Abstract
Cardiotonic glycosides are extracted mostly from leaves of Digitalis plants. Commercial production of bioactive secondary metabolites by traditional agriculture is an inefficient process and can be affected by climatic and soil conditions. Strategies, based on in vitro culture methods, have been extensively studied to improve the production of specific plant derived chemicals. The aim of the present research was to obtain biomass of D. purpurea using the temporary immersion system (TIS) and to determine the content of cardiotonic glycosides (digitoxin, digoxin and lanatoside C) as secondary metabolites of commercial value for the pharmaceutical industry. Shoots were cultured in 1,000 ml TIS during 28 days. The effect of four immersion frequencies (once every 2, 4, 6, and 12 h) was studied. Biomass accumulation was influenced by immersion frequency. The maximum biomass accumulation (values in respect of fresh and dry weight) was obtained with immersions every 4 h (six immersions per day). HPLC analysis revealed the presence of digoxin and digitoxin for all immersion frequencies. No lanatoside C was detected in the biomass cultured in TIS. Digoxin concentrations varied depending on the frequencies tested. In contrast, the digitoxin content showed no dependency on the immersion frequency. Net production of digoxin and digitoxin per TIS were found to be higher with immersions every 4 h. The best net production of digitoxin and digoxin per TIS were 167.6 and 119.9 μg, respectively. The development of organ culture based on temporary immersion system can be a reliable method for the steady production of biomass for cardiotonic glycosides production, which is reported for the first time for Digitalis genus in this communication.
Similar content being viewed by others
Abbreviations
- 6-BAP:
-
6-Benzylaminopurine
- DW:
-
Dry weight
- FW:
-
Fresh weight
- IAA:
-
Indole-3-acetic acid
- TIS:
-
Temporary immersion system
- MS:
-
Murashige and Skoog medium
References
Arencibia A, Bernal A, Yang L, Cortegaza L, Carmona E, Pérez A, Hu CH-J, Li Y-R, Zayas C, Santana I (2008) New role of phenylpropanoid compounds during sugarcane micropropagation in temporary immersion bioreactors (TIBs). Plant Sci 175:487–496
Berthouly M, Etienne H (2005) Temporary immersion system: a new concept for use liquid medium in mass propagation. In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, New York, pp 165–195
Bourgaud F, Gravot A, Milesi S, Gontier E (2001) Production of plant secondary metabolites: a historical perspective. Plant Sci 161:839–851
Cabrera M, Gómez R, Basail M, Santos A, Medero V, López J, Rayas A, García M, Ventura J (2005) Production of yam microtubers using a temporary immersion system. Plant Cell Tiss Org Cult 83(1):103–107
Cheel J, Schmeda-Hirschmann G, Jordan M, Theoduloz C, Rodríguez J, Gerth A, Wilken D (2007) Free radical scavenging activity and secondary metabolites from in vitro cultures of Sanicula graveolens. Z Naturforsch 62c:555–562
Debjani D, Brattati D (2005) Elicitation of diosgenin production in Dioscorea floribunda by ethylene-generating agent. Fitoterapia 76:153–156
Eisenbeiβ M, Kreis W, Reinhard E (1999) Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures. Plant Physiol Biochem 37:13–23
Gavidia I, Pérez-Bermúdez P (1997) Cardenolides of Digitalis obscura: the effect of phosphate and manganese on growth and productivity of shoot-tip cultures. Phytochemistry 45:81–85
Gavidia I, Del Castillo L, Pérez-Bermúdez P (1996) Selection and long-term cultures of high-yielding Digitalis obscura plants: RAPD markers for analysis of genetic stability. Plant Sci 121:197–205
Gedeon R (1982) Propagating foxglove from sterile seeds or shoot apices US Patent # GB2099851 [online] http://v3.espacenet.com/ textdoc?DB = EPODOC&IDX = GB2099851
Georgiev V, Ilieva M, Bley T, Pavlov A (2008) Betalain production in plant in vitro systems. Acta Physiol Plant 30:581–593
Giri A, Dhingra V, Giri CC, Singh A, Ward O, Narasu M (2001) Biotransformations using plant cells, organ cultures and enzyme systems: current trends and future prospects. Biotechnol Adv 19:175–199
Hagimori M, Matsumoto T, Obi Y (1982) Studies on the production of Digitalis cardenolides by plant tissue culture. II Effects of light and plant growth substances on digitoxin formation by undifferentiated cells and shoot-forming cultures of Digitalis purpurea L. grown in liquid media. Plant Physiol 69:653–656
Hagimori M, Matsumoto T, Obi Y (1983) Effects of mineral salts, initial pH and precursors on digitoxin formation by shoot-forming cultures of Digitalis purpurea L. grown in liquid media. Agric Biol Chem 47:565–571
Hajari E, Berjak P, Pammenter NW, Watt MP (2008) A protocol for the production of adventitious shoot explants for cryopreservation of Ekebergia capensis using a temporary immersion system (RITA®). S Afr J Bot 74:387
Jeong CH-S, Chakrabarty D, Hahn EJ, Lee H, Paek KY (2006) Effects of oxygen, carbon dioxide and ethylene on growth and bioactive compound production in bioreactor culture of ginseng adventitious roots. Biochem Eng J 27:252–263
Jiménez E, Pérez N, de Feria M, Barbón R, Capote A, Chávez M, Quiala E, Pérez JC (1999) Improved production of potato microtubers using a temporary immersion system. Plant Cell Tiss Org Cult 59:19–23
Kreis W, May U, Reinhard E (1986) UDP-Glucose: digitoxin 16′-O-glucosyltransferase from suspension cultured Digitalis lanata cells. Plant Cell Rep 5:442–445
Lindemann P, Luckner M (1997) Biosynthesis of pregnane derivatives in somatic embryos of Digitalis lanata. Phytochemistry 46:507–513
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Navarro E, Alonso P, Alonso S, Trujillo J, Pérez C, Toro MV, Ayuso MJ (2000) Cardiovascular activity of a methanolic extract of Digitalis purpurea spp. Heywoodii. J Ethnopharmacol 71:437–442
Nebauer SG, Del Castillo L, Segura J (1999) Cardenolide variation within and among natural populations of Digitalis obscura. J Plant Physiol 154:426–430
Niemenak N, Saare-Surminski K, Rohsius CH, Omokolo D, Lieberei R (2008) Regeneration of somatic embryos in Theobroma cacao L in temporary immersion bioreactor and analices of free amino acids in different tissues. Plant Cell Rep 27:667–676
Paek KY, Chakrabarty D, Hahn EJ (2005) Application of bioreactor systems for large scale production of horticultural and medicinal plants. Plant Cell Tiss Org Cult 81:287–300
Palacio L, Baeza C, Cantero JJ, Cusidó R, Goleniowsi ME (2008) In Vitro propagation of “Jarilla” (Larrea divaricata CAV.) and secondary metabolite production. Biol Pharm Bull 31:2321–2325
Quiala E, Barbón R, Jiménez E, de Feria M, Chávez M, Capote A, Pérez N (2006) Biomass production of Cymbopogon citratus (D.C) Stapf., a medicinal plant, in temporary immersion systems. In Vitro Cell Dev Biol-Plant 42:298–300
Roca-Pérez L, Boluda R, Gavidia I, Pérez-Bermúdez P (2004) Seasonal cardenolide production and Dop5βr gene expression in natural populations of Digitalis obscura. Phytochemistry 65:1869–1878
Roels S, Escalona M, Cejas I, Noceda C, Rodríguez R, Canal MJ, Sandoval J, Debergh P (2005) Optimization of plantain (Musa AAB) micropropagation by temporary immersion system. Plant Cell Tiss Org Cult 82:57–66
Russowski D, Maurmann N, Rech SB, Fett-Neto A (2006) Role of light and medium composition on growth and valepotriate contents in Valeriana glechomifolia whole plant liquid cultures. Plant Cell Tiss Org Cult 86:211–218
Schwinger RHG, Bundgaard H, Muller-Ehmsen J, Kjeldsen K (2003) The Na, K-ATPase in the failing humann heart. Cardiovasc Res 57:913–920
Seitz HU, Gärtner D (1994) Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L. Plant Cell Tiss Org Cult 38:337–344
Shimomura K, Yoshimatsu K, Sauerwein M, Christen P, Toda Y, Aoki T (1992) Production of biologically active compounds by transformed cultures. In: Oono R, Hirabayashi T, Kiruchi S, Handa H, Kahwara S (eds) Plant tissue culture and gene manipulation for breeding and formation of phytochemicals. National Institute of Agrobiological Resources, Tsukuba, Japan, pp 293–296
Silvestrini A, Pasqua G, Botta B, Monacelli B, van der Heijden R, Verpoorte R (2002) Effect of alkaloid precursor feeding on a Camptotheca acuminate cell line. Plant Physiol Biochem 40:749–753
Teisson C, Alvard D, Berthouly M, Cote F, Escalant J, Etienne H, Lartand M (1996) Simple apparatus to perform plant tissue culture by temporary immersion. Acta Hort 440:521–526
Tisserat B, Vaughn S (2008) Growth, morphogenesis, and essential oil production in Mentha spicata L. plantlets in vitro. In Vitro Cell Dev Biol-Plant 44:40–50
Vanisree M, Tsay HS (2004) Plant cell cultures—an alternative and efficient source for the production of biologically important secondary metabolites. Int J Appl Sci Eng 2(1):29–48
Wichtl M, Mangkudidjojo M, Wichtl-Bleier W (1982) Hochleistungs-flüssigkeits-chromatographische analyse von digitalis-blattextrakten. J Chromatogr 234:503–508
Wilken D, Jiménez E, Hohe A, Jordan M, Gómez R, Schmeda G, Gerth A (2005) Comparison of secondary plant metabolite production in cell suspension, callus culture and temporary immersion system. In: Hvoslef-Eide AK, Preil W (eds) Liquid culture systems for in vitro plant propagation. Springer, New York, pp 525–537
Yang SH, Yeh D-M (2008) In vitro leaf anatomy, ex vitro photosynthetic behaviors and growth of Calathea orbifolia (Linden) Kennedy plants obtained from semi-solid medium and temporary immersion systems. Plant Cell Tiss Org Cult 93:201–207
Zhao J, Zhu W, Hu Q (2001) Effects of stress factors, bioregulators and synthetic precursor on indole alkaloid production in compact callus clusters cultures of Catharanthus roseus. Appl Microbiol Biotechnol 55:693–698
Acknowledgments
The authors wish to thank the support of the EU through the ALFA Network CARIBIOTEC (project AML/B7-311/97/0666/II-0201), the German Ministry for Education and Research (BMBF) and the Cuban Ministry of Science, Technology and Environment (CITMA). Also to Emeritis Prof. Dr. ir. Oswald Van Cleemput from the Faculty of Bioscience Engineering at Gent University for the revision of the manuscript.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Pérez-Alonso, N., Wilken, D., Gerth, A. et al. Cardiotonic glycosides from biomass of Digitalis purpurea L. cultured in temporary immersion systems. Plant Cell Tiss Organ Cult 99, 151–156 (2009). https://doi.org/10.1007/s11240-009-9587-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-009-9587-x