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Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey
Plant Physiology and Biochemistry 73 (2013) 139e143
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey G. Sahin*, S.K. Verma, E. Gurel Abant Izzet Baysal University, Department of Biology, 14280 Bolu, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2013 Accepted 10 September 2013 Available online 24 September 2013
Elimination of calcium (Ca), magnesium (Mg) or both from the medium of callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach increased cardenolides production. Callus was induced from hypocotyl segments from one-month old seedlings were cultured on MS medium containing 0.5 mg ml1 thidiazuron (TDZ) and 0.25 mg ml1 indole acetic acid (IAA). After 30 days of culture, callus was transferred in hormone-free MS medium (MSO) as well as Ca or Mg or both were completely eliminated from same medium. The amount of five cardenolides from D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach were compared. Higher amounts of five cardenolides and total cardenolides were obtained when callus of four Digitalis species were incubated on MS medium lacking both Ca and Mg. The mean contents of total cardenolides obtained were in the order of D. lamarckii (2017.97 mg g1) > D. trojana (1385.75 mg g1) > D. cariensis (1038.65 mg g1) > D. davisiana (899.86 mg g1) when both Ca and Mg were eliminated from the medium, respectively. This protocol is useful for development of new strategies for the large-scale production of cardenolides. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Calcium Magnesium Cardenolides accumulation Digitalis cariensis Boiss. ex Jaub. et Spach Digitalis davisiana Heywood Digitalis lamarckii Ivanina Digitalis trojana Ivanina
1. Introduction Digitalis, commonly known as foxgloves, belongs to the family Plantaginaceae. The genus consists of about 20 species of herbaceous perennials, shrubs and biennials. Members of the genus Digitalis L. are medicinally and economically important plants as they contain cardiac glycosides that are used as heart medicines [1] and effective agents in cancer chemotherapy [2e4]. Because of their importance, studies have focused on in vitro culture of several Digitalis species including Digitalis davisiana [5], Digitalis lanata [6], Digitalis obscura [7], Digitalis purpurea [8,9] and Digitalis thapsi [10e13]. Digitalis species is distributed in Europe, Western Asia and the Mediterranean region. D. davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach, which are the most widespread members of the nine Digitalis species growing in Turkey and are endemic to Turkey [14,15]. D. trojana contains the highest amount of cardiac glycoside in leaves among the endemic Digitalis species in Turkey [16]. Largescale plant cell and tissue culture has been considered as an
Abbreviations: IAA, indole-3-acetic acid; TDZ, thidiazuron; MS, Murashige and Skoog; Lan C, lanatoside C. * Corresponding author. Tel.: þ90 5544509688; fax: þ90 3742534642. E-mail address: [email protected] (G. Sahin). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.09.007
alternative technique for the production of bioactive secondary metabolites since their production by traditional agriculture is inefficient [17]. The production of cardenolides using tissue culture techniques for Digitalis species has been studied for several years, however, the productivity has not yet reached economically desired levels. Most scientists have reported that tissue cultures established from Digitalis or other cardenolide containing plants either did not produce cardenolides or contained only at trace amounts. On the other hand, to improve the production of such secondary metabolites, some strategies have been developed [18]. One of them is the employment of various kinds of abiotic stress factors such as changing nutrient conditions, temperature range, UV and chemical application [19]. The aim of this work was to achieve the enhancement of cardenolide accumulation in D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach, using calcium and magnesium deprived cultures and to determine the content of digitoxin, digoxin, lanatoside C, gitoxigenin and digoxigenin as secondary metabolites of commercial interest for the pharmaceutical industry. 2. Results Callus was readily initiated within one week when hypocotyl segments excised from one-month old seedlings were cultured on
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Fig. 1. Experimental design of D. lamarckii. Germinated seedlings in vitro (A), hypocotyl segments (5e8 mm) from one-month old seedlings were cultured on MS medium containing 0.5 mg ml1 TDZ and 0.25 mg ml1 IAA (B), growth of callus derived from hypocotyl explants (C), deficiency symptoms of Ca (D), Mg (E), or both Ca & Mg (F) in the callus tissue of D. lamarckii.
MS medium supplemented with 0.5 mg ml1 TDZ combined with 0.25 mg ml1 IAA. Following a 30 days culture on this medium, calli were transferred to Ca-, Mg- or both Ca and Mg-eliminated medium for 15 days. Elimination of Ca or Mg or both from the medium slightly reduced the growth of callus without affecting cell viability. The colour of the callus changed from green in the control treatment to yellowebrown on Ca- or Mg-eliminated media, and gradually from yellowebrown to brown on both Ca and Mgeliminated media after 15 days of culture (Fig. 1). As seen in the last column of Table 1, the elimination of Ca, Mg or both from callus cultures of four Digitalis species clearly promoted the total accumulation of cardenolides. Ca and Mg elimination stimulated the cardenolide accumulation in D. lamarckii compared to control group. Deficiency of both Ca and Mg from the medium was followed by a significant increase of Lan C (1962.4 7.84 mg g1dry weight, dw). The non-treated group
produced 251.2 4.62 mg g1, dw Lan C while those cultured on medium lacking either Ca or Mg producing 883.66 9.71 mg g1, dw or 561.53 4.55 mg g1, dw Lan C, respectively. The deficiency of both macronutrients resulted in the highest level of digitoxin (38.55 2.03 mg g1, dw), digoxigenin (9.74 0.26 mg g1, dw) and gitoxigenin (7.68 0.40 mg g1, dw). For D. trojana, Ca and Mg elimination also seemed to enhance the cardenolide accumulation. When both Ca and Mg were eliminated from the medium, the level of Lan C increased significantly (1344 6.97 mg g1, dw). 225.63 7.58 mg g1, dw Lan C was produced in the non-treated group compared to those cultured on medium lacking either Ca or Mg, which produced 890.83 9.6 mg g1 or 562.56 7.00 mg g1, dw Lan C, respectively. Moreover, the highest digitoxin (26.65 4.43 mg g1, dw) and digoxigenin (8.64 0.38 mg g1, dw) levels were obtained when both Ca and Mg were eliminated from the medium. There was not a
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Table 1 Analysis of five different cardenolides in the control (non-treated) and stress-treated callus tissues of four endemic Digitalis species. Data are means SD from three replicates. Values followed by different letters in the same column are significantly different (P < 0.05). Species
Types of stress
Amount of cardenolides (mg g1, dw) Digoxigenin
D. lamarckii
D. trojana
D. davisiana
D. cariensis
Control Ca Mg Ca & Mg Control Ca Mg Ca & Mg Control Ca Mg Ca & Mg Control Ca Mg Ca & Mg
ijk
7.37 0.34 9.5efg 0.45 9.07fgh 0.20 9.74ef 0.26 7.07jk 0.09 8.42ghi 0.14 8.36ghi 0.22 8.64fghi0.38 6.23k 2.17 9.11efgh0.99 7.82hij 0.50 11.16cd 0.39 10.37de 0.65 13.16b 0.34 11.74c 0.55 19.35a 0.33
Gitoxigenin e
5.82 0.24 6.38de 0.35 6.63de 0.26 7.68cd 0.4 5.94e 0.216 6.45de 0.42 6.45de 0.20 6.46de 0.50 7.63cd 2.10 15.20a 1.13 8.91c 1.83 15.95a 1.06 6.17de 0.31 8.91c 0.31 8.63c 0.88 11.90b 0.33
Lan C
Digoxin k
251.20 4.62 883.66d 9.71 561.53g 4.55 1962a 7.845 225.63l 7.58 890.83d 9.60 562.56g 7.00 1344b 6.97 274.41j 2.74 463.28h 5.48 317.08i 2.55 843.95e 3.37 251.35k 5.50 684.18f 9.76 462.23h 5.56 968.18c 9.49
Digitoxin g
11.24 0.78 32.1bc0.53 23.47e 3.92 38.55a 2.03 11.69g 1.40 19.33f 1.43 14.47g 3.78 26.65de 4.43 11.51g 0.58 13.77g 1.26 12.91g 1.86 28.8cd 0.28 11.64g 0.69 34.10b 0.47 30.85bc0.68 39.22a 2.55
Total 275.64 931.64 600.70 2017.97 250.33 925.03 591.84 1385.75 299.78 501.36 346.73 899.86 279.53 740.35 513.45 1038.65
LOD: Limit of detection.
significant difference observed in gitoxigenin level between nontreated and treated-groups. With the elimination of both Ca and Mg from the medium, the amount of Lan C was increased to 843.95 3.37 mg g1, dw in D. davisiana. The non-treated callus produced 274.417 2.74 mg g1, dw Lan C while those cultured on medium lacking either Ca or Mg producing 463.28 5.48 or 317.08 2.55 mg g1, dw Lan C, respectively. On the basis of the analysis, the highest digitoxin (28.80 0.20 mg g1, dw), digoxigenin (11.16 0.39 mg g1, dw) and gitoxigenin (15.95 1.06 mg g1, dw) levels were estimated when both Ca and Mg eliminated from the medium. In D. cariensis, Ca and Mg elimination gave rise to an enhancement in the cardenolide levels whereas the levels were very low in non-treated groups. Lan C was significantly increased to 968.18 9.49 mg g1, dw when both Ca and Mg were eliminated from the medium. The non-treated callus produced 251.35 5.50 mg g1, dw Lan C while those cultured on medium lacking either Ca or Mg producing 684.18 9.76 mg g1 or 462.23 5.56 mg g1, dw Lan C, respectively. The highest digitoxin (39.22 2.55 mg g1, dw), digoxigenin (19.35 0.33 mg g1, dw) and gitoxigenin (11.90 0.33 mg g1, dw) levels were recorded with the elimination of both macronutrients. 3. Discussion The secondary plant metabolites play a major role in the adaptation of plants to their environment. Therefore, strategies such as changing nutrients, growth regulators, elicitors, biosynthetic precursor feeding and bioreactors have been extensively used to improve the production of bioactive secondary metabolites [7,8]. The stimuli are noticed by receptors, which then result in the activation of the secondary messengers. These then transmit the signals into the cell through the signal transduction pathways leading to gene expression and biochemical changes. Different macro elements and their varying concentrations used in the culture medium play an important role in the secondary metabolite production. Gavidia and Perez-Bermudez [20] reported that changes in the concentrations of phosphate or manganese ions in the nutrient media did not significantly affect the production of cardenolides. The same group [7] also reported that cardenolide production was increased in shoot-tip cultures of D. obscura with reduced level of major salt concentration. Moreover, it has been shown that lower phosphate levels lead to higher metabolite
accumulation in the in vitro cultures of different medicinal plants, including Nicotiana tabacum, Arabidopsis thaliana [21], Datura stramonium [22], Catharanthus roseus [23] and Peganum harmala [24]. In our studies, elimination of calcium and magnesium ions alone or together significantly increased cardenolide production in all Digitalis species tested. Data for all the five cardiac glycosides of the four Turkish endemic Digitalis species investigated are presented in Table 1. Genotypic variation was clear in terms of the five cardiac glycosides as well as total cardenolides content. The predominant cardiac glycoside was lanatoside C, which was followed by digitoxin, digoxigenin, gitoxigenin and digoxin. In all treatments as well as control group of all species, no digoxin was detected. It is well known that lanatoside C is transformed into digoxin by deglucosylation using digilanidase present in the leaves and subsequent deacetylation [25]. It has been reported that undifferentiated callus cultures, embryogenic cell cultures, suspension cultures, root and shoot cultures of Digitalis genus produce low amounts of cardenolides [8]. Gurel et al. [5] reported that consistently increasing amounts of cardenolide (mainly digoxin) were detected while callus redifferentiate into organized tissues. It has been also reported that calcium is necessary for secondary metabolite production [26]. However, several other studies demonstrated that calcium negatively affects the production of cardenolides [8,11e13,27], alkaloids [28], and flavonolignans [27]. In the present work, it should be pointed out that Ca and Mg elimination stimulated the cardenolide accumulation, although the production of cardenolides was very low in the control group. While on the contrary, both of the deficiencies of these macronutrients resulted in the highest level of cardenolides in four Turkish endemic species. The amount of total cardenolides estimated in callus obtained from four Digitalis callus cultured for 15 days in a medium containing no calcium and magnesium was 2017.97 mg g1 in D. lamarckii, 1385.75 mg g1 in D. trojana, 899.86 mg g1 in D. davisiana and 1038.65 mg g1 in D. cariensis, respectively. Moreover, this is the highest value of total cardenolides production found in cultured cells, although most workers have reported that undifferentiated cultured cells either did not produce cardenolides [29,30] or contained only trace amounts [10,31]. Similar results were also observed in undifferentiated cultures of D. thapsi, an endemic species to Portugal and West Spain. However, other studies showed that the elimination of calcium ions from
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undifferentiated cultures of D. thapsi promoted the accumulation of cardenolides [11,12]. Similar results have been obtained in Silybum marianum cultures [27], the elimination of this salt increased the different components of silymarin. There is evidence that in the absence of calcium, H2O2 and cardenolide accumulation were enhanced using by chelator and the channel blocker, moreover, it was also observed that extracellular calcium is necessary for the formation of reactive oxygen species (ROS) and phytoalexin production in many plant systems [13,32]. ROS play important roles in plant defense system and act as intermediate signalling molecules to regulate the expression of genes. In our studies, elimination of calcium ions significantly induced cardenolide production in all Digitalis species tested. This was probably due to the reason that enhanced ROS generation due to macronutrient deficiencies was responsible for transcriptional activation of genes responsible for cardenolide production [33]. It was also known that various elicitors induced a coordinated regulation of enzyme activities [34,35]. Vögeli and Chappell [36] reported that the isoprenoid metabolism in plants is regulated both positively and negatively. Considering the presence of a common pool of isoprenoid intermediates, in our study the calcium deficiency might have induced a different regulation of enzyme activity related to cardenolide production. There are no reports of Digitalis tissue cultures in which improvements in cardenolide production have been achieved by Mg elimination. On the other hand, earlier studies reported that an increase in phenolic compounds resulting from potassium, sulphur and magnesium deficiencies [37]. Putrescine accumulation was also reported in response to K and Mg deficiencies. Magnesium deficiency is also known to increase putrescine levels. It was known that the accumulation of putrescine is a universal response to stress in plants, regulating the structure and function of biological macromolecules as well as their synthesis in vivo [38]. In our studies, magnesium deficiency significantly increased cardenolide production in all Digitalis species tested, but more refined information is necessary to determine the possible role of Mg in the biosynthesis of cardiac glycosides. However, the increment of cardenolides level promoted by Mg elimination could be possibly associated with putrescine accumulation and significant modifications of enzyme activities related to cardenolide production. 4. Materials and methods 4.1. Plant materials Seeds of four endemic Digitalis species (D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach) were collected in August to September, 2010. Seeds of D. davisiana and D. cariensis from Alanya-Mahmutlar (N36 31.9160 , E03214.4020 ) (N36 30.7670, E032 12.6950 , 03.09.2010) and seeds of D. lamarckii around the Ankara-Kızılcahamam (N40 37.7090 , E032 26.2650 ) as well as D. trojana were collected from the National Park of Ida Mountains (N39 38.8850 , E026 57.4020 ). Identification of species was made according to Davis [14], and voucher specimens (IEker-2693; IEker-2694; IEker-2691; IEker2692) were deposited at the Abant Izzet Baysal University Herbarium (Bolu, Turkey).
agar. Medium (pH 5.8) autoclaved at 121 C and 1.06 kg cm2 pressure for 15 min. The culture was transferred to 16 h light : 8 h dark photoperiod at a relative humidity of 60%. 4.3. Callus initiation In order to obtain callus, hypocotyl explants excised from germinated seedlings were cultured on MS medium containing different auxin and cytokinin concentrations (0.5 mg ml1 TDZ and 0.25 mg ml1 IAA) [5]. After 30 days of culture, callus was transferred to MS medium lacking either Ca or Mg or both of them so that to create nutrient stress for 15 days. 4.4. Extraction of cardenolides Extraction of cardenolides was determined according to the method of Wiegrebe and Wichtl [40]. Callus materials were ground in liquid nitrogen, then 50 mg of material was transferred to the centrifuge tube containing 1 ml 70% (v/v) methanol. After 15 min treatment in an ultrasonic bath at 70e75 C, the extract was rapidly cooled on ice for 5 min and then centrifuged at 12,000 rpm for 5 min. The supernatant was thoroughly mixed with 250 ml of 15% (w/v) lead acetate and centrifuged for 5 min at 12,000 rpm. After elucidating the lead acetate residue, 500 ml of 4% NaH2PO4 (w/ v) was added and centrifuged again at 12,000 rpm for 8 min. The supernatant was transferred into the 2 ml centrifuge tube, diluted to a final volume of 2 ml with water and mixed by vortex for 1 min. Then, the extract was divided into two 1.5 ml new tubes. Then, 500 ml of chloroform/isopropanol (3/2, v/v) was added each tubes and were centrifuged for 5 min at 12,000 rpm. The lower phase were transferred into 2 ml centrifuge tubes as the first extraction. The remaining methanolic solutions were used for the second extraction by adding 500 ml of chloroform. Then, the lower phases were transferred to tubes, which was the second extraction. Extractions were evaporated to dryness under gentle stream of nitrogen and finally the volume was completed to 0.5 ml with HPLC grade methanol and passed through a PTFE millipore membrane filter (0.22 mm). 4.5. HPLC analysis of cardenolides After extraction and evaporation, 20 ml aliquot of pretreated extract described above were injected in an Agilent 1100 HPLC system with a UV-DAD detector operating at 220 nm and a GL Sciences Inc. Inertsil ODS-3 column (4.6 150 mm). All measurements were carried out a flow rate of 1.2 ml/min at 40 C. For the calibration curves, concentrations of 5, 10, 20, 30 and 40 mg ml1 digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin were used (R values were 0.99 for digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin). Cardenolides were eluted with acetonitrile (A) and water (B) gradients as follows: 0e20 min 20% (A), 80% (B); 20e23.40 min 30% (A), 70% (B); 23.40e30 min 25% (A), 75% (B) and 30e40 min 40% (A), 60%(B). Average peak area of the glycoside in samples were detected and calculated using ChemStation LC/MS software against standard glycosides. Every sample group was repeated three times.
4.2. Surface sterilization and culture conditions 4.6. Statistical analysis Seeds of four endemic Digitalis species were surface disinfected with 20% (v/v) commercial bleach and finally rinsed with sterile distilled water for three times. Seeds were aseptically cultured on 100 15 mm Petri dishes containing 30 ml of the Murashige and Skoog [39] medium consisting of 3% (w/v) sucrose and 0.8% (w/v)
The measurements were made in triplicate and they were statistically evaluated using SPSS and Duncan’s multiple range test. Values (P 0 05) indicated whether statistically significant differences existed between treatments and control at a given time.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Calcium and magnesium elimination enhances accumulation of cardenolides in callus cultures of endemic Digitalis species of Turkey G. Sahin*, S.K. Verma, E. Gurel Abant Izzet Baysal University, Department of Biology, 14280 Bolu, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 July 2013 Accepted 10 September 2013 Available online 24 September 2013
Elimination of calcium (Ca), magnesium (Mg) or both from the medium of callus cultures of Digitalis davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach increased cardenolides production. Callus was induced from hypocotyl segments from one-month old seedlings were cultured on MS medium containing 0.5 mg ml1 thidiazuron (TDZ) and 0.25 mg ml1 indole acetic acid (IAA). After 30 days of culture, callus was transferred in hormone-free MS medium (MSO) as well as Ca or Mg or both were completely eliminated from same medium. The amount of five cardenolides from D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach were compared. Higher amounts of five cardenolides and total cardenolides were obtained when callus of four Digitalis species were incubated on MS medium lacking both Ca and Mg. The mean contents of total cardenolides obtained were in the order of D. lamarckii (2017.97 mg g1) > D. trojana (1385.75 mg g1) > D. cariensis (1038.65 mg g1) > D. davisiana (899.86 mg g1) when both Ca and Mg were eliminated from the medium, respectively. This protocol is useful for development of new strategies for the large-scale production of cardenolides. Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords: Calcium Magnesium Cardenolides accumulation Digitalis cariensis Boiss. ex Jaub. et Spach Digitalis davisiana Heywood Digitalis lamarckii Ivanina Digitalis trojana Ivanina
1. Introduction Digitalis, commonly known as foxgloves, belongs to the family Plantaginaceae. The genus consists of about 20 species of herbaceous perennials, shrubs and biennials. Members of the genus Digitalis L. are medicinally and economically important plants as they contain cardiac glycosides that are used as heart medicines [1] and effective agents in cancer chemotherapy [2e4]. Because of their importance, studies have focused on in vitro culture of several Digitalis species including Digitalis davisiana [5], Digitalis lanata [6], Digitalis obscura [7], Digitalis purpurea [8,9] and Digitalis thapsi [10e13]. Digitalis species is distributed in Europe, Western Asia and the Mediterranean region. D. davisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojana Ivanina and Digitalis cariensis Boiss. ex Jaub. et Spach, which are the most widespread members of the nine Digitalis species growing in Turkey and are endemic to Turkey [14,15]. D. trojana contains the highest amount of cardiac glycoside in leaves among the endemic Digitalis species in Turkey [16]. Largescale plant cell and tissue culture has been considered as an
Abbreviations: IAA, indole-3-acetic acid; TDZ, thidiazuron; MS, Murashige and Skoog; Lan C, lanatoside C. * Corresponding author. Tel.: þ90 5544509688; fax: þ90 3742534642. E-mail address: [email protected] (G. Sahin). 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.09.007
alternative technique for the production of bioactive secondary metabolites since their production by traditional agriculture is inefficient [17]. The production of cardenolides using tissue culture techniques for Digitalis species has been studied for several years, however, the productivity has not yet reached economically desired levels. Most scientists have reported that tissue cultures established from Digitalis or other cardenolide containing plants either did not produce cardenolides or contained only at trace amounts. On the other hand, to improve the production of such secondary metabolites, some strategies have been developed [18]. One of them is the employment of various kinds of abiotic stress factors such as changing nutrient conditions, temperature range, UV and chemical application [19]. The aim of this work was to achieve the enhancement of cardenolide accumulation in D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach, using calcium and magnesium deprived cultures and to determine the content of digitoxin, digoxin, lanatoside C, gitoxigenin and digoxigenin as secondary metabolites of commercial interest for the pharmaceutical industry. 2. Results Callus was readily initiated within one week when hypocotyl segments excised from one-month old seedlings were cultured on
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Fig. 1. Experimental design of D. lamarckii. Germinated seedlings in vitro (A), hypocotyl segments (5e8 mm) from one-month old seedlings were cultured on MS medium containing 0.5 mg ml1 TDZ and 0.25 mg ml1 IAA (B), growth of callus derived from hypocotyl explants (C), deficiency symptoms of Ca (D), Mg (E), or both Ca & Mg (F) in the callus tissue of D. lamarckii.
MS medium supplemented with 0.5 mg ml1 TDZ combined with 0.25 mg ml1 IAA. Following a 30 days culture on this medium, calli were transferred to Ca-, Mg- or both Ca and Mg-eliminated medium for 15 days. Elimination of Ca or Mg or both from the medium slightly reduced the growth of callus without affecting cell viability. The colour of the callus changed from green in the control treatment to yellowebrown on Ca- or Mg-eliminated media, and gradually from yellowebrown to brown on both Ca and Mgeliminated media after 15 days of culture (Fig. 1). As seen in the last column of Table 1, the elimination of Ca, Mg or both from callus cultures of four Digitalis species clearly promoted the total accumulation of cardenolides. Ca and Mg elimination stimulated the cardenolide accumulation in D. lamarckii compared to control group. Deficiency of both Ca and Mg from the medium was followed by a significant increase of Lan C (1962.4 7.84 mg g1dry weight, dw). The non-treated group
produced 251.2 4.62 mg g1, dw Lan C while those cultured on medium lacking either Ca or Mg producing 883.66 9.71 mg g1, dw or 561.53 4.55 mg g1, dw Lan C, respectively. The deficiency of both macronutrients resulted in the highest level of digitoxin (38.55 2.03 mg g1, dw), digoxigenin (9.74 0.26 mg g1, dw) and gitoxigenin (7.68 0.40 mg g1, dw). For D. trojana, Ca and Mg elimination also seemed to enhance the cardenolide accumulation. When both Ca and Mg were eliminated from the medium, the level of Lan C increased significantly (1344 6.97 mg g1, dw). 225.63 7.58 mg g1, dw Lan C was produced in the non-treated group compared to those cultured on medium lacking either Ca or Mg, which produced 890.83 9.6 mg g1 or 562.56 7.00 mg g1, dw Lan C, respectively. Moreover, the highest digitoxin (26.65 4.43 mg g1, dw) and digoxigenin (8.64 0.38 mg g1, dw) levels were obtained when both Ca and Mg were eliminated from the medium. There was not a
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Table 1 Analysis of five different cardenolides in the control (non-treated) and stress-treated callus tissues of four endemic Digitalis species. Data are means SD from three replicates. Values followed by different letters in the same column are significantly different (P < 0.05). Species
Types of stress
Amount of cardenolides (mg g1, dw) Digoxigenin
D. lamarckii
D. trojana
D. davisiana
D. cariensis
Control Ca Mg Ca & Mg Control Ca Mg Ca & Mg Control Ca Mg Ca & Mg Control Ca Mg Ca & Mg
ijk
7.37 0.34 9.5efg 0.45 9.07fgh 0.20 9.74ef 0.26 7.07jk 0.09 8.42ghi 0.14 8.36ghi 0.22 8.64fghi0.38 6.23k 2.17 9.11efgh0.99 7.82hij 0.50 11.16cd 0.39 10.37de 0.65 13.16b 0.34 11.74c 0.55 19.35a 0.33
Gitoxigenin e
5.82 0.24 6.38de 0.35 6.63de 0.26 7.68cd 0.4 5.94e 0.216 6.45de 0.42 6.45de 0.20 6.46de 0.50 7.63cd 2.10 15.20a 1.13 8.91c 1.83 15.95a 1.06 6.17de 0.31 8.91c 0.31 8.63c 0.88 11.90b 0.33
Lan C
Digoxin k
251.20 4.62 883.66d 9.71 561.53g 4.55 1962a 7.845 225.63l 7.58 890.83d 9.60 562.56g 7.00 1344b 6.97 274.41j 2.74 463.28h 5.48 317.08i 2.55 843.95e 3.37 251.35k 5.50 684.18f 9.76 462.23h 5.56 968.18c 9.49
Digitoxin g
11.24 0.78 32.1bc0.53 23.47e 3.92 38.55a 2.03 11.69g 1.40 19.33f 1.43 14.47g 3.78 26.65de 4.43 11.51g 0.58 13.77g 1.26 12.91g 1.86 28.8cd 0.28 11.64g 0.69 34.10b 0.47 30.85bc0.68 39.22a 2.55
Total 275.64 931.64 600.70 2017.97 250.33 925.03 591.84 1385.75 299.78 501.36 346.73 899.86 279.53 740.35 513.45 1038.65
LOD: Limit of detection.
significant difference observed in gitoxigenin level between nontreated and treated-groups. With the elimination of both Ca and Mg from the medium, the amount of Lan C was increased to 843.95 3.37 mg g1, dw in D. davisiana. The non-treated callus produced 274.417 2.74 mg g1, dw Lan C while those cultured on medium lacking either Ca or Mg producing 463.28 5.48 or 317.08 2.55 mg g1, dw Lan C, respectively. On the basis of the analysis, the highest digitoxin (28.80 0.20 mg g1, dw), digoxigenin (11.16 0.39 mg g1, dw) and gitoxigenin (15.95 1.06 mg g1, dw) levels were estimated when both Ca and Mg eliminated from the medium. In D. cariensis, Ca and Mg elimination gave rise to an enhancement in the cardenolide levels whereas the levels were very low in non-treated groups. Lan C was significantly increased to 968.18 9.49 mg g1, dw when both Ca and Mg were eliminated from the medium. The non-treated callus produced 251.35 5.50 mg g1, dw Lan C while those cultured on medium lacking either Ca or Mg producing 684.18 9.76 mg g1 or 462.23 5.56 mg g1, dw Lan C, respectively. The highest digitoxin (39.22 2.55 mg g1, dw), digoxigenin (19.35 0.33 mg g1, dw) and gitoxigenin (11.90 0.33 mg g1, dw) levels were recorded with the elimination of both macronutrients. 3. Discussion The secondary plant metabolites play a major role in the adaptation of plants to their environment. Therefore, strategies such as changing nutrients, growth regulators, elicitors, biosynthetic precursor feeding and bioreactors have been extensively used to improve the production of bioactive secondary metabolites [7,8]. The stimuli are noticed by receptors, which then result in the activation of the secondary messengers. These then transmit the signals into the cell through the signal transduction pathways leading to gene expression and biochemical changes. Different macro elements and their varying concentrations used in the culture medium play an important role in the secondary metabolite production. Gavidia and Perez-Bermudez [20] reported that changes in the concentrations of phosphate or manganese ions in the nutrient media did not significantly affect the production of cardenolides. The same group [7] also reported that cardenolide production was increased in shoot-tip cultures of D. obscura with reduced level of major salt concentration. Moreover, it has been shown that lower phosphate levels lead to higher metabolite
accumulation in the in vitro cultures of different medicinal plants, including Nicotiana tabacum, Arabidopsis thaliana [21], Datura stramonium [22], Catharanthus roseus [23] and Peganum harmala [24]. In our studies, elimination of calcium and magnesium ions alone or together significantly increased cardenolide production in all Digitalis species tested. Data for all the five cardiac glycosides of the four Turkish endemic Digitalis species investigated are presented in Table 1. Genotypic variation was clear in terms of the five cardiac glycosides as well as total cardenolides content. The predominant cardiac glycoside was lanatoside C, which was followed by digitoxin, digoxigenin, gitoxigenin and digoxin. In all treatments as well as control group of all species, no digoxin was detected. It is well known that lanatoside C is transformed into digoxin by deglucosylation using digilanidase present in the leaves and subsequent deacetylation [25]. It has been reported that undifferentiated callus cultures, embryogenic cell cultures, suspension cultures, root and shoot cultures of Digitalis genus produce low amounts of cardenolides [8]. Gurel et al. [5] reported that consistently increasing amounts of cardenolide (mainly digoxin) were detected while callus redifferentiate into organized tissues. It has been also reported that calcium is necessary for secondary metabolite production [26]. However, several other studies demonstrated that calcium negatively affects the production of cardenolides [8,11e13,27], alkaloids [28], and flavonolignans [27]. In the present work, it should be pointed out that Ca and Mg elimination stimulated the cardenolide accumulation, although the production of cardenolides was very low in the control group. While on the contrary, both of the deficiencies of these macronutrients resulted in the highest level of cardenolides in four Turkish endemic species. The amount of total cardenolides estimated in callus obtained from four Digitalis callus cultured for 15 days in a medium containing no calcium and magnesium was 2017.97 mg g1 in D. lamarckii, 1385.75 mg g1 in D. trojana, 899.86 mg g1 in D. davisiana and 1038.65 mg g1 in D. cariensis, respectively. Moreover, this is the highest value of total cardenolides production found in cultured cells, although most workers have reported that undifferentiated cultured cells either did not produce cardenolides [29,30] or contained only trace amounts [10,31]. Similar results were also observed in undifferentiated cultures of D. thapsi, an endemic species to Portugal and West Spain. However, other studies showed that the elimination of calcium ions from
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undifferentiated cultures of D. thapsi promoted the accumulation of cardenolides [11,12]. Similar results have been obtained in Silybum marianum cultures [27], the elimination of this salt increased the different components of silymarin. There is evidence that in the absence of calcium, H2O2 and cardenolide accumulation were enhanced using by chelator and the channel blocker, moreover, it was also observed that extracellular calcium is necessary for the formation of reactive oxygen species (ROS) and phytoalexin production in many plant systems [13,32]. ROS play important roles in plant defense system and act as intermediate signalling molecules to regulate the expression of genes. In our studies, elimination of calcium ions significantly induced cardenolide production in all Digitalis species tested. This was probably due to the reason that enhanced ROS generation due to macronutrient deficiencies was responsible for transcriptional activation of genes responsible for cardenolide production [33]. It was also known that various elicitors induced a coordinated regulation of enzyme activities [34,35]. Vögeli and Chappell [36] reported that the isoprenoid metabolism in plants is regulated both positively and negatively. Considering the presence of a common pool of isoprenoid intermediates, in our study the calcium deficiency might have induced a different regulation of enzyme activity related to cardenolide production. There are no reports of Digitalis tissue cultures in which improvements in cardenolide production have been achieved by Mg elimination. On the other hand, earlier studies reported that an increase in phenolic compounds resulting from potassium, sulphur and magnesium deficiencies [37]. Putrescine accumulation was also reported in response to K and Mg deficiencies. Magnesium deficiency is also known to increase putrescine levels. It was known that the accumulation of putrescine is a universal response to stress in plants, regulating the structure and function of biological macromolecules as well as their synthesis in vivo [38]. In our studies, magnesium deficiency significantly increased cardenolide production in all Digitalis species tested, but more refined information is necessary to determine the possible role of Mg in the biosynthesis of cardiac glycosides. However, the increment of cardenolides level promoted by Mg elimination could be possibly associated with putrescine accumulation and significant modifications of enzyme activities related to cardenolide production. 4. Materials and methods 4.1. Plant materials Seeds of four endemic Digitalis species (D. davisiana Heywood, D. lamarckii Ivanina, D. trojana Ivanina and D. cariensis Boiss. ex Jaub. et Spach) were collected in August to September, 2010. Seeds of D. davisiana and D. cariensis from Alanya-Mahmutlar (N36 31.9160 , E03214.4020 ) (N36 30.7670, E032 12.6950 , 03.09.2010) and seeds of D. lamarckii around the Ankara-Kızılcahamam (N40 37.7090 , E032 26.2650 ) as well as D. trojana were collected from the National Park of Ida Mountains (N39 38.8850 , E026 57.4020 ). Identification of species was made according to Davis [14], and voucher specimens (IEker-2693; IEker-2694; IEker-2691; IEker2692) were deposited at the Abant Izzet Baysal University Herbarium (Bolu, Turkey).
agar. Medium (pH 5.8) autoclaved at 121 C and 1.06 kg cm2 pressure for 15 min. The culture was transferred to 16 h light : 8 h dark photoperiod at a relative humidity of 60%. 4.3. Callus initiation In order to obtain callus, hypocotyl explants excised from germinated seedlings were cultured on MS medium containing different auxin and cytokinin concentrations (0.5 mg ml1 TDZ and 0.25 mg ml1 IAA) [5]. After 30 days of culture, callus was transferred to MS medium lacking either Ca or Mg or both of them so that to create nutrient stress for 15 days. 4.4. Extraction of cardenolides Extraction of cardenolides was determined according to the method of Wiegrebe and Wichtl [40]. Callus materials were ground in liquid nitrogen, then 50 mg of material was transferred to the centrifuge tube containing 1 ml 70% (v/v) methanol. After 15 min treatment in an ultrasonic bath at 70e75 C, the extract was rapidly cooled on ice for 5 min and then centrifuged at 12,000 rpm for 5 min. The supernatant was thoroughly mixed with 250 ml of 15% (w/v) lead acetate and centrifuged for 5 min at 12,000 rpm. After elucidating the lead acetate residue, 500 ml of 4% NaH2PO4 (w/ v) was added and centrifuged again at 12,000 rpm for 8 min. The supernatant was transferred into the 2 ml centrifuge tube, diluted to a final volume of 2 ml with water and mixed by vortex for 1 min. Then, the extract was divided into two 1.5 ml new tubes. Then, 500 ml of chloroform/isopropanol (3/2, v/v) was added each tubes and were centrifuged for 5 min at 12,000 rpm. The lower phase were transferred into 2 ml centrifuge tubes as the first extraction. The remaining methanolic solutions were used for the second extraction by adding 500 ml of chloroform. Then, the lower phases were transferred to tubes, which was the second extraction. Extractions were evaporated to dryness under gentle stream of nitrogen and finally the volume was completed to 0.5 ml with HPLC grade methanol and passed through a PTFE millipore membrane filter (0.22 mm). 4.5. HPLC analysis of cardenolides After extraction and evaporation, 20 ml aliquot of pretreated extract described above were injected in an Agilent 1100 HPLC system with a UV-DAD detector operating at 220 nm and a GL Sciences Inc. Inertsil ODS-3 column (4.6 150 mm). All measurements were carried out a flow rate of 1.2 ml/min at 40 C. For the calibration curves, concentrations of 5, 10, 20, 30 and 40 mg ml1 digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin were used (R values were 0.99 for digoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin). Cardenolides were eluted with acetonitrile (A) and water (B) gradients as follows: 0e20 min 20% (A), 80% (B); 20e23.40 min 30% (A), 70% (B); 23.40e30 min 25% (A), 75% (B) and 30e40 min 40% (A), 60%(B). Average peak area of the glycoside in samples were detected and calculated using ChemStation LC/MS software against standard glycosides. Every sample group was repeated three times.
4.2. Surface sterilization and culture conditions 4.6. Statistical analysis Seeds of four endemic Digitalis species were surface disinfected with 20% (v/v) commercial bleach and finally rinsed with sterile distilled water for three times. Seeds were aseptically cultured on 100 15 mm Petri dishes containing 30 ml of the Murashige and Skoog [39] medium consisting of 3% (w/v) sucrose and 0.8% (w/v)
The measurements were made in triplicate and they were statistically evaluated using SPSS and Duncan’s multiple range test. Values (P 0 05) indicated whether statistically significant differences existed between treatments and control at a given time.
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