Comparative Analysis of Chemical Composition and Biological Activities of Ajuga genevensis L. in in vitro Culture and Intact Plants

Authors: Naira Sahakyan, Margarit Petrosyan, Armen Trchounian

Abstract:

One of the tasks in contemporary biotechnology, pharmacology and other fields of human activities is to obtain biologically active substances from plants. They are very essential in the treatment of many diseases due to their actually high therapeutic value without visible side effects. However, sometimes the possibility of obtaining the metabolites is limited due to the reduction of wild-growing plants. That is why the plant cell cultures are of great interest as alternative sources of biologically active substances. Besides, during the monitored cultivation, it is possible to obtain substances that are not synthesized by plants in nature. Isolated culture of Ajuga genevensis with high growth activity and ability of regeneration was obtained using MS nutrient medium. The agar-diffusion method showed that aqueous extracts of callus culture revealed high antimicrobial activity towards various gram-positive (Bacillus subtilis A1WT; B. mesentericus WDCM 1873; Staphylococcus aureus WDCM 5233; Staph. citreus WT) and gram-negative (Escherichia coli WKPM M-17; Salmonella typhimurium TA 100) microorganisms. The broth dilution method revealed that the minimal and half maximal inhibitory concentration values against E. coli corresponded to the 70 μg/mL and 140 μg/mL concentration of the extract respectively. According to the photochemiluminescent analysis, callus tissue extracts of leaf and root origin showed higher antioxidant activity than the same quantity of A. genevensis intact plant extract. A. genevensis intact plant and callus culture extracts showed no cytotoxic effect on K-562 suspension cell line of human chronic myeloid leukemia. The GC-MS analysis showed deep differences between the qualitative and quantitative composition of callus culture and intact plant extracts. Hexacosane (11.17%); n-hexadecanoic acid (9.33%); and 2-methoxy-4-vinylphenol (4.28%) were the main components of intact plant extracts. 10-Methylnonadecane (57.0%); methoxyacetic acid, 2-tetradecyl ester (17.75%) and 1-Bromopentadecane (14.55%) were the main components of A. genevensis callus culture extracts. Obtained data indicate that callus culture of A. genevensis can be used as an alternative source of biologically active substances.

Keywords: Ajuga genevensis, antibacterial activity, antioxidant activity, callus cultures.

Digital Object Identifier (DOI): doi.org/10.5281/zenodo.1125105

References:

[1] S. Hellwig, J. Drossard, R.M. Twyman, and R. Fischer, “Plant cell cultures for the production of recombinant proteins,” Nat Biotech, 2004, vol. 22, pp. 1415-1422.
[2] N. Darbinian-Sarkissian, A. Darbinyan, J. Otte, S. Radhakrishnan, B.E. Sawaya et al., “p27SJ, a novel protein in St John's Wort, that suppresses expression of HIV-1 genome,” Gene Therapy. 2006, vol.1, pp.288–295.
[3] M. Petrosyan, Y. Sherbakova, N. Sahakyan, Z. Vardanyan, A. Poladyan, Yu. Popov, A. Trchounian, “Alkanna orientalis (L.) Boiss. plant isolated cultures and antimicrobial activity of their extracts: Phenomenon, dependence on different factors and effects on some membrane-associated properties of bacteria,” Plant Cell, Tissues and Organ Culture - J Plant Biotechnol, 2015, vol.122, N 3, pp.727-738.
[4] H. Md. Sarfaraj, F. Sheeba, A. Saba, Md. Rahman, A. Akhlaquer et al., “Current approaches toward production of secondary plant metabolites,” J Pharm & Bioall Scien, 2012, vol. 4, N 1, pp.10-20.
[5] M. Z. Abdin, M. Izrar, R.R. U, S.K. Jain, “Artemisinin, a novel antimalarial drug: biochemical and molecular approaches for enhanced production,” Planta Med, 2003, vol. 69, pp. 289–299.
[6] S. M. K. Rates, “Plants as source of drugs,” Toxicol, 2001, vol. 39, pp. 603-613.
[7] I. Smetanska, “Production of secondary metabolites using plant cell cultures,” Adv Biochem Eng Biotechnol, 2008, vol. 111, pp. 187-228.
[8] D.J. N ewman, G. Cragg, “Natural products as sources of new drugs over the last 25 years,” J Nat Prod, 2007, vol.70, pp.461–477.
[9] J.Coll, “NMR shift data of neo-clerodane diterpenes from the genus Ajuga,” Phytochem Anal, 2002, vol.13, pp. 372-380.
[10] J. H. Ben, A. Chaari, A. Bakhrouf, Z. Mighri, “Structure–antibacterial activity relationship of secondary metabolites from Ajuga pseudoiva Rob. leaves,” J Nat Prod Res, 2006, vol. 20, pp. 299–304.
[11] A. Chaari, H.B. Jannet, Z. Mighri, M.C. Lallemand, “7-0-6`-0-malonylcachinesidic acid, a new macrocyclic iridoid ester of malonic acid from Tunisian plant Ajuga pseudoiva,” J Nat Prod, 2002, vol. 65, pp. 618-620.
[12] R. Lafont, L. Dinan, “Practical uses for ecdysteroids in mammals including humans: an update,” J. Insect. Sci, 2003, vol. 3, p. 30.
[13] A. Ványolós, A. Simon, G. Tóth, L. Polgá, Z. Kele et al., “C-29 ecdysteroids from Ajuga reptans var. reptans,” J Nat. Prod, 2009, vol. 72, N 5, pp. 929-932.
[14] J. Coll, A. T. Yudelsy, “Neo-Clerodane diterpenoids from Ajuga: structural elucidation and biological activity,” Phytochem Rev, 2008, vol. 7, pp. 25–49.
[15] H. C. Inan, F. Keller, “Purification and characterization of the raffinose oligosaccharide chain elongation enzyme, galactan: galactan galactosyltransferase (GGT), from Ajuga reptans leaves,” Physiol Plant, 2002, vol. 114, pp. 361-371.
[16] R. Singh, S. M. Patil, G. Pal, M. Ahmad, “Evaluation of in vivo and in vitro anti-inflammatory activity of Ajuga bracteosa Wall ex Benth,” Asian Pac J Trop Dis, 2012, vol.1, pp. 404-407.
[17] A. Pal, F. A. Toppo, P. K. Chaurasiya, P. K. Singour, R. S. Pawar, “In-vitro cytotoxicity study of methanolic fraction from Ajuga Bracteosa wall ex. benth on MCF-7 breast adenocarcinoma and hep-2 larynx carcinoma cell lines,” Pharmacogn Res, 2014, vol. 6, N 1, pp. 87-91.
[18] N. Sh. Ramazanov, “Phytoecdysteroids and other biologically active compounds from plants of the genus Ajuga,” Chem Nat Comp, 2005, vol. 41, pp. 361-369.
[19] B. L. Ayari, L. Riahi, S. Ziadi, H. Chograni, A. Mliki, “Evaluation of antioxidant and antimicrobial activities of Tunisian Ajuga iva L. essential oils,” Revue F. S. B. 2013, pp. 203-201.
[20] J. N. Gitua, D. R. Muchiri, X. A. Nguyen, “In vivo antimalarial activity of Ajuga remota water extracts against Plasmodium berghei in mice,” Southeast Asian J Trop Med Publ Health, 2012, vol. 43, N 3, pp. 545-548.
[21] G. Xinbo, H. L. Rui, F. Xueqing, S. Xiaofen, T. Kexuan, “Over-expression of l-galactono-c-lactone dehydrogenase increases vitamin C, total phenolics and antioxidant activity in lettuce through bio-fortification,” Plant Cell, Tissues and Organ Culture - J Plant Biotechnol, 2013, vol. 114, pp. 225–236.
[22] R.M. Kuriba, “Antifungal activity of Ajuga remota” Fitoterapia, 2001, vol. 72, pp. 177-178.
[23] M. Makni, A. Hadaar, W. Kriaa, N. Zeghal, “Antioxidant, free radical scavenging, and antimicrobial activities of Ajuga iva leaf extracts,” Int J Food Prop, 2013, vol. 16, pp. 756-765.
[24] D.S reeramulu, C. V. K. Reddy, A. Chauhan, N. Balakrishna, M. Raghunath, “Natural antioxidant activity of commonly consumed plant foods in India: Effect of domestic processing,” Oxid Med Cell Longev, 2013, vol. 1, pp. 1-12.
[25] J. K. Willcox, S. L. Ash, G. L. Catignani, “Antioxidants and prevention of chronic diseases,” Crit Rev Food Sci Nutr, 2004, vol. 44, pp. 275-295.
[26] T. Murashige, F. Skoog, “A revised medium for rapid growth and with tohoco tissues cultures,” Physiol Plantarum, 1962, vol. 15, pp. 475-477.
[27] A. W. Bauer, W. M. M. Kirby, J. C. Sheriss, M. Turck, “Antibiotic susceptibility testing by standardized single method,” Am J Clin. Pathol, 1966, vol. 45, pp. 493–496.
[28] P. Kanoktip, S. Ratchada, N. Ikuo, M. Masahiro, S. Kanyaratt, “Tetraploid induction of Mitracarpus hirtus L. by colchicine and its characterization including antibacterial activity,” Plant Cell, Tissues and Organ Culture - J Plant Biotechnol, 2014, vol. 117, N3, 380-391.
[29] A. A. Adesokan, M. A. Akanji, M. T. Yakubu, “Antibacterial potentials of aqueous extract of Enantia chlorantha stem bark,” Afr J Biotechnol, 2007, vol. 6, pp. 2502-2505.
[30] M. Griffiths, H. Sundaram, “Drug design and testing: profiling of antiproliferative agents for cancer therapy using a cell-based methyl-(3H)-thymidine incorporation assay,” Methods Mol Biol, 2011, vol. 731, pp. 451-465.
[31] S. R. Georgetti, R. Casagrande, F. T. Vicentini, W. A. Verri, M. J. Vieira Fonseca, “Evaluation of the antioxidant activity of soybean extract by different in vitro methods and investigation of this activity after its incorporation in topical formulations,” Eur J Pharm Biopharm, 2006, vol. 64, pp. 99-106.
[32] A. Doyle, J. B. Griffiths, “Cell and Tissue culture: Laboratory Procedures in Biotechnology,” Chichester: JohnWiley&Sons Inc., UK, 1998, pp. 174-178.
[33] NIST Standard Reference Database 1Ahttp://www.nist.gov/srd/upload/NIST1a11Ver2-0Man.pdf
[34] N. Zh. Sahakyan, M. Т. Petrosyan, V. V. Volodin, S. O. Volodina, J. A. Aghajanyan, Yu. G. Popov, “Isolated culture of Ajuga genevensis L. as a potential source of biological active substances,” New Armenian Med J, 2008, vol. 2, N 4, 65-74.