Isolation, Purification and Characterization of Novel Phytomitogen from Nigella sativa Seeds

Haleem, Azhar M.

doi:10.30684/etj.v38i1B.568

	Isolation, Purification and Characterization of Novel Phytomitogen from Nigella sativa Seeds
Engineering and Technology Journal
Article 4, Volume 38, 1B, 2020, Pages 26-33 PDF (755.2 K)
DOI: 10.30684/etj.v38i1B.568
Author
Azhar M. Haleem
Environmental Research Center, University of Technology, Baghdad, Iraq
Abstract
The objective of the present study was to extract and purified proliferative factor for peripheral blood lymphocytes from Nigella sativa seeds. Dimeric 44, 32 and 26 -kDa glucosamine-specific lectins were extracted and purified from seeds of N. sativa L. by using different ionic strength solvents (deionized distilled water, phosphate buffer slain and 0.1 M HCl) purification procedure included, precipitation in saturated ammonium sulphate, then Sephadex G-100 gel filtration was used for further purification, finally electrophoresis by polyacrylamide gel to estimate the molecular weight in the presence of sodium dodecylsulphate. Single absorbance peak at 280 nm was recorded in each extracted method. The temperature and pH value on mitogenic activity was studied in temperature ranged from 4°C-60°C, the mitogenic activity reduced with gradual increase of temperatures, 50% of activity lost at temperature greater than 40°C, while the optimum pH for mitogenic activity ranged from 5-9, at pH value less than 3, and greater than 12 the activity disappeared. Interestingly, lectin extracted from N. sativa seeds have high activity especially that extracted with 0.1 M HCl and showed stability at temperature from 4°C to 10°C and pH from 5 to 9. The pure lectin is a homodimeric molecule of 26 kDa with subunit molecular mass of 12.9 kDa. The novel lectin exceed the PHA-Sigma in mitogenic activity.
Keywords
Mitogen; Nigella sativa; Phytomitogen; Protein extraction

References
[1] B. Liu, H.J. Bian, J.K. Bao, “Plant lectins: potential antineoplastic drugs from bench to clinic,” Cancer Lett 287:1–12, 2010. [2] M.A. Khan, “Chemical composition and medicinal properties of Nigella Sativa Linn,” Inflammo. Pharmacology. Vol. 7, 1, 15-35, 1999. [3] P. Pellerin, “Comparing extraction by traditional solvents with supercritical extraction from an economic and environmental standpoint,” Proceedings of 6 th International Symposium on Supercritical Fluids, Versailles, France, 28-30 April 111-120. 2003. [4] K.M. Fararh, Y. Atoji, Y. Shimizu, T. Takewaki, “Isulinotropic properties of Nigella sativa oil in streptozotocin plus nicotinamide diabetic hamster,” Res Vet Sci. 73, 279-82. 2003. [5] M. El-Dakhakhny N. Mady, N Lembert, H P T Ammon, “The hypoglycemic effect of Nigella sativa oil is mediated by extrapancreatic actions,” Planta Med. 68, 465-466, 2002. [6] A. Wajs, R. Bonikowski, D. Kalemba, “Composition of essential oil from seeds of Nigella sativa L. cultivated in Poland,” J. Flavour and Fragrance, 23, 126-132.2008. [7] P. Paarakh, “Nigella Sativa Linn. – A comprehensive review. 2010,” Indian Journal of Natural Products and Resources, 1, nr 4, 409-429.2010. [8] A. Rym, F. Benkaci, M. Zerrouki, G. Eppe, “ Biological activities of the essential oil of Nigella sativa seeds isolated by Composition and accelerated microwave steam distillation with cryogenic grinding,” American Journal of Essential Oils and Natural Products, 1, 3, 23-33, 2014. [9] E. Halawani, “Antibacterial activity of thymoquinone and thymohydroquinone of Nigella sativa L. and their interaction with some antibiotics,” Adv Biol Res. 3, 5-6, 148-152, 2009. [10] M. A. Jebor and Y. H. Jalil, “Extraction, Purification and characterization of a lectin from Phaseolus vulgaris L. cv. white seeds (white kidney bean),” Medical Journal of Babylon. 9:4. 2014. [11] M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding,” Analytical Biochemistry 72, 248-254. 1976. [12] V. Dhuna, J. S. Bains, S. S. Kamboj, J. S. Shanmugavel and A. K. Saxena, “Purification and characterization of a lectin from Arisa ema tortuosum schott having in-vitro anticancer activity against human cancer cell lines,” Journal of Biochemistry and Molecular Biology. 38, 5, 526-532.2005. [13] E. H. Konozy, E.S. Bernardes, C. Rosa, V. Faca, L. J. Greene, R. J. Warda, “Isolation, purification, and physicochemical characterization of a D-galactose-binding lectin from seeds of Erythrina speciosa”Arch Biochem Biophys, 410, 2, 222-229. 2003. [14] R. Verma and A. Babu “Human chromosomes: Manual of basic techniques,” Pregramon press, New York, 1989. [15] Q. Silvia, C. Nanne and L . González, “Characterization and mitogenic effect, Rev. Biol. Trop. 46, 4, 1039-1046, 1998. [16] D. J. Hall, J. J. O’Leary, A. Rosenberg, “Effects of temperature on aggregation and the mitogen-induced exit of lymphocytes from the resting state,” Journal of Cellular Physiology, 121, 1, 206-214. 1984. [17] S.C. Yau, J.H. Wong, E. F. Fang, W. Pan, T. N. Bun, “Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti proliferative activity towards tumor cells,” Plosone.7 , 6 , e38961, 2012. [18] J.M. Van Damme, W. J. Peumans, A. Barre and P. Rougé, “Classification of plant lectins in families of structurally and evolutionary related proteins,” Adv Exp Med Biol 491, 27–54, 1998. [19] A. Kaur, S. S. Kamboj, J. Singh , A.K. Saxena , and V. Dhuna, “Isolation of a novel N-acetyl-D-lactosamine specific lectin from Alocasia cucullata,” Biotechnology Letters. 27, 1815-1820, 2005. [20] J. S. Bains, V. Dhuna, J. Singh,, S. Kamboj, K. KaurNijjar and J. N. Aqrewala ,“ Novel lectins from rhizomes of two acorus species with mitogenic activity and inhibitory potential towards murine cancer cell lines,” International Immuno-pharmacology, 5, 9, 1470-1478, 2005. [21]C. E. Hayes and I. J. Goldstein “An alphad-galactosyl-binding lectin from Bandeiraea simplicifolia seeds. Isolation by affinity chromatography and characterization,” The Journal of Biological Chemistry. 249(6):1904-1914, 1974. [22] E.J. Van Damme, K. Smeets, S. Torrekens, F. van Leuven , I.J. Goldstein, W. J. Peumans, “The closely related homomeric and heterodimeric mannosebinding lectins from garlic are encoded by one-domain and two-domain lectin genes, respectively,” European Journal of Biochemistry, 206, 2, 413-420, 1992.
Statistics Article View: 285 PDF Download: 247