Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(3): 75-86, 2019 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-769 Citation: N.K. Bhagyanathan, J.E. Thoppil (2019) Active chemical con- stituents of Cynanchum viminale and its cytotoxic effects via apoptotic signs on Allium cepa root meristematic cells. Caryologia 72(3): 75-86. doi: 10.13128/ caryologia-769 Published: December 13, 2019 Copyright: © 2019 N.K. Bhagyana- than, J.E. Thoppil. This is an open access, peer-reviewed article pub- lished by Firenze University Press (http://www.fupress.com/caryologia) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distri- bution, and reproduction in any medi- um, 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. Active chemical constituents of Cynanchum viminale and its cytotoxic effects via apoptotic signs on Allium cepa root meristematic cells Neethu Kannan Bhagyanathan*, John Ernest Thoppil Cell and Molecular Biology Division, Department of Botany, University of Calicut, Kera- la, India *Corresponding author: neethu_dob@uoc.ac.in Abstract. The present study evaluates the cytotoxic efficacy of methanolic extract of C. viminale on A. cepa root meristematic cells. DAPI staining was used to study the chromosomal aberrations indued by extract of C. viminale. Evans blue staining method was employed to estimate the cell death of root cells of A. cepa. The plant extract was found to impart severe cytological damges, specifically chromosomal aberrations at interphase and prophase stage of cell division. Various apoptotic signs such as apop- totic body formation, nuclear budding, micronucleus, nuclear disintegration, nuclear breakage etc. were observed in meristematic cells of A. cepa. The results suggest the cytotoxic, preferably genotoxic effect of methanolic extract of C. viminale as evidenced by various apoptotic symptoms on A. cepa root cells. Keywords. Cynanchum viminale, aberrations, Allium cepa, cytotoxicity, apoptosis INTRODUCTION Plants have long been used for millennia in traditional medicine against various ailments. Instead of a conventional single compound-single target approach, a consortium of bioactive molecules against multiple targets is gaining more attention nowadays. The synergistic action of various phyto- chemical compounds acts on various target domains, thus increasing ther- apeutic efficacy and eliminating the side effects (Cilla et al. 2015). The Sar- costemma genus (preferably Sarcostemma acidum) is considered as Somalata or Somavalli, also known as moon plant. It is a xerophytic, perennial leaf- less, jointed trailing shrub with green, cylindrical, fleshy glabrous, twining branches having milky white latex, leaves reduced to scales, opposite, flowers white or pale greenish white. The decoction of the plant is useful to gargle for throat and mouth infection, gonorrhoea, muscle pain etc. Recent molecu- lar studies resulted in the taxonomic dissolution of Sarcostemma into Cynan- chum (Meve & Liede- Schumann, 2012). Allium cepa bioassay is an efficient procedure for assessing chromosome damages induced by plant extracts. It is considered as a preliminary cyto- 76 Neethu Kannan Bhagyanathan, John Ernest Thoppil toxic screening test which shows high sensitivity and good correlation with mammalian test systems. It is also an important tool for environmental monitoring stud- ies, employed to assess the impacts caused by xenobiot- ics (Leme & Marin-Morales, 2009; Khanna & Sharma, 2013). The present study is an attempt to evaluate the phy- tochemical constituents of methanolic extract of C. vimi- nale by GC/MS analysis and its cytotoxic screening with special emphasis on apoptotic signs. MATERIALS AND METHODS Plant material Cynanchum viminale (L.) Bassi (1768: 17) subsp. viminale was collected from Karnataka, India (Coor- dinates: 11.8083° N, 76.6927° E). The specimen was authenticated and a voucher specimen (CALI No. 123742) was deposited at the Herbarium of Department of Botany, University of Calicut, Malappuram, Kerala, India. Plant extract preparation 10 g of the ground plant materials were subjected to sequential extraction in n-hexane to remove non-polar components followed by 100 mL methanol. The extract thus obtained is then completely evaporated to remove the trace amount of methanol so as to avoid toxicity. Stock solution was prepared in water and different con- centrations of plant extracts (200, 400, 600, 800 and 1000 µg/mL) were then made from it. GC/MS analysis Chemical composition was determined by GC– MS (Shimadzu QP-2010 Plus with Thermal Desorption System TD 20, fitted with a 60 m × 0.25 mm × 0.25 m WCOT column coated with diethylene glycol (AB- Innowax 7031428, Japan). Helium was used as a carrier gas at a flow rate of 1.21 mL/min at a column pressure of 77.6 kPa. Both injector and detector temperatures were maintained at 260 °C. Samples (6 μL) were inject- ed into the column with a split ratio of 10:0. Compo- nent separation was achieved following a linear tem- perature program of 70-260 °C at 3 ◦C/min and then held at 260 °C for 6 min, with a total run time of 44.98 min. The MS parameters used were: electron ionization (EI) voltage 70 eV, peak width 2 s, mass range 40-850 m/z and detector voltage 1.5 V. The constituents were identified by comparison of their linear retention indi- ces. The MS fragmentation pattern was checked with those of other compounds of known composition, with pure compounds and by matching the MS fragmenta- tion patterns with National Institute of Standards and Technolog y (NIST) mass spectra libraries and with those in the literature (Adams, 2001). Finally, their quantification was performed on the basis of their GC peak areas. Cytotoxic screening on A. cepa Prior to initiating the test, the outer dry scales of onion bulbs were removed without destroying the root primordia. They were allowed for rooting by placing in distilled water for 1-2 days. Germinated bulbs with healthy roots (1-2 cm) were collected at a period of maximum mitotic activity (between 9 am and 10 am on sunny days) and washed with distilled water. The bases of bulbs were kept in vials containing different concentrations of plant extracts (200, 400, 600, 800 and 1000 μg/mL) in such a way that only roots were sus- pended in extracts. Positive and negative controls were also kept viz., hydrogen peroxide (2%) and distilled water. Root tips were collected from the different vials at 12 h, 24 h, and 48 h intervals. The collected sam- ples were washed in distilled water and immediately fixed in modified Carnoy’s fluid for 1 h. Then the root tips were subjected to hydrolysis with 1N HCl for 5-10 min and washed in distilled water followed by incuba- tion in PBS for 15 minutes. Staining was done in DAPI staining solution for 30 minutes in dark condition and washed in PBS by a modified method (Begum & Alam, 2016). Root tips were squashed and mounted in 50% glycerol. Slides were then prepared and the number of damaged cells and total cells were scored in 6 different fields of view using 40X of the fluorescent microscope (Leica DFC 450C, Germany) for cy togenetic effects. Mitotic index (%) and aberration percentage (%) were calculated using the following formulae and values were expressed as mean±SE from at least three inde- pendent experiments: Mitotic Index (%) = Number of dividing cells Total number of cells ×100 Aberration percentage (%) = Number of aberrated cells Total number of cells ×100 77Active chemical constituents of Cynanchum viminale In situ visualization of cell death For the assessment of cell death, control and treated bulbs with intact roots were placed in Evans blue stain- ing solution for 15 min, followed by washing of the roots in running tap water for 30 min (Baker & Mock, 1994). Subsequently, 10 root tips measuring equal length (10 mm) from control and the treated groups were excised and soaked in 3 mL of N, N-dimethylformamide for 1 h at room temperature. The absorbance of Evans blue released was measured spectrophotometrically at 600 nm (Elico SL 218, India). RESULTS GC/MS analysis The volatile composition of methanolic extract of C. viminale was determined by GC/MS. A total of 26 compounds were detected in the methanolic extracts of C. viminale by GC/MS. These compounds belonged to various classes viz., terpenoids, aldehydes, fatty acids, phenolics, fatty acid esters etc. The compounds identi- fied in the methanolic extract of C. viminale by GC-MS analysis are enlisted in Table 1 and gas chromatogram is given as Fig. 1. The major compounds detected were carvone (31.57%), hexadecanoic acid (29.56%) and 9-cis- octadecenoic acid (10.57%). Terpenes were the predomi- nant class of compounds present in the extract; also aldehydes and alcohols in significant quantities. 2-hex- yl-2-decenal, pentadecanal, and myristaldehyde were the aldehydes present in the extract. Coniferyl alcohol, 2,4,4-trimethyl-2-penten-1-ol, (E)-2-nonenol and 1-hep- tanol were the alcohols present in the extract. Nona- noic acid methyl ester, heptadecanoic acid methyl ester, isopropyl pentadecanoate and methyl docosanoate were the fatty acid esters present in the extract. Phenolic compounds like p-vinylguaiacol, 3-tert-butyl-4-meth- oxyphenol and allylsyringol were detected in negligible amounts. An alkaloid, 6-bromo-5-methoxy-Nb methox- ycarbonyltryptamine was also detected in the analysis. The extract contained fatty acids such as myristic acid, Table 1. Chemical composition of C. viminale as analysed by GC/MS. Sl No. RT Compounds Class Content (%) 1 6.58 Limonene Terpene 8.06 2 9.68 Carvone Terpene 31.57 3 10.98 p-vinylguaiacol Phenol 0.37 4 14.25 3-tert-butyl-4-methoxyphenol Phenol 0.43 5 15.85 Allylsyringol Phenol 0.96 6 16.33 Coniferyl alcohol Alcohol 5.34 7 16.35 2-nitropropane Alkane 0.12 8 17.24 Myristaldehyde Aldehyde 1.64 9 17.69 Nitrous acid, butyl ester Carboxylic acid ester 0.13 10 18.12 Nonanoic acid, methyl ester Fatty acid ester 0.47 11 18.45 Myristic acid Fatty acid 0.56 12 18.55 Hexadecanoic acid Fatty acid 29.56 13 18.82 2-methyl 1-butanol nitrite Organic compound 0.1 14 18.91 Acetic acid, methyl ester Carboxylic acid ester 0.11 15 18.95 6-bromo-5-methoxy-Nb methoxycarbonyltryptamine Alkaloid 0.81 16 19.97 Phytol Diterpene alcohol 0.95 17 20.28 9-cis-octadecenoic acid Fatty acid 10.57 18 22.33 Pentadecanal Aldehyde 0.37 19 22.63 Isopropyl pentadecanoate Fatty acid ester 0.9 20 24.11 2,4,4-trimethyl-2-penten-1-ol Alcohol 0.33 21 24.23 Methyl docosanoate Fatty acid ester 0.12 22 24.27 (E)-2-nonenol Alcohol 0.25 23 24.58 4-methyl pentanoic acid Carboxylic acid 0.66 24 36.20 1,5-diazabicyclo[5.4.0]undec-5-ene Amide 0.73 25 37.33 2-hexyl-2-decenal Aldehyde 0.34 26 39.38 1-heptanol Alcohol 4.55 78 Neethu Kannan Bhagyanathan, John Ernest Thoppil Fig. 1. GC chromatogram of methanolic extract of C. viminale [Total ion current (TIC) chromatogram]. 79Active chemical constituents of Cynanchum viminale 9-cis-octadecenoic acid, tridecanoic acid and hexade- canoic acid. Among these, hexadecanoic acid was the predominant one. Negligible quantity of carboxylic acid esters viz., nitrous acid butyl ester and acetic acid methyl ester were also present in the extract. Other compounds belonging to amides, alkanes, alkenes, alkynes and het- erocyclic organic compounds etc. were also detected in trace quantity. Cytotoxic evaluation on A. cepa root meristem Cytotoxic potential of C. viminale in terms of mitot- ic index and chromosomal aberrations were tested on A. cepa root tips. The concentrations of methanolic plant extracts of 200, 400, 600, 800 and 1000 μg/mL as well as incubation period of 12 h, 24 h, and 48 h were taken as the experimental conditions. Time-and dose-dependent increase in chromosome aberrations were observed in A. cepa as visualized by DAPI staining. Effect on mitotic index Reduction in mitotic index is an important factor concerning the cytotoxicity of plant extracts on A. cepa. At 12 h period of incubation, the percentage of divid- ing cells was 86.66 ± 1.07 in the 200 μg/mL concentra- tion of C. viminale (Fig. 2b). On increasing concentra- tion, mitotic index is found to be gradually declined with respect to concentration and exposure time. At the highest concentration, 1000 μg/mL of C. viminale, mitotic index was observed as 31.11 ± 2.24%. In C. vimi- nale extract treatment, mitotic index was found to be even lower than the positive control. The decrease in mitotic index was positively correlated with an increas- ing concentration of plant extracts. In addition to con- centration, the time period is an important factor in genotoxicity and reduction of mitotic index. At the final time period of 48 h, mitotic index (5.47 ± 0.62%) was declined to much lower percentage in all concentra- tions tested than other two time periods considered. In the case of positive control, mitotic index was found to sharply decreased to 2.55 ± 0.56% at 48 h where was in negative control group, no reduction in mitotic index was observed. The progressive reduction in the number of dividing cells at increasing concentrations of plant extracts suggests that the plant extract has a mitodepres- sive effect on the cell division of A. cepa. Effect on chromosomal aberrations Chromosomal aberration percentage is also an endpoint parameter considered for cytotoxicity assays. Time-and dose-dependent increase in chromosome aberrations was observed in A. cepa exposed to plant extracts (Fig. 2a). At the lowest concentration 200 μg/ mL, chromosome aberrations were 13.98 ± 1.74%. As observed in the case of mitotic index of A. cepa root cells treated with plant extract, dose- and time-depend- ent variation of chromosome aberrations were also observed. During 12 h treatment period and at the high- Fig. 2. a: Determination of chromosomal aberrations on A. cepa by C. viminale; b: Mitotic index; c: Spectrophotometric determination of cell death by Evans blue staining. 80 Neethu Kannan Bhagyanathan, John Ernest Thoppil est concentration of methanolic extracts of C. viminale, chromosome aberrations observed were 46.88 ± 0.68%. Hydrogen peroxide was used as the positive control, which induced serious clastogenic aberrations in A. cepa root cells in the form of prominent nuclear lesions. However, the plant extracts at 600, 800 and 1000 μg/mL concentrations induced more aberrations than the posi- tive control. In the case of chromosomal aberrations, it was increased up to 86.24 ± 0.68 % for C. viminale at 1000 μg/mL concentration for 48 h. Wide spectra of chromosomal aberrations were induced by the plant extract, more specifically numer- ous apoptotic symptoms were found to be prominent. The major chromosomal aberrations observed in the study was lesions, nuclear budding, nuclear peak, nucle- ar extrusion, nuclear fragmentation, bridged binucleate cell, giant cells, nuclear disintegration, nuclear erosion, hyperchromasia, fragmentation, cytoplasmic vacuolation etc. (Fig. 3-4). Nuclear buds were observed as frequent chromosomal aberration observed in higher concen- tration of plant extract and its various stages of devel- opment were also observed (Fig. 5). It is noteworthy to observe the apoptotic symptoms such as apoptotic bod- ies, nuclear disintegration, micronucleus etc. in A. cepa cells treated with different concentrations of C. viminale plant extract. Most of the damages were multiple aber- rations such as bridged binucleate cell, giant cell with cytoplasmic shrinkage, shrunken and twisted cell with nuclear diminution, double budding and lesion, chro- mosome fragmentation in the hypoploid cell etc. which indicated the acute cytotoxic potential of the species of Cynanchum. These results suggested the significant cyto- toxic potential or more specifically, genotoxic potential of methanolic extracts of C. viminale on A. cepa meris- tematic cells mediated by apoptotic signs. In-situ visualization of cell death Visualization of cell death of A. cepa root cells was performed by Evans blue staining and their correspond- ing estimation of cell death was carried out spectropho- tometrically at 12, 24 and 48 h of treatment periods. N, N-dimethylformamide was the solvent used to release Evans blue from root cells and the solvent containing Evans blue was then quantified by noting their absorb- ance. Spectrophotometric determination of cell death suggested that severe cytotoxicity was observed in the higher concentration of plant extracts at 48 h of the incubation period. At 12 h of incubation of A. cepa root cells with methanolic extracts of C. viminale, absorbance was found to be gradually increasing with respect to the concentration. Furthermore, cell death was highest in positive control and minimum for negative control. Dose and time served as an important factor concern- ing the cell death of A. cepa by methanolic extracts of C. viminale. Dosage and exposure time was found to be directly proportional to cell death. Incubation of A. cepa root cells with methanolic extracts of C. viminale for 24 h resulted in the cell death of maximum absorbance 0.48 ± 0.02 (Fig. 2c). Finally, incubation of A. cepa with methanolic extracts of C. viminale for 48 h caused a pro- found cell death. The absorbance read was 0.51 ± 0.01, which corresponds to the cell death. Negative control exerted negligible cell death and positive control treated A. cepa showed extremely severe cell death in terms of 0.73 ± 0.03 absorbance. DISCUSSION Methanol has a higher dielectric constant than etha- nol; which enables to extract more polar compounds in comparison with ethanol. As a safety concern, metha- nol content is completely removed by evaporating the extract and thus the further studies were carried out with various concentration of extracts prepared in water. The GC/MS analysis revealed the presence of 26 con- stituents in the methanolic extract of C. viminale (Table 1). The peak with a maximum area of intensity of 31.57% corresponds to carvone followed by hexadecanoic acid (29.56%) and 9-cis-octadecenoic acid (10.57%). Carvone is a monoterpene found as an important constituent of essential oil of spearmint, clove, syzygium etc. (Kokkini et al. 1995; Chaieb et al. 2007) and found to have insecti- cidal and genotoxic activity. Apart from these, limonene is another monoterpene that occupied 8.06% of the total area. The cytotoxic activity of limonene was evaluated in amelanotic melanoma C32, renal cell adenocarcinoma ACHN, hormone-dependent prostate carcinoma LNCaP, and MCF-7 breast cancer cell lines by the sulfo rhoda- mine B assay (Loizzo et al. 2007). p-vinyl guaiacol is a phenolic component present in 0.37% peak area. Also, the compound is a major constituent of almost all essen- tial oils from plants (Bituminaria, Ferula, Torreya etc.) as reported before. 3-tert-butyl-4-methoxyphenol is a phe- nolic constituent and a potential antioxidant compound which was recognized from the essential oil of Dictam- nus dasycarpus which showed significant antimicrobial activity and cytotoxicity towards ACHN, MCF-7, ZR-75- 30, MDA-MB-435S, Hep-G2 and Bel-7402 cell lines (Lei et al. 2008). Coniferyl alcohol is present as 5.34% of the peak area of the total area of intensity. It is synthesized via 81Active chemical constituents of Cynanchum viminale Fig. 3. Chromosomal aberrations induced by extract of C. viminale on A. cepa. a: apoptotic breakage of nucleus at interphase; b: cytoplas- mic vacuolation; c: apoptotic fragmentation of nucleus; d: binucleate cell showing micronuclei; e: binucleate cell with lesions; f: nuclear disintegration; g: apoptotic nuclear disintegration; h: nuclear peak; i: shrunken and twisted cell with nuclear diminution; j: nuclear disinte- gration; k: trinucleate cell; l: trinucleate cell showing different stages of nuclear budding; m: nuclear budding and micronucleus; Bar: 10 μm. 82 Neethu Kannan Bhagyanathan, John Ernest Thoppil Fig. 4. Chromosomal aberrations induced by extract of C. viminale on A. cepa. a: giant cell; b: giant cell with cytoplasmic shrinkage; c: for- mation of apoptotic bodies in the nucleus; d: bridged binucleate cell; e: nuclear extrusion; f: apoptotic body formation; g: nuclear and cyto- plasmic lesions; h: cytomictic transfer of nuclear material; i: nuclear disintegration; j: binucleate cell; k: nuclear lesion; l: double budding and lesion; m: giant cell showing nuclear disintegration and lesion; n: double nuclear lesions; Bar: 10 μm. 83Active chemical constituents of Cynanchum viminale the  phenylpropanoid biochemical pathway and it is an intermediate in the biosynthesis of eugenol, stilbe- noids, and coumarin (Kadir et al. 2015). Myristic acid is another fatty acid component present in 0.56% in the whole content of plant extract. Earlier the antioxidant and larvicidal activity against malaria and filariasis vec- tors were studied using the bioactive fraction of myristic acid from Ammannia baccifera aerial extract (Suman et al. 2013). Phytol is another compound detected (0.95%) and it is a diterpene alcohol found ubiquitously in many plant species. It was well documented that phytol is hav- ing cancer preventive properties irrespective of their concentration in the plant (Hema et al. 2011). In this study, C. viminale contained a bioactive fatty acid hexadecanoic acid which was found to be predomi- nant (29.56%). Hexadecanoic acid, a palmitic acid type compound was detected in extracts of various plants and have shown to possess hemolytic, antioxidant, antican- cer, nematicide, 5-alpha reductase inhibition properties etc. (Jananie et al. 2011; Kalaivani et al. 2012). A sys- tematic study with respect to the fatty acid composition of Sarcostemma viminale has been carried out earlier, where hexadecanoic acid and octadecanoic acid were the major components (Girme et al. 2014). 9-cis-octadeceno- ic acid, another fatty acid present as 10.57% of the total content of volatile compounds present in C. viminale. The derivatives or their esters have the potential to act against cancer or prevent cancer at the very initial stage itself (Farina & Chodahry, 2011). The present study evaluates cytotoxic efficacy of C. viminale mediated by acute apoptotic symptoms in A. cepa root cells. Several researchers had demonstrated the efficacy of A. cepa bioassay for validating the cytotoxic potential of plants. The present observations showed a sharp decline in the mitotic index of A. cepa root cells as a result of treatment with different concentrations of the Fig. 5. Various stages of nuclear budding induced by extract of C. viminale. a: initiation of bud; b: growth of bud; c: protruding as fully formed bud; d: bud with a basal notch; e: detachment of bud from the nucleus; f: micronucleus. 84 Neethu Kannan Bhagyanathan, John Ernest Thoppil plant extract, which is a clear indication of the mitotic depressive effect of the crude plant extracts. The positive control used for the study was H2O2, a serious clastogen which directly interacts with genetic material and result in prominent nuclear lesions in A. cepa which suggest that it interfere with cell cycle mechanism at the ini- tial stage itself; so cells couldn’t be passed onto the next stages of cell cycle. The aberrations induced by plant extract had the potential to affect all phases of cell cycle. Henceforth, these results suggest that the tested concentrations of C. viminale extract is inhibitory, turbagenic and mito- depressive on cell division of A. cepa, which is in agree- ment with Akintonwa et al. (2009). The genotoxic effect of C. viminale was evidenced by a remarkable lower- ing of mitotic division in vegetative cells of A. cepa. In the experiments, mitotic activity showed a tendency to decrease to 5.47 ± 0.62% respectively for C. viminale at the highest concentration (1000 µg/mL) of plant extract at 48 h treatment. This reduction in the mitotic activity could be attributed to inhibition of DNA synthesis or block- age of the cell cycle in G2 phase, thus preventing the cells from entering into mitosis (Sudhakar et al. 2001). Many serious chromosoma l aberrations were observed as a result of treatment with various concen- trations of plant extract. Of these, 90% of the damages were contributed to the genotoxic aberrations. Treat- ment of A. cepa with C. viminale extract resulted in vari- ous apoptotic symptoms like nuclear buds, micronuclei, nuclear fragmentation, nuclear blebbing etc. Most of the damages were nucleotoxic, whereas other aberra- tions were caused by the disturbance on the formation of spindle fibers during cell division. Nuclear buds are one of the prominent aberrations observed in the bioas- say experiment. Four models have been proposed for the generation of nuclear buds. Nuclear buds are formed in the S-phase, representing the expulsion of excess genet- ic material derived from the polyploidization process, which may subsequently lead to micronucleation (Fer- nandes et al. 2007; Lindberg et al. 2007)micronucleus-like bodies attached to the nucleus by a thin nucleoplasmic connection, have been proposed to be generated simi- larly to micronuclei during nuclear division or in S-phase as a stage in the extrusion of extra DNA, possibly giving rise to micronuclei. To better understand these phenom- ena, we have characterized the contents of 894 nuclear buds and 1392 micronuclei in normal and folate-deprived 9-day cultures of human lymphocytes using fluorescence in situ hybridization with pancentromeric and pantelom- eric DNA probes. Such information has not earlier been available for human primary cells. Surprisingly, there appears to be no previous data on the occurrence of tel- omeres in micronuclei (or buds, whose chromatin repli- cation has failed. Nuclear bud formation from broken anaphase bridges (Gisselsson and Pettersson 2000) would appear to be an clear explanation, assuming that the typ- ically stalked structure of a bud results from the collapse of the bridge when it is resolved. The mechanisms responsible for micronucleus have not been yet fully understood. It may have originated during anaphase from lagging acentric chromosomes or chromatid fragments caused by misrepair of DNA breaks or unrepaired DNA breaks (Fenech et al. 2011; Bonciu et al. 2018). Nuclear blebs were also observed in A. cepa cells, consisting of nuclear material, with bud-shaped excrescences on the main nucleus, protruded from the nucleus, but without an obvious constriction or bridge between the protruding nuclear material and nucleus (Wang et al. 2014). Nuclear lesions and erosions are a type of nuclear disintegration, observed frequently in A. cepa cells as a result of treatment with C. viminale extract. These may suggest the direct action of phytochemical components on DNA synthesis and it is a cytological evi- dence for the inhibitory action on DNA biosynthesis and nuclear poisoning (Saghirzadeh et al. 2008; Ngozi, 2011). Nuclear erosion, which may result from the disintegra- tion of chromatid proteins, represents irreversible toxicity (Karaismailoglu et al. 2013). Nuclear extrusion or some- times nucleolar extrusion was another type of clastogenic event frequently observed in A. cepa cells. It is known that the nuclear pore complex (NPC) was the most impor- tant channel for nuclear material transport. The phe- nomenon that the nucleolar material was extruded from the nucleus into the cytoplasm could be explained by the fact that the proteins were affected after plant extract treatment, causing the NPC to lose selectivity (Qin et al. 2010). The fragmentation of nuclei may indicate cell death process and this may ultimately result in aneuploidy and then to cell death. This pattern of nuclear degeneration of nucleus were also observed in programmed cell death in the nucellus of Tillandsia presenting various signals of degeneration like deformed shape, chromatin conden- sation, plasmalemma detachment etc. (Brighigna et al. 2006). Binucleate and trinucleate cells were the frequent aberrations observed in the study, due to the inhibi- tion of cytokinesis in any of the control points of the cell cycle (Özkara et al. 2015). Moreover, shrunken root cells, nuclear blebs, marked nuclear chromatin condensation, fragmentation etc. clearly indicate the possibilities to tend towards apoptosis. These clastogenic, as well as apoptotic signs of aberrations, provide a clue that the plant C. vimi- nale can be effectively utilized for anticancer studies. In addition, it is interesting to highlight the high fre- quency of multiple chromosomal aberrations [bridged 85Active chemical constituents of Cynanchum viminale binucleate cell, giant cell with cytoplasmic shrinkage, chromosome fragmentation in a hypoploid cell, giant cell showing nuclear disintegration and lesion, double nuclear lesions etc.] in cells of A. cepa rather than single aberration by treatment with C. viminale extracts. The above results point to the phytochemicals present in the extracts which might have disrupted the cell cycle mecha- nism since various cytotoxic compounds such as carvone, limonene etc. were detected in GC/MS analysis. They might have possibly interfered with the normal cell cycle process and led to cell death. The present results thus sup- port the notion that the cytotoxic effect of plant extracts is due to the synergistic action of a broad array of phyto- chemicals, the total activity of which may result in health benefits. Moreover, multiple aberrations of chromosomes might have attributed by the multiple compound-multiple target mechanism of interaction between phytochemical constituents of C. viminale and A. cepa cells. Cytotoxic efficacy of C. viminale was then confirmed by estimating the cell death of A. cepa root cells. Evans blue staining method works on the basis of its penetra- tion to non-viable cells (Panda et al. 2011). Evans blue staining of treated and control roots of A. cepa points is considered as an indirect evidence of cell death by visu- alising the intensity of Evans blue taken up by roots, sug- gesting the loss of viability of cells. The intensity of dye absorbed by root cells was directly proportional to the cell death; this could be seen within few minutes after the treatment, in corroboration with the result reported earlier (Achary et al. 2008). Cell death can be positively correlated with an increase in the concentration of plant extract and increase in duration of treatment. CONCLUSION The cytotoxic effects were found to increase propor- tionately with the concentration of plant extract. The chromosomal aberrations observed in this study are evi- dently caused by the chemical constituents in the extract since no aberration was observed in the negative control. The above obtained cytotoxic results may account for the severe cell death and this observation provides a plausi- ble basis for its further use in anti-proliferative studies on in vitro cancer cell lines. However, the mechanism of action remains to be investigated in plant test system and further studies are necessary to clarify the fact. ACKNOWLEDGEMENT The first author gratefully acknowledges the Depart- ment of Science and Technology, Government of India (C/2003/1FD/2014-15) for the financial assistance in the form of INSPIRE fellowship. Thanks are also due to Advanced Instrumentation Research Facility, Jawaharlal Nehru University for the GC-MS facility. REFERENCES Adams RP 2001. Identification of Essential Oils by Cap- illary Gas Chromatography/Mass Spectroscopy, Allured, Carol Stream, IL, USA. Akintonwa A, Awodele O, Afolayan G, Coker HAB 2009. Mutagenic screening of some commonly used medic- inal plants in Nigeria. J Ethnopharmacol 125: 461– 470. Begum K, Alam S 2016. Karyomorphological analysis with differential staining of nine Cicer arietinum L. varieties. Bangl J Bot 45(2), 327–334. Baker C, Mock N 1994. An improved method for moni- toring cell death in cell suspension and leaf disc assays using Evans blue. Plant Cell, Tissue and Organ Culture, 39(1), 7–12. Bonciu E, Firbas P, Fontanetti CS, Wusheng J, Karaismailoğlu MC, Liu D, Menicucci F, Pesnya DS, Popescu A, Romanovsky AV, Schiff S. 2018. An eval- uation for the standardization of the Allium cepa test as cytotoxicity and genotoxicity assay. Caryologia. 71(3): 191-209. Chaieb K, Hajlaoui H, Zmantar T, Kahla-Nakbi A Ben, Rouabhia M, Mahdouani K, Bakhrouf A 2007. The chemical composition and biological activity of clove essential oil,Eugenia caryophyllata (Syzigium aromat- icum L. Myrtaceae): a short review. Phyther Res 21: 501–506. doi: 10.1002/ptr.2124 Cilla A, Attanzio A, Barberá R, Tesoriere L, Livrea MA 2015. Anti-proliferative effect of main dietary phy- tosterols and β-cryptoxanthin alone or combined in human colon cancer Caco-2 cells through cytosolic Ca+ 2– and oxidative stress-induced apoptosis. J Functl Foods 12: 282–293. Farina Asghar S, Choudahry MI 2011. Gas chromatogra- phy-mass spectrometry (GC-MS) analysis of petrole- um ether extract (oil) and bio-assays of crude extract of Iris germanica. Int J Genet Mol Biol 3: 95–100. Fenech M, Kirsch-Volders M, Natarajan AT, Surralles J, Crott JW, Parry J, Norppa H, Eastmond DA, Tucker JD, Thomas P 2011. Molecular mechanisms of micro- nucleus, nucleoplasmic bridge and nuclear bud for- mation in mammalian and human cells. Mutagenesis 26: 125–132. Fernandes TCC, Mazzeo DEC, Marin-Morales MA 2007. Mechanism of micronuclei formation in polyploidi- 86 Neethu Kannan Bhagyanathan, John Ernest Thoppil zated cells of Allium cepa exposed to trifluralin her- bicide. Pestic Biochem Physiol 88: 252–259. Girme AS, Bhalke RD, Nirmal SA, Chavan MJ 2014. Chromatographic and chemical analysis of Sar- costemma viminale R. Br. Orient Pharm Exp Med 14: 279–284. doi: 10.1007/s13596-014-0157-3 Gisselsson D, Pettersson L 2000. Chromosomal breakage- fusion-bridge events cause genetic intratumor hetero- geneity. Proceedings of the National Academy of Sci- ences, 97(10), 5357-5362 Hema. R, Kumaravel. S, Alagusundaram. K 2011. GC/MS Determination of Bioactive Components of Murraya koenigii. J Am Sci 7: 2009–2012. Jananie R, Priya V, Vijayalakshmi K 2011. Determination of bioactive components of Cynodon dactylon by GC-MS analysis. New York Sci J 4: 16–20. Kadir R, Awang K, Khamaruddin Z, Soit Z 2015. Chemi- cal compositions and termiticidal activities of the heartwood from Calophyllum inophyllum L. An Acad Bras Cienc 87: 743–751. Kalaivani CS, Sathish SS, Janakiraman N, Johnson M 2012. GC-MS studies on Andrographis paniculata (Burm.f.) Wall. ex Nees - A medicinally important plant. Int J Med Arom Plants 2: 69–74. Karaismailoglu, M. C., Inceer, H., & Ayaz, S. H. 2013. Effects of Quizalofop-p-ethyl herbicide on the somat- ic chromosomes of Helianthus annus (sunflower). Ekoloji, 89, 49–56. Khanna N, Sharma S 2013. Allium cepa root chromosom- al aberration assay: A review. Ind J Pharm Biol Res 1(3), 105–119. Kokkini S, Karousou R, Lanaras T 1995. Essential oils of spearmint (Carvone-rich) plants from the island of Crete (Greece). Biochem Syst Ecol 23: 425–430. Lei J, Yu J, Yu H, Liao Z 2008. Composition, cytotoxic- ity and antimicrobial activity of essential oil from Dictamnus dasycarpus. Food Chem 107: 1205–1209. Leme DM, Marin-Morales MA 2009. Allium cepa test in environmental monitoring: A review on its applica- tion. Mutat Res – Reviews in Mutat Res 682(1), 71–81. Lindberg HK, Wang X, Järventaus H, Falck GCM, Norp- pa H, Fenech M 2007. Origin of nuclear buds and micronuclei in normal and folate-deprived human lymphocytes. Mutat Res - Fundam Mol Mech Muta- gen 617: 33–45. Loizzo MR, Tundis R, Menichini F, Saab AM, Statti GA, Menichini F 2007. Cytotoxic activity of essential oils from Labiatae and Lauraceae families against in vitro human tumor models. Anticancer Res 27: 3293–3299. Meve U, Liede-Schumann S 2012. Taxonomic dissolu- tion of Sarcostemma (Apocynaceae: Asclepiadoideae). Kew Bullet 67(4), 751–758. Achary MMV, Jena S, Panda KK, Panda BB 2008. Alu- minium induced oxidative stress and DNA damage in root cells of Allium cepa L. Ecotoxicol Environ Saf 70: 300–310. Ngozi E 2011. Mutagenicity testing of pharmaceutical effluents on Allium cepa root tip meristems.  J Toxicol Envtl Health Sci 3(2), 44–51. Özkara A, Akyıl D, Eren Y, Erdoğmuş SF 2015. Poten- tial cytotoxic effect of Anilofos by using Allium cepa assay. Cytotechnol 67: 783–791. Panda K, Achary V, Krishnaveni R, Padhi B 2011. In vitro biosynthesis and genotoxicity bioassay of silver nano- particles using plants. Toxicol. In vitro. 25(5): 1097- 105. Qin R, Jiao Y, Zhang S, Jiang W, Liu D 2010. Effects of aluminum on nucleoli in root tip cells and selected physiological and biochemical characters in Allium cepa var. agrogarum L. BMC Plant Biol 10: 225. Saghirzadeh M, Gharaati MR, Mohammadi S, Ghiassi- Nejad M 2008. Evaluation of DNA damage in the root cells of Allium cepa seeds growing in soil of high background radiation areas of Ramsar - Iran. J Environ Radioact 99: 1698–1702. Sudhakar R, Gowda KNN, Venu G 2001. Mitotic abnor- malities induced by silk dyeing industry effluents in the cells of Allium cepa. Cytologia (Tokyo) 66: 235– 239. Suman TY, Elumalai D, Kaleena PK, Rajasree RSR 2013. GC-MS analysis of bioactive components and synthe- sis of silver nanoparticle using Ammannia baccifera aerial extract and its larvicidal activity against malar- ia and filariasis vectors. Ind Crops Prod 47: 239–245. Wang QL, Zhang LT, Zou JH, Liu DH, Yue JY 2014. Effects of cadmium on root growth, cell division and micronuclei formation in root tip cells of Allium cepa var. agrogarum L. ΦYTON 83: 291–298. Substantia An International Journal of the History of Chemistry Vol. 2, n. 1 - March 2018 Firenze University Press