Hydrogen peroxide-induced antioxidant activities and cardiotonic glycoside accumulation in callus cultures of endemic Digitalis species

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Plant Physiology and Biochemistry 82 (2014) 89e94

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Hydrogen peroxide-induced antioxidant activities and cardiotonicglycoside accumulation in callus cultures of endemic Digitalis species

Gunce Sahin Cingoz*, Sandeep Kumar Verma, Ekrem GurelAbant Izzet Baysal University, Department of Biology, Bolu 14280, Turkey

a r t i c l e i n f o

Article history:Received 17 April 2014Accepted 18 May 2014Available online 28 May 2014

Keywords:Antioxidant activityCardenolides accumulationDigitalis L.FoxgloveHydrogen peroxide

Abbreviations: H2O2, hydrogen peroxide; IAA,lanatoside C; MS, Murashige and Skoog; TDZ, thidiaz* Corresponding author. Tel.: þ90 5544509688; fax

E-mail address: [email protected] (G.S. Cing

http://dx.doi.org/10.1016/j.plaphy.2014.05.0080981-9428/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

The effect of hydrogen peroxide (H2O2) on callus cultures of four Digitalis species (Digitalis lamarckii,Digitalis trojana, Digitalis davisiana and Digitalis cariensis) increased catalase (CAT), superoxide dismutase(SOD), total phenolic, proline activity and cardiotonic glycoside production. Callus derived from hypo-cotyl explants was cultured on Murashige and Skoog medium supplemented with 0.25 mg L�1 indole-3-acetic acid (IAA) and 0.5 mg L�1 thidiazuron (TDZ). After a month of culture, callus was transferred to MSmedium containing 10 mM H2O2 and then incubated for 6 h. The amount of five cardenolides (LanatosideC, Digitoxin, Digoxigenin, Gitoxigenin and Digoxin) as well as CAT, SOD, total phenolic, proline activityfrom Digitalis species were compared. No digoxin was detected in all treatments and control groups. Thetotal cardenolides estimated were in the order of D. lamarckii (586.65 mg g�1 dw), D. davisiana(506.79 mg g�1 dw), D. cariensis (376.60 mg g�1 dw) and D. trojana (282.39 mg g�1 dw). It was clear thatH2O2 pre-treatment resulted in an increase in enzymatic and nonenzymatic antioxidants. However, asignificant negative relationship between cardenolides production and overall activities of CAT, SOD,total phenolic and proline was evident. The described protocol here will be useful for the development ofnew strategies for a large-scale production of cardenolides.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

The genus Digitalis L., is a member of the Plantaginaceae, areregarded as having an economically important role in medicinesince they contain important cardioactive compounds used to treatheart problems, myocardial infarction, edema, angina, cardiacdysfunction, hypertrophy and arterial hypertension. Digitalis spe-cies are distributed in Europe, Western Asia and the Mediterraneanregion. Anatolia has five endemic Digitalis species, namely Digitalisdavisiana Heywood, Digitalis lamarckii Ivanina, Digitalis trojanaIvanina and Digitalis cariensis Boiss, which are the most widespreadmembers of the nine Digitalis species growing in Turkey (Daviset al., 1988; Verma et al., 2012). In addition, among the endemicDigitalis species in Turkey, D. trojana contains the highest amount ofcardiac glycoside in the leaves (Verma et al., 2012).

Large-scale production of bioactive secondary metabolites viatraditional agriculture is not efficient. Mostly, strategies related

indole-3-acetic acid; Lan C,uron.: þ90 3742534642.oz).

served.

with in vitro culture methods have been studied to improve theproduction of these metabolites. With the addition of many bioticand abiotic agents, several reports demonstrate that an increasedaccumulation of secondary metabolite is promoted. However, withspecific elicitor, there are no reports on Digitalis callus cultures inwhich improvement in cardenolide production has been achieved.Under biotic and abiotic stress, the accumulation of ROS and H2O2activates plant defense mechanisms as a signal (Doke et al., 1994;Prasad et al., 1994; Foyer et al., 1997). Some workers reportedthat plant defense and the related pathways such as antioxidantenzymes, glutathione-S transferase and phenylalanine ammonialyase, defense proteins and transcription factors are also influencedby H2O2 (Kovtun et al., 2000; Hung et al., 2005). However, H2O2 as astress signal molecule, has several functions in plant growth anddefense mechanism against environmental stresses. Moreover,oxidative species and H2O2 can react to produce additional acti-vated oxygen species that are damaging certain essential cellularcompartments. Oxygen radicals and H2O2 can be used in a benefi-cial manner in several metabolic processes (Inz�e and Montagu,1995). Furthermore, many biotic and abiotic agents promote anincreased accumulation of secondary metabolites in plant tissuecultures (Wolters and Eilert, 1983). Hitherto, there is little infor-mation on the use of H2O2 as an elicitor production of cardenolides

G.S. Cingoz et al. / Plant Physiology and Biochemistry 82 (2014) 89e9490

in Digitalis thapsi (Paranhos et al., 1999) and Digitalis lanata (P�erez-Alonso et al., 2012), but there are no reports about cardenolideproduction and CAT, SOD, total phenolic, proline activity in calluscultures of D. lamarckii, D. trojana, D. davisiana and D. cariensis. Theaim of the present workwas to evaluate the effect of H2O2 exposureof callus tissue on the enhancement of cardenolide production andCAT, SOD, total phenolic, proline activities. Here, we report for thefirst time, a significant relationship between the cardenolidesaccumulation and activities of CAT, SOD, total phenolic, proline incallus cultures of four Digitalis species under oxidative stress byH2O2.

2. Materials and method

2.1. Plant material and culture conditions

Digitalis species callus cultures were initiated from seeds thatwere germinated in vitro. Seeds were collected around the Ankara-Kızılcahamam (N40�37.7090, E032�26.2650) in August toSeptember, 2010 and voucher specimens (IEker-2694) weredeposited at the Abant Izzet Baysal University Herbarium (Bolu,Turkey). Seeds of Digitalis species were disinfected in with 20% (v/v) sodium hypochlorite solutions and cultured in petri dishescontaining 30 mL MS medium (Murashige and Skoog, 1962) con-taining 3% (w/v) sucrose and 0.8% (w/v) agar. Seedlings weregrown until they have attained 3e5 cm of length. Callus wasinduced from 5 to 8 mm length of hypocotyl segments excisedfrom 1 month old seedlings when cultured on MS medium con-taining 0.5 mg L�1 TDZ and 0.25 mg L�1 IAA. After 30 days ofculture, callus was transferred to MS medium with 0.0 and 10 mMH2O2 for creating oxidative stress for 6 h. All cultures were incu-bated under a 16 h light:8h dark photoperiod from cool whitefluorescent lamps.

2.2. Extraction of cardenolides

Extraction of cardenolides was determined according to themethod of (Wiegrebe andWichtl, 1993). Callus materials of Digitalisspecies were ground in liquid nitrogen, then 50 mg of material wastransferred to the centrifuge tube containing 1 mL 70% methanol.After 15 min treatment in an ultrasonic bath at 70e75 �C, theextract was rapidly cooled on ice for 5 min and then centrifuged at12,000 rpm for 5 min. The supernatant was thoroughly mixed with250 mL of lead acetate (15%) and centrifuged for 5 min at12,000 rpm. After elucidating the lead acetate residue, 500 mL of 4%NaH2PO4 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 vortexfor 1 min. Then, the extract was divided into two 1.5 mL new tubes.Next, 500 mL of chloroform:isopropanol (3:2) was added each tubesand were centrifuged for 5 min at 12,000 rpm. The lower phasewastransferred into 2 mL centrifuge tubes as the first extraction. Theremaining methanolic solutions were used for the second extrac-tion by adding 500 mL of chloroform. Then, the lower phases weretaken to the tubes. It was called as second extraction. Afterward,Na2SO4 was added to the tubes and mixed by hand and centrifugedat 12,000 rpm for 1 min in order to remove water. Extractions wereevaporated to dryness under gentle stream of nitrogen and finallythe volume was completed to 0.5 mL with HPLC grade methanoland passed through a PTFE millipore membrane filter (0.22 mm).

2.3. HPLC analysis of cardenolides

After extraction and evaporation, 20 ml aliquot of pretreatedextract described above were injected in an Agilent 1100 HPLC

system with a UV-DAD detector operating at 220 nm and an GLSciences Inc. Inertsil ODS-3 column (4.6 � 150 mm). All measure-ments were carried out a flow rate of 1.2 mL/min at 40 �C. For thecalibration curves, concentrations of 5, 10, 20, 30 and 40 mg/Ldigoxigenin, gitoxigenin, lanatoside C, digoxin and digitoxin wereused (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) and30e40 min 40% (A), 60% (B). Average peak area of the glycoside insamples was detected and calculated using ChemStation LC/MSsoftware against standard glycosides. Every sample group wasrepeated three times.

2.4. Enzyme extraction and protein determination

After being ground with liquid nitrogen, 0.1 g of callus materialwas homogenized in 2 mL of ice cold 50 mM K-phosphate buffer(pH 7.0) which contained 2 mM NaeEDTA and 1% (w/v) polyvinyl-polypyrrolidone (PVP). The homogenate was centrifuged at12,000 rpmmin�1 and 4 �C for 10min. After that, tissue extract wasstored at �80 �C for subsequent analyses of SOD and CAT. Thesoluble protein content was determined according to Lowrymethod (Classics Lowry et al., 1951) with bovine serum albumin asa standard curve.

2.5. Catalase (CAT; EC 1.11.1.6) activity

CAT activity was determined by using the method of Lartillotet al. (1988). Briefly CAT activity was determined spectrophoto-metrically at 240 nm using a specific absorption coefficient at0.0392 cm3 mmol�1 H2O2 and CAT activity was calculated as mmolH2O2 decomposed/mg protein/min.

2.6. Superoxide dismutase (SOD; EC 1.11.1.1) activity

SOD activity involved inhibition of nitroblue tetrazolium (NBT)reduction was determined using xanthineexanthine oxidase as asuperoxide generator (Sun et al., 1988). SOD assay reagent con-tained the following reagents in a 200 mL beaker, 40 mL of0.3 mmol/L xanthine solution, 20 mL of 0.6 mmol/L EDTA solution,20 mL of 150 mmol/L nitroblue tetrazolium solution, 12 mL of400 mmol/L Na2CO3 solution, and 6 mL of bovine serum albumin(1 g L�1). 0.05 mL enzyme extract was added into the each testtubes containing 1.425 mL of the SOD assay reagent. Tubes werekept into a water bath at 25 �C. Xanthine oxidase (0.025 mL) so-lution was put into the each tubes and incubated for 20 min, thenthe reaction was terminated by adding 0.05 mL of 0.8 mmol/LCuCl2 solution to each tubes. The production of formazan wasdetermined at 560 nm spectrophotometrically. One unit of SOD isdefined as the amount of protein that inhibits the rate of NBTreduction by 50%.

2.7. Total phenolic assay

The content of total phenolics in extracts was determinedfollowing the modified method of FolineCiocalteu (Marigo andBoudet, 1979). 20 mL extract was placed in a reaction test tubecontaining 1.58 mL of water and 100 mL of FolineCiocalteu reagent.The test tube was allowed to stand for 5 min, and then 300 mL 20%Na2CO3 was added into the tube. After 20 min at 40 �C, absorbancewas measured at 750 nm. Total phenolic content was expressed asmg gallic acid (GA) equivalents/g dry weight.

G.S. Cingoz et al. / Plant Physiology and Biochemistry 82 (2014) 89e94 91

2.8. Proline analysis

Proline was determined according to the method of Bates et al.(1973). For proline estimation callus tissues (0.5 g) were extractedwith 5 mL 3% sulfosalicylic acid and centrifuged at 5000 rpmmin�1

for 20 min. A 2 mL sample of the supernatant was added in a testtube containing 2 mL of ninhydrin and 2 mL of glacial acetic acid.After 1 h at 100 �C, the reaction terminated in an ice bath. The re-action mixture was extracted with 4 mL toluene and mixed withvortex for 20 s. The chromophore in which contained toluene wastransferred into the new test tube and warmed to room tempera-ture. Proline content was calculated spectrophotometrically asmmol g�1 dw against standard proline at 520 nm.

2.9. Statistical analysis

Each treatment was made in triplicate and the data were sta-tistically evaluated using SPSS and Duncan's multiple range test.Values of P � 0.05 indicated significance among treatments andcontrol at a given time.

3. Results

Callus was easily initiated within one week when hypocotylexplant excised from one-month old seedlings was cultured on MSmedium supplemented with 0.25 mg L�1 IAA and 0.5 mg L�1 TDZ.Following a 30 days culture on this medium, callus was transferredto MS medium with or without 10 mM H2O2 for 6 h. Here, weexamined the effect of H2O2 as an oxidative stress on callus culturewhich induced antioxidant activities and cardiotonic glycosideaccumulation in four Digitalis species (D. lamarckii, D. trojana,D. davisiana, D. cariensis).

3.1. Cardenolide content

Data for five different cardenolides of four endemic TurkishDigitalis species are presented in Table 1. The predominantcardiotonic glycoside was lanatoside C (Lan C) followed by digi-toxin, digoxigenin, gitoxigenin and digoxin. Among the all treat-ments as well as in control groups, there was no digoxin detected.Lan C accumulation in control (non-treated callus) ranged from159.51 mg g�1 dw for D. trojana to 273.62 mg g�1 dw for D. lamarckiiwith an overall mean of 234.99 mg g�1 dw. Digitoxin ranged from8.51 mg g�1 dw for D. lamarckii to 18.51 mg g�1 dw for D. trojanawithan overall mean of 12.23 mg g�1 dw. Digoxigenin ranged from5.82 mg g�1 dw forD. lamarckii to 13.09 mg g�1 dw forD. trojanawithan overall mean of 9.2 mg g�1 dw. Gitoxigenin ranged from

Table 1Effect of exogenous hydrogen peroxide (10 mM, H2O2) on cardenolide content of Turkiscultures. Values are means ± SD of two independent experiments performed in triplicat

Digitalis species H2O2 (mM) Amount of cardenolides (mg/g, dw)

Digoxigenin Gitoxigenin

D. lamarckii 0 5.82f ± 0.6 (1.9)* 6.33d ± 1.4 (2.1)10 10.03cd ± 0.7 (1.7) 8.53b ± 0.1 (1.5)

D. trojana 0 13.09cb ± 0.4 (6.6) 5.81d ± 0.3 (2.9)10 16.58a ± 0.1 (5.9) 6.92bcd ± 0.2 (2.4)

D. davisiana 0 6.97hi± 0.1 (2.6) 6.89defg± 0.2(2.6)10 11.71c ± 0.7 (2.3) 8.39bcd ± 0.7 (1.6)

D. cariensis 0 10.87ef± 0.3 (3.7) 7.65ef± 0.6 (2.6)10 11.86d ± 0.4 (3.1) 8.46de ± 0.8 (2.2)

Mean 0 9.2 (3.5) 6.67 (2.5)10 12.5 (2.9) 8.07 (1.8)

* Values given in parentheses indicate the percentage of the total cardenolides content.** LOD: limit of detection.

5.81 mg g�1 dw for D. trojana to 7.65 mg g�1 dw for D. cariensis withan overall mean of 6.67 mg g�1 dw. Total cardenolides contentranged from 196.92 mg g�1 dw for D. trojana to 294.28 mg g�1 dw forD. lamarckii with an overall mean of 263.07 mg g�1 dw.

We further determined the effect of H2O2 on callus induced LanC ranged from 229.80 mg g�1 dw for D. trojana to 551.80 mg g�1 dwfor D. lamarckiiwith an overall mean of 397.97 mg g�1 dw. Digitoxinranged from 15.08 mg g�1 dw for D. cariensis to 29.09 mg g�1 dw forD. trojana with an overall mean of 19.51 mg g�1 dw. Digoxigeninranged from 10.03 mg g�1 dw for D. lamarckii to 16.58 mg g�1 dw forD. trojana with an overall mean of 12.5 mg g�1 dw. Gitoxigeninranged from 6.92 mg g�1 dw for D. trojana to 8.53 mg g�1 dw for D.lamarckii. Total cardenolides content ranged from 282.39 mg g�1 dwfor D. trojana to 586.65 mg g�1 dw for D. lamarckii followed by506.79 mg g�1 dw for D. davisiana with an overall mean value of438.90 mg g�1 dw.

3.2. Antioxidative enzymes activity

3.2.1. Catalase (CAT) and superoxide dismutase (SOD) activityOxidative stress gave rise to a noticeable increase in CAT activity

in callus cultures (Fig. 1A). The CAT activity in control group rangedfrom 11.51 mmol H2O2/min/mg protein for D. lamarckii to17.42 mmol H2O2/min/mg protein for D. davisiana followed by17.38 mmol H2O2/min/mg protein for D. trojana with an overallmean of 14.02 mmol H2O2/min/mg protein. When incubating calluswith 10 mM H2O2 induced CAT activity ranged from21.31 mmol H2O2/min/mg protein for D. cariensis to34.04 mmol H2O2/min/mg protein for D. trojana followed by34.04 mmol H2O2/min/mg protein for D. davisiana with an overallmean of 29.09 mmol H2O2/min/mg protein. Exposure of H2O2 wasfollowed by a remarkable increase in SOD activity as compared tothe control group (Fig. 1B). In control, SOD activity ranged 0.13 U/mg protein for D. davisiana to 0.5 U/mg protein for D. cariensiswithoverall mean of 0.282 U/mg protein. While that cultured exposewith H2O2 induced SOD activity ranged from 0.33 U/mg protein forD. davisiana to 1.3 U/mg protein for D. cariensiswith overall mean of0.78 U/mg protein.

3.2.2. Total phenolic and proline contentsTreatment of callus samples with H2O2 for 6 h drastically

increased total phenolic and proline content. With regards to thecontrol cultures for total phenolic ranged 186.95 mg g�1 GA, dw forD. lamarckii to 248.20 mg g�1 GA, dw for D. cariensis with overallmean of 213.5 mg g�1 GA, dw. Proline content ranged 0.73 mmol/g,dw for D. trojana to 0.93 mmol g�1, dw for D. cariensis with overallmean of 0.85 mmol g�1, dw. Under oxidative stress, total phenolic

h Digitalis endemic species (D. lamarckii, D. trojana, D. davisiana, D. cariensis) calluse and are significantly different in each column (P < 0.05).

Total

Lanatoside C Digoxin Digitoxin

273.62K ± 6.5 (93) <LOD** 8.51fg ± 0.1 (2.9) 294.28551.80c ± 2.4 (94) <LOD 16.29d ± 5.2 (2.8) 586.65159.51k ± 2.3 (81) <LOD 18.51e ± 1.9 (9.3) 196.92229.80i ± 4.5 (81) <LOD 29.09bc± 1.5 (10.3) 282.39245.74j ± 5.7 (91) <LOD 9.79j ± 0.2 (3.6) 269.35

469.09cd ± 9.5 (93) <LOD 17.60d ± 0.8 (3.5) 506.79261.11i ± 5.1 (89) <LOD 12.11fg ± 0.7 (4.1) 291.74341.20g ± 7.7 (91) <LOD 15.08de ± 0.2 (4.0) 376.60

234.99 (89.3) <LOD 12.23 (4.6) 263.07397.97 (90.8) <LOD 19.51 (4.4) 438.09

0mM 10mM

0

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AT

Act

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2O2/

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D.lamarckiiD.trojanaD.davisianaD.cariensis

0mM 10mM

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Treatment (H2O2)

D.lamarckiiD.trojanaD.davisianaD.cariensis

0mM 10mM

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200

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D.lamarckiiD.trojanaD.davisianaD.cariensis

0mM 10mM

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A

B

C

D

Fig. 1. (AeD) The CAT activity (A), SOD activity (B), total phenolic content (C), proline content (D) of Digitalis species (D. lamarckii, D. trojana, D. davisiana, D. cariensis) callus culturestreated with hydrogen peroxide (10 mM, H2O2) to exposure to oxidative stress for 6 h. Data represented are means of three separate experiments ± S.D.

G.S. Cingoz et al. / Plant Physiology and Biochemistry 82 (2014) 89e9492

content was markedly enhanced with the ranged 235.70 mg g�1 GA,dw for D. lamarckii to 412.37 mg g�1 GA, dw for D. cariensis with anoverall mean of 359.03 mg g�1 GA, dw (Fig. 1C). The exposure ofoxidative stress gave rise to a marked increase of the proline con-tent ranged from 2.46 mmol/g, dw for D. lamarckii to 5.42 mmol g�1,dw with overall mean of 3.67 mmol g�1, dw (Fig. 1D).

4. Discussion

For the first time, this study reports, H2O2-induced antioxidantactivities and cardiotonic glycoside accumulation in four Digitalisspecies (D. lamarckii, D. trojana, D. davisiana and D. cariensis). Todevelop an efficient in vitro protocol for callus induction, TDZ andIAA combination were used. After the exposure of H2O2 for 6 h oncallus culture, further estimation of antioxidants activities andcardiotonic glycoside accumulation were carried out. For thisexpriement, hypocotyl explant were excised from one moth of oldseedling in vitro germinated.

Some researchers have found that exogenous application ofH2O2 is capable of inducing secondary metabolite formation.Araceli et al. (2007) reported that H2O2 induced capsidiol accu-mulation in pepper fruits (Capsicum annuum L.). Huerta-Herediaet al. (2009) found that in Uncaria tomentosa cell suspension cul-tures, H2O2 stimulated the production of monoterpenoid oxindolealkaloids. Five studies have provided detailed information

concerning oxidative stress and biosynthesis of secondary metab-olites (Yu et al., 2002; Chong et al., 2004). Reactive oxygen species(ROS) may serve as signaling molecules upon induction of somedefense responses in plants like production of secondary metabo-lites (Yu et al., 2002). Paranhos et al. (1999) concluded that additionof exogenous H2O2 in cell cultured of D. thapsi stimulated carde-nolide production, the intensity of the response being higher after24 h at a concentration of 1 mM. In the current study we found thatD. lamarckii (586.65 mg g�1) produced highest amount of totalcardenolides followed by D. davisiana (469.09 mg g�1) after 6 hincubation in MS medium containing 10 mM H2O2. The predomi-nant cardiac glycoside was Lan C (551.80 mg g�1 in D. lamarckii)followed by digitoxin (29.09 mg g�1 in D. trojana), digoxigenin(16.58 mg g�1 in D. trojana), gitoxigenin (8.53 mg g�1 in D. lamarckii).No digoxin was detected in callus cultures of Digitalis species.However, a role for H2O2 in cardenolide formation cannot be yetascribed. Moreover, this is the highest value of total cardenolidesproduction found in cultured cells, although most workers havereported that undifferentiated cultured cells either did not producecardenolides (Hirotani and Furuya, 1977) or contained only traceamount (Corchete et al., 1990; Lui and Staba, 1979). In our previousstudies (Sahin et al., 2013), it was demonstrated that, higheramount of five cardenolides and total cardenolides were obtainedwhen callus of Digitalis species was incubated on MS mediumlacking both calcium (Ca) and magnesium (Mg). The mean content

G.S. Cingoz et al. / Plant Physiology and Biochemistry 82 (2014) 89e94 93

of total cardenolides obtained was in the order of D. lamarckii(2017.97 mg g�1) > D. trojana (1385.75 mg g�1) > D. cariensis(1038.65 mg g�1) > D. davisiana (899.86 mg g�1) when both Ca andMg were eliminated from the medium respectively. The predomi-nant cardiac glycoside was Lan C (1962 mg g�1 in D. lamarckii) fol-lowed by digitoxin (39.22 mg g�1 in D. cariensis), digoxigenin(19.35 mg g�1 in D. cariensis), gitoxigenin (15.95 mg g�1 in D.davisiana). No digoxin was detected in callus cultures of Digitalisspecies when MS medium lacking both Ca and Mg. Here it shouldbe point out that MS medium lacking both Ca and Mg is moreeffective in terms of Lan C and total cardenolides production thanpretreatment with H2O2. There is evidence that in the absence ofCa, H2O2 and cardenolide accumulation were enhanced using bychelator and the channel blocker (Paranhos et al., 1999). Hagimoriet al. (1983) reported that undifferentiated callus cultures,embryogenic cell cultures, suspension cultures, root and shootcultures of Digitalis genus produce low amount of cardenolides.Gurel et al. (2011) reported that consistently increasing amounts ofcardenolides (mainly digoxin) were detected while callus rediffer-entiates into organized tissues.

SOD and CAT considered as primary antioxidant enzymes as theyare involved in the direct elimination of ROS. SOD catalyzes the dis-mutation of superoxide radicals and CAT catalyzes the reduction ofhydrogen peroxides and protects tissues from highly reactive hy-droxyl radicals. Both are imperative defense enzyme against oxygenradicals in any stressful conditions (McCord and Fridovich, 1969).Therefore, treatmentwithH2O2 causes to a small increase in the SODactivity in the pea seedlings however did not influence CAT activity(Moskova et al., 2009). In contrast, SOD and CATactivity increased inwheat seedlings treated with exogenous H2O2 (Li et al., 2011). Heet al. (He and Gao, 2009) showed that H2O2 pretreatment enhancedhigher expression of CAT inTriticum aestivum seedlings. Liu et al. (Luiand Staba, 1979) proposed that the unified effect of osmotic stressand exogenous H2O2 gave rise to highest antioxidant activities incucumber ecotypes. A similar observation was also found in ourexperiment that SOD (1.3 U/mg protein for D. cariensis) and CAT ac-tivity (17.42mmolH2O2/min/mgprotein forD. davisiana) significantlyincreased in Digitalis species callus cultures pretreated with H2O2.The present data suggested that H2O2 could trigger the activation ofantioxidants and related genes as a stress signal molecule.

Araceli et al. (2007) reported that accumulation of soluble phe-nolics under biotic and abiotic stress was accompanied withincreased phenyl ammonia lyase (PAL). Although, numerous reportsproposed the effects of exogenous application of H2O2 to improveantioxidant enzymes and secondary metabolite production, nostudy has been performed to clear that relationship between totalphenolic content. In present study, total phenolic content of Digitalisspecies was enhanced (412.37 mg g�1 GA, dw for D. cariensis) byexogenous addition of H2O2. Verslues et al. (2007) showed thatincreased H2O2 levels stimulated gene expression and prolineaccumulation induced by ABA in Arabidopsis, indicating that H2O2may join in proline biosynthesis as a signal molecule. The minorincrease in proline concentration was observed in leaf samples ofOryza sativa treated with H2O2 (Upadhyaya et al., 2007). Ozden et al.(2009) found that H2O2 treatment caused to an increase in endog-enous proline accumulation in leaves of grapevine. He et al. (He andGao, 2009) showed that H2O2 pretreatment significantly enhancedfree proline content in T. aestivum L. seedlings. A similar observationwas also made by Yang et al. (2009) who reported that exogenousH2O2 treatment led to a significant accumulation of proline in maizeseedlings. Our present results clearly showed that H2O2 treatmentinduced a rapid accumulation of proline in Digitalis species calluscultures (5.42 mmol/g, dw D. cariensis followed by 4.13 mmol/g, dwD. davisiana). The present data suggested that H2O2 could transformenzyme activities of proline metabolism.

In particular, recent studies have emphasized that relationshipbetween cardenolides production and CAT, SOD, total phenolic,proline activity in Digitalis species. However, highest total carde-nolides production was found in D. lamarckii and lowest total car-denolides was found in D. cariensis, while an overall activities ofCAT, SOD, total phenolic and proline were lowest in D. lamarckii andhighest in D. cariensis, respectively. It should be noted that, carde-nolides production had a significant negative relationship with CAT,SOD, total phenolic and proline in D. species.

In conclusion, our results indicate that the pretreatment of H2O2may be an effective method to increase the cardenolides and otherrelated secondary metabolites in Digitalis species. The result asso-ciated with oxidative stress, highest amount of total cardenolidesand Lan C was found as 586.65 mg g�1 (D. lamarckii) followed by506.79 mg g�1 (D. davisiana) and 551.80 mg g�1 (D. lamarckii) fol-lowed by 469.09 mg g�1 (D. davisiana), respectively. From this study,it is also observed that CAT, SOD, total phenolic and proline have asignificant negative relationship with cardenolides production, thatindicate the reduction of CAT, SOD, total phenolic and proline ac-tivity under 10 mM H2O2 concomitantly enhances the productionof total cardenolides under this condition. Further studies to becarried out toward cardenolides production and relation betweenantioxidant activities during oxidative stress.

Acknowledgments

The authors are grateful to the Scientific Research Projects(BAP 2010.03.01.341) Commission of Abant Izzet Baysal Universityfor a research grant.

Authors contribution

Gunce Sahin Cingoz designed and performed experiments,analyzed the data, and wrote this paper. Sandeep Kumar Vermahelped for experiment design and writing the manuscript. EkremGurel guided the research. All authors have contributed to, seenand approved the manuscript.

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