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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Cytotoxic and genotoxic activity of some Helleborus species a
a
a
Jasmina Čakar , Anja Haverić , Sanin Haverić , Milka Maksimović
b
c
& Adisa Parić a
Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Zmaja od Bosne 8, Sarajevo, Bosnia and Herzegovina b
Department of Chemistry, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina c
Department of Biology, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina Published online: 03 Mar 2014. To cite this article: Jasmina Čakar, Anja Haverić, Sanin Haverić, Milka Maksimović & Adisa Parić (2014) Cytotoxic and genotoxic activity of some Helleborus species, Natural Product Research: Formerly Natural Product Letters, 28:12, 883-887, DOI: 10.1080/14786419.2014.889135 To link to this article: http://dx.doi.org/10.1080/14786419.2014.889135
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2014 Vol. 28, No. 12, 883–887, http://dx.doi.org/10.1080/14786419.2014.889135
Cytotoxic and genotoxic activity of some Helleborus species
Downloaded by [Florida International University] at 02:52 04 September 2014
Jasmina Cˇakara*, Anja Haveric´a, Sanin Haveric´a, Milka Maksimovic´b and Adisa Paric´c a Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Zmaja od Bosne 8, Sarajevo, Bosnia and Herzegovina; bDepartment of Chemistry, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina; cDepartment of Biology, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina
(Received 26 November 2013; final version received 27 January 2014) Despite their known toxic properties, various Helleborus species are used as medicaments in folk medicine to treat some diseases and health conditions. As the main mechanism of many cytostatic drugs is based on their cytotoxic activity, there is potential for the toxicity of hellebore to be used in anticancer therapy. This study tested the geno- and cytotoxic effects of extracts of three hellebore taxa (Helleborus odorus, Helleborus multifidus and Helleborus hercegovinus) on meristemic onion (Allium cepa L.) cells and human lymphocytes. Treatments with Helleborus extracts induced cytotoxic and cytostatic effects in meristemic onion cells as well as in cultivated cytokinesis-blocked human lymphocytes. Cytokinesis-block micronucleus cytome assay indicated that treatments with hellebore extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of their antiproliferative activity is apoptosis induction. Keywords: cytotoxic activity; genotoxic activity; cytostatic activity; Helleborus; mitotic index; nuclear division index
1. Introduction Plants are the source of numerous compounds used for the prevention and treatment of diseases. Many of these compounds are also toxic, depending on the dosage. These contrary properties are also highly dependent on the choice of test systems and protocols, on the interactions between specific test chemicals and on the physiology and life cycle stage of the cell or organism (Musarraet et al. 2006). Some plant extracts act by disrupting and/or inhibiting the cell cycle, which frequently results in apoptosis-induced cell death, which may be the mechanism of their antiproliferative activity. Therefore, phytopreparations that have cytotoxic activity could potentially be used as medications for a number of health conditions that primarily feature uncontrolled cell proliferations. Numerous studies point to the bioactive properties of extracts of some Helleborus species (Bu¨ssing & Schweizer 1998; Lindholm et al. 2002; Jesse et al. 2009). Extracts of some Helleborus species have immunostimulatory and anti-inflammatory properties (Linke et al. 1998). Lindholm et al. (2002) state that Helleborus cyclophyllus Boiss. possesses different anticancer properties. Also, it has been demonstrated that the aqueous extract of Helleborus niger L. induces apoptosis in various types of tumour cell lines (Jesse et al. 2009). Aqueous extracts of H. niger L. have immunomodulating properties by inducing sister chromatid exchange in lymphocyte cultures of healthy individuals (Bu¨ssing & Schweizer 1998). The authors of this study reported that Helleborus multifidus Vis., Helleborus hercegovinus Martinis and Helleborus odorus Waldst. & Kit possess strong antioxidative and antitumour
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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potential (Cˇakar et al. 2011). The aim of this study was to investigate the genotoxic and cytotoxic potential of three Helleborus taxa, which are spread over a wide geographical area of Bosnia and Herzegovina, including H. hercegovinus, whose taxonomic status is still questionable (Martinis 1973; Tutin 1996).
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2. Results and discussion 2.1. Effects on onion meristemic cells The extracts of all three Helleborus taxa, in all tested concentrations (0.5, 1, 2, 10 and 100 mL/ mL), alter the mitotic activity of meristematic onion cells compared with the control (Figures S1 and S2) based on the dosage (Figures S3 and S4). The mitotic index (MI) decreased significantly following treatment with the two highest concentrations of all tested extracts, when compared with the control treatment ( p , 0.01). Ethanol extract treatments significantly decrease the MI in comparison to aqueous extracts ( p , 0.05). Significant differences in MI values were observed after treatments with root and leaf extracts ( p , 0.001). It was observed that the MI, except for treatment with the ethanol extract of leaves, fell below 23% when treated with the highest concentrations, which is lower than the cytotoxic limit value (50%) (Sharma 1983) and which suggests sublethal consequences of applying these concentrations (Antonsie-Wiez 1990). The reduction of the mitotic activity could be due to the inhibition of DNA and nucleus-protein synthesis, or due to a blocking in the G2 phase of the cell cycle, thereby preventing the cell from entering mitosis (Sudhakar et al. 2001). The decreased MI in Allium cepa roots treated with hellebore extracts is probably caused either by disturbances in the cell cycle or by a chromatin dysfunction. Cytogenetic analysis indicated that extracts of the three Helleborus taxa, in all tested concentrations, change the frequency of mitotic phases when compared with the control (Figures S5 and S6). All tested Helleborus extract treatments induced abnormalities in all mitotic phases and had destructive effects on the kinetics, structure and organisation of A. cepa chromosomes in meristematic cells (Figure S7). The ethanol extracts induced an inhibition of the cell cycle in interphases (Figure S7(a)). The frequency of aberrant phases after treatments with the highest concentration of extracts (except for aqueous extracts of H. odorus) was significantly higher than the control treatment ( p , 0.001). Ethanol extracts induced aberrations even at lower concentrations (2 mL/mL and 1 mL/mL), while it was only at the highest concentration that the aqueous extracts induced a significantly higher frequency of aberrations, when compared with the control treatment ( p , 0.01). The most frequent abnormalities were related to the formation and functioning of the mitotic spindle, reflected in the irregular distribution of chromosomes, the multi-polarity and the formation of anaphase bridges. The percentage of aberrant cells with abnormal kinetics (Figures S7(b) – (d)) was higher in cells treated with ethanol extracts compared with aqueous extracts ( p ¼ 0.001). The telophase chromosome bridges (Figures S7(f),(g)) and agglutinated anaphase and telophase chromosomes (Figures S7 (g),(h)) were recorded in the treatments with the highest concentrations of hellebore extracts. Generally, the agglutinated ‘sticky’ chromosomes are the most frequently observed aberrations after treatments with different substances; they indicate clastogenic properties of the tested compounds (Rank 2003), resulting in cell death (Odeigah et al. 1997). As a result of the disruption of spindle functioning (Onfelt 1987), due to the tubulin depolymerisation (Novichkova et al. 2003), C-mitoses were recorded at the highest frequencies following treatment with ethanol extract of H. odorus root, where nearly 32% of all analysed cells had a distinctive C-mitotic status (Figures S7(i),(j)). It is very likely that extracts of the three Helleborus taxa affect the organisation of the microtubule, resulting in the inhibition of spindle formation and the appearance of C-mitosis. The highest frequency of interphase nuclei with buds (Figure S7(k)) was observed in onion root cells treated with 100 mL/mL of ethanol extract of H. odorus. It was found that the ethanol extracts at the highest applied concentrations also induce
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the formation of micronuclei (MNi; Figure S7(l)) but the frequency of registered MNi did not significantly differ in comparison with the control. ANOVA revealed no significant differences in genotoxic, cytotoxic and cytostatic effects on A. cepa cells between the three Helleborus taxa extracts.
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2.2. Effects on human lymphocytes All root extracts and leaf ethanol extracts at the highest applied concentrations of all three Helleborus taxa expressed strong cytotoxic and cytostatic activity on cytokinesis-blocked lymphocytes. These extracts blocked cell proliferation and caused the destruction of lymphocytes. Therefore, an analysis was conducted only following treatments with leaf extracts (Table S1). The relative frequency of MNi was in correlation with the applied concentrations, and a significant positive correlation was observed for aqueous extracts of H. multifidus and H. hercegovinus leaf extracts ( p , 0.05). Also, the frequency of MNi in binuclear cells treated with an aqueous extract at a concentration of 2.0 mL/mL was significantly different ( p , 0.001) compared with control treatments (Figures S8(a),(b)). Human lymphocytes therefore exhibited a greater sensitivity to Helleborus extract treatment compared with meristematic onion cells. MNi are the most representative indicators of cell damages and are, therefore, critical in the evaluation of genotoxic/cytotoxic effects of certain preparations (Ma et al. 1995). MNi can originate as a result of the disruption of spindle apparatus (Soliman 2001) or as a result of acentric fragments or lagging chromosomes that have failed to incorporate in the nuclei of daughter cell during telophase (Albertini et al. 2000; Krishna & Hayashi 2000). High frequencies of MNi in binuclear human lymphocyte cells suggest different mechanisms of action of hellebore extracts in these two cell types. This is supported by the fact that the complex metabolic system present in mammalian cells, which (de)activates the genotoxic potential of the tested substances, differs from the (de)activation system found in plants (Leme & MarinMorales 2009). In addition, Feretti et al. (2008) reported no correlation between the results obtained from the plant test systems and cultured human cells that could be attributed to the different mechanisms of detoxification process of exogenous substances in plant and mammalian cells. Consequently, a comprehensive evaluation of the different aspects of action of the tested substances provides a detailed outline of their toxic effects. The highest relative frequency (0.8%) of nuclear buds (Figure S8(c)) was registered in the treatment with H. hercegovinus aqueous extract at the highest applied concentration. The relative frequency of nuclear bridges (Figure S8(d)) in aqueous extracts was relatively low (0.2 –0.7%). The differences in the relative frequencies of nucleoplasmic bridges between control lymphocyte cultures and cultures treated with Helleborus aqueous extracts were statistically significant only for the treatment with 2 mL/mL of H. odorus extract ( p , 0.05). Ethanol extract treatments produced high frequencies of necrotic and apoptotic cells (Figures S8(e),(f)). Accordingly, the nuclear division indices were very low for the tested concentration (Figures S9 and S10) which indicates a highly cytotoxic and cytostatic activity of ethanol extracts in cytokinesis-blocked lymphocyte cultures. Cytological effects of aqueous extracts were in general significantly lower than those of ethanol extracts ( p , 0.01). A significantly negative correlation with the tested concentrations was observed for the aqueous extracts of H. odorus ( p , 0.05) and H. hercegovinus ( p , 0.01) (Figures S9 and S10). Treatment with the highest concentration of aqueous extracts of all three taxa also induced a C-mitosis (Figure S11). Based on these findings, it can be presumed that treatments with hellebore extracts have both a clastogenic and aneugenic effects, and that an induction of apoptosis is the main mechanism of antiproliferative activity of extracts of the three Helleborus taxa. These findings confirm earlier research that explains the mechanisms of apoptosis induction in five tumour cell lines treated with aqueous extracts of H. niger (Jesse et al. 2009). Further studies demonstrated that apoptosis was the result
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of activation of capsase-3 that is present in mammalian cells, and that it catalyses the degradation of many essential cell enzymes resulting in cell death (Porter & Ja¨nicke 1999). Many drugs with antiproliferative activity act in a way to disturb the dynamics of tubulin polymerisation in microtubule, thereby blocking mitosis in metaphase/anaphase transitions that finally result in cell death induced by apoptosis (Antonsie-Wiez 1990; Jordan 2002). The authors of this study previously reported strong antiproliferative properties of the three hellebore taxa against Burkitt’s lymphoma B tumour cells (Cˇakar et al. 2011) and that strong genotoxic, cytotoxic and cytostatic potential of hellebore extracts could be responsible for their antitumour activity. 3. Conclusion Treatments with all tested Helleborus extracts induced cytotoxic and cytostatic effects in meristemic A. cepa L. cells and in cultivated cytokinesis-blocked human lymphocytes. The rate of cytotoxicity of extracts was directly dependent on the extract concentrations, on the type of extract (roots/leaves), the solvent used (water/ethanol), the test organisms and the assay used. Cytokinesis-block micronucleus cytome assay revealed that treatments with Helleborus extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of the antiproliferative activity of the extracts is apoptosis induction. Today, when phytotherapy is becoming more accepted as a method for prevention and treatment of many diseases, an interest in new phytopreparates with potential therapeutic activity is growing dramatically. In this context, this study builds on the authors’ previous work on bioactive properties of the three hellebore taxa, and the achieved results provide a clearer picture of the complex bioactive action of H. odorus, H. multifidus and H. hercegovinus extracts which could enable their efficient and practical use in the future. Supplementary material Experimental details relating to this article are available online, alongside Table S1 and Figures S1 –S11. Acknowledgements The authors are thankful to Prof. Dr Edina Muratovic´, Faculty of Natural Sciences and Mathematics, University of Sarajevo, for the collection and determination of plant material. This research was financially supported by the Ministry of Education, Science and Youth of Canton Sarajevo, Bosnia and Herzegovina (under Grant No. 11-14-19953.1/07).
References Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F, Natarajan AT, Norppa H, Shuker DE, Tice R, Waters MD, Aitio A. 2000. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat Res. 463:111–172. Antonsie-Wiez D. 1990. Analysis of the cell cycle in the root meristem of Allium cepa under the influence of Leda krin. Folia Histochem Cytobiol. 26:79–96. Bu¨ssing A, Schweizer K. 1998. Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro. J Ethnopharm. 59:9–146. ˇ akar J, Paric´ A, Vidic D, Haveric´ A, Haveric´ S, Maksimovic´ M, Bajrovic´ K. 2011. Antioxidant and antiproliferative C activities of Helleborus odorus Waldst. & Kit, H. multifidus Vis. and H. hercegovinus Martinis. Nat Prod Res. 25:1969–1974. Feretti D, Zerbini I, Ceretti E, Villlarini M, Zani C, Moretti M, Fatigoni C, Orizio G, Donato F, Monarca S. 2008. Evaluation of chlorite and chlorate genotoxicity using plant bioassays and in vitro DNA damage tests. Water Res. 42:4075–4082.
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Jesse P, Mottke G, Eberle J, Seifert G, Henze G, Prokop A. 2009. Apoptosis-inducing activity of Helleborus niger in ALL and AML. Pediatr Blood Cancer. 52:464–469. Jordan MA. 2002. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem Anticancer Agents. 2:1–17. Krishna G, Hayashi M. 2000. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat Res. 455:155–166. Leme DM, Marin-Morales MA. 2009. Allium cepa test in environmental monitoring: a review on its application. Mutat Res. 682:71–81. Lindholm P, Gullbo J, Claeson P, Go¨rannson U, Johansson S, Backlund A, Larsson R, Bohlin L. 2002. Selective cytotoxicity evaluation in anticancer drug screening of fractionated plant extracts. J Biomol Screen. 7:333–340. Linke S, Dufter C, Kerek F, Jung M, Watzlik A, Jung T, Opelz G, Terness P. 1998. Functional characterization of HP12: a novel immunosuppressant purified from Helleborus species. Transplant Proc. 30:4106–4107. Ma TH, Xu Z, Xu C, McConnell H, Rabago EV, Arreola GA, Zhang H. 1995. The improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmental pollutants. Mutat Res. 334:185– 195. Martinis Z. 1973. Taxa nova et combinations novae generic Helleborus L. Flora Jugoslaviae. Suppl Fl Anal Jugoslav. 1:13–16. Musarraet J, Aqil F, Ahmand I. 2006. Mutagenicity and antimutagenicity of medicinal plants. In: Ahmad I, Aqil E, Owais M, editors. Modern phytomedicine: turning medicinal plants into drug. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co.; p. 271 –286. Novichkova EA, Onishchenko GE, Shtil AA. 2003. Microtubule depolymerization by vincristine causes cell death after transition from C mitosis to interphase. Cell Biol. 393:840– 843. Odeigah PGC, Makinwa J, Lawal B, Oyeniyi R. 1997. Genotoxicity screening of leachates from solid industrial waste evaluated Allium test. ATLA. 25:311–321. Onfelt A. 1987. Spindle disturbances in mammalian cells. III. Toxicity, c-mitosis and aneuploidy with 22 different compounds. Specific and unspecific mechanisms. Mutat Res Environ Mutagen Relat Subj. 182:135–154. Porter AG, Ja¨nicke RU. 1999. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6:99–104. Rank J. 2003. The method of Allium anaphase-telophase chromosome aberration assay. Ekologika. 1:38–42. Sharma CBSR. 1983. Plant meristems as monitors of genetic toxicity of environmental chemicals. Current Sci. 52:1000–1002. Soliman MI. 2001. Genotoxicity testing of Neem plant (Azadirachta indica A. Juss) using the Allium cepa chromosome aberration assay. J Biol Sci. 1:1021–1027. Sudhakar R, Ninge Gowda KN, Venu G. 2001. Mitotic abnormalities induced by silk dyeing industry effluents in the cells of Allium cepa. Cytologia. 66:235–239. Tutin TG. 1996. Genus Helleborus L. In: Tutin TG, Burges NA, Chater AO, Edmodson J, Heywood VH, Moore DM, Valentine DH, Walters SM, Webb DA, editors. Flora Europaea. Vol. 1. Cambridge: Cambridge University Press; p. 249– 251.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Cytotoxic and genotoxic activity of some Helleborus species a
a
a
Jasmina Čakar , Anja Haverić , Sanin Haverić , Milka Maksimović
b
c
& Adisa Parić a
Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Zmaja od Bosne 8, Sarajevo, Bosnia and Herzegovina b
Department of Chemistry, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina c
Department of Biology, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina Published online: 03 Mar 2014. To cite this article: Jasmina Čakar, Anja Haverić, Sanin Haverić, Milka Maksimović & Adisa Parić (2014) Cytotoxic and genotoxic activity of some Helleborus species, Natural Product Research: Formerly Natural Product Letters, 28:12, 883-887, DOI: 10.1080/14786419.2014.889135 To link to this article: http://dx.doi.org/10.1080/14786419.2014.889135
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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Natural Product Research, 2014 Vol. 28, No. 12, 883–887, http://dx.doi.org/10.1080/14786419.2014.889135
Cytotoxic and genotoxic activity of some Helleborus species
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Jasmina Cˇakara*, Anja Haveric´a, Sanin Haveric´a, Milka Maksimovic´b and Adisa Paric´c a Institute for Genetic Engineering and Biotechnology, University of Sarajevo, Zmaja od Bosne 8, Sarajevo, Bosnia and Herzegovina; bDepartment of Chemistry, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina; cDepartment of Biology, Faculty of Natural Sciences and Mathematics, University of Sarajevo, Bosnia and Herzegovina
(Received 26 November 2013; final version received 27 January 2014) Despite their known toxic properties, various Helleborus species are used as medicaments in folk medicine to treat some diseases and health conditions. As the main mechanism of many cytostatic drugs is based on their cytotoxic activity, there is potential for the toxicity of hellebore to be used in anticancer therapy. This study tested the geno- and cytotoxic effects of extracts of three hellebore taxa (Helleborus odorus, Helleborus multifidus and Helleborus hercegovinus) on meristemic onion (Allium cepa L.) cells and human lymphocytes. Treatments with Helleborus extracts induced cytotoxic and cytostatic effects in meristemic onion cells as well as in cultivated cytokinesis-blocked human lymphocytes. Cytokinesis-block micronucleus cytome assay indicated that treatments with hellebore extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of their antiproliferative activity is apoptosis induction. Keywords: cytotoxic activity; genotoxic activity; cytostatic activity; Helleborus; mitotic index; nuclear division index
1. Introduction Plants are the source of numerous compounds used for the prevention and treatment of diseases. Many of these compounds are also toxic, depending on the dosage. These contrary properties are also highly dependent on the choice of test systems and protocols, on the interactions between specific test chemicals and on the physiology and life cycle stage of the cell or organism (Musarraet et al. 2006). Some plant extracts act by disrupting and/or inhibiting the cell cycle, which frequently results in apoptosis-induced cell death, which may be the mechanism of their antiproliferative activity. Therefore, phytopreparations that have cytotoxic activity could potentially be used as medications for a number of health conditions that primarily feature uncontrolled cell proliferations. Numerous studies point to the bioactive properties of extracts of some Helleborus species (Bu¨ssing & Schweizer 1998; Lindholm et al. 2002; Jesse et al. 2009). Extracts of some Helleborus species have immunostimulatory and anti-inflammatory properties (Linke et al. 1998). Lindholm et al. (2002) state that Helleborus cyclophyllus Boiss. possesses different anticancer properties. Also, it has been demonstrated that the aqueous extract of Helleborus niger L. induces apoptosis in various types of tumour cell lines (Jesse et al. 2009). Aqueous extracts of H. niger L. have immunomodulating properties by inducing sister chromatid exchange in lymphocyte cultures of healthy individuals (Bu¨ssing & Schweizer 1998). The authors of this study reported that Helleborus multifidus Vis., Helleborus hercegovinus Martinis and Helleborus odorus Waldst. & Kit possess strong antioxidative and antitumour
*Corresponding author. Email: [email protected] q 2014 Taylor & Francis
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ˇ akar et al. J. C
potential (Cˇakar et al. 2011). The aim of this study was to investigate the genotoxic and cytotoxic potential of three Helleborus taxa, which are spread over a wide geographical area of Bosnia and Herzegovina, including H. hercegovinus, whose taxonomic status is still questionable (Martinis 1973; Tutin 1996).
Downloaded by [Florida International University] at 02:52 04 September 2014
2. Results and discussion 2.1. Effects on onion meristemic cells The extracts of all three Helleborus taxa, in all tested concentrations (0.5, 1, 2, 10 and 100 mL/ mL), alter the mitotic activity of meristematic onion cells compared with the control (Figures S1 and S2) based on the dosage (Figures S3 and S4). The mitotic index (MI) decreased significantly following treatment with the two highest concentrations of all tested extracts, when compared with the control treatment ( p , 0.01). Ethanol extract treatments significantly decrease the MI in comparison to aqueous extracts ( p , 0.05). Significant differences in MI values were observed after treatments with root and leaf extracts ( p , 0.001). It was observed that the MI, except for treatment with the ethanol extract of leaves, fell below 23% when treated with the highest concentrations, which is lower than the cytotoxic limit value (50%) (Sharma 1983) and which suggests sublethal consequences of applying these concentrations (Antonsie-Wiez 1990). The reduction of the mitotic activity could be due to the inhibition of DNA and nucleus-protein synthesis, or due to a blocking in the G2 phase of the cell cycle, thereby preventing the cell from entering mitosis (Sudhakar et al. 2001). The decreased MI in Allium cepa roots treated with hellebore extracts is probably caused either by disturbances in the cell cycle or by a chromatin dysfunction. Cytogenetic analysis indicated that extracts of the three Helleborus taxa, in all tested concentrations, change the frequency of mitotic phases when compared with the control (Figures S5 and S6). All tested Helleborus extract treatments induced abnormalities in all mitotic phases and had destructive effects on the kinetics, structure and organisation of A. cepa chromosomes in meristematic cells (Figure S7). The ethanol extracts induced an inhibition of the cell cycle in interphases (Figure S7(a)). The frequency of aberrant phases after treatments with the highest concentration of extracts (except for aqueous extracts of H. odorus) was significantly higher than the control treatment ( p , 0.001). Ethanol extracts induced aberrations even at lower concentrations (2 mL/mL and 1 mL/mL), while it was only at the highest concentration that the aqueous extracts induced a significantly higher frequency of aberrations, when compared with the control treatment ( p , 0.01). The most frequent abnormalities were related to the formation and functioning of the mitotic spindle, reflected in the irregular distribution of chromosomes, the multi-polarity and the formation of anaphase bridges. The percentage of aberrant cells with abnormal kinetics (Figures S7(b) – (d)) was higher in cells treated with ethanol extracts compared with aqueous extracts ( p ¼ 0.001). The telophase chromosome bridges (Figures S7(f),(g)) and agglutinated anaphase and telophase chromosomes (Figures S7 (g),(h)) were recorded in the treatments with the highest concentrations of hellebore extracts. Generally, the agglutinated ‘sticky’ chromosomes are the most frequently observed aberrations after treatments with different substances; they indicate clastogenic properties of the tested compounds (Rank 2003), resulting in cell death (Odeigah et al. 1997). As a result of the disruption of spindle functioning (Onfelt 1987), due to the tubulin depolymerisation (Novichkova et al. 2003), C-mitoses were recorded at the highest frequencies following treatment with ethanol extract of H. odorus root, where nearly 32% of all analysed cells had a distinctive C-mitotic status (Figures S7(i),(j)). It is very likely that extracts of the three Helleborus taxa affect the organisation of the microtubule, resulting in the inhibition of spindle formation and the appearance of C-mitosis. The highest frequency of interphase nuclei with buds (Figure S7(k)) was observed in onion root cells treated with 100 mL/mL of ethanol extract of H. odorus. It was found that the ethanol extracts at the highest applied concentrations also induce
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the formation of micronuclei (MNi; Figure S7(l)) but the frequency of registered MNi did not significantly differ in comparison with the control. ANOVA revealed no significant differences in genotoxic, cytotoxic and cytostatic effects on A. cepa cells between the three Helleborus taxa extracts.
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2.2. Effects on human lymphocytes All root extracts and leaf ethanol extracts at the highest applied concentrations of all three Helleborus taxa expressed strong cytotoxic and cytostatic activity on cytokinesis-blocked lymphocytes. These extracts blocked cell proliferation and caused the destruction of lymphocytes. Therefore, an analysis was conducted only following treatments with leaf extracts (Table S1). The relative frequency of MNi was in correlation with the applied concentrations, and a significant positive correlation was observed for aqueous extracts of H. multifidus and H. hercegovinus leaf extracts ( p , 0.05). Also, the frequency of MNi in binuclear cells treated with an aqueous extract at a concentration of 2.0 mL/mL was significantly different ( p , 0.001) compared with control treatments (Figures S8(a),(b)). Human lymphocytes therefore exhibited a greater sensitivity to Helleborus extract treatment compared with meristematic onion cells. MNi are the most representative indicators of cell damages and are, therefore, critical in the evaluation of genotoxic/cytotoxic effects of certain preparations (Ma et al. 1995). MNi can originate as a result of the disruption of spindle apparatus (Soliman 2001) or as a result of acentric fragments or lagging chromosomes that have failed to incorporate in the nuclei of daughter cell during telophase (Albertini et al. 2000; Krishna & Hayashi 2000). High frequencies of MNi in binuclear human lymphocyte cells suggest different mechanisms of action of hellebore extracts in these two cell types. This is supported by the fact that the complex metabolic system present in mammalian cells, which (de)activates the genotoxic potential of the tested substances, differs from the (de)activation system found in plants (Leme & MarinMorales 2009). In addition, Feretti et al. (2008) reported no correlation between the results obtained from the plant test systems and cultured human cells that could be attributed to the different mechanisms of detoxification process of exogenous substances in plant and mammalian cells. Consequently, a comprehensive evaluation of the different aspects of action of the tested substances provides a detailed outline of their toxic effects. The highest relative frequency (0.8%) of nuclear buds (Figure S8(c)) was registered in the treatment with H. hercegovinus aqueous extract at the highest applied concentration. The relative frequency of nuclear bridges (Figure S8(d)) in aqueous extracts was relatively low (0.2 –0.7%). The differences in the relative frequencies of nucleoplasmic bridges between control lymphocyte cultures and cultures treated with Helleborus aqueous extracts were statistically significant only for the treatment with 2 mL/mL of H. odorus extract ( p , 0.05). Ethanol extract treatments produced high frequencies of necrotic and apoptotic cells (Figures S8(e),(f)). Accordingly, the nuclear division indices were very low for the tested concentration (Figures S9 and S10) which indicates a highly cytotoxic and cytostatic activity of ethanol extracts in cytokinesis-blocked lymphocyte cultures. Cytological effects of aqueous extracts were in general significantly lower than those of ethanol extracts ( p , 0.01). A significantly negative correlation with the tested concentrations was observed for the aqueous extracts of H. odorus ( p , 0.05) and H. hercegovinus ( p , 0.01) (Figures S9 and S10). Treatment with the highest concentration of aqueous extracts of all three taxa also induced a C-mitosis (Figure S11). Based on these findings, it can be presumed that treatments with hellebore extracts have both a clastogenic and aneugenic effects, and that an induction of apoptosis is the main mechanism of antiproliferative activity of extracts of the three Helleborus taxa. These findings confirm earlier research that explains the mechanisms of apoptosis induction in five tumour cell lines treated with aqueous extracts of H. niger (Jesse et al. 2009). Further studies demonstrated that apoptosis was the result
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of activation of capsase-3 that is present in mammalian cells, and that it catalyses the degradation of many essential cell enzymes resulting in cell death (Porter & Ja¨nicke 1999). Many drugs with antiproliferative activity act in a way to disturb the dynamics of tubulin polymerisation in microtubule, thereby blocking mitosis in metaphase/anaphase transitions that finally result in cell death induced by apoptosis (Antonsie-Wiez 1990; Jordan 2002). The authors of this study previously reported strong antiproliferative properties of the three hellebore taxa against Burkitt’s lymphoma B tumour cells (Cˇakar et al. 2011) and that strong genotoxic, cytotoxic and cytostatic potential of hellebore extracts could be responsible for their antitumour activity. 3. Conclusion Treatments with all tested Helleborus extracts induced cytotoxic and cytostatic effects in meristemic A. cepa L. cells and in cultivated cytokinesis-blocked human lymphocytes. The rate of cytotoxicity of extracts was directly dependent on the extract concentrations, on the type of extract (roots/leaves), the solvent used (water/ethanol), the test organisms and the assay used. Cytokinesis-block micronucleus cytome assay revealed that treatments with Helleborus extracts induce genotoxic effects in human lymphocytes, and that the significant mechanism of the antiproliferative activity of the extracts is apoptosis induction. Today, when phytotherapy is becoming more accepted as a method for prevention and treatment of many diseases, an interest in new phytopreparates with potential therapeutic activity is growing dramatically. In this context, this study builds on the authors’ previous work on bioactive properties of the three hellebore taxa, and the achieved results provide a clearer picture of the complex bioactive action of H. odorus, H. multifidus and H. hercegovinus extracts which could enable their efficient and practical use in the future. Supplementary material Experimental details relating to this article are available online, alongside Table S1 and Figures S1 –S11. Acknowledgements The authors are thankful to Prof. Dr Edina Muratovic´, Faculty of Natural Sciences and Mathematics, University of Sarajevo, for the collection and determination of plant material. This research was financially supported by the Ministry of Education, Science and Youth of Canton Sarajevo, Bosnia and Herzegovina (under Grant No. 11-14-19953.1/07).
References Albertini RJ, Anderson D, Douglas GR, Hagmar L, Hemminki K, Merlo F, Natarajan AT, Norppa H, Shuker DE, Tice R, Waters MD, Aitio A. 2000. IPCS guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutat Res. 463:111–172. Antonsie-Wiez D. 1990. Analysis of the cell cycle in the root meristem of Allium cepa under the influence of Leda krin. Folia Histochem Cytobiol. 26:79–96. Bu¨ssing A, Schweizer K. 1998. Effects of a phytopreparation from Helleborus niger on immunocompetent cells in vitro. J Ethnopharm. 59:9–146. ˇ akar J, Paric´ A, Vidic D, Haveric´ A, Haveric´ S, Maksimovic´ M, Bajrovic´ K. 2011. Antioxidant and antiproliferative C activities of Helleborus odorus Waldst. & Kit, H. multifidus Vis. and H. hercegovinus Martinis. Nat Prod Res. 25:1969–1974. Feretti D, Zerbini I, Ceretti E, Villlarini M, Zani C, Moretti M, Fatigoni C, Orizio G, Donato F, Monarca S. 2008. Evaluation of chlorite and chlorate genotoxicity using plant bioassays and in vitro DNA damage tests. Water Res. 42:4075–4082.
Downloaded by [Florida International University] at 02:52 04 September 2014
Natural Product Research
887
Jesse P, Mottke G, Eberle J, Seifert G, Henze G, Prokop A. 2009. Apoptosis-inducing activity of Helleborus niger in ALL and AML. Pediatr Blood Cancer. 52:464–469. Jordan MA. 2002. Mechanism of action of antitumor drugs that interact with microtubules and tubulin. Curr Med Chem Anticancer Agents. 2:1–17. Krishna G, Hayashi M. 2000. In vivo rodent micronucleus assay: protocol, conduct and data interpretation. Mutat Res. 455:155–166. Leme DM, Marin-Morales MA. 2009. Allium cepa test in environmental monitoring: a review on its application. Mutat Res. 682:71–81. Lindholm P, Gullbo J, Claeson P, Go¨rannson U, Johansson S, Backlund A, Larsson R, Bohlin L. 2002. Selective cytotoxicity evaluation in anticancer drug screening of fractionated plant extracts. J Biomol Screen. 7:333–340. Linke S, Dufter C, Kerek F, Jung M, Watzlik A, Jung T, Opelz G, Terness P. 1998. Functional characterization of HP12: a novel immunosuppressant purified from Helleborus species. Transplant Proc. 30:4106–4107. Ma TH, Xu Z, Xu C, McConnell H, Rabago EV, Arreola GA, Zhang H. 1995. The improved Allium/Vicia root tip micronucleus assay for clastogenicity of environmental pollutants. Mutat Res. 334:185– 195. Martinis Z. 1973. Taxa nova et combinations novae generic Helleborus L. Flora Jugoslaviae. Suppl Fl Anal Jugoslav. 1:13–16. Musarraet J, Aqil F, Ahmand I. 2006. Mutagenicity and antimutagenicity of medicinal plants. In: Ahmad I, Aqil E, Owais M, editors. Modern phytomedicine: turning medicinal plants into drug. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co.; p. 271 –286. Novichkova EA, Onishchenko GE, Shtil AA. 2003. Microtubule depolymerization by vincristine causes cell death after transition from C mitosis to interphase. Cell Biol. 393:840– 843. Odeigah PGC, Makinwa J, Lawal B, Oyeniyi R. 1997. Genotoxicity screening of leachates from solid industrial waste evaluated Allium test. ATLA. 25:311–321. Onfelt A. 1987. Spindle disturbances in mammalian cells. III. Toxicity, c-mitosis and aneuploidy with 22 different compounds. Specific and unspecific mechanisms. Mutat Res Environ Mutagen Relat Subj. 182:135–154. Porter AG, Ja¨nicke RU. 1999. Emerging roles of caspase-3 in apoptosis. Cell Death Differ. 6:99–104. Rank J. 2003. The method of Allium anaphase-telophase chromosome aberration assay. Ekologika. 1:38–42. Sharma CBSR. 1983. Plant meristems as monitors of genetic toxicity of environmental chemicals. Current Sci. 52:1000–1002. Soliman MI. 2001. Genotoxicity testing of Neem plant (Azadirachta indica A. Juss) using the Allium cepa chromosome aberration assay. J Biol Sci. 1:1021–1027. Sudhakar R, Ninge Gowda KN, Venu G. 2001. Mitotic abnormalities induced by silk dyeing industry effluents in the cells of Allium cepa. Cytologia. 66:235–239. Tutin TG. 1996. Genus Helleborus L. In: Tutin TG, Burges NA, Chater AO, Edmodson J, Heywood VH, Moore DM, Valentine DH, Walters SM, Webb DA, editors. Flora Europaea. Vol. 1. Cambridge: Cambridge University Press; p. 249– 251.
