Aqueous Extracts of Dried Fruits of Zanthoxylum armatum DC., (Rutaceae) Induce Cellular and Nuclear Damage Coupled with Inhibition of Mitotic Activity in Vivo

Eros Vasil Kharshiing

Department of Botany, St. Edmund’s College, Shillong, India..

DOI: 10.4236/ajps.2012.311200                

Abstract

Bioactive compounds derived from plant natural compounds have proven to be valuable sources of metabolites which can seldom be obtained from other sources. Plants belonging to the genus Zanthoxylum have been valued across various cultures for their curative properties. Zanthoxylum armatum DC., belonging to the family Rutaceae is extensively used in traditional practices in North-Eastern India and neighbouring regions including South-East Asia. However, the potential cytogenetic effects of Zanthoxylum armatum under in vivo conditions, and their causative mechanisms have not yet been studied in detail. The current study was undertaken to evaluate the cytotoxic and genotoxic potential of aqueous extracts of fruits of Z. armatum under in vivo conditions using the Allium test. Physiological and cellular data indicate that the extracts induce clumped chromosomes at metaphase stage of cell division coupled with mitotic arrest. Electron microscopy data reveal membrane damage of cellular organelles, chromatin condensation and chromatin marginalisation in cell of roots incubated in the extracts. The extracts also induce concentration dependent protein precipitation and genomic DNA degradation.

Keywords

Clumped Chromosomes; Chromatin Condensation and Marginalisation; TEM; Toxicity; Zanthoxylum armatum

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	D. J. Newman and G. M. Cragg, “Natural Products as Sources of New Drugs over the last 25 Years,” Journal of Natural Products, Vol. 70, No. 3, 2007, pp. 461-477. doi:10.1021/np068054v
[2]	G. M. Cragg, D. G. I. Kingston and D. J. Newman, “Anticancer Agents from Natural Products,” In: D. G. I. Kingston, G. M. Cragg and D. J. Newman, Eds., CRC Taylor & Francis, New York, 2005, pp. 1-3.
[3]	P. B. Kaufman, A. Kirakosyan, M. McKenzie, P. Dayanandan, J. E. Hoyt and C. Li, “The Uses of Plant Natural Products by Humans and Risks Associated with Their Use,” In: L. J. Cseke, A. Kirakosyan, P. B. Kaufman, S. Warber, J. A. Duke and H. L. Brielmann, Eds., Natural Products from Plants, CRC Taylor & Francis, New York, 2006, pp. 442-468. doi:10.1201/9781420004472.ch12
[4]	S. M. Colegate and R. J. Molyneux, “An Introduction and Overview,” In: S. M. Colegate and R. J. Molyneux, Eds., Bioactive Natural Products: Detection, Isolation, and Structural Determination, 2nd Edition, CRC Press, New York, 2007, pp. 1-3.
[5]	C. Tringali, “Bioactive Compounds from Natural Sources: Isolation, Characterization and Biological Properties,” CRC Taylor & Francis, New York, 2001, pp. 9-10.
[6]	J. S. Negi, V. K. Bisht, A. K. Bhandari, P. Singh and R. C. Sundriyal, “Chemical Constituents and Biological Activities of the Genus Zanthoxylum: A Review,” African Journal of Pure and Applied Chemistry, Vol. 5, No. 12, 2011, pp. 412-416.
[7]	L. O. J. Pati?o, R. J. A. Prieto and S. L. E. Cuca, “Bioactive Compounds in Phytomedicine: Zanthoxylum Genus as Potential Source of Bioactive Compounds,” In: I. Rasooli, Ed., InTech, 2012, pp. 185-218.
[8]	R. Diéguez, G. Garrido, S. Prieto, Y. Iznaga, L. González, J. Molina, M. Curini , F. Epifano and M. C. Marcotullio, “Antifungal Activity of Some Cuban Zanthoxylum Species,” Fitoterapia, Vol. 74, No. 4, 2003, pp. 384-386. doi:10.1016/S0367-326X(03)00048-0
[9]	S. K. Adesina, “The Nigerian Zanthoxylum: Chemical and Biological Values,” African Journal of Traditional, Complimentary and Alternative Medicines, Vol. 2, No. 3, 2005, pp. 282-301.
[10]	S. Rochfort, J. Anthony, J. A. Parker and F. R. Dunshea, “Plant Bioactives for Ruminant Health and Productivity,” Phytochemistry, Vol. 69, No. 2, 2008, pp. 299-322. doi:10.1016/j.phytochem.2007.08.017
[11]	S. S. Pereira, L. S. Lopes, R. B. Marques, K. A. Figueiredo, D. A. Costa, M. H. Chaves and F. R. C. Almeida, “Anti-nociceptive Effect of Zanthoxylum rhoifolium Lam. (Rutaceae) in Models Acute Pain in Rodents,” Journal of Ethnopharmacology, Vol. 129, 2010, pp. 227-231. doi:10.1016/j.jep.2010.03.009
[12]	S. Bhattacharya, M. K. Zaman and P. K. Haldar, “Anti-bacterial Activity of Stem Bark and Root of Indian Zanthoxylum nitidum,” Asian Journal of Pharmaceutical and Clinical Research, Vol. 2, No. 1, 2009, pp. 30-24.
[13]	L.-F. Shyur, J.-H. Tsung, J.-H. Chen, C.-Y. Chiu and C.-P. Lo, “Antioxidant Properties of Extracts from Medicinal Plants Popularly Used in Taiwan,” International Journal of Appllied Science and Engineering, Vol. 3, No. 3, 2005, pp. 195-202.
[14]	I. Elekwa, M. O. Monanu and E. O. Anosike, “Effects of Aqueous Extracts of Zanthoxylum macrophylla Roots on Membrane Stability of Human Erythrocytes of Different Genotypes,” Biokemistri, Vol. 17, No. 1, 2005, pp. 7-12.
[15]	E. Yamazaki, “Extraction of Antioxidants from Zanthoxylum piperitum DC. Fruit-Inhibition of Glycation with Polyphenols from Zanthoxylum piperitum DC. Fruit,” Reports of the Mie Prefectural Science and Technology, Promotion Center Industrial Research Division, Vol. 26, 2002, pp. 1-5.
[16]	A. H. Cahyana and L. Mardiana “The Use of Chemical Components Isolated from Andaliman (Zanthoxylum acanthopodium DC) Essential Oil as a Source of Natural Antioxidant,” Journal of Food Science and Technology, Vol. 1, No. 1, 2003, pp. 106-111.
[17]	Y.-C. Chung, C.-T. Chien, K.-Y. Teng and S.-T. Chou, “Antioxidative and Mutagenic Properties of Zanthoxylum ailanthoides Sieb & Zucc.,” Food Chemistry, Vol. 97, 2006, pp. 418-425. doi:10.1016/j.foodchem.2005.05.019
[18]	T. P. Singh and O. M. Singh, “Phytochemical and Phar-macological Profile of Zanthoxylum armatum DC.—An Overview,” Indian Journal of Natural Products and Resources, Vol. 2, No. 3, 2011, pp. 275-285.
[19]	Barkatullah, M. Ibrar, N. Muhammad and L. Tahir, “Antimicrobial Evaluation, Determination of Total Phenolic and Flavoniod Contents in Zanthoxylum armatum DC,” Journal of Medicinal Plants Research, Vol. 6, No. 11, 2012, pp. 2105-2110.
[20]	G. Pachon, H. Rasoanaivo, A. Azqueta, J. C. Rakotozafy, A. Raharisololalao, A. L. De Cerain, J. De Lapuente, M. Borràs, S. Moukha, J. J. Centelles, E. E. Creppy and M. Cascante, “Anticancer Effect of a New Benzophenanthridine Isolated from Zanthoxylum madagascariense (Rutaceline),” In Vivo, Vol. 21, No. 2, 2007, pp. 417-422.
[21]	G. Cebrián-Torrejón, S. A. Kahn, N. Lagarde, F. Castel-lano, K. Leblanc, J. Rodrigo, V. Molinier-Frenkel, A. Rojas de Arias, M. E. Ferreira, C. Thirant, A. Fournet, B. Figadère, H. Chneiweiss and E. Poupon, “Antiproliferative Activity of Trans-Avicennol from Zanthoxylum chiloperone var. Angustifolium against Human Cancer Stem Cells,” Journal of Natural Products, Vol. 75, No. 2, 2012, pp. 257-261. doi:10.1021/np2004165
[22]	J. Y. Seo, M. Y. Park, T. Y. Jung, H. Y. Choi, D. Kim, H. S. Lee and S. K. Ku, “Genotoxicity Testing of Aqueous Extracts of Mahwangyounpae-Tang, a Polyherbal Formula,” Food and Chemical Toxicology, Vol. 46, 2008, pp. 3827-3831. doi:10.1016/j.fct.2008.10.005
[23]	C. J. van den Boutvan den Beukel, O. J. Hamza, M. J. Moshi, M. I. Matee, F. Mikx, D. M. Burger, P. P. Koopmans, P. E. Verweij, W. G. Schoonen and A. J. van der Ven, “Evaluation of Cytotoxic, Genotoxic and CYP450 Enzymatic Competition Effects of Tanzanian Plant Extracts Traditionally Used for Treatment of Fungal Infections,” Basic & Clinical Pharmacology & Toxicology, Vol. 102, No. 6, 2008, pp. 515-526. doi:10.1111/j.1742-7843.2008.00225.x
[24]	J. Demma, E. Engidawork and B. Hellman, “Potential Genotoxicity of Plant Extracts Used in Ethiopian Traditional Medicine,” Journal of Ethnopharmacology, Vol. 122, No. 1, 2009, pp. 136-142. doi:10.1016/j.jep.2008.12.013
[25]	N. A. Spiridonov, D. A. Konovalov and V. V. Arkhipov, “Cytotoxicity of Some Russian Ethnomedicinal Plants and Plant Compounds,” Phytotherapy Research, Vol. 19, No. 5, 2005, pp. 428-432. doi:10.1002/ptr.1616
[26]	N. J. Lee, J. H. Choi, B. S. Koo, S. Y. Ryu, Y. H. Han, S. I. Lee and D. U. Lee, “ Antimutagenicity and Cytotoxicity of the Constituents from the Aerial Parts of Rumex acetosa,” Biological and Pharmaceutical Bulletin, Vol. 28, No. 11, 2005, pp. 2158-2161. doi:10.1248/bpb.28.2158
[27]	M. Gálvez, C. Martín-Cordero, M. López-Lázaro, F. Cortés and M. J. Ayuso, “Cytotoxic Effect of Plantago spp. on Cancer Cell Lines,” Journal of Ethnopharmacology, Vol. 88, No. 2-3, 2003, pp. 125-130. doi:10.1016/S0378-8741(03)00192-2
[28]	S. R. Hynniewta and Y. Kumar, “Herbal Remedies among the Khasi Traditional Healers and Village Folk in Meghalaya,” Indian Journal of Traditional Knowledge, Vol. 7, No. 4, 2008, pp. 581-586.
[29]	V. Jaiswal, “Culture and Ethnobotany of Jaintia Tribal Community of Meghalaya, Northeast India—A Review,” Indian Journal of Traditional Knowledge, Vol. 9, No. 1, 2010, pp. 38-44.
[30]	H. P. S. Makkar, M. Bluemmel, N. K. Borowy and K. Becker, “Gravimetric Determination of Tannins and Their Correlations with Chemical and Protein Precipitation Methods,” Journal of the Science of Food and Agriculture, Vol. 61, No. 2, 1993, pp. 161-165. doi:10.1002/jsfa.2740610205
[31]	Y. Choudhury and R. N. Sharan, “Ultrastructural Alterations in Liver of Mice Exposed Chronically and Transgenerationally to Aqueous Extract of Betel Nut: Implications in Betel Nut-Induced Carcinogenesis,” Microscopy Research and Techique, Vol. 73, No. 5, 2010, pp. 530- 539.
[32]	M. Bradford, “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding,” Analalytical Bio-chemistry, Vol. 72, 1976, pp. 248-254. doi:10.1016/0003-2697(76)90527-3
[33]	M. Kri?man, J. Jak?e, D. Bari?evi?, B. Javornik and M. Pro?ek, “Robust CTAB-Activated Charcoal Protocol for Plant DNA Extraction,” Acta Agriculturae Slovenica, Vol. 87, No. 2, 2006, pp. 427-433.
[34]	F. Haber and J. Weiss, “On the Catalysis of Hydroperoxide,” Naturwissenschaften, Vol. 20, 1932, pp. 948-950. doi:10.1007/BF01504715
[35]	A. S. Multani, C. Li, M. Ozen, M. Yadav, D. F. Yu, S. Wallace and S. Pathak, “Paclitaxel and Water-Soluble Poly(L-Glutamic Acid)-Paclitaxel, Induce Direct Chromosomal Abnormalities and Cell Death in a Murine Metastatic Melanoma Cell Line,” Anticancer Research, Vol. 17, No. 6D, 1997, pp. 4269-4274.
[36]	W. C. Chou, A. L. Hawkins, J. F. Barrett, C. A. Griffin and C. V. Dang, “Arsenic Inhibition of Telomerase Transcription Leads to Genetic Instability,” Journal of Clinical Investigation, Vol. 108, No. 10, 2001, pp. 1541-1547
[37]	S. Y. Lyu, S. H. Choi and W. B. Par, “Korean Mistletoe Lectin-Induced Apoptosis in Hepatocarcinoma Cells Is Associated with Inhibition of Telomerase via Mitochondrial Controlled Pathway Independent of p53,” Archives of Pharmacal Research, Vol. 25, No. 1, 2002, pp. 93-101. doi:10.1007/BF02975269
[38]	S. H. Choi, S. Y. Lyu and W. B. Park, “Mistletoe Lectin Induces Apoptosis and Telomerase Inhibition in Human A253 Cancer Cells through Dephosphorylation of Akt.,” Archives of Pharmacal Research, Vol. 27, No. 1, 2004, pp. 68-76. doi:10.1007/BF02980049
[39]	M. Pourhassan, N. Zarghami, M. Rahmati, A. Alibakhshi and J. Ranjbari, “The Inhibitory Effect of Curcuma longa Extract on Telomerase Activity in A549 Lung Cancer Cell Line,” African Journal of Biotechnology, Vol. 9, No. 6, 2010, pp. 912-919.
[40]	K. Riha, J. Fajkus, J. Siroky and B. Vyskot, “Developmental Control of Telomere Lengths and Telomerase Activity in Plants,” The Plant Cell, Vol. 10, No. 10, 1998, pp. 1691-1698.
[41]	H. Kosugi, T. Kato and K. Kikugawa, “Formation of Yellow, Orange, and Red Pigments in the Reaction of Alk-2-Enals with 2-Thiobarbituric Acid,” Analytical Bio-chemistry, Vol. 165, No. 2, 1987, pp. 456-464. doi:10.1016/0003-2697(87)90296-X
[42]	Q. Sun, C. Faustman, A. Senecal, A. L. Wilkinson and H. Furr, “Aldehyde Reactivity with 2-Thiobarbituric Acid and TBARS in Freeze-Dried Beef during Accelerated Storage,” Meat Science, Vol. 57, No. 1, 2001, pp. 55-60. doi:10.1016/S0309-1740(00)00076-0
[43]	E. J. Cho, T. Yokozawa, D. Y. Rhyu, H. Y. Kim, N. Shi-bahara and J. C. Park, “The Inhibitory Effects of 12 Medicinal Plants and Their Component Compounds on Lipid Peroxidation,” The American Journal of Chinese Medicine, Vol. 31, No. 6, 2003, pp. 907-917. doi:10.1142/S0192415X03001648
[44]	J. M. Hur, J. G. Park, K. H. Yang, J. C. Park, J. R. Park, S. S. Chun, J. S. Choi and J. W. Choi, “Effect of Methanol Extract of Zanthoxylum piperitum Leaves and of Its Compound, Protocatechuic Acid, on Hepatic Drug Metabolizing Enzymes and Lipid Peroxidation in Rats,” Bioscience Biotechnology Biochemistry, Vol. 67, No. 5, 2003, pp. 945-950. doi:10.1271/bbb.67.945
[45]	N. Yao, Y. Tada, P. Park, H. Nakayashiki, Y. Tosa and S. Mayama, “Novel Evidence for Apoptotic Cell Response and Differential Signals in Chromatin Condensation and DNA Cleavage in Victorin-Treated Oats,” Plant Journal, Vol. 28, No. 1, 2001, pp. 13-26. doi:10.1046/j.1365-313X.2001.01109.x
[46]	K. Zheng, J.-W. Pan, L. Ye, Y. Fu, H.-Z. Peng, B.-Y. Wan, Q. Gu, H.-W. Bian, N. Han, J.-H. Wang, B. Kang, J.-H. Pan, H.-H. Shao, W.-Z. Wang and M.-Y. Zhu, “Pro-grammed Cell Death-Involved Aluminum Toxicity in Yeast Alleviated by Antiapoptotic Members with Decreased Calcium Signals,” Plant Physiology, Vol. 143, No. 1, 2007, pp. 38-49. doi:10.1104/pp.106.082495
[47]	E. O’Brien, B. G. Murray, B. C. Baguley, B. A. Morris and I. B. Ferguson, “Major Changes in Chromatin Condensation Suggest the Presence of an Apoptotic Pathway in Plant Cells,” Experimental Cell Research, Vol. 241, No. 1, 1998, pp. 46-54. doi:10.1006/excr.1998.4036
[48]	S. Chakraborty, M. Roy, A. K. Taraphdar and R. K. Bhat-tacharya, “Cytotoxic Effect of Root Extract of Tiliacora racemosa and Oil of Semecarpus anacardium Nut in Human Tumour Cells,” Phytotherapy Research, Vol. 18, No. 8, 2004, pp. 595-600. doi:10.1002/ptr.1501.