Hairy roots of Hypericum perforatum L. - Springer Link

Cent. Eur. J. Biol. • 8(10) • 2013 • 1010-1022 DOI: 10.2478/s11535-013-0224-7

Central European Journal of Biology

Hairy roots of Hypericum perforatum L.: a promising system for xanthone production Research Article

Oliver Tusevski1, Jasmina Petreska Stanoeva2, Marina Stefova2, Dzoko Kungulovski3, Natalija Atanasova Pancevska3, Nikola Sekulovski4, Saso Panov4, Sonja Gadzovska Simic1,* Department of Plant Physiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia 1

Department of Analytical Chemistry, Institute of Chemistry, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia 2

Department of Microbiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia 3

Department of Molecular Biology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia 4

Received 22 April 2013; Accepted 03 June 2013

Abstract: H ypericum perforatum L. is a common perennial plant with a reputed medicinal value. Investigations have been made to develop an efficient protocol for the identification and quantification of secondary metabolites in hairy roots (HR) of Hypericum perforatum L. HR were induced from root segments of in vitro grown seedlings from H. perforatum, after co-cultivation with Agrobacterium rhizogenes A4. Transgenic status of HR was confirmed by PCR analysis using rolB specific primers. HR had an altered phenolic profile with respect to phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones comparing to control roots. Phenolics in control and HR cultures were observed to be qualitatively and quantitatively distinct. Quinic acid was the only detectable phenolic acid in HR. Transgenic roots are capable of producing flavonol glycosides such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. The HPLC analysis of flavonoid aglycones in HR resulted in the identiﬁcation of kaempferol. Transformed roots yielded higher levels of catechin and epicatechin than untransformed roots. Among the twenty-eight detected xanthones, four of them were identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in HR. Altogether, these results indicated that H. perforatum HR represent a promising experimental system for enhanced production of xanthones. Keywords: A grobacterium rhizogenes A4 • Phenolic acids • Flavonol glycosides • Flavan-3-ols • Flavonoid aglycones • Xanthones © Versita Sp. z o.o.

1. Introduction Hypericum perforatum L. (Saint John’s wort) is a medicinal plant considered as an important natural source of secondary metabolites with a wide range of pharmacological attributes. It contains naphthodianthrones, acylphloroglucinols, flavonoids, biflavones, phenylpropanes, xanthones and an essential oil rich in sesquiterpenes [1]. Flavonoids,

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naphthodianthrones and phloroglucinols are distributed in the aerial parts of the plant, whereas xanthones are mainly produced in the roots [2]. Flavonol derivatives, naphthodianthrones and phloroglucinols are used for the treatment of mild and moderate depression [3]. Xanthones are a class of polyphenolics that exhibit well-documented pharmacological properties, such as monoamine oxidase inhibition, and antioxidant, antimicrobial, * E-mail: [email protected]

O. Tusevski et al.

cytotoxic and hepatoprotective activity [4]. To meet the increasing demand for Hypericum utilized in the pharmaceutical industry [5], the emphasis in recent research has been focused on the development of new in vitro culture techniques as a useful alternative to improve the yield of bioactive metabolites in plant material. Plant genetic transformation offers an opportunity to introduce new qualities into medicinal and aromatic plants. Agrobacterium rhizogenes-mediated hairy root (HR) cultures represent an attractive experimental system for the production of high-value secondary metabolites, including pharmaceuticals and other biologically active substances of commercial importance [6,7]. Namely, HR cultures may synthesize higher levels of secondary metabolites or amounts comparable to those of the intact plant and offer a promising approach to the production of novel metabolites [8]. The first step towards the application of transformation procedures to few Hypericum species has been encountered. Until now, only A. rhizogenes[9-11] and biolistic-mediated [12] transformation methods have been applied. Wild agropine strain A. rhizogenes ATCC 15834 was used in the first successful transformation of H. Perforatum [9]. Also, an efficient transformation protocol of this species was reported with A. rhizogenes A4M70GUS [10]. Recently, two other Hypericum species (H. tomentosum and H. tetrapterum) were successfully transformed with A. rhizogenes ATCC 15834 and A4 [11]. HR cultures of H. perforatum exhibited high potential for spontaneous regeneration into whole transgenic plants [9,10]. Selected Hypericum HR regenerated plants have been evaluated for their bioactive secondary metabolites [9,13,14]. However, no study has been published on the identification and quantification of secondary metabolites in H. perforatum HR cultures. The objectives of this study were to establish an efficient A. rhizogenes A4-mediated transformation system that would result in the rapid formation of HR cultures for the purposes of studying the production and accumulation of bioactive compounds. Phenolic compounds in the control roots and transformed HR were analyzed using high-performance liquid chromatography (HPLC) coupled with diode-array detection (DAD) for routine analysis and tandem mass spectrometry (MSn) with electrospray ionization (ESI) as a more sophisticated means for identifying phenolic compounds. All present derivatives of phenolic acids, flavonol glycosides, flavonoid aglycones, flavan-3-ols and xanthones were identified from corresponding UV and MS spectra and quantified by HPLC-DAD.

2. Experimental Procedures 2.1 Plant material

Seeds from H. perforatum were collected from wild plants growing in a natural population in the National Park Pelister at about 1394 m. Voucher specimen number (060231) of H. perforatum is deposited in the Herbarium at the Faculty of Natural Sciences and Mathematics, University “Ss. Cyril and Methodius”Skopje, Republic of Macedonia (MKNH). As for a previous study [15], seeds were washed with 70% ethanol for 30 sec, surface sterilized with 1% NaOCl for 15 min, rinsed 3 times in sterile deionized water and cultured on MS macro and oligoelements [16], B5 vitamin solution [17], supplemented with 3% sucrose and solidified with 0.7% agar. No growth regulator was added. The medium was adjusted to pH 5.8 before autoclaving (20 min at 120°C). In vitro cultures were maintained in a growth chamber at 25±1°C under a photoperiod of 16 h light, irradiance at 50 mmol m2 s-1 and 50 to 60% relative humidity.

2.2 Preparation of Agrobacterium rhizogenes A4 suspension The wild type Agrobacterium rhizogenes agropine strain A4 (obtained from INRA, Versailles, France) was used for H. perforatum transformation experiments [18]. The procedure for A. rhizogenes A4 culture preparation was based on the method of Di Guardo et al., [9] with the following modifications. A. rhizogenes A4 was grown on nutrient agar medium (15 g l-1 peptone, 3 g l-1 beef extract, 5 g l-1 NaCl, 0.3 g l-1 KH2PO4 and 15 g l-1 agar). The suspension culture was prepared by growing a single bacterial colony in 10 ml of nutrient broth medium at 28ºC with continuous rotary shaking (120 rpm) for 24 h. Subsequently, 1 ml of the bacterial suspension was transferred into 9 ml fresh nutrient broth medium and maintained under similar conditions for 12 h or until bacterial concentration of approximately 4.2x109 colony-forming units (CFU) per ml medium was achieved. Overnight-grown bacterial suspension was diluted 1:20 (v/v) in sterile water (0.1 absorbance at 660 nm) and used for transformation protocol.

2.3 Transformation protocol and establishment of hairy roots A. rhizogenes A4-mediated transformation protocol was performed by Di Guardo et al., [9] with the following modifications. Root segments (1-2 cm) without apical tip were excised from 4 week-old in vitro seedlings and gently wounded with a sterile lancet blade. Root explants were soaked for 15 min in bacterial suspension and blotted on sterile filter paper. Control root explants 1011

Xanthone production in Hypericum perforatum hairy roots

were soaked in sterile distilled water. Fifty root explants were used in each treatment and this experiment was repeated three times. Infected and control explants were than placed on MS/B5 hormone-free medium in the dark at 25±1°C. After 2 days, the explants were transferred to hormone-free medium supplemented with 200 mg l-1 cefotaxime. The transformation frequency was calculated in percentage ((final number of explants forming HR/initial number of infected explants) x100) after 30 days of culture. Within 3-4 weeks, numerous HR emerged from the wounded sites. When the HR reached about 4-5 cm in length, they were excised from the explant tissue and subcultured on fresh MS/B5 medium. After repeated transfer to fresh medium rapidly growing HR cultures were obtained. Thereafter, putative HR lines were selected by Di Guardo et al. [9]. These HR lines were subcultured monthly on MS/B5 medium and concentration of the antibiotic cefotaxime was gradually decreased (200, 100, 50 mg l-1) in the next three subcultures down to the antibiotic free medium in the fourth subculture. The HR cultures were then harvested, frozen in liquid nitrogen, lyophilized and stored at -80°C, until analysis.

2.4 Molecular analysis

Genomic DNA from transformed and non-transformed roots of H. perforatum was isolated using the cetyltrimethylammonium bromide (CTAB) method [19], with minor modifications. Non-transformed root DNA was used as a negative control, while plasmid DNA from A. rhizogenes A4 served as a positive control for polymerase chain reaction (PCR) analysis. The presence of the integrated genes in the genome of the putative transformed roots was determined by PCR amplification of rolB gene. The primers used for the amplification of a 348 bp DNA fragment of the rolB gene in the given instant were as follows: 5’-AAAGTCTGCTATCATCCTCCTATG-3’ and 5’-AAAGAAGGTGCAAGCTACCTCTCT-3’, according to the sequence of rolB gene from A. rhizogenes A4 [20]. Bacterial contamination of plant tissue was excluded by testing the amplification of a 421 bp DNA fragment of the virC1 gene which is located outside the bacterial T-DNA and is not transferred to the plant genome using the following primers: 5’-CTCGCTCAGCAGCAGTTCAATG-3’ and 5’-ACGGCAAACGATTGGCTCTC-3’ [21]. The PCR reactions were performed in a total 10 ml volume and contained 30-50 ng of DNA, 0.5 mM of each primer, 0.2 mM dNTP, 1 unit Taq DNA polymerase, 1xPCR buffer and 3 mM MgCl2. The PCR mixture was incubated in a DNA thermal cycler (Perkin Elmer 2400, USA). PCR conditions for rolB and virC1 fragment amplification 1012

were: 95ºC for 5 min (initial denaturation), 35 cycles of 95ºC for 30 sec, 64ºC for 1 min and 72ºC for 1 min and a final extension at 72ºC for 7 min. PCR amplification products were analysed by electrophoretic separation on 2% (w/v) agarose gel in TE buffer (40 mM Tris acetate, 1 mM EDTA, pH 8.3) and were detected by fluorescence under UV light after staining with ethidium bromide.

2.5 HPLC/DAD/ESI-MSn analysis

The phenolic profile was investigated in 30-day-old control and HR cultures. For this purpose, one HR line exhibiting the highest growth potential was selected for HPLC analysis. Phenolic compounds extraction from freeze-dried lyophilized and powdered root cultures was performed as previously reported [22,23]. Three independent HPLC analyses were performed for control and HR cultures. The HPLC system was equipped with an Agilent 1100 series diode array and mass detector in series (Agilent Technologies, Waldbronn, Germany). It consisted of a G1312A binary pump, a G1313A autosampler, a G1322A degasser and G1315B photo-diode array detector, controlled by ChemStation software (Agilent, v.08.03). Chromatographic separations were carried out on 150x4.6 mm, 5 mm XDB-C18 Eclipse column (Agilent, USA). The mobile phase consisted of two solvents: water-formic acid (A; 99:1, v/v) and methanol (B) in the following gradient program: 90% A and 10% B (0-20 min), 80% A and 20% B (20-30 min), 65% A and 35% B (30-50 min), 50% A and 50% B (50-70 min), 20% A and 80% B (70-80 min) and continued with 100% B for a further 10 min. Each run was followed by an equilibration period of 10 min. The flow rate was 0.4 mL/min and the injection volume 10 ml. All separations were performed at 38°C. Formic acid (HCOOH) and methanol (CH3OH) were HPLC grade solvents (Sigma-Aldrich, Germany). HPLCwater was purified by a Purelab Option-Q system (Elga LabWater, UK). The commercial standards chlorogenic acid, rutin, quercetin, kaempferol, catechin, epicatechin and xanthone (Sigma-Aldrich, Germany) were used as reference compounds. The reference compounds were dissolved in 80% methanol in water. The concentration of the stock standard solutions was 1 mg ml-1 and they were stored at -20ºC. Spectral data from all peaks were accumulated in range 190-600 nm, and chromatograms were recorded at 260 nm for xanthones, at 280 nm for flavan-3-ols, at 330 nm for phenolic acids, and at 350 nm for flavonols. Peak areas were used for quantification at wavelengths where each group of phenolic compounds exhibited an absorption maximum. The HPLC system was connected to the Agilent G2445A ion-trap mass spectrometer equipped with electrospray ionization (ESI) system and controlled by LCMSD software

O. Tusevski et al.

(Agilent, v.6.1.). Nitrogen was used as nebulizing gas at a pressure-level of 65 psi and the flow was adjusted to 12 L·min-1. Both the heated capillary and the voltage were maintained at 350°C and 4 kV, respectively. MS data were acquired in the negative ionization mode. The full scan mass covered the mass range from m/z 100 to 1200. Collision-induced fragmentation experiments were performed in the ion trap using helium as a collision gas, with voltage ramping cycle from 0.3 up to 2 V. Maximum accumulation time of the ion trap and the number of MS repetitions to obtain the MS average spectra was set at 300 ms and 3, respectively. Identification of the component peaks was performed by the UV/Vis, MS and MS2 spectra and retention times of the abovementioned available standards.

2.6 Statistical analysis

The experiments were independently repeated two times under the same conditions and all analyses were performed in triplicate. Secondary metabolite contents were expressed as mg 100 g-1 dry weight (DW). Standard error of mean value was showed as ±S.D. The statistical analyses including calculations of means and standard deviations were performed using Excel (Microsoft Office, 2003).

3. Results 3.1 Establishment of hairy roots

HR cultures of H. perforatum were initiated by inoculation of root explants with A. rhizogenes A4. After

one week of bacterial infection, some root segments subsequently regenerated adventitious roots from wounded sites on explants. The adventitious roots elongated within the next 3 weeks reaching up to 4-5 cm in length and showing high level of lateral branching. In contrast, control root segments rarely produced adventitious roots and further elongation of these roots was very slow (Figure 1A). Fifteen independent HR lines were selected on the basis of their active growth and formation of lateral roots. Transformation of HR lines was confirmed by PCR analysis and transformation frequency was recorded 1 month past the fourth subculture on antibiotic-free medium. The percentage of HR induction from infected root explants was 33%. HR cultures grew rapidly in the dark and showed characteristics of transformed roots. Namely, the HR cultures were thin and whitish in colour showing plagiotropic growth with active branching and a vigorous production of elongated lateral roots (Figure 1B). On the other hand, the non-transformed roots grew slowly without branching or displaying altered geotropism (Figure 1A). The phenotype of HR cultures was stable for over one year of maintenance on a hormone-free medium in in vitro conditions. There was no variability in the morphology and growth patterns among individual HR clones, despite the fact that each HR clone arose from a separate transformation event. It was seen that the growth of HR was generally most vigorous between the 3rd and 4th weeks of the cultivation period (1 month), but their growth declined after the 5th week. For this reason, 4-week-old HR cultures were further evaluated for PCR and HPLC analysis.

Figure 1. C ontrol roots (A) and hairy roots (B) of H. perforatum cultivated on solid hormone-free MS/B5 medium. 1013



The transgenic nature of the selected HR cultures was confirmed by PCR analysis of the presence of rolB sequences from TL-DNA of A. rhizogenes Ri plasmid. PCR analyses (Figure 2) performed on HR led to the amplification of the expected rolB fragments (348 bp), which were identical to those of the positive control (pRi A4). No such product was obtained from the nontransformed roots (negative control). To confirm the transformation and exclude any possibility of bacterial contamination, primers directed against a virC1 gene, which is not transferred into the HR were used. No product was obtained either from the non-transformed or from the tested transformed roots when using the virC1 primers. The virC1 amplification band (421 bp) was visualised only in A. rhizogenes DNA samples (Figure 2). Negative results from the attempted amplification of the virC1 gene suggested that HR cultures were bacteriafree and the Ri TL-DNA was successfully incorporated into the genome of H. perforatum HR cultures.

3.3 HPLC/DAD/ESI-MSn analysis

The HPLC/DAD/ESI-MSn technique was used to analyse the secondary metabolite profile of H. perforatum HR cultures. Five groups of phenolic compounds such as phenolic acids, flavonol glycosides, flavan-3-ols, flavonoid aglycones and xanthones were recorded in HR cultures (Table 1). Their identification was based on the typical UV/Vis spectral data and LC/MS in the negative ionization mode [M–H]– with the subsequent MS2, MS3 and MS4 analysis for further identification with reference to similar data previously reported [24-33]. The HPLC analysis of secondary metabolites revealed marked differences between control roots (Figure 3A) and HR cultures (Figure 3B). Phenolic acids. HPLC chromatograms confirmed the presence of 5 phenolic acids (F1, F2, F4, F6 and F15) in root extracts (Table 1, Figure 3). Compound F1

Figure 2.

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with a molecular ion [M–H]– at m/z 191 was identified as quinic acid, taking in account its MSn fragmentation pattern [24]. Quinic acid (F1) was the only detectable phenolic acid in HR cultures. A 6-fold increase of quinic acid was observed in HR cultures compared to control rootsFour peaks, F2, F4, F6 and F15 were detected in control roots with identical UV spectra at 240–246 nm and 320–325 nm and by a sharp diagnostic shoulder at 290–300 nm typical for compounds containing a caffeoyl group [25]. The full mass spectrum of 3-caffeoylquinic acid (F2) exhibited an intense [M–H]– ion at m/z 353 with fragment ions corresponding to quinic acid (base peak m/z 191) and caffeic acid (m/z 179) moieties. 3-p-coumaroylquinic acid (F4) and 3-feruloylquinic acid (F6) were readily distinguished by their cinnamic acidderived MS2 base peaks at m/z 163 and at m/z 193, respectively. Compound F15 with a molecular ion [M–H]– at m/z 359 was identified as rosmarinic acid. In the MS2 spectra of the [M–H]– ion of the compound F15 exhibited ions at m/z 179 and 161 derived from neutral loss of caffeic acid (180 amu) or 3,4-dihydroxyphenyllactic acid (198 amu). Flavonol glycosides and flavonoid aglycones. In H. perforatum HR, the flavonol glycosides and flavonoid aglycones were observed to be qualitatively and quantitatively distinct from those of the corresponding control roots (Table 1, Figure 3). A major group of identified compounds belonged to flavonols according to their characteristic UV spectra of flavonols glycosylated at C3 (257, 265sh, 355 nm). The detected compound F9 can be identified as C-glycoside of quercetin. The deprotonated molecular ion [M–H]– of compound F9 was detected at m/z 421. It showed an MS2 fragmentation characteristic of mono-C-hexosyl flavones, with losses of 90 and 120 amu [26], giving m/z ions characteristic for quercetin. The compound F11 had UV-spectrum and MS data consistent with those of kaempferol 3-rhamnoside. This compound gave deprotonated molecular ion [M–H]– at m/z 431 and its MS2 gave a single ion at m/z 285. The

Gel electrophoresis of PCR products amplified from H. perforatum genomic DNA. A. PCR performed with rolB primers; the black arrow indicates the 348 bp amplification product. B. PCR performed with virC1 primers; the black arrow indicates the 421 bp amplification product. A.r: positive control (pRi A4); HR: hairy roots; M: molecular weight marker; NC: negative control (non transformed roots).

O. Tusevski et al.

Compounds

tR (min)

UV (nm)

[M–H] – (m/z)

–MS2 [M–H]– (m/z)

Control roots mg 100g-1 DW±S.D.

Hairy roots mg 100g-1 DW±S.D.

Phenolic acids F1

quinic acid

3.9

262, 310

191

173, 127

26.26±3.19

166.77±1.20

F2

3-caffeoylquinic acid

13.7

240, 294sh, 326

353

191, 179, 135

18.24±3.01

ND

F4

3-p-coumaroylquinic acid

19.9

314

337

191, 163

4.39±0.09

ND

F6

3-feruloylquinic acid

25.3

314

367

193

15.54±2.17

ND

359

223, 197, 179, 161

7.63±1.46

ND

F15

rosmarinic acid

49.7

238, 294sh, 324

F9

quercetin 6-C-glucoside

33.9

256, 356

421

331, 301

36.64±1.75

2.99±0.79

F11

kaempferol 3-O-rhamnoside

37.3

256, 264 352

431

285

9.03±0.53

ND

F12

isorhamnetin O-hexoside

38.1

254, 356

477

316, 315, 271

ND

11.80±0.94

F13

kaempferol hexoside

41.2

256, 266, 350

447

285

8.01±0.97

ND

F14

rutin (quercetin 3-O-rutinoside)

44.9

263, 298sh, 356

609

301

5.21±0.78

14.72±2.16

F16

kaempferol 3-O-rutinoside

52.2

256, 266, 350

593

285

10.20±1.32

ND

F3

catechin

19.5

280

289

245, 205

ND

27.28±3.20

F7

(epi)catechin

29.9

280

289

245, 205

24.24±1.55

184.85±12.92

151.27±5.31

146.95±9.13

Flavonol glycosides

Flavan-3-ols

F5

proanthocyanidin dimer

24.5

280

577

559, 451, 425, 407, 289

F8


33.4

280

577

559, 451, 425, 407, 289

135.34±1.76

41.43±1.03

577

559, 451, 425, 407, 289

71.15±1.30

29.24±2.41

273, 229, 179, 151

5.63±0.11

ND

ND

3.92±0.38

F10


36.8

280

Flavonoid aglycones F17

quercetin

57.1

256, 372

301

F18

kaempferol

59.5

256, 266, 350

285

421

331, 301, 258

1242.75±65.10

1383.25±88.91

Xanthones X1

mangiferin

37.3

238, 256, 312, 362

X2

xanthone derivative 1

45.8

208, 257, 322, 374

441

423, 397, 373, 305, 257

ND

109.47±9.81

X3


46.2

242, 306

367

287

ND

635.06±18.52

X4

1,3,5,6-tetrahydroxyxanthone dimmer

50.2

252, 284, 328

517

499, 468, 446, 391, 365

ND

821.61±28.39

X5

1,3,6,7-tetrahydroxyxanthone dimmer

53.9

238, 254, 312, 364

517

517, 469, 447, 379, 257

ND

522.56±25.44

X6

1,3,5,6-tetrahydroxyxanthone

55.4

250, 282, 328

259

229, 213, 187

92.61±11.77

190.17±20.73

259

231, 215, 187, 147

96.07±6.03

167.14±9.52

353

273

94.16±10.69

ND

X7

1,3,6,7-tetrahydroxyxanthone

55.8

236, 254, 314, 364

X8


59.2

244, 280, 316

Table 1.

HPLC/DAD/ESI-MSn data of the major identified phenolic compounds in H. perforatum control and hairy roots.a

ND not detected, DW dry weight, sh shoulder, tr retention time. MS2 ions in bold indicate the base peak. For information on peak numbers see Figure 3.

a

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Compounds

tR (min)

UV (nm)

[M–H] – (m/z)

–MS2 [M–H]– (m/z)

Control roots mg 100g-1 DW±S.D.

Hairy roots mg 100g-1 DW±S.D.

X9

mangiferin C-prenyl isomer

73.5

238, 260, 312, 372

489

399, 327

343.56±14.90

433.68±82.56

X10

1,3,6,7-tetrahydroxyxanthone 8-prenyl xanthone

73.9

248, 312, 366

327

325, 297, 258,201

392.91±33.68

547.65±15.21

X11


74.9

242, 260, 320, 368

327

325, 297, 258, 201

512.15±42.44

368.17±21.70

X12

1,3,7-trihydroxy-2-(2-hydroxy-3methyl-3-butenyl) xanthone

75.3

238, 260, 314, 388

327

309, 257

239.94±12.69

588.66±49.31

X13

toxyloxanthone

76.2

242, 262, 330, 384

325

307, 283, 272

305.39±41.07

577.03±5.09

X14

1,3,7-trihydroxy-6-methoxy-8prenyl xanthone

76.5

240, 260, 318, 370

341

326, 311, 297, 285

343.66±10.68

650.13±34.77

X15


76.7

248, 312, 368

327

325, 283, 271

825.69±44.10

1402.03±85.98

X16

γ-mangostin isomer

77.1

254, 286, 324

395

326, 283, 271

ND

1226.31±185.52

X17

1,3,6-trihydroxy-7-methoxy-8prenyl xanthone

77.2

240, 256, 312, 370

341

293, 256

936.51±74.91

3240.28±140.14

X18

γ-mangostin isomer

78.9

260, 316, 370

395

351, 339, 326, 283

2642.86±191.86

3629.15±338.08

X19

trihydroxy-1-metoxy-C-prenyl xanthone

79.4

260, 286, 314

341

326

1212.21±95.11

11314.34±469.01

X20


79.9

260, 308, 374

295

277, 251, 195, 171

990.04±185.83

ND

X21

γ-mangostin

80.0

246, 262, 320

395

351, 339, 326, 283

ND

7861.71±415.11

X22

banaxanthone D

80.2

244, 268, 332

461

393, 341, 297

1928.08±165.48

1784.69±88.90

ND

2266.19±191.89

X23


80.5

254, 310

355

340, 325, 297, 285, 271

X24

garcinone E

81.2

256, 286, 332

463

394, 351, 339, 297, 285

1147.34±40.77

8229.95±537.14

X25


82.2

262, 288, 322

393

/

ND

421.44±36.66

824.95±93.58

ND

X26

banaxanthone E

82.6

252, 302, 330

477

419, 393, 339, 297

X27

garcinone C

83.9

286, 340

413

369, 344, 301, 233

ND

1185.94±149.05

X28


84.4

254, 284, 326

481

412, 397, 327, 271, 234

98.98±1.69

562±38.99

Table 1.

continued

HPLC/DAD/ESI-MSn data of the major identified phenolic compounds in H. perforatum control and hairy roots.a

ND not detected, DW dry weight, sh shoulder, tr retention time. MS2 ions in bold indicate the base peak. For information on peak numbers see Figure 3.

a

compound F12 had molecular ion [M–H]– at m/z 477. MS2 spectra of this compound showed fragmentation ions at m/z 315 (loss of 162 amu), suggesting presence of hexose residue. So, compound F12 was tentatively identified as isorhamnetin O-hexoside. The compound F13 was identified as kaempferol derivative with glycosilation in position 3 according to its UV-spectra (256, 266, 350 nm). The MS and MS2 spectra were consistent with the presence of a hexose residue and confirmed the kaempferol aglycone. Therefore, this 1016

compound was identified as kaempferol hexoside. Compounds F14 and F16 had molecular ions [M–H]– at m/z 609 and 593, and their MS2 gave a single ion at m/z 301 and 285, respectively, indicating quercetin and kaempferol derivatives with rutinose at C3 [27]. The absence of intermediate fragmentation between the deprotonated molecular ion and the aglycone ion is indicative of an interglycosidic linkage 1→6 [28]; therefore these compounds were putatively identified as quercetin 3-O-rutinoside (rutin) and kaempferol

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Figure 3. H PLC/DAD data of the major identified phenolic compounds in H. perforatum control roots (A) and hairy roots (B). Compound symbols correspond to those indicated in Table 1.

3-O-rutinoside. Three compounds (F9, F12, and F14) could be distinguished in HR cultures that belong to the group of flavonol glycosides. A 2.8-fold increase of rutin (F14) was observed in HR compared to control roots. In contrast, quercetin 6-C-glucoside (F9) was in lower amounts compared with those in control roots. Isorhamnetin O-hexoside (F12) was de novo synthesized in transformed roots while kaempferol 3-rhamnoside (F11), kaempferol hexoside (F13) and kaempferol rutinoside (F16) were not detectable in HR cultures. Two compounds in the extracts were detected as flavonoid aglycones (F17, F18) but only F18 was identified in HR while F17 was observed in control samples. The peaks at m/z 301 and 285 correspond to quercetin (F17) and kaempferol (F18), respectively. Flavan-3-ols. The HPLC analysis confirmed the presence of 5 flavan-3-ols (F3, F5, F7, F8 and F10) in HR extracts (Table 1, Figure 3). The mass spectrum in full scan mode showed the deprotonated molecules [M–H]– of catechin and epicatechin at m/z 289 (compounds F3, F7), with characteristic MS2 ions at m/z 245 and 205 and UV maximum at 278 nm. Compounds F5, F8 and

F10 had [M–H]– at m/z 577 and main fragmentation with loss of 152 amu, characteristic fragmentation pathway by retro Diels-Alder reaction [29] and were recognized as proanthocyanidin dimers. Regarding the group of flavan-3-ols in HR cultures, catechin (F3) was de novo synthesized while compound epicatechin (F7) was 8-fold increased, compared to control roots. In contrast, proanthocyanidin dimers (F5, F8 and F10) were generally in lower quantities in HR cultures as compared to control roots. Xanthones. Twenty-eight xanthones were detected in the methanolic extracts from in vitro biomass of H. perforatum transformed and untransformed roots and 22 of them were fully identified by ESI-MS (Table 1, Figure 3). These included simple oxygenated xanthones or derivatives with prenyl, pyran or methoxy groups. Xanthones in HR cultures could be distinguished in five groups: (i) compounds whose quantity increased (xanthones X6, X7, X10, X12, X13, X14, X15, X17, X19, X24, X28), (ii) compounds whose quantity decreased (xanthone X11), (iii) compounds whose quantity was not significantly modified (xanthones X1, X9, X18, X22), (iv) 1017


compounds that were not detectable (xanthones X8, X20, X26), and (v) compounds that were de novo synthesized (xanthones X2, X3, X4, X5, X16, X21, X23, X25, X27). The compound X1 was putatively identified as mangiferin. HPLC–MS/MS analysis of this compound gave a molecular ion m/z [M–H]– of 421 and major –MS2 fragments at m/z 331 [M–H–90]– and 301 [M–H–120]–, thus proving that this compound loses the characteristics of C-hexosyl compounds [26]. Compounds X4, X6, and X11 showed UV spectral characteristics of the 1,3,5,6 oxygenated xanthones, with band IV reduced to shoulder while most of the other identified xanthones had UV spectra similar to mangiferin typical of the 1,3,6,7 oxygenation pattern with a very well-defined band IV [30]. Compounds X6 and X7 were identified as 1,3,5,6-tetrahydroxyxanthone and 1,3,6,7-tetrahydroxyxanthone aglycones, respectively (single intense molecular ion [M–H]– at m/z 259). Compounds X4 and X5 gave molecular ions [M–H]– at m/z 517. Major –MS2 fragments at m/z 365 and 257, respectively, characterized them as dimers of 1,3,5,6-tetrahydroxyxanthone and 1,3,6,7-tetrahydroxyxanthone. Compound X9 was putatively identified as mangiferin-C-prenyl isomer. HPLC–MS/MS analysis of this compound gave molecular ions [M–H]– at m/z 489 and major MS2 fragments at m/z 399 [M–H–90]–, 369 [M–H–120]– with loss of the characteristics of C-hexosyl compounds [28] and 327 as a base peak (1,3,6,7-tetrahydroxyxanthone-C-prenyl residue). Compounds X10 and X15 had UV spectra characteristic of 1,3,6,7-oxygenated xanthones and molecular ions [M–H]– at 327. So, these compounds were identified as 1,3,6,7-tetrahydroxyxanthone-Cprenyl isomers. It is commonly argued in literature that in some Hypericum species the C-prenyl moiety can be in position 2 or 8 [31]. They can be tentatively termed 1,3,6,7-tetrahydroxy-8-prenyl xanthone and 1,3,6,7-tetrahydroxy-2-prenyl xanthone. Compound X11 had the same fragmentation pattern as X10 and X15 but different UV spectra, characteristic of 1,3,5,6-tetrahydroxyxanthone, and was therefore termed 1,3,5,6-tetrahydroxy-8-prenyl xanthone [32]. Compound X12 gave molecular ion [M–H]– at m/z 327, but showed a different fragmentation pattern in comparison with the other compounds with the same mass. In the MS2 it exhibited a loss of a hydroxyl group [M–H2O]– to give the base peak at m/z 309, indicating that the OH group is not linked to the xanthone aglycone, but to the prenyl group. In the next MS3 step, after the loss of the prenyl moiety, a base peak at m/z 257 was detected. In line with this behaviour and literature data, it is evident that this compound is 1,3,7-trihydroxy-2-(2-hydroxy-3-methyl-3butenyl)-xanthone [33]. Compound X13 gave a [M–H]– peak at m/z 325. The UV spectrum was characteristic 1018

of 1,3,5,6-tetraoxygenated xanthone. A distinct shoulder at 365 nm revealed conjugation with a pyran ring. MSn and UV spectra were in complete agreement with those of toxyloxanthone, previously reported by Dias et al. [32]. Xanthones X14 and X17 were identiﬁed as 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone and 1,3,6-trihydroxy-7-methoxy-8-prenyl xanthone (molecular ions [M–H]– at m/z 341) using previously published data [27,31,32]. Compounds X16 and X18 were putatively identified as isomers of γ-mangostin (1,3,6,7-tetrahydroxyxanthone-C-bis-prenyl), since they have a similar molecular ion [M–H]– of 395 but different UV spectra and retention times. Compound X19 had a similar fragmentation pattern to compound X14, thus indicating that compound X19 is similar in nature to compound X14. We can tentatively term compound X19 as trihydroxy-1-metohy-C-prenyl xanthone. Comparisons to previously published data for UV and MS spectra indicate that compound X21 is γ-mangostin (molecular ion [M–H]– at m/z 395). Compounds X22, X24, X26 and X27 gave deprotonated molecular ions [M–H]– at m/z 461, 463, 477 and 413, respectively. Their MS2 spectra were generated by the loss of a prenyl residue C4H8 (56 amu) and two prenyl residues (112 amu). So, compounds X22, X24, X26 and X27 were identified as banaxanthone D, garcinone E, banaxanthone E and garcinone C, respectively. Several other peaks (X2, X3, X20, X23, X25 and X28) were categorized as xanthone derivatives, but were not fully identified.

4. Discussion 4.1 Establishment of hairy roots

In the present study, we have successfully described a method for an A. rhizogenes A4 mediated transformation of H. perforatum. The results showed that root segments, as primary explants, displayed susceptibility to an A. rhizogenes infection, which resulted in the development of HR cultures. Namely, HR formation with pRiA4 occurred at a transformation frequency of about 33%. Recent studies on different primary explants infected with A. rhizogenes reported lower HR transformation rates. Efficient transformation with A. rhizogenes A4M70GUS was observed in 21% of infected shoots [10]. Di Guardo et al. [9] showed that 25% of leaf explants and only 13% of root segments had been successfully transformed with A. rhizogenes ATCC 15834. These authors suggested that the transformation of leaf segments was more troublesome and occurred only on a medium supplemented with indole-3-acetic acid and zeatin. Phytohormones promote cell division of the host target tissue and it is reasonable that

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wound sites associated with actively dividing cells are capable of undergoing a successful transformation [9]. As presently established, efficient Agrobacteriummediated transformation occurred when H. perforatum root segments were maintained on a hormone-free medium. Therefore, it is possible to consider that root segments are promising explants and better target sites for a higher transformation rate. Present results confirmed that transformed roots of H. perforatum had characteristic traits of HR previously described by Tepfer [34]. Namely, H. perforatum HR phenotype includes a high degree of lateral branching, plagiotropism, and an exponential growth pattern on hormone-free medium. A slow growth of HR was recorded in the first week of culture, followed by a gradual increase of biomass in the next 3 weeks. Thereafter, the retarded growth phase began and it reached stationary/ declining trend on 5th week, when HR started to senesce due to the nutrient depletion. In addition, H. perforatum HR lines showed a homogeneous morphology and similar growth patterns among individual root clones. The uniformity of HR phenotypes obtained in this study is curious, because the HR morphological traits depend of particular expression levels of various rol genes within the clones, differences in length or copy number of inserted T-DNA, positional effects or by an epigenetic control [7].


T-DNA of agropine type of Agrobacterium Ri plasmid consists of TL-DNA and TR-DNA which is separated by 16-18 kb non-transferred DNA sequence [35]. Both TL-DNA and TR-DNA are transferred and integrated independently into the host plant genome, but the transfer of TL-DNA is essential for HR formation. White et al. [35] identified the rol loci on TL-DNA to be the most important virulent factors and indicated that rolB gene has a main role in pathogenicity. In our study, the integration of TL-DNA region in H. perforatum HR genome was confirmed by showing the presence of rolB gene segment. In other studies, the transgenic nature of H. perforatum HR cultures was verified by the amplification of rolC gene [9], while transgenosis of H. tetrapterum and H. tomentosum was confirmed by the presence of rolABCD genes [11]. Considering that the rol genes are essential genetic determinants, it is reasonable to assume that these gene loci have a large impact on secondary metabolism in transformed plant cells [36].

4.3 Production of phenolic compounds

The main advantage of using HR lies in their differentiated nature, genetic and biochemical stability,

rapid growth and capability for enhanced production of secondary metabolites [37]. So far, phenolic profile of H. perforatum HR cultures has not been the subject of extended research. Therefore, in the present study we used HPLC/DAD/ESI-MSn method to thoroughly analyse HR extracts for the production of various groups of phenolics. The results revealed the presence of phenolic acids, flavonol glycosides, flavonoid aglycones, flavan-3-ols, and xanthones in root extracts. The HPLC profiles obtained in the course of this work clearly evidenced a distinct phenolic production between control roots and HR cultures. As established, while HR did not exhibit a superior potential for the accumulation of various phenolic acids, it is noteworthy to mention in this study that they did exhibit the potential to accumulate quinic acid. Quinic acid is the most important component as a key intermediate in the biosynthesis of aromatic compounds. The condensation between quinic acid and caffeic acid leads to the formation of chlorogenic acid in the shikimic acid pathway. Chlorogenic acid is an important antioxidative compound recently produced by H. perforatum adventitious roots cultivated in bioreactor [38], shoot cultures [39] and transgenic plantlets [13]. With regard to the class of flavonol glycosides, our results showed that HR have the capability to produce quercetin derivatives such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin O-hexoside. However, there is no available study for the potential of H. perforatum root cultures to produce flavonol derivatives. Several differences can be pointed out when comparing the composition of flavonol glycosides in HR extracts with those of H. perforatum in vitro cultures. In our previous work [22,23], we indicated that H. perforatum cells, calli and shoots demonstrate a considerable potential for producing quercetin, isoquercitrin and quercitrin upon elicitation with jasmonic acid and salicylic acid. The LC-MS screening of twelve H. perforatum HR transgenic plant lines showed a large variability in the content of rutin, hyperoside, quercetrin and quercetin [13]. Moreover, the abovementioned flavonol glycosides had been identified in H. perforatum regenerated plantlets [40] and H. undulatum shoot cultures [41]. HPLC-MS analysis of flavonoid aglycones in HR cultures resulted in the identification of kaempferol but the aglycone quercetin was not detected. Kaempferol and quercetin are typical flavonoid aglycones in H. perforatum wild plants, which are considered to have strong antioxidant properties and neuroprotective action [42]. The absence of aglycone quercetin in HR extracts represents a potentially interesting finding; since it is well known that quercetin is a biologically 1019


active flavonoid that interacts synergistically with other bioactive substances [43]. One of the main achievements in this study was the identification of flavan-3-ols (catechins) as the major flavonoid fraction in root extracts. Namely, HR cultures were better producers of both catechin and epicatechin than control roots. Furthermore, catechin and epicatechin play important role as antioxidants and can exert marked medicinal effects [44]. H. perforatum in vitro cultures had never been reported to posses catechin derivatives. Nevertheless, catechin, epicatechin and proanthocyanidin dimers had been previously identified in shoots and calli of H. erectum [45] and H. undulatum shoot cultures [41]. Our data demonstrated that xanthones correspond to the major peaks recorded in the chromatograms of H. perforatum root extracts. It is worth noting that transformed roots synthesized and stored significant quantities of xanthones compared to control roots. Among the twenty-eight detected xanthones, eleven were up-regulated in HR cultures. Moreover, four xanthones identified as 1,3,5,6-tetrahydroxyxanthone, 1,3,6,7-tetrahydroxyxanthone, γ-mangostin and garcinone C were de novo synthesized in transformed roots. Such an accumulation of xanthones in HR cultures could be related to a stress-induced response due to the infection with A. rhizogenes A4. The possible importance of xanthones as defence compounds is also reported in H. perforatum cells elicited with Colletotrichum gloeosporioides [27], A. tumefaciens [31] and chitosan [46]. Taken together, these compelling results support the hypothesis that xanthones belong to the chemical defence arsenal employed by H. perforatum to combat biological stress factors due to the transformation process. Recent studies showed that Hypericum in vitro cultures have the potential to accumulate xanthones and their production could be manipulated by the hormonal supplementation [47] or/and by the culture type [40]. It is probable that phytohormones either facilitate or hamper the expression and activity of specific xanthone

enzymes that influence xanthone accumulation in calli and suspended cells of H. perforatum and H. androsaemum [47]. The presence of xanthones was also confirmed in H. perforatum undifferentiated calli [40,48]. However, callus cultures are not a valid choice for large-scale production due to the lack of available technology and due to their low productivities [47]. To this view, H. perforatum root cultures elicited with chitosan and supplemented with indol-3-butiryc acid represent a valuable tool for obtaining extracts with stable quantities of xanthones [2,49]. These authors suggested that root cultures grow continuously on nutrient media supplemented with auxins, but sometimes repetitive subcultures may induce loss of morphogenetic potential, resulting in poor or negligible secondary metabolite production. On the other hand, our results showed that H. perforatum HR successfully grow on hormone-free media and represent a continuous source for highlevel secondary metabolite production. Therefore, we can consider that H. perforatum HR cultures are a promising biotechnological system for mass-production of xanthones.

5. Conclusions In conclusion, we have developed an efficient transformation system for H. perforatum, which leads to the formation of HR cultures producing various groups of phenolic compounds. A distinct phenolic profile between control and HR cultures was shown as detailed for the first time. HR cultures showed biosynthetic potential for the production of specific secondary metabolites such as quinic acid, quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin), isorhamnetin O-hexoside, kaempferol, catechin and epicatechin. More importantly, HR cultures synthesized and stored significant quantities of xanthones. Therefore, H. perforatum HR cultures represent a promising experimental system for studying the regulation of xanthone biosynthesis.

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