Investigation of Relationship Between Chemical Stress Factors and Certain Metabolites Including Cardenolides in Callus Cultures of Endemic Turkish Digitalis L. Species

Year 2017, Volume: 4 Issue: 3, Special Issue 1, 27 - 36, 25.11.2017

Gunce Sahin Sandeep Kumar Verma Ekrem Gurel

https://doi.org/10.21448/ijsm.356249

Cited By: 1

Abstract

The aim of the present research is to obtain
relationship between different stress treatments [Cu (copper) and Hg (mercury)]
and content of cardiac glycosides (digoxigenin, gitoxigenin, lanatoside C,
digoxin and digitoxin) as secondary metabolites of commercial value for the
pharmaceutical industry and to determine the antioxidant metabolites against
stress conditions in callus cultures of endemic Turkish Digitalis species. The effects of different stress treatments on
cardiotonic glycoside accumulation in D.
lamarckii Ivanina, D. trojana
Ivanina, D. davisiana Heywood and D. cariensis Boiss. ex Jaub. et Spach
were investigated using HPLC. HPLC analysis revealed that all stress conditions
were significantly effective at 5% significance level according to their
control groups. The predominant cardiac glycoside was lanatoside C (Lan C)
followed by digitoxin, digoxigenin, gitoxigenin and digoxin. No digoxin was
detected in all treatments as well as in control groups. For the calibration
curves, concentrations of 5, 10, 20, 30 and 40 mg/l digoxigenin, gitoxigenin,
lanatoside C, digoxin and digitoxin were used (R²= 0.99).
Cardenolides were eluted with acetonitrile (A) and water (B) gradients as
follows: 0 to 20 min 20% (A), 80% (B); 20 to 23.40 min 30% (A), 70% (B); 23.40
to 30 min 25% (A), 75% (B) and 30 to 40 min 40% (A), 60%(B). Average peak area
of the glycoside in samples was automatically calculated and monitored by
ChemStation LC/MS software against that of standards. Enhanced production of
cardenolides was achieved from callus cultures elicited with 50 μm CuSO₄
and HgCl₂. Higher amounts of cardenolides were obtained when callus
of four Digitalis species were
elicited with CuSO4. Results demonstrated that catalase (CAT, EC 1.11.1.6),
superoxide dismutase (SOD, EC 1.15.1.1) activities, the total contents of
phenolics and proline were markedly stimulated under stress conditions. All
these results indicated that treatments have induced changes in the redox state
of callus cells and suggest that this alteration change cardenolides
accumulation and antioxidative status in Digitalis
L. callus cultures.

Keywords

Antioxidant, cardiac glycosides, Digitalis L., heavy metal stress

Investigation of Relationship Between Chemical Stress Factors and Certain Metabolites Including Cardenolides in Callus Cultures of Endemic Turkish Digitalis L. Species

Abstract

The aim of the present research is to obtain relationship between different stress treatments [Cu (copper) and Hg (mercury)] and content of cardiac glycosides (digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin) as secondary metabolites of commercial value for the pharmaceutical industry and to determine the antioxidant metabolites against stress conditions in callus cultures of endemic Turkish Digitalis species. The effects of different stress treatments on cardiotonic glycoside accumulation in D. lamarckii Ivanina, D. trojana Ivanina, D. davisiana Heywood and D. cariensis Boiss. ex Jaub. et Spach were investigated using HPLC. HPLC analysis revealed that all stress conditions were significantly effective at 5% significance level according to their control groups. The predominant cardiac glycoside was lanatoside C (Lan C) followed by digitoxin, digoxigenin, gitoxigenin and digoxin. No digoxin was detected in all treatments as well as in control groups. For the calibration curves, concentrations of 5, 10, 20, 30 and 40 mg/l digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin were used (R2= 0.99). Cardenolides were eluted with acetonitrile (A) and water (B) gradients as follows: 0 to 20 min 20% (A), 80% (B); 20 to 23.40 min 30% (A), 70% (B); 23.40 to 30 min 25% (A), 75% (B) and 30 to 40 min 40% (A), 60%(B). Average peak area of the glycoside in samples was automatically calculated and monitored by ChemStation LC/MS software against that of standards. Enhanced production of cardenolides was achieved from callus cultures elicited with 50 μm CuSO4 and HgCl2. Higher amounts of cardenolides were obtained when callus of four Digitalis species were elicited with CuSO4. Results demonstrated that catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) activities, the total contents of phenolics and proline were markedly stimulated under stress conditions. All these results indicated that treatments have induced changes in the redox state of callus cells and suggest that this alteration change cardenolides accumulation and antioxidative status in Digitalis L. callus cultures.

Keywords

Antioxidant, cardiac glycosides, Digitalis L., heavy metal stress

References

• Baytop, T. (1999). Türkiye’de Bitkilerle Tedavi. Istanbul: Nobel Tıp Kitabevi.
• Newman, R.A., Yang P., Pawlus A.D., & Block K.I. (2008). Cardiac glycosides as novel cancer therapeutic agents. Molecular Interventions, 8, 36-49.
• Verma, S.K., Sahin, G., Yucesan, B., Sahbaz, N., Eker, I., Gurel, S., & Gurel, E. (2012). Direct somatic embryogenesis from hypocotyl segments of Digitalis trojana Ivan and subsequent plant regeneration. Industrial Crops and Products, 40, 76-80.
• Radman, R., Saez, T., Bucke, C., & Keshavarz, T. (2003). Elicitation of plants and microbial cell systems. Biotechnology and Applied Biochemistry, 37, 91-102.
• Zhao, J., Davis, L.C., & Verpoorte, R. (2005). Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances, 23, 283-333.
• Kim, O.T., Bang, K.H., Kim, Y.C., Hyun, D.Y., Kim, M.Y., & Cha, S.W. (2009). Upregulation of ginsenoside and gene expression related to triterpene biosynthesis in ginseng hairy root cultures elicited by methyl jasmonate. Plant Cell Tissue and Organ Culture, 98, 25–33.
• Lin, C.C., Chen, L.M., Liu, Z.H. (2005). Rapid effect of copper on lignin biosynthesis in soybean roots. Plant Science, 168, 855-861.
• Maksymiec, W. (2007). Signalling responses in plants to heavy metal stress. Acta Physiologiae Plantarum, 29, 177-187.
• Agrawal, V., & Sharma, K. (2006). Phytotoxic effects of Cu, Zn, Cd and Pb on in vitro regeneration and concomitant protein changes in Holarrhena antidysenterica. Biologia Plantarum, 50, 307-310.
• Parmar, G.N., & Chanda, V.S. (2005). Effects of Mercury and Chromium on Peroxidase and IAA Oxidase Enzymes in the Seedlings of Phaseolus vulgaris. Turkish Journal of Biology, 29, 15-21.
• Shanker, A.K., Cervantes, C., Loza-Tavera, H., & Avudainayagam, S. (2005). Chromium toxicity in plants. Environment International, 31, 739-753.
• Swaran, J.S.F. (2009). Structural, chemical and biological aspects of antioxidants for strategies against metal and metalloid exposure. Oxidative Medicine and Cellular Longevity, 2, 191-206.
• Cho, U., & Park, J. (2000). Mercury induced oxidative stress in tomato seedlings. Plant Science, 156, 1-9.
• Wiegrebe, H., & Wichtl, M. (1993) High-performance liquid chromatographic determination of cardenolides in Digitalis leaves after solid-phase extraction. Journal of Chromatography A, 630, 402-407.
• Cingoz, S.G., Verma, S.K., & Gürel, E. (2014). Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species. Plant Physiology and Biochemistry, 82, 89-94.
• Sun, Y., Oberley, L.W., & Li, Y. (1988). A simple method for clinical assay of superoxide dismutase. Clinical Chemistry, 34, 497-500.
• Lartillot, S., Kedziora, P., & Athias, A. (1988). Purification and characterization of a new fungal catalase. Preparative Biochemistry and Biotechnology, 18, 241-246.
• Lowry, O.H., Rosebrough, N.J., Farr, A.L., & Randall, R.J. (1951). Protein measurement with the Folin Phenol Reagent. The Journal of Biological Chemistry, 193, 265-75.
• Marigo, G. (1973). Sur une méthode de’fractionnement et d’ estimation des composés phénoliques chez les vegétaux. Analusis, 2, 106-110.
• Bates, L.S., Waldren, R.P., & Teare, I.D. (1973). Rapid determination of free proline for water-stress studies. Plant Soil, 39, 205-207.
• Koricheva, J., Larssson, S., Haukioja, E., & Keinanen, M. (1998). Regulation of woody plant secondary metabolism by resource availability: hypothesis testing by means of meta-analysis. Okios, 83, 121-226.
• Eisenbeiß, M., Kreis, W., & Reinhard E. (1999). Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures. Plant Physiology and Biochemistry, 37,13–23.
• Gavidia, I., Del Castillo Agudo, L., & Perez-Bermudez, P. (1996). Selection and long-term cultures of high-yielding Digitalis obscura plants: RAPD markers for analysis of genetic stability. Plant Science, 121, 197-205.
• Gavidia, I., & Perez-Bermudez, P. (1997). Digitalis obscura cardenolides effect of macronutrient concentration and N source on growth and productivity of shoot–tip cultures. Phytochemistry, 46, 273-238.
• Roca-Perez, L., Boluda, R., & Perez-Bermudez, P. (2004). Soil–plant relationships, micronutrient contents and cardenolide production L. in natural populations of Digitalis obscura. Journal of Plant Nutrition and Soil Science,167, 79-84.
• Stuhlfauth, T., Klug, K., & Fock, H.P. (1987). The production of secondary metabolites by Digitalis lanata during CO2 enrichment and water stress. Phytochemistry, 26, 2735-2739.
• Brugidou, C., Jacques, M., Cosson, L., Jarreau, F.X., & Ogerau, T. (1988). Growth and digoxin content in Digitalis lanata in controlled conditions and natural environment. Planta Medica, 54, 262–265.
• Sahin, G., Verma, S.K., & Gürel, E. (2013). Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey. Plant Physiology and Biochemistry, 73, 139-143.
• Palacio, L., Baeza, C., Cantero, J.J., Cusido´, R., & Goleniowsi, M.E. (2008). In Vitro propagation of “Jarilla” (Larrea divaricata CAV.) and secondary metabolite production. Biological and Pharmaceutical Bulletin, 31, 2321-2325.
• Maksymiec, W., & Krupa, Z. (2006). The effects of short term exposition to Cd, excess Cu ions and jasmonate on oxidative stress appearing in Arabidopsis thaliana. Environmental and Experimental Botany, 57, 187-194.
• Smith, R.A., Drummond, S., Haines, A., Porter, J.R., & Hock, R.S (2001). Induction of umbelliferone in sweet potato cell suspension culture using mercuric chloride. Biotechnological Letters, 23, 1397-1400.
• Korsangruang, S., Soonthornchareonnon, N., Chintapakorn, Y., Saralamp, P., & Prathanturarug, S. (2010). Effects of abiotic and biotic elicitors on growth and isoflavonoid accumulation in Pueraria candollei var. candollei and P. Candollei var. mirifica cell suspension cultures. Plant Cell Tissue and Organ Culture, 103, 333-342. [33]. Perez-Bermudez, P., Garcıa, A.A.M., Tunon, I., & Gavidia, I. (2010). Digitalis purpurea P5βR2, encoding steroid 5β-reductase, is a novel defense-related gene involved in cardenolide biosynthesis. New Phytologist, 185, 687–700. [34]. Hung, S.H., Yu, C.W., Lin, C.H. (2005). Hydrogen peroxide functions as a stress signal in plants. Botanical Bulletin Academia Sinica, 46, 1-10. [35]. Ikeda, Y., Fujii, Y., & Yamazaki, M. (1992). Determination of Lanatoside C and Digoxin in Digitalis lanata by HPLC and its Application to Analysis of the Fermented Leaf Powder. Journal of Natural Products, 55, 748-752. [36]. Romero-Puertas, M.C., Perazzolli, M., Zago, E.D., & Delledonne, M. (2004). Nitric oxide signalling functions in plant-pathogen interactions. Cellular Microbiology, 6, 795-803. [37]. Mittler R. (2002). Oxidative stress, antioxidants and stress tolerance. Trends in Plant Science, 7, 405-410. [38]. Chen, C.T., Chen, L.M., Lin, C.C., & Kao, C.H. (2001). Regulation of Proline Accumulation in Detached Rice Leaves Exposed to Excess Copper. Plant Science, 160, 283-290. [39]. Alia, A., Saradhi, P.P., & Mohanty P. (1991). Proline enhances primary photochemical activities in isolated thylakoid membranes of Brassica juncea by arresting photo inhibitory damage. Biochemical and Biophysical Research Communications, 181, 1238-1244. [40]. Schat, H., Sharma, S.S., & Vooijs, R. (1997). Heavy metal-induced accumulation of free proline in metal-tolerant and a nontolerant ecotype of Silene vulgaris. Physiologia Plantarum, 101, 477-482. [41]. Lutts, S., Kinet, J.M., & Bouharmont, J. (1996). Effects of various salts and of mannitol on ion and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) callus cultures. Journal of Plant Physiology, 149, 186-195. [42]. Siripornadulsil, S., Train, S., Verma, D.P.S., & Sayre, R.T. (2002). Molecular mechanisms of proline-mediated tolerance to toxic heavy metals in transgenic microalgae. Plant Cell, 14, 2837-2847.
• Zengin, F.K., & Munzuroğlu, Ö. (2005). Effects of some heavy metals on content of chlorophyll, proline and some antioxidant chemicals in bean (Phaseolus vulgaris L.) seedlings. Acta Biologica Cracoviensia Series Botanica, 47, 157-164.
• Grant, J.J., Yun, B.W., & Loake, G.J. (2000). Oxidative burst and cognate redox signaling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me-JA but is dependent on MAPKK activity. The Plant Journal, 24, 569-582.
• Jung, C.H., Maeder, V., Funk, F., Frey, B., Sticher, H., & Frosserd, E. (2003). Release of phenols from Lupinus albus L. roots exposed to Cu and their possible role in Cu detoxification. Plant Soil, 252, 301-312.
• Ganeva, G., & Zozikova, E. (2007). Effect of increasing Cu2+ concentrations on growth and content of free phenols in two lines of wheat (Triticum aestivum) with different tolerance. General and Applied Plant Physiology, 33, 75-82.
• Esteban, E., Morenoa, E., Penalosa, J., Cabrero, J.I., Millan, R., & Zornoza, P. (2008). Short and long-term uptake of Hg in white lupin plants: Kinetics and stress indicators. Environmental and Experimental Botany, 62, 316-322.