Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 72(3): 87-95, 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-770 Citation: S. Chatterjee, S. Ray (2019) Clastogenic and cytotoxic effects of aerial parts’ aqueous extract of Syn- edrella nodiflora (L.) Gaertn. on Wistar rat bone marrow cells. Caryologia 72(3): 87-95. doi: 10.13128/caryolo- gia-770 Published: December 13, 2019 Copyright: © 2019 S. Chatterjee, S. Ray. 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 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. Clastogenic and cytotoxic effects of aerial parts’ aqueous extract of Synedrella nodiflora (L.) Gaertn. on Wistar rat bone marrow cells Saumabha Chatterjee1,2, Sanjib Ray2* 1 Department of Zoology, Durgapur Govt. College, Durgapur-713214 2 Molecular Biology and Genetics Unit, Department of Zoology, The University of Burd- wan, Golapbag, Burdwan-713104, West Bengal, India *Corresponding author: ray.sanjibray@gmail.com Abstract. Synedrella nodiflora is a traditionally used medicinal plant. The aim of the present study was to analyze the clastogenic and cytotoxic effects (CCEs) of aerial parts’ aqueous extract of S. nodiflora (AAESN) on Wistar rat bone marrow cells (WRB- MCs). The CCEs of AAESN were analyzed with light and fluorescence microscopes respectively. The data indicate a dose-dependent (100-500 mg/kg body weight [bw]) increase in the aberrant cell frequencies, chromosomal structural aberrations and a sig- nificant (p<0.001) increase in apoptosis in the AAESN treated WRBMCs. The chromo- somal aberration per 100 cells (apoptosis %) were calculated as 2.01±0.241 (5.02±1.72), 4.76±0.05 (46.73±2.34), 5.37±0.32 (66.92±2.92) and 6.58±0.14 (76.79±0.73) respec- tively for the AAESN doses of 0, 100, 300 and 500 mg/kg bw. In conclusion, the AAESN may contain phytochemicals with clastogenic and cytotoxic efficacy on WRB- MCs, indicating, having the anticancer as well as carcinogenic potentials. Therefore, it demands further an elaborate study to explore the active principle(s) and a proper care should be taken while it is prescribed in traditional medicine. Keywords. Apoptosis, Clastogenic, Cytotoxic, Synedrella nodiflora, Genotoxic, Anti- cancer, Secondary metabolites. 1. INTRODUCTION Cancer is an important worldwide health problem and the antiprolifera- tive pharmacological activities of plant-derived secondary metabolites appear to explain the anticancer effects (Figueroa-Hernandez et al. 2005). A varie- ty of bioactive components were isolated from the different medicinal herbs (Cragg et al. 1996). Alkaloids, fixed oils and fats, polyphenols, flavonoids, saponins, glycosides, terpenoids etc., having medicinal value, were extracted from a wide variety of plant species (Tamilarashi et al. 2000). Many plant- based active compounds act as antitumor and apoptotic cell death inducer in tumors (Sato et al. 1994). Acetogenins like uvaribonin, 22-epicalmistrin, and chalcone showed significant antiproliferative activity against a panel of can- cer cell lines (Pettit et al. 2008). Generally, the cell cycle components are the 88 Saumabha Chatterjee, Sanjib Ray prime targets of most of the efficient anticancer agents (Li et al., 2002). The discovery of competent anticancer drugs like vincristine and vinblastine were isolated from Catharanthus roseous; Paclitaxel (Taxol®) extracted from Taxus brevifolia represent trustworthy proof that plants are potential sources of novel anticancer chemothera- peutic drugs (Cragg et al., 1996). The antiproliferative, clastogenic, and cytotoxic pharmacological activities of plant-derived secondary metabolites appear to elucidate the chemo-preventive or anticancer effects. Therefore, searching for the phytochemicals having the clastogenic and cytotoxic effects (CCEs) are of renewed interest in drug discovery for cancer treatment. The CCEs of a medicine provide a glimpse into its mechanism of action. A clastogen can cause chromo- somal structural alterations like chromatid break, dele- tion, sister chromatid exchanges, sister chromatid union, dicentric chromosome, acentric fragments, micronuclei etc. that subsequently may lead to the various cytotoxic effects including cell killing, apoptosis, and necrosis. Moreover, the medicinal plants having these effects, one can also assume their toxicity risk factor for its indis- criminate use in the traditional therapeutic purpose (Thybaud et al. 2007). Cancer chemotherapeutic drugs are generally cytogenotoxic, hence subjected to non-tar- get destruction but non-cancerous cells can revive bet- ter than the cancerous one (Choudhury et al. 2000; Palo et al. 2009). Since these clastogenic chemotherapeutic drugs might enhance the chance of secondary cancer development, dose optimization, target specification, and combination chemotherapy with the other antioxi- dants are highly recommended (Pandit and Choudhury, 2011). Many anti-cancer agents cause DNA damage at a very high level leading to the cell cycle checkpoint acti- vation and programmed cell death (Helleday et al. 2008). Administration of the many plant-derived anticancer agents, including paclitaxel, can affect spindle stability leading to abnormal mitosis and chromosomal aberra- tion (Dumontet and Jordan, 2010). Synedrella nodiflora (L.) Gaertn. (Family: Asterace- ae) is an ephemeral flowering weed. It is indigenous to tropical America and also distributed in India, Malaysia, Bangladesh, China, Japan, and other Indopacific coun- tries (Wiart 2006). In India, S. nodiflora leaves are tradi- tionally applied for the remedy of rheumatism. In Gha- na, oral application of warm aqueous juice of this plant results in the remedy of epilepsy. In Malaysia, it is used externally as a medicine for the treatment of inflamma- tion, headache, and earache. The leaves are also used for the treatment of hiccup, stomachache, and threatened abortion cases (Rathi and Gopalkrishnan 2005; Rah- matullah et al. 2010; Bhogaonkar et al. 2011). The toxi- cological (Olukunle and Abatan 2008; Dutta et al. 2012), insecticidal (Rathi and Gopalkrishnan, 2005), larvicidal (Ghayal et al. 2010), antibacterial, antioxidant (Wijaya et al. 2011), antidiarrhoeal, hypoglycaemic (Zahan et al., 2012), anti-inflammatory properties (Haque et al. 2012) of this plant have been reported. Our previous study revealed the antiproliferative activity of the aerial parts’ aqueous extract of S. nodiflora (AAESN) on root apical meristem cells and Wistar rat bone marrow cells (WRBMCs) as well as the presence of different phyto- chemicals like alkaloids, flavonoids, terpenoids, tannins, phlobatannins, and saponins in the AAESN (Ray et al. 2013b). However, the CCEs of AAESN on the mamma- lian system has not been well-studied. Thus, the present study is focused on the assessment of the clastogenic and cytotoxic effects of the aerial parts’ aqueous extract of S. nodiflora on WRBMCs in vivo condition. Nabeel et al. (2008) described the cytogenetic effect of the aqueous extract of Arum maculatum on the Bone Marrow Cells of the Swiss male mice. Some of the parameters record- ed by the scientists were also taken into consideration in this study. The novel aspects of this study are that it explored the CCEs of AAESN, a source of future anti- cancer chemotherapeutic drugs, and raised the question against its indiscriminate use in traditional medicine. 2. MATERIALS AND METHODS 2.1. Chemicals Colchicine, glacial acetic acid, and methanol were obtained from BDH Chemicals Ltd., UK. EDTA was procured from Gibco, Grand Island, N.Y, USA. Ethidi- um bromide and acridine orange were purchased from Sigma, St. Louis, M.O., USA and S.D. Fine-Chem. Ltd., Mumbai, India respectively. Other chemicals used in this work were of analytical grade from reputed manu- facturers. 2.2. Plant products collection, storage, and extract prepara- tion Plant aerial parts collection, storage, and extract preparation procedures were described in detail in our earlier report (Ray et al. 2013b) and briefly the fresh aerial parts’ of S. nodiflora were collected from Golap- bag campus of The University of Burdwan, taxonomi- cally authenticated by Prof. Ambarish Mukherjee, and the voucher specimen (No.BUGBSC013) is maintained in the Department ( Figure 1). The dried and pulver- ized plant product was boiled in double-distilled water 89Clastogenic and cytotoxic effects of Synedrella nodiflora (1:10, W/V) in a water bath for 30 min. The extract was allowed to cool to room temperature, filtered by What- man filter paper No. 1 (Sigma-Aldrich, Inc., St. Louis, MO, USA), and then refrigerated at -20°C for further use. For the measurement of extract value (17.64%w/w) and extract concentration (11.3 mg/mL), 10 mL of extract was kept for evaporation to complete dehydra- tion in a hot air oven at 60 ºC. 2.3. Experimental animals Male Wistar-albino rats (age 4-6 weeks; weight 40–60 g) were purchased from local vendors and main- tained in the Departmental animal house (in com- munity cages) at room temperature (25±2oC), con- trolled illumination (12 h light and 12 h dark cycle), and with standard rat diet and water. The rules of the “Institutional Animal Care and Use Committee” were strictly followed throughout the whole experi- ment and the required total 24 rats were euthanized with prior approval from the Dissection Monitoring Committee (DMC) of The University of Burdwan (No: R-S/N-1/646, Dated 30-03-2016; Under Ref. No. BU- DMC/2016/01/05(a) Dated 13.07.2016). 2.4. Treatment and clastogenicity analysis The AAESN (100, 300 and 500 mg/kg body weight [bw]) was injected into the peritoneal cavity of the male Wistar rats (Ray et al. 2013b). Control rat groups were injected an equal volume of double distilled water. At each data point, six rats were used. After 12 h of AAESN injection, colchicine (10 mg/kg bw), a standard meta- phase arresting agent, was injected into the peritoneal cavity of the rats irrespective of control and treatment groups for 3 h (Ray et al 2013b). Then the animals were euthanized by cervical dislocation just before the femur bones were dissected out (Ray et al. 2013b) and the WRBMCs were fixed in aceto-methanol (1:3) after the required hypotonic (0.56% KCl) treatment. Control group was considered for nullifying the sole toxic effect of col- chicine. The detailed procedure of metaphase plate prepa- ration and Giemsa staining procedure were described earlier (Ray et al. 2013b). Briefly, the femur bones were dissected out, the bone marrow cells were collected in 15 mL centrifuge tubes by flushing with pre-warmed (37°C) 2.5 mL of 0.56% aqueous KCl solution with 5 mL hypo- dermic syringe, the cells were maintained in a hypotonic solution for 30 min at 37°C in a water bath and then the cells were fixed with aceto-methanol (methanol 3 parts and acetic acid 1 part). Metaphase plate preparation was done through flame-drying technique and 2% Giemsa (staining duration: 35 min) was used for staining. The Giemsa stained slides were mounted with a coverslip in synthetic medium and the different types of chromosom- al abnormalities like chromatid break, terminal deletion, fragmented chromosome, centric fusion, centromeric association, ring chromosome, chromatid gap, chromo- somal association, end to end association etc. were scored (Kumpawat et al. 2003). 2.5. Fluorescence microscopic cytotoxicity analysis The acridine orange-ethidium bromide (AO-EB) double staining procedure (Bustillo et al. 2009) was used to determine cytotoxic effects of AAESN in terms of early and late phases of apoptosis with the fluorescence microscope by observing changes in nuclear morphol- ogy and apoptotic blebbing. It is the established fact that acridine orange can infiltrate in both the live and dead cells while ethidium bromide enters only in dead cells. The AO-EB staining strategy causes color differentiation with blue-filter excitation; the living cells (stained only with acridine orange) give green fluorescence, the early apoptotic cells (permitting limited penetration of ethid- ium bromide) show green to yellowish nuclei with peri- nuclear chromatin condensation, the late apoptotic cells show a dark red color with fragmented or condensed chromatin, and the necrotic cells give red color with large nucleus having no condensed chromatin. Here, the AAESN treatment and WRBMCs collec- tion procedures were same as described for clastogenic- ity analysis, except, the harvested cells were washed in 1X PBS instead of hypotonic KCl solution. After cen- Fig. 1. Showing the aerial parts (leaf, stem, and flower) of Synedrel- la nodiflora (L.) Gaertn. 90 Saumabha Chatterjee, Sanjib Ray trifugation at 1000 rpm for 5 minutes and discarding the supernatant, the precipitates were stained with acri- dine orange-ethidium bromide mix (conc. 100 µg/mL) in 1:1 ratio for 5 minutes in 2 mL Eppendorf tubes. Then, cells were washed thrice with 1X PBS repeating the cen- trifugation steps. The cells were resuspended in 100 µl 1X PBS and then 20 µL cell suspensions were taken on grease free slide, the percentages of dead cells (apoptotic vs necrotic) and live cells were scored under Leica fluo- rescence microscope. 2.5. Scoring and Statistical analysis All the results were expressed as Mean±SEM. Aber- rant cell percentage were analyzed through one way ANOVA (d.f.=11) followed by Tukey-Kramer tests. The differences between the untreated and treated groups for cellular viability and chromosomal abnormalities were analyzed with the 2x2 contingency χ2-test and were considered statistically significant at p<0.05, p<0.01 and p<0.001. 3. RESULTS 3.1. Clastogenic effects of AAESN Metaphase chromosomal abnormalities were scored in all the AAESN treated groups of rats and were com- pared with the untreated controls. The various clasto- genic effects were observed in the AAESN treated rats. The number of cell abnormality percentage was cal- culated as 4.76±0.03, 5.37±0.32, and 6.58±0.14 after AAESN treatment respectively with 100, 300, and 500 mg/kg bw as compared with the control (2.01±0.24 %) (Table 1). Among the different clastogenic effects scored, chromatid break, terminal deletion, centric fusion, cen- tromeric association, ring chromosome, chromosomal association, and an end to end association followed a dose-dependent increase in frequencies. Here, the chro- mosomal association (0.98±0.01) percentage was found to be the highest frequency of chromosomal abnormality followed by centric fusion (0.89±0.03), fragmented chro- mosome (0.88±0.01), end to end association (0.86±0.02), centromeric association (0.83±0.01), chromatid break (0.54±0.03), ring chromosome (0.51±0.06), and terminal deletion (0.45±0.02) after the treatment with 500 mg/kg bw of AAESN (Figure 2 and Table S1). 3.2. Fluorescence microscopic analysis for cytotoxicity Apoptotic cells were examined under a f luores- cence microscope after acridine orange and ethidium bromide (AO-EB) combined staining of WRBMCs. A dose-dependent increase in the apoptotic cells (%) was observed in AAESN treated samples. As compared with the untreated cells, a significantly (p<0.001) increased percentages of early and late apoptotic cells, and necrotic cells were observed in AAESN treated WRBMCs. The dose-dependent response was more prevalent in apop- totic cell death than that of necrosis. The maximum per- centages (85.40±1.71) of viable cells were counted in the Table 1. Pooled data showing the AAESN induced aberrant cell percentage of WRBMCs. AAESN treatment Dose (mg/kg bw) TM Ab.Cell(%)= (TAC/TMC)x100 Range Mean±SEM (% increase) 00 100 1.72-2.48 2.01±0.24#♯ᵟ 100 96 4.69-4.84 4.76±0.05*♯ᵟ (136.8) 300 94 4.81-5.93 5.37±0.32*#ᵟ (167.2) 500 109 6.42-6.86 6.58±0.14*#♯ (227.4) *Significant at p < 0.05 as compared to the control, # at p < 0.05 as compared to the 100 mg/kg AAESN treatment, ♯ at p < 0.05 as compared to the 300 mg/kg AAESN treatment, ᵟ at p < 0.05 as com- pared to the 500 mg/kg AAESN treatment by one way ANOVA (d.f.=11) followed by Tukey-Kramer Procedure. bw; body weight, TM; Total no. of metaphases counted for studying chromosomal abnormality, Ab.Cell %; aberrant cell per percentage; TAC; Total number of aberration count, TMC; Total metaphase count. 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 * * * * *** * * * * ** *** ** * * ** * * * ** ** 00 100 300 500 D .A b/ 10 0 C el ls Doses of AAESN (mg/kg bw) CB TD FC CF CtA RC CA EA Fig. 2. Influence of AAESN on WRBMCs in terms of different chromosomal abnormalities. *Significant at p<0.05, **at p<0.01 and ***at p<0.001 as compared to the control by 2x2 contingency χ2-test (d.f.=1). The data were represented as Mean±SEM. D.Ab./Cell: Dif- ferent abnormalities per cell scored; CB: Chromatid break; TD: Ter- minal deletion; FC: fragmented chromosome; CF: Centric fusion; CtA: Centromeric association; RC: Ring chromosome; CA: Chro- mosomal association; EA: End to end Association. 91Clastogenic and cytotoxic effects of Synedrella nodiflora untreated samples and the maximum percentages of ear- ly apoptotic (20.92±0.34), late apoptotic (55.87±0.69) and the total apoptotic cell frequency (76.79±0.73) were cal- culated in AAESN (500 mg/kg bw) treated samples. The maximum percentage (5.34±0.57) of necrotic cells were scored at a dose of 300 mg/kg bw of AAESN for 500 mg/ kg of AAESN treatment (Figure 3 and Table S2). 4. DISCUSSION The leaves of Synedrella nodiflora are traditionally used for the remedy of rheumatism, epilepsy, inflam- mation, headache, stomachache, and earache (Rathi and Gopalkrishnan 2005; Rahmatullah et al. 2010; Bhogaonkar et al. 2011). In the present study, we have tested in vivo CCEs of AAESN on the WRBMCs. The rapidly dividing bone marrow cells are the ideal mod- el to demonstrate antiproliferative activity of herbal extracts (Ray et al., 2013 a, b). The WRBMCs are also considered as a basic model for clastogenicity testing and many authors have used it to study the side-effects of anti-cancer/anti-inf lammatory medicines (Pearse et al. 2009; Pandit and Choudhury 2011; Haque et al. 2012; Ray et al. 2013a; Ray et al. 2013b; Gomaa 2018). Generally, the bone-marrow mito-depressive drugs show anticancer activity (Prasanthi, 2016). The previ- ous studies indicated the mutagenic activity of pyre- throids on murine bone marrow cells, human peripheral blood lymphocytes, and in aquatic animals (Barrueco et al. 1992; Oraby 1997). The general non-steroidal anti- inflammatory drugs (NSAIDs) may also inhibit the pro- liferation of bone marrow cells without any intervention of hormonal activities in murine models (Chang et al., 2007). The study of the bone marrow suppression is des- ignated as a common toxicity assessment of cytotoxic agents and this toxicity assay had been included in the preclinical study of the four-stage trial system (Heidel- berg and Fox, 1990). Our earlier study indicated a dose-dependent decrease in metaphase frequency (Ray et al. 2013b) and in the present study a dose-dependent increase in chro- mosomal abnormalities (both total and differential counts) and apoptotic cells percentage in WRBMCs. Sev- eral anticancer agents put forth their influence through cell cycle events (Salmon et al., 1984). The antiprolifera- tive activities of the several herbal extracts are related to their ability to obstruct DNA synthesis (Akinboro and Bakare 2007; Mercykutty and Stephen 1980). Toona sin- ensis leaf aqueous extract has been reported to have an anti-proliferative influence on human lung cancer cells (Laosinwattana et al. 2007, 2009). In the present study, clastogenicity of AAESN on the WRBMCs revealed that several types of chromosomal abnormalities including chromosomal fragmentation, chromatid break, ring chromosome formation, centro- meric association etc. were induced by AAESN. Kump- awat et al. (2003) showed that raw betel-nut extract introduced clastogenicity on mouse bone marrow cells and human peripheral blood lymphocytes. There are similar kinds of study reports where they explored the genotoxic activity of plant extracts on WRBMCs and human peripheral blood lymphocytes (Pandit and Choudhury 2011; Sakamoto-Hojo et.al. 2017). We previously reported the cytogenotoxic altera- tion of onion apical meristem cells that were exerted by the aerial parts’ aqueous extract of Ampelocissus lati- folia (Chaudhuri and Ray 2014). Nefic (2008) described the effect of ascorbic acid on human peripheral blood lymphocytes, where vitamin-C at a higher concentra- tion (1,000μg/mL) could induce mitotic arrest and chro- mosomal abnormalities. The similar kinds of dose-opti- mization studies were performed using the methanolic extracts of Artemisia annua and Pyracantha coccinea on Allium cepa root apical meristem cells (Karaismailo- glu MC 2014, 2017). Pandit and Choudhury (2011) nar- rated the clastogenic effect of a chemotherapeutic drug on mouse bone marrow cells. Gewirtz (1999) revealed cytogenotoxic effect of anthracyclin antibiotics due to suppression of Topoisomerase-II and thus hindering Topoisomerase-II mediated DNA cleavage and re-liga- tion. Moreover, it also triggers ROS generation (Doro- sho, 1983). 0 20 40 60 80 100 ** ** * ** * ** * ** * ** * ** * ** * ** * ** * * ** * ** * * ** * 50030010000 C el l ( % ) Doses of AAESN (mg/kg) NVC EAC LAC TAC NC Fig. 3. Shows AAESN induced cytotoxicity observed on WRBMCs under a fluorescent microscope. NVC: normal viable cells; EAC: early apoptotic cells; LAC: late apoptotic cells, TAC: total apoptotic cells. NC: necrotic cells. Experiments were done in three sets and data were represented as Mean±SEM. *Significant at p<0.05, **at p<0.01 and ***at p<0.001 as compared to the control by 2x2 contin- gency χ2-test (d.f.=1). 92 Saumabha Chatterjee, Sanjib Ray Many anti-cancer agents cause DNA damage at a high level leading to checkpoint activation and pro- grammed cell death (Helleday et al. 2008). Adminis- tration of anticancer agents like paclitaxel can affect spindle stability leading to abnormal mitosis and chro- mosomal aberration (Dumontet and Jordan, 2010). One noticeable thing is that colchicine, a mitostatic drug, was also used here to arrest the cells at metaphase but it was equally administered to both control and treatment groups. Here, the treatment group showed a significantly higher level of clastogenic alterations of chromosomes in comparison to the control group in a dose-dependent manner. So, apart from colchicine, there must be extra clastogenic effects of AAESN. We previously report- ed cell cycle retardation effect as well as the increased prophase-metaphase frequency on Allium cepa root api- cal meristem cells treated by the same extract. Recently, Bonciu et al. (2018) used Allium cepa root apical meris- tem cells as a genotoxicity test system. The mito-retard- ing effect was also noticed in case of WRBMCs in that study in a dose-dependent manner, apart from the influ- ence of colchicine in both control and treatment groups (Ray et al., 2013b). Hence, the strong probability of interaction of the phytochemical(s) present in AAESN with mitotic spindle could not be ignored. However, a detailed mechanism of clastogenic action of AAESN is subjected to further detailed study. The AO-EB combined staining assay data indicate a dose-dependent increase in the apoptotic cell frequen- cy to a much greater extent than that of necrotic cells. Treatment with AAESN caused the characteristic chang- es related to apoptotic morphologies in WRBMCs indi- cating that S. nodiflora is an extensive source of natural bioactive substances with apoptotic cell death-inducing activity on WRBMCs. Another important observation was that the necrotic cells’ increased frequency did not follow a dose-response relationship and the data, in turn, suggest an apoptotic potential of AAESN. The maximum percentage (5.34±0.57) of necrotic cells were scored at a dose of 300 mg/kg bw of AAESN, indicating, unlike apoptosis, the necrotic cell frequencies did not follow a dose-dependent response pattern. There are similar reports showing the apoptotic cell death-inducing effects of some of the anticancer agents like isodeoxyelephantopin (Farha et al., 2013) and farnesiferol c (Hasanzadeh et al., 2017). Previously, we also described the cytotoxic effect of aerial parts’ aque- ous extract of A. latifolia on apical meristem cells using AO-EB staining method (Chaudhuri and Ray 2014). Chu et al. (2014) showed the antiproliferative and cytotoxic effect of Camptothecin-20(s)-O-(2-pyrazolyl-1) acetic ester (CPT6) on breast tumor MCF-7 cells by increased sub G1 cell population and apoptosis induction among the treatment groups. Farha et al. (2013) reported iso- deoxyelephantopin (IDOE) mediated apoptosis on naso- pharyngeal carcinoma (KB) cells by obtaining more apoptotic morphologies through AO-EB combined staining assay in IDOE treated KB cells. Our results are in agreement with those of Ichikawa who reported the apoptosis-inducing effects of isodeoxyelephantopin in various cells (Ichikawa et al., 2006). Our previous study revealed the presence of alka- loids, tannins, terpenoids, f lavonoids, phlobatannins, and saponins as active ingredients in this extract (Ray et al. 2013b). These active ingredients might have inter- acted with the cell cycle machinery and/or with DNA thus inducing cell cycle delay, clastogenicity, and apopto- sis. The AAESN-induced abnormal mitosis in WRBMCs might lead to the mitotic catastrophe through apoptosis and necrosis. Mitotic catastrophe is an intrinsic mechanism that senses mitotic failure / abnormal mitosis and responds by driving a cell to an irreparable antiproliferative fate of death or senescence (Margaret 2015; Vakifahmetoglu et al. 2008). Here, the mitotic catastrophe induced by AAESN was more prone to result in apoptosis rather than necrosis which is more desired outcome in can- cer chemotherapy. Our results related to toxicity of this plant are in agreement with the histopathological toxic- ity and brine shrimp lethality of S. nodiflora (Olukunle et al. 2008; Dutta et al. 2012). 5. CONCLUSION The phytochemicals present in aerial parts’ aque- ous extract of Synedrella nodifolia could induce cyto- toxicit y, clastogenicit y and mitotic catastrophe in WRBMCs and thus indicate its potential use in cancer chemotherapy in the near future. There is ample scope to explore the active principle(s) of AAESN and the detailed molecular mechanism of the CCEs. 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Supplementary pooled data of Fig.2 showing the AAESN induced different categories of aberrant cell percentage of WRBMCs. Doses (mg/ kg bw) of AAESN TM D.Ab/Cell (%) CB TD FC CF CtA RC CA EA 00 100 0.14±0.04 0.05±0.04 0.48±0.05 0.15±0.03 0.31±0.04 0.1±0.03 0.27±0.05 0.11±0.03 100 96 0.43±0.11b 0.09±0.00 0.93±0.01b 0.60±0.05c 0.49±0.03 0.37±0.04c 0.87±0.03c 0.5±0.03c 300 94 0.46±0.11c 0.27±0.05c 0.87±0.05a 0.65±0.05c 0.59±0.02a 0.46±0.08c 0.96±0.04c 0.63±0.08c 500 109 0.54±0.04c 0.45±0.03c 0.88±0.01b 0.89±0.04c 0.83±0.02c 0.51±0.08c 0.98±0.01c 0.86±0.03c aSignificant at p<0.001, bat p<0.01 and cat p<0.05 as compared to the control by 2x2 contingency χ2-test (d.f.=1). TM; Total no. of meta- phases counted for studying chromosomal abnormality, D.Ab./Cell: Different abnormalities per cell scored; CB: Chromatid break; TD: Ter- minal deletion; FC: fragmented chromosome; CF: Centric fusion; CtA: Centromeric association; RC: Ring chromosome; CA: Chromosomal association; EA: End to end Association. Table S2. Supplementary pooled data of Fig. 3 showing AAESN induced apoptosis and necrosis in WRBMCs in vivo. Doses (mg/ kg bw) of AAESN TC NVC EAC LAC TAC NC TC Mean±SEM TC Mean±SEM TC Mean±SEM TC Mean±SEM TC Mean±SEM 00 1728 1476 85.40±1.71 102 6.26±0.78 150 8.75±1.23 252 15.02±1.72 0 - 100 2015 1035 51.44±2.00a 240 11.93±0.89c 700 34.80±1.54a 940 46.73±2.34a 40 2.00±0.33c 300 1677 468 27.92±1.16a 258 15.43±0.86a 861 51.49±2.12a 1119 66.92±2.92a 90 5.34±0.57a 500 2066 404 19.57±0.45a 432 20.92±0.34a 1154 55.87±0.69a 1586 76.79±0.73a 76 3.68±0.24b Conc.: Concentration; TC: total cells; NVC: normal viable cells; EAC: early apoptotic cells; LAC: late apoptotic cells, NC: necrotic cells, TAC: total apoptotic cells aSignificant at p<0.001 as compared to the control by 2x2 contingency χ2-test (d.f.=1). Experiments were done in triplicate and data were represented as Mean±SEM. Substantia An International Journal of the History of Chemistry Vol. 2, n. 1 - March 2018 Firenze University Press