Molecular and cytotoxicity investigations of Phytolacca americana (l.) root, leaf, and berry extracts
Background: Pokeweed anti-viral protein (PAP) and lectin are two of the toxic components of pokeweed, Phytolacca americana, suspected of affecting free grazing livestock and small herbivorous animals.
Objectives: This research aimed to investigate the antimicrobial activity of the pokeweed extracts against two bacterial strains, the gram negative Escherichia coli and the gram positive Staphylococcus aureus, to investigate the toxicity of the extracts to cells of tobacco, Nicotiana tabacum, callus, and to investigate the presence of selected toxic constituents present in pokeweed.
Methodology: Pokeweed plants were identified and brought to the laboratory and separated into roots and leaves. The berries were collected later in the growing season. Aqueous extracts were obtained by homogenizing the plant parts separately in sterile water followed by centrifugation. The supernatants were filter-sterilized and used for bacterial and tobacco callus growth inhibition assays. Total cytoplasmic proteins were also obtained by homogenizing the plant parts separately in protein extraction buffer and centrifuging. The supernatants were investigated for the presence of various toxins suspected of being present in pokeweed, using western blot analyses.
Results and Conclusions: Pokeweed constituents possess growth inhibitory effects to gram negative E. coli and to N. tabacum callus but not of the gram positive S. aureus, and that all three plant parts studied were rich in lectin and lectin-like constituents such as PL-A, PL-C, and PL-G. No PL-B was detected in any of the plant extracts.
 Aron, G. and J. Irvin. 1980. Inhibition of herpes simplex virus multiplication by the pokeweed antiviral protein. Antimicrobial Agents and Chemotherapy. (17), 1032-1033. http://dx.doi.org/10.1128/AAC.17.6.1032.
 Biswal, B., D. Saha, A. Jena, S. B. Jana, A. Koley, D. Sur, and J. C. Mohanty. 2011. Anti-Bacterial Activity of Leaf Extract of Derris indica. International Journal of Research in Pharmaceutical and Biomedical Sciences. 2 (1), 271-273.
 Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. (72), 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3.
 Chapagain, B. P., Z. Wiesman, and L. Tsror (Lahkim). 2007. In vitro study of the antifungal activity of saponin-rich extracts against prevalent phytopathogenic fungi. Industrial Crops and Products (26), 109â€“115. http://dx.doi.org/10.1016/j.indcrop.2007.02.005.
 Chrispeel, M. J. and N. V. Raikhelb. 1991. Lectin genes, and their role in plant defense. The Plant Cell. (3):1-9. http://dx.doi.org/10.1105/tpc.3.1.1.
 Duke, J.A. 2002. Handbook of Medicinal Herbs. 2nd Ed. CRC Press. Boca Raton, FL., USA http://dx.doi.org/10.1201/9781420040463.
 Escalante, A. M., C. B. Santecchia, S. N. LoÂ´pez, M. A. Gattuso, A. G. Ravelo, F. D. Monache, M. G. Sierra, and S. A. Zacchino. 2002. Isolation of antifungal saponins from Phytolacca tetramera, an Argentinean species in critic risk. Journal of Ethnopharmacology (82), 29-34. http://dx.doi.org/10.1016/S0378-8741(02)00145-9.
 Gamborg, O. L., T. Murashige, T. A. Thorpe, and I. K. Vasil. 1976. Nutrient requirements of suspension culture of soybean root cells. Experim. Cell. Res. (50), 151-158.
 Irvin, J. D. 1975. Purification and partial characterization of the anti-viral protein from Phytolacca Americana which inhibits eukaryotic protein synthesis. The archives of biochemistry and Biophysics. (169), 522-528. http://dx.doi.org/10.1016/0003-9861(75)90195-2.
 Irvin, J. D., T. Kelly, J. D. Robertus.1980. Purification and properties of a second antiviral protein from Phytolacca americana which inactivates eukaryotic ribosomes. Archi. Biochem. Biophys 200(2), 418-425. http://dx.doi.org/10.1016/0003-9861(80)90372-0.
 Irvin, J. D. and F.M. Uckun. 1992. Pokeweed antiviral protein: Ribosome inactivation and therapeutic applications. Pharmacology & Therapeutics (55), 279-302. http://dx.doi.org/10.1016/0163-7258(92)90053-3.
 Karadi, R. V., A. Shah, P. Parekh, and P. Azmi. 2011. Antimicrobial Activities of Musa paradisiaca and Cocos nucifera. International Journal of Research in Pharmaceutical and Biomedical Sciences. 2 (1), 264-267.
 Kino, M., K. Yamaguchi, H. Umekawa, G. Funatsu. 1995. Purification and characterization of three mitogenic lectins from the roots of pokeweed (Phytolacca americana). Bioscience, Biotechnology, and Biochemistry. (59), 683-688.
 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. (2277), 680-685. http://dx.doi.org/10.1038/227680a0.
 Lodge, J. K., W. K. Kaniewski, and N.E. Tumer. 1993. Broad-spectrum virus resistance in transgenic plants expressing pokeweed antiviral protein. Proceedings of the Nation. Academy of Science of the United States of America (90), 7089-7093. http://dx.doi.org/10.1073/pnas.90.15.7089.
 Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. (15), 473â€“497. http://dx.doi.org/10.1111/j.1399-3054.1962.tb08052.x.
 Opassiri, R., J. R. K. Cairns, T. Akiyama, O.W. Aswapati, J. Svasti, A. Esen.2003. Characterization of a rice Î²-glucosidase highly expressed in flower and germinating shoots. Plant Science. (165), 627-638. http://dx.doi.org/10.1016/S0168-9452(03)00235-8.
 Owen, M. D. K. 1988. "Pokeweed (Phytolacca americana L.)," Publication Pm-746 of the Iowa State University Extension Service, Ames, IA. Iowa State University.
 Rajamohan, F., T. K. Venkatachalam, J. D. Irvin, and F. M. Uckun.1999. Pokeweed Antiviral Protein Isoforms PAP-I, PAP-II, and PAP-III Depurinate RNA of Human Immunodeficiency Virus (HIV)-1. Biochemical and Biophysical Research Communications (260), 453â€“458. http://dx.doi.org/10.1006/bbrc.1999.0922.
 Ravikiran, G., A. B. Raju, and Y. Venugopal. 2011. Phytolacca Americana: a review. International Journal of Research in Pharmaceutical and Biomedical Sciences. 3(2), 942-946.
 Ready, M. P., D.T Brown, and J.D. Robertus. 1986. Extracellular localization of pokeweed antiviral protein. Proceedings National Academy Science, USA. (83), 5053-5056. http://dx.doi.org/10.1073/pnas.83.14.5053.
 Sussner, U., G. Abel, R. Schulte, and W. Kreis. 2004. Isolation and characterization of a cysteine protease (phytolacain G), from Phytolacca americana roots. Planta Medica. (70), 942-947. http://dx.doi.org/10.1055/s-2004-832621.
 Steel, R. G. D. and J. H. Torrie. 1980. Principles and procedures of statistics: a biometrical approach. 2nd Ed. McGraw-Hill Inc., New York.
 Sindambiwe, J.B., M. Calomme, S. Geerts, L. Pieters, A. J. Vlietinck, D. A. Vanden Berghe. 1998. Evaluation of biological activities of triterpenoid saponins from Maesa lanceolata. Journal of Natural Products. (61), 585â€“590. http://dx.doi.org/10.1021/np9705165.
 Sparg, S. G., M.E. Light, J. van Staden. 2004. Review: Biological activities and distribution of plant saponins. Journal of Ethnopharmacology. (94), 219â€“243. http://dx.doi.org/10.1016/j.jep.2004.05.016.
 Strauss A., S. M. Spengel, and W. Schaffner. 1994. Saponins From Root cultures of Phytolacca acinosa. Phytochemistry 38(4), 861-865. http://dx.doi.org/10.1016/0031-9422(94)00749-J.
 Suga, Y., Y. Maruyama, S. Kawanishi, and J. Shoji. 1978. Studies on the constituents of phytolaccaceous plants. I. On the structures of phytolaccasaponin B, E and G from the roots of Phytolacca americana L. Chem. Pharm. Bull. (26), 520â€“525. http://dx.doi.org/10.1248/cpb.26.520.
 United States FDA Poisonous, 2015. Plants Database, http://www.accessdata.fda.gov/scripts/plantox/
 Upadhyay, R. K. 2015. Antimicrobial Activity of Fruit Latexes from Ten Laticiferous Plants. Am. J. Plant Sci. (6), 483-499.