPs when compared with negative control. DISCUSSION Physicochemical characterization of C. vitalba extract and its nanoformulations The band at 3347 cm-1 revealed by the FTIR analy- sis corresponded to the -OH groups of phenolic com- pounds and -NH stretching of the proteins (Borchert et al. 2005; Prakash et al. 2013). The reduced intensity and shifting in IR to 3359 cm-1 spectrum of AgNPs indicat- ed the involvement of –OH group in the biosynthesis of AgNPs (Kumar et al. 2016). Similarly, the band at 1636 cm-1 revealed for Cv sample was shifted in the FTIR spectrum of the AgNPs, inferring that the –OH group of the phenolic compounds and carboxylate groups of the extract might have bond to silver ions. At the same time, shifting and decreasing bands intensity of peaks 1267 cm-1 and at 1046 cm-1 found for the sample CvAg are related to the C–O linkages or C=O stretching from phenolic and ceto compounds (Fig. 1). After 2 hours of incubation in the dark, at room temperature, the reaction mixture changed from green- ish-brown to light brown (Fig. 3). The color change of the reaction solution has often been mentioned as a marker of successful AgNPs biosynthesis (Chhatre et al. 2012; Şuţan et al. 2016; Reddy et al. 2021; Lalsangpuii et al. 2022). The shoulder peak in the range of 320-330 of the UV-Vis spectra of Cv sample (Figure 4), suggests the presence of flavonoids as a major phenolic compound of aqueous decoction of C. vitalba (Arabshahi-Delouee and Urooj 2007). These results are in accordance with the method of preparing extracts, knowing that hot water is used for the extraction of phenols and f lavonoids (Valencia-Avilés et al. 2018). The maximum absorbance Figure 7. EDS-mapping on the microparticle: Ni grid sample holder (left) and Ag mapping (right). Table 1. The effects of C. vitalba extract and its nanostructured mixture on mitotic index, mitotic cycle and nuclear abnormalities of the A. cepa meristematic roots cells (a, b, c, d, e – the interpretation of the significance of the differences by means of the Duncan test, p < 0.05). Experimental variants MI (%) Prophase (%) Metaphase (%) Anaphase (%) Telophase (%) Binucleate cells (%) Giant cells (%) Anaphase bridge (%) C12 8.4±0.7a 35.7±4.4d 27±3.3bc 21.6±3.2a 15.7±0.5abc 0e 0c 15±5.1a C24 5.5±0.8b 29.2±2.4d 34.6±7ab 18.6±3.4ab 17.7±5.2ab 0.1e 0c 6.7±3.6ab C48 5.8±1.1b 26.8±5.1d 41.9±1.7a 20±4.6a 11.3±1.4bc 0e 0c 14.8±5.2a Cv12 2.1±0.1c 62±1.2bc 21.3±1.8c 13.4±1.8abc 3.3±1.6cd 0.2±0.1cd 0c 0b Cv24 1.7±0.1c 70.9±5.2abc 15.6±1.6c 9.7±1.7bcd 3.8±1.9cd 0.3±0.1bc 0c 0b Cv48 1.3±0.1c 59.4±6.8c 25.5±4.4bc 8.6±4.8cde 6.5±3.3cd 0.5b 0.1±0.1ab 0b CvAg12 1.3c 77.8±4.8b 19.6±2.7c 2.6±2.6de 0 d 0.7±0.1a 0.1±0.1ab 0b CvAg24 1±0.2c 82.2±6.7a 15.2±4.3c 2.6±2.6de 0 d 0.2cd 0.2±0.1a 0b CvAg48 0.6c 77.8±5.6b 22.2±5.6bc 0e 0 d 0.1±0.1de 0.2±0.1a 0b 75In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba aqueous decoction Figure 8. Mitosis, chromosomal aberrations and nuclear abnormalities observed in the root cells of A. cepa L. treated with C. vitalba L. extracts. (a) - normal prophase, metaphase and telophase (C); (b) – normal metaphase and anaphase (C); (c) - giant cells (CvAg24); (d) – anaphase bridge and laggards (C); (e) – binucleate cell (Cv12); (f) – plasmolysis (Cv48). Figure 9. Variation of A. cepa meristematic root cell death (left) and root cell viability (right) after treatment with C. vitalba aqueous extracts prior to and after AgNPs biosynthesis (a, b, c – the interpretation of the significance of the differences by means of the Duncan test, p < 0.05). 76 Nicoleta Anca Şuţan et al. at 436 nm characteristic for CvAg sample (Fig. 4B) is an indicator of the presence of AgNPs. The data in the lit- erature suggest that the presence of peaks closer to the wavelength of 400 nm are characteristic of NPs with dimensions of about 10 nm (Martínez-Castañón et al. 2008). Moreover, the position of the peaks specific to AgNPs is also influenced by the shape of the particles, the interaction between them, as well as the density of free electrons and the dispersion medium (Desai et al. 2012; Agnihotri et al. 2014). The data obtained are con- sistent with the established literature, polyphenols being frequently considered as reducing agents of silver ions (Tyagi et al. 2021). Some authors appreciate that the absence of ions from AgNO3 initially added to the extract is an indica- tion of their reduction and of the successful AgNPs syn- thesis (Kambale et al. 2020; Kthiri et al. 2021). The observed microparticle embedded in a com- plex biological matrix may be due to the agglomera- tion of several AgNPs (Taurozzi et al. 2011). The iden- tification of these microparticles suggests the action of secondary metabolites as capping agents for AgNPs (Kambale et al. 2020). A stabilized coating of biosyn- thesized AgNPs using plant extracts were previously noticed (Jacob et al. 2019). The high free energy from the surface of NPs causes the selective and progres- sive adsorption of biomolecules on their surface when they come in contact with complex biological liquids (Monopoli et al. 2012). The composition of this bio- molecular capping depends on the size, charge, shape of NPs, on the nature of the biological fluids in which NPs are dispersed, hydrophobicity of NPs surface and surface roughness (Piloni et al. 2019; Marichal et al. 2020). Thus, NPs acquire an identity, which must be taken into account in assessing their biological fates and functions (Wypij et al. 2021). Cytotoxicity, genotoxicity and phytotoxicity of C. vitalba extracts prior to and after AgNPs biosynthesis The Allium test was proposed as a standardized method in environmental monitoring, being a fast, low- cost, sensitive test and having a good correlation with other test systems (Fiskesjö 1985). A. cepa is a genetic model frequently used to evaluate the cytogenotoxicity (mitotic index, chromosomal and nuclear abnormalities) and mutagenicity (micronucleus) of chemicals and their mechanism of action on the mitotic apparatus, allow- ing the detection of clastogenic and/ or aneugenic effects (Wieczerzak et al. 2016; Bonciu et al. 2018). In our study, the severe inhibition of mitosis can be attributed to the application of the treatment with the whole and undiluted extract of C. vitalba, suggesting a high toxicity on onion meristematic cells, even only after 12h. The bioactive compounds of the buttercup family, most of them secondary metabolites, induce cell cycle arrest, apoptosis, and inhibit cell proliferation (Segneanu et al. 2015; Hao et al. 2017). Triterpenoid, saponins, phe- nolic acids have been mentioned by Łaska et al. (2021) as potent suppressors of HeLa cells growth and prolifera- tion. It has been suggested that active protoanemonin, released by splitting the glycoside ranunculin, can alkylate proteins and DNA (Wink 2010). Treatment with C. vitalba extract and its nanoformula- tion inhibited mitosis in onion root tips, blocking cells in prophase and significantly reducing the frequency of other phases of mitosis, in a time-dependent manner (Table 1). It is important to note that after 48 hours of incubation in CvAg48, the frequency of anaphase and telophase was zero. These results may be attributed either to suppressing DNA synthesis preventing the cells entering mitosis or to altera- tion of prophase and prometaphase stages (El-Ghamery et al. 2000). These results confirm the severe cytotoxic effects of the tested extracts and nanoformulations on meristemtic Figure 10. The phytotoxicity of C. vitalba extract and its nanostructured mixture on T. aestivum seeds: (A) root and stem growth (B) dry and fresh biomass of germinated seeds (a, b, c – the interpretation of the significance of the differences by means of the Duncan test, p < 0.05). 77In vitro cytotoxic activity of phytosynthesized silver nanoparticles using Clematis vitalba aqueous decoction root cells of A. cepa. Recent studies showed the specificity of AgNPs towards ds DNA, to which it binds and deter- mines its destabilization (Pramanik et al. 2016). Giant cells may be polyploid formed by endomitosis or endoreduplication (Bonciu et al. 2018) or may suggest altered signals for cell growth (Hammann et al. 2020). The cause of binucleate cell formation may be due to inhibition of cytokinesis after telophase (Nefic et al. 2013) or cell plate formation (De Keijzer et al. 2014). However, both types of mitotic abnormalities suggest a disruption of the functioning of the microtubules that make up the mitotic spindle and cell plate, on the basis of which C. vitalba decoct can be classified as aneugenic agent. It has been proved that during plasmolytic process, cortical microtubules and actin microfilaments are sub- jected to architectural changes depending on the severity of water flow (Lang et al. 2014). Microtubules and actin filaments play important roles in establishing the division plan by forming the preprophase band and by forming the phragmoplast that directs the vesicles to form a new cell wall (Rasmussen et al. 2013). Other studies revealed that some substances allow cell plate initiation, but is ultimately disintegrated and the phragmoplast microtu- bules break down (Valster and Hepler 1997). Disrupting the organization of these structures may be the cause of nuclear aberrations and mitotic inhibition. Evans blue staining was applied to assess cell mem- brane integrity. Damaged cell membranes are unable to exclude dye and cells are stained blue. Based on the direct correlation between extracted Evans blue optical density and cell membrane damage, Evans blue staining is a method used to evaluate the cytotoxicity of external stimuli (Roy et al. 2019). Geisler-Lee et al. (2013, 2014) found that AgNPs were progressively accumulated in root cells. Following this bioaccumulation, sever cytotoxicity may be due to AgNPs coating developed during the bottom-up approach of their biosynthesis. It has been suggested that coating of AgNPs affects cellular responses and alters cell fate (Riaz Ahmed et al. 2017). Cytotoxicity caused by biosyn- thesized AgNPs in broth of Pandanus odorifer (Forssk.) Kuntze (Panda et al. 2011) or by citrate-stabilized AgNPs were previously mentioned (Gorczyca et al. 2022). In contrast to the Evans test, TTC is used to assess cell viability. TTC is reduced by the mitochondrial dehydrogenase (active only in the living cells) to red formazan. Therefore, formazan stains only the viable cells and the optical density of the extracted formazan decrease in damaged cells (Roy et al. 2019). The amount of formazan produced directly relates to mitochondrial electron activity, and is proportional to the amount of oxidative damage. In our study, the results suggest high enzymatic activity following stress (Towill and Mazur 1975) imposed by AgNPs and reduced oxidative damage of respiratory processes, perhaps due to adaptive tolerance capacity (De Ronde and van der Mescht 1997). Furthermore, a non-enzymatic reduction of TTC may have been generated under the conditions of incubating onion roots in CvAg, such as incubation time, temperature and pH (Burdock et al. 2011). Nanoparticle-induced phytotoxicity can be assessed by morphophysiological (germination rate, root/stem growth potential, biomass, transpiration rate, clorophyll con- tent, water absorbtion capacity, etc.), cellular or molecular changes (Yan and Chen 2019; Heikal and Şuţan 2021). In our study, improving the growth of roots and stems, but also fresh biomass compared with control may be due to the process of overcompensation hormesis, which is an adaptive response to disruption of homeo- stasis induced by low levels of stress (Calabrese and Bald- win 2002). Testing the phytotoxicity of different concen- trations of uncoated AgNPs with an average size of 13.8 ± 2.5 nm on vegetative growth stages of T. aestivum, Cui et al. (2014) reported similar results. The mode of inter- action and cytogenotoxic effects of NPs depend on many factors, such as the path of synthesis, concentration, expo- sure time, species, growth stage, etc. (Cui et al. 2014; Hei- kal and Şuţan 2021). The particularities of biosynthesized metallic nanoparticles can significantly influence their biological effects. Their cytogenotoxic action depends on numerous factors, such as dosage and exposure duration but also on their chemical composition, surface proper- ties, size and shape. The biosynthesized AgNPs triggered various toxic effects (inhibition of mitosis, disruption of cellular metabolism, impairment of root and stem growth and biomass) that could be attributed to numerous fac- tors, such as dosage and exposure duration but also on their chemical composition, surface properties, size and shape (Heikal and Şuţan, 2021). Furthermore, the pres- ence of a surface coating based on the premise that bio- synthesized NPs in C. vitalba extracts presents various capping biomolecules that influence their morphology, size and stability, which leads to the diversification of bio- nano interactions. Thus, further studies comparing the phytotoxicity of NPs with and without surface coating, as well as identification the secondary metabolites bound on their surface and the way of modulating their own bioac- tivity, should be performed. CONCLUSIONS The bottom-up approach of AgNPs biosynthesis was successfully performed using C. vitalba aqueous decoc- 78 Nicoleta Anca Şuţan et al. tion, which proved to be rich in polyphenols. Phytosyn- tesized AgNPs had approximately spherical shape, sizes ranging from 1-15 nm. The formation of a biomolecular capping induced changes in the size of the green synthe- sized AgNPs. Severe mitoinhibitory effects of C. vitalba extracts were amplified after phytosynthesis of AgNPs. 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Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 76, Issue 2 - 2023 Firenze University Press Molecular classification of Barbeyaceae (Barbeya oleoides Schweinf.) using four different DNA barcodes Fatima Omari Alzahrani, Sami Asir Al-Robai Mitotic metaphase karyotype of the mosquito Anopheles arabiensis Patton (Diptera: Culicidae) from Kassala State, eastern Sudan Asma Mahmoud Hamza1,*, Sumaya Hussein Elboshra2 Karyotypic analysis of Crucian carp, Carassius carassius (Linnaeus, 1758) from cold waters of Kashmir Himalayas Gousia Jan1, Asim Iqbal Bazaz2, Azra Shah1, Saima Andleeb1, Irfan Ahmad1,*, Durdana Qazi1, Oyas Asimi3, Bilal A. 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