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

Central European Journal of Biology

Hairy roots of 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,*

1Department of Physiology, Institute of Biology, Faculty of Natural Sciences and Mathematics, “Ss. Cyril and Methodius” University, 1000 Skopje, Macedonia

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

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

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

Received 22 April 2013; Accepted 03 June 2013

Abstract: 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) ofHypericum 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: Agrobacterium rhizogenes A4 • Phenolic acids • Flavonol glycosides • Flavan-3-ols • Flavonoid aglycones • Xanthones

© Versita Sp. z o.o.

naphthodianthrones and phloroglucinols are 1. Introduction distributed in the aerial parts of the plant, whereas Hypericum perforatum L. (Saint John’s wort) is a xanthones are mainly produced in the roots [2]. medicinal plant considered as an important natural Flavonol derivatives, naphthodianthrones and source of secondary metabolites with a wide phloroglucinols are used for the treatment of mild range of pharmacological attributes. It contains and moderate depression [3]. Xanthones are a naphthodianthrones, acylphloroglucinols, flavonoids, class of polyphenolics that exhibit well-documented biflavones, phenylpropanes, xanthones and an pharmacological properties, such as monoamine essential oil rich in sesquiterpenes [1]. Flavonoids, oxidase inhibition, and antioxidant, antimicrobial,

* E-mail: [email protected] 1010 O. Tusevski et al.

cytotoxic and hepatoprotective activity [4]. To meet 2. Experimental Procedures the increasing demand for Hypericum utilized in the pharmaceutical industry [5], the emphasis in recent 2.1 Plant material research has been focused on the development of Seeds from H. perforatum were collected from wild new in vitro culture techniques as a useful alternative growing in a natural population in the National to improve the yield of bioactive metabolites in plant Park Pelister at about 1394 m. Voucher specimen material. number (060231) of H. perforatum is deposited in Plant genetic transformation offers an opportunity the Herbarium at the Faculty of Natural Sciences and to introduce new qualities into medicinal and aromatic Mathematics, University “Ss. Cyril and Methodius”- plants. Agrobacterium rhizogenes-mediated hairy root Skopje, Republic of Macedonia (MKNH). As for a (HR) cultures represent an attractive experimental previous study [15], seeds were washed with 70% system for the production of high-value secondary ethanol for 30 sec, surface sterilized with 1% NaOCl metabolites, including pharmaceuticals and other for 15 min, rinsed 3 times in sterile deionized water biologically active substances of commercial and cultured on MS macro and oligoelements [16], B5 importance [6,7]. Namely, HR cultures may synthesize vitamin solution [17], supplemented with 3% sucrose higher levels of secondary metabolites or amounts and solidified with 0.7% agar. No growth regulator was comparable to those of the intact plant and offer added. The medium was adjusted to pH 5.8 before a promising approach to the production of novel autoclaving (20 min at 120°C). In vitro cultures were metabolites [8]. The first step towards the application of maintained in a growth chamber at 25±1°C under a transformation procedures to few Hypericum species photoperiod of 16 h light, irradiance at 50 mmol m2 s-1 has been encountered. Until now, only A. rhizogenes- and 50 to 60% relative humidity. [9-11] and biolistic-mediated [12] transformation methods have been applied. Wild agropine strain 2.2 Preparation of Agrobacterium rhizogenes A. rhizogenes ATCC 15834 was used in the first A4 suspension successful transformation of H. Perforatum [9]. Also, The wild type Agrobacterium rhizogenes agropine an efficient transformation protocol of this species was strain A4 (obtained from INRA, Versailles, France) was reported with A. rhizogenes A4M70GUS [10]. Recently, used for H. perforatum transformation experiments two other Hypericum species (H. tomentosum and H. [18]. The procedure for A. rhizogenes A4 culture tetrapterum) were successfully transformed with A. preparation was based on the method of Di Guardo rhizogenes ATCC 15834 and A4 [11]. HR cultures of et al., [9] with the following modifications. H. perforatum exhibited high potential for spontaneous A. rhizogenes A4 was grown on nutrient agar medium regeneration into whole transgenic plants [9,10]. (15 g l-1 peptone, 3 g l-1 beef extract, 5 g l-1 NaCl, 0.3 g l-1 -1 Selected Hypericum HR regenerated plants have been KH2PO4 and 15 g l agar). The suspension culture was evaluated for their bioactive secondary metabolites prepared by growing a single bacterial colony in 10 ml [9,13,14]. However, no study has been published of nutrient broth medium at 28ºC with continuous rotary on the identification and quantification of secondary shaking (120 rpm) for 24 h. Subsequently, 1 ml of the metabolites in H. perforatum HR cultures. bacterial suspension was transferred into 9 ml fresh nutrient The objectives of this study were to establish an broth medium and maintained under similar conditions efficient A. rhizogenes A4-mediated transformation for 12 h or until bacterial concentration of approximately system that would result in the rapid formation of HR 4.2x109 colony-forming units (CFU) per ml medium was cultures for the purposes of studying the production achieved. Overnight-grown bacterial suspension was and accumulation of bioactive compounds. Phenolic diluted 1:20 (v/v) in sterile water (0.1 absorbance at compounds in the control roots and transformed 660 nm) and used for transformation protocol. HR were analyzed using high-performance liquid chromatography (HPLC) coupled with diode-array 2.3 Transformation protocol and establishment detection (DAD) for routine analysis and tandem of hairy roots mass spectrometry (MSn) with electrospray A. rhizogenes A4-mediated transformation protocol was ionization (ESI) as a more sophisticated means performed by Di Guardo et al., [9] with the following for identifying phenolic compounds. All present modifications. Root segments (1-2 cm) without apical derivatives of phenolic acids, flavonol glycosides, tip were excised from 4 week-old in vitro seedlings flavonoid aglycones, flavan-3-ols and xanthones and gently wounded with a sterile lancet blade. Root were identified from corresponding UV and MS explants were soaked for 15 min in bacterial suspension spectra and quantified by HPLC-DAD. 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: 95ºC for 5 min (initial denaturation), 35 cycles of were used in each treatment and this experiment was 95ºC for 30 sec, 64ºC for 1 min and 72ºC for 1 min and repeated three times. Infected and control explants a final extension at 72ºC for 7 min. PCR amplification

were than placed on MS/B5 hormone-free medium in products were analysed by electrophoretic separation on the dark at 25±1°C. After 2 days, the explants were 2% (w/v) agarose gel in TE buffer (40 mM Tris acetate, transferred to hormone-free medium supplemented with 1 mM EDTA, pH 8.3) and were detected by fluorescence 200 mg l-1 cefotaxime. The transformation frequency under UV light after staining with ethidium bromide. was calculated in percentage ((final number of explants forming HR/initial number of infected explants) x100) 2.5 HPLC/DAD/ESI-MSn analysis after 30 days of culture. Within 3-4 weeks, numerous The phenolic profile was investigated in 30-day-old HR emerged from the wounded sites. When the HR control and HR cultures. For this purpose, one HR line reached about 4-5 cm in length, they were excised exhibiting the highest growth potential was selected for

from the explant tissue and subcultured on fresh MS/B5 HPLC analysis. Phenolic compounds extraction from medium. After repeated transfer to fresh medium rapidly freeze-dried lyophilized and powdered root cultures growing HR cultures were obtained. Thereafter, putative was performed as previously reported [22,23]. Three HR lines were selected by Di Guardo et al. [9]. These independent HPLC analyses were performed for control

HR lines were subcultured monthly on MS/B5 medium and HR cultures. The HPLC system was equipped and concentration of the antibiotic cefotaxime was with an Agilent 1100 series diode array and mass gradually decreased (200, 100, 50 mg l-1) in the next detector in series (Agilent Technologies, Waldbronn, three subcultures down to the antibiotic free medium Germany). It consisted of a G1312A binary pump, a in the fourth subculture. The HR cultures were then G1313A autosampler, a G1322A degasser and G1315B harvested, frozen in liquid nitrogen, lyophilized and photo-diode array detector, controlled by ChemStation stored at -80°C, until analysis. software (Agilent, v.08.03). Chromatographic separations were carried out on 150x4.6 mm, 5 mm 2.4 Molecular analysis XDB-C18 Eclipse column (Agilent, USA). The mobile Genomic DNA from transformed and non-transformed phase consisted of two solvents: water-formic acid (A; roots of H. perforatum was isolated using the 99:1, v/v) and methanol (B) in the following gradient cetyltrimethylammonium bromide (CTAB) method program: 90% A and 10% B (0-20 min), 80% A and 20% [19], with minor modifications. Non-transformed B (20-30 min), 65% A and 35% B (30-50 min), 50% A root DNA was used as a negative control, while and 50% B (50-70 min), 20% A and 80% B (70-80 min) plasmid DNA from A. rhizogenes A4 served as a and continued with 100% B for a further 10 min. Each positive control for polymerase chain reaction (PCR) run was followed by an equilibration period of 10 min. analysis. The presence of the integrated genes in The flow rate was 0.4 mL/min and the injection volume the genome of the putative transformed roots was 10 ml. All separations were performed at 38°C. Formic

determined by PCR amplification of rolB gene. The acid (HCOOH) and methanol (CH3OH) were HPLC primers used for the amplification of a 348 bp DNA grade solvents (Sigma-Aldrich, Germany). HPLC- fragment of the rolB gene in the given instant were water was purified by a Purelab Option-Q system (Elga as follows: 5’-AAAGTCTGCTATCATCCTCCTATG-3’ LabWater, UK). The commercial standards chlorogenic and 5’-AAAGAAGGTGCAAGCTACCTCTCT-3’, acid, rutin, quercetin, kaempferol, catechin, epicatechin according to the sequence of rolB gene from A. and xanthone (Sigma-Aldrich, Germany) were used as rhizogenes A4 [20]. Bacterial contamination of plant reference compounds. The reference compounds were tissue was excluded by testing the amplification of dissolved in 80% methanol in water. The concentration a 421 bp DNA fragment of the virC1 gene which of the stock standard solutions was 1 mg ml-1 and they is located outside the bacterial T-DNA and is not were stored at -20ºC. Spectral data from all peaks were transferred to the plant genome using the following accumulated in range 190-600 nm, and chromatograms primers: 5’-CTCGCTCAGCAGCAGTTCAATG-3’ and were recorded at 260 nm for xanthones, at 280 nm for 5’-ACGGCAAACGATTGGCTCTC-3’ [21]. The PCR flavan-3-ols, at 330 nm for phenolic acids, and at 350 nm reactions were performed in a total 10 ml volume and for flavonols. Peak areas were used for quantification at contained 30-50 ng of DNA, 0.5 mM of each primer, wavelengths where each group of phenolic compounds 0.2 mM dNTP, 1 unit Taq DNA polymerase, 1xPCR buffer exhibited an absorption maximum. The HPLC system

and 3 mM MgCl2. The PCR mixture was incubated in was connected to the Agilent G2445A ion-trap mass a DNA thermal cycler (Perkin Elmer 2400, USA). PCR spectrometer equipped with electrospray ionization conditions for rolB and virC1 fragment amplification (ESI) system and controlled by LCMSD software

1012 O. Tusevski et al.

(Agilent, v.6.1.). Nitrogen was used as nebulizing gas one week of bacterial infection, some root segments at a pressure-level of 65 psi and the flow was adjusted subsequently regenerated adventitious roots from to 12 L·min-1. Both the heated capillary and the voltage wounded sites on explants. The adventitious roots were maintained at 350°C and 4 kV, respectively. MS elongated within the next 3 weeks reaching up to data were acquired in the negative ionization mode. The 4-5 cm in length and showing high level of lateral full scan mass covered the mass range from m/z 100 branching. In contrast, control root segments rarely to 1200. Collision-induced fragmentation experiments produced adventitious roots and further elongation were performed in the ion trap using helium as a of these roots was very slow (Figure 1A). Fifteen collision gas, with voltage ramping cycle from 0.3 up independent HR lines were selected on the basis of to 2 V. Maximum accumulation time of the ion trap their active growth and formation of lateral roots. and the number of MS repetitions to obtain the MS Transformation of HR lines was confirmed by PCR average spectra was set at 300 ms and 3, respectively. analysis and transformation frequency was recorded Identification of the component peaks was performed by 1 month past the fourth subculture on antibiotic-free the UV/Vis, MS and MS2 spectra and retention times of medium. The percentage of HR induction from infected the abovementioned available standards. root explants was 33%. HR cultures grew rapidly in the dark and showed characteristics of transformed roots. 2.6 Statistical analysis Namely, the HR cultures were thin and whitish in colour The experiments were independently repeated two showing plagiotropic growth with active branching times under the same conditions and all analyses were and a vigorous production of elongated lateral roots performed in triplicate. Secondary metabolite contents (Figure 1B). On the other hand, the non-transformed were expressed as mg 100 g-1 dry weight (DW). roots grew slowly without branching or displaying Standard error of mean value was showed as ±S.D. altered geotropism (Figure 1A). The phenotype of HR The statistical analyses including calculations of means cultures was stable for over one year of maintenance and standard deviations were performed using Excel on a hormone-free medium in in vitro conditions. There (Microsoft Office, 2003). 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. 3. Results It was seen that the growth of HR was generally most vigorous between the 3rd and 4th weeks of the cultivation 3.1 Establishment of hairy roots period (1 month), but their growth declined after the 5th HR cultures of H. perforatum were initiated by week. For this reason, 4-week-old HR cultures were inoculation of root explants with A. rhizogenes A4. After further evaluated for PCR and HPLC analysis.

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

1013 Xanthone production in Hypericum perforatum hairy roots

3.2 Molecular analysis with a molecular ion [M–H]– at m/z 191 was identified The transgenic nature of the selected HR cultures was as quinic acid, taking in account its MSn fragmentation confirmed by PCR analysis of the presence of rolB pattern [24]. Quinic acid (F1) was the only detectable

sequences from TL-DNA of A. rhizogenes Ri plasmid. phenolic acid in HR cultures. A 6-fold increase of quinic PCR analyses (Figure 2) performed on HR led to the acid was observed in HR cultures compared to control amplification of the expected rolB fragments (348 bp), rootsFour peaks, F2, F4, F6 and F15 were detected in which were identical to those of the positive control control roots with identical UV spectra at 240–246 nm (pRi A4). No such product was obtained from the non- and 320–325 nm and by a sharp diagnostic shoulder at transformed roots (negative control). To confirm the 290–300 nm typical for compounds containing a caffeoyl transformation and exclude any possibility of bacterial group [25]. The full mass spectrum of 3-caffeoylquinic contamination, primers directed against a virC1 gene, acid (F2) exhibited an intense [M–H]– ion at m/z 353 which is not transferred into the HR were used. No with fragment ions corresponding to quinic acid (base product was obtained either from the non-transformed or peak m/z 191) and caffeic acid (m/z 179) moieties. from the tested transformed roots when using the virC1 3-p-coumaroylquinic acid (F4) and 3-feruloylquinic acid primers. The virC1 amplification band (421 bp) was (F6) were readily distinguished by their cinnamic acid- visualised only in A. rhizogenes DNA samples (Figure 2). derived MS2 base peaks at m/z 163 and at m/z 193, Negative results from the attempted amplification of the respectively. Compound F15 with a molecular ion [M–H]– virC1 gene suggested that HR cultures were bacteria- at m/z 359 was identified as rosmarinic acid. In the MS2 – free and the Ri TL-DNA was successfully incorporated spectra of the [M–H] ion of the compound F15 exhibited into the genome of H. perforatum HR cultures. ions at m/z 179 and 161 derived from neutral loss of caffeic acid (180 amu) or 3,4-dihydroxyphenyllactic acid (198 amu). 3.3 HPLC/DAD/ESI-MSn analysis Flavonol glycosides and flavonoid aglycones. In H. The HPLC/DAD/ESI-MSn technique was used to analyse perforatum HR, the flavonol glycosides and flavonoid the secondary metabolite profile of H. perforatum HR aglycones were observed to be qualitatively and cultures. Five groups of phenolic compounds such quantitatively distinct from those of the corresponding as phenolic acids, flavonol glycosides, flavan-3-ols, control roots (Table 1, Figure 3). A major group of flavonoid aglycones and xanthones were recorded in identified compounds belonged to flavonols according to HR cultures (Table 1). Their identification was based their characteristic UV spectra of flavonols glycosylated on the typical UV/Vis spectral data and LC/MS in the at C3 (257, 265sh, 355 nm). The detected compound negative ionization mode [M–H]– with the subsequent F9 can be identified as C-glycoside of quercetin. The MS2, MS3 and MS4 analysis for further identification with deprotonated molecular ion [M–H]– of compound F9 was reference to similar data previously reported [24-33]. detected at m/z 421. It showed an MS2 fragmentation The HPLC analysis of secondary metabolites revealed characteristic of mono-C-hexosyl flavones, with losses marked differences between control roots (Figure 3A) of 90 and 120 amu [26], giving m/z ions characteristic for and HR cultures (Figure 3B). quercetin. The compound F11 had UV-spectrum and MS Phenolic acids. HPLC chromatograms confirmed data consistent with those of kaempferol 3-rhamnoside. the presence of 5 phenolic acids (F1, F2, F4, F6 and This compound gave deprotonated molecular ion [M–H]– F15) in root extracts (Table 1, Figure 3). Compound F1 at m/z 431 and its MS2 gave a single ion at m/z 285. The

Figure 2. Gel electrophoresis of PCR products amplified fromH. 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).

1014 O. Tusevski et al.

t [M–H] – –MS2 [M–H]– Control roots mg Hairy roots mg Compounds R UV (nm) (min) (m/z) (m/z) 100g-1 DW±S.D. 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 223, 197, 179, F15 rosmarinic acid 49.7 238, 294sh, 324 359 7.63±1.46 ND 161 Flavonol glycosides

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

Flavan-3-ols

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±12.92 559, 451, 425, F5 proanthocyanidin dimer 24.5 280 577 151.27±5.31 146.95±9.13 407, 289 559, 451, 425, F8 proanthocyanidin dimer 33.4 280 577 135.34±1.76 41.43±1.03 407, 289 559, 451, 425, F10 proanthocyanidin dimer 36.8 280 577 71.15±1.30 29.24±2.41 407, 289 Flavonoid aglycones 273, 229, 179, F17 quercetin 57.1 256, 372 301 5.63±0.11 ND 151 F18 kaempferol 59.5 256, 266, 350 285 ND 3.92±0.38

Xanthones 238, 256, 312, X1 mangiferin 37.3 421 331, 301, 258 1242.75±65.10 1383.25±88.91 362 208, 257, 322, 423, 397, 373, X2 xanthone derivative 1 45.8 441 ND 109.47±9.81 374 305, 257 X3 xanthone derivative 2 46.2 242, 306 367 287 ND 635.06±18.52

1,3,5,6-tetrahydroxyxanthone 499, 468, 446, X4 50.2 252, 284, 328 517 ND 821.61±28.39 dimmer 391, 365

1,3,6,7-tetrahydroxyxanthone 238, 254, 312, 517, 469, 447, X5 53.9 517 ND 522.56±25.44 dimmer 364 379, 257

X6 1,3,5,6-tetrahydroxyxanthone 55.4 250, 282, 328 259 229, 213, 187 92.61±11.77 190.17±20.73 236, 254, 314, 231, 215, 187, X7 1,3,6,7-tetrahydroxyxanthone 55.8 259 96.07±6.03 167.14±9.52 364 147 X8 xanthone derivative 3 59.2 244, 280, 316 353 273 94.16±10.69 ND

Table 1. HPLC/DAD/ESI-MSn data of the major identified phenolic compounds in H. perforatum control and hairy roots.a

a 2 ND not detected, DW dry weight, sh shoulder, tr retention time. MS ions in bold indicate the base peak. For information on peak numbers see Figure 3.

1015 Xanthone production in Hypericum perforatum hairy roots

t [M–H] – –MS2 [M–H]– Control roots mg Hairy roots mg Compounds R UV (nm) (min) (m/z) (m/z) 100g-1 DW±S.D. 100g-1 DW±S.D.

238, 260, 312, X9 mangiferin C-prenyl isomer 73.5 489 399, 327 343.56±14.90 433.68±82.56 372 1,3,6,7-tetrahydroxyxanthone 325, 297, X10 73.9 248, 312, 366 327 392.91±33.68 547.65±15.21 8-prenyl xanthone 258,201

1,3,5,6-tetrahydroxyxanthone 242, 260, 320, 325, 297, 258, X11 74.9 327 512.15±42.44 368.17±21.70 8-prenyl xanthone 368 201

1,3,7-trihydroxy-2-(2-hydroxy-3- 238, 260, 314, X12 75.3 327 309, 257 239.94±12.69 588.66±49.31 methyl-3-butenyl) xanthone 388 242, 262, 330, X13 toxyloxanthone 76.2 325 307, 283, 272 305.39±41.07 577.03±5.09 384 1,3,7-trihydroxy-6-methoxy-8- 240, 260, 318, 326, 311, 297, X14 76.5 341 343.66±10.68 650.13±34.77 prenyl xanthone 370 285

1,3,6,7-tetrahydroxyxanthone X15 76.7 248, 312, 368 327 325, 283, 271 825.69±44.10 1402.03±85.98 2-prenyl xanthone

X16 γ-mangostin isomer 77.1 254, 286, 324 395 326, 283, 271 ND 1226.31±185.52

1,3,6-trihydroxy-7-methoxy-8- 240, 256, 312, X17 77.2 341 293, 256 936.51±74.91 3240.28±140.14 prenyl xanthone 370 351, 339, 326, X18 γ-mangostin isomer 78.9 260, 316, 370 395 2642.86±191.86 3629.15±338.08 283 trihydroxy-1-metoxy-C-prenyl X19 79.4 260, 286, 314 341 326 1212.21±95.11 11314.34±469.01 xanthone 277, 251, 195, X20 xanthone derivative 3 79.9 260, 308, 374 295 990.04±185.83 ND 171 351, 339, 326, X21 γ-mangostin 80.0 246, 262, 320 395 ND 7861.71±415.11 283 X22 banaxanthone D 80.2 244, 268, 332 461 393, 341, 297 1928.08±165.48 1784.69±88.90 340, 325, 297, X23 xanthone derivative 4 80.5 254, 310 355 ND 2266.19±191.89 285, 271 394, 351, 339, X24 garcinone E 81.2 256, 286, 332 463 1147.34±40.77 8229.95±537.14 297, 285 X25 xanthone derivative 5 82.2 262, 288, 322 393 / ND 421.44±36.66 419, 393, 339, X26 banaxanthone E 82.6 252, 302, 330 477 824.95±93.58 ND 297 369, 344, 301, X27 garcinone C 83.9 286, 340 413 ND 1185.94±149.05 233 412, 397, 327, X28 xanthone derivative 6 84.4 254, 284, 326 481 98.98±1.69 562±38.99 271, 234

n a continuedTable 1. HPLC/DAD/ESI-MS data of the major identified phenolic compounds in H. perforatum control and hairy roots.

a 2 ND not detected, DW dry weight, sh shoulder, tr retention time. MS ions in bold indicate the base peak. For information on peak numbers see Figure 3.

compound F12 had molecular ion [M–H]– at m/z 477. compound was identified as kaempferol hexoside. MS2 spectra of this compound showed fragmentation Compounds F14 and F16 had molecular ions [M–H]– ions at m/z 315 (loss of 162 amu), suggesting presence at m/z 609 and 593, and their MS2 gave a single ion of hexose residue. So, compound F12 was tentatively at m/z 301 and 285, respectively, indicating quercetin identified as isorhamnetin O-hexoside. The compound and kaempferol derivatives with rutinose at C3 [27]. F13 was identified as kaempferol derivative with The absence of intermediate fragmentation between glycosilation in position 3 according to its UV-spectra the deprotonated molecular ion and the aglycone ion (256, 266, 350 nm). The MS and MS2 spectra were is indicative of an interglycosidic linkage 1→6 [28]; consistent with the presence of a hexose residue and therefore these compounds were putatively identified confirmed the kaempferol aglycone. Therefore, this as quercetin 3-O-rutinoside (rutin) and kaempferol

1016 O. Tusevski et al.

Figure 3. HPLC/DAD data of the major identified phenolic compounds inH. 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) F10 had [M–H]– at m/z 577 and main fragmentation with could be distinguished in HR cultures that belong to loss of 152 amu, characteristic fragmentation pathway the group of flavonol glycosides. A 2.8-fold increase of by retro Diels-Alder reaction [29] and were recognized rutin (F14) was observed in HR compared to control as proanthocyanidin dimers. Regarding the group of roots. In contrast, quercetin 6-C-glucoside (F9) was flavan-3-ols in HR cultures, catechin (F3) was de novo in lower amounts compared with those in control synthesized while compound epicatechin (F7) was roots. Isorhamnetin O-hexoside (F12) was de novo 8-fold increased, compared to control roots. In contrast, synthesized in transformed roots while kaempferol proanthocyanidin dimers (F5, F8 and F10) were 3-rhamnoside (F11), kaempferol hexoside (F13) and generally in lower quantities in HR cultures as compared kaempferol rutinoside (F16) were not detectable in HR to control roots. cultures. Two compounds in the extracts were detected Xanthones. Twenty-eight xanthones were detected as flavonoid aglycones (F17, F18) but only F18 was in the methanolic extracts from in vitro biomass of H. identified in HR while F17 was observed in control perforatum transformed and untransformed roots and samples. The peaks at m/z 301 and 285 correspond to 22 of them were fully identified by ESI-MS (Table 1, quercetin (F17) and kaempferol (F18), respectively. Figure 3). These included simple oxygenated xanthones Flavan-3-ols. The HPLC analysis confirmed the or derivatives with prenyl, pyran or methoxy groups. presence of 5 flavan-3-ols (F3, F5, F7, F8 and F10) in HR Xanthones in HR cultures could be distinguished in extracts (Table 1, Figure 3). The mass spectrum in full five groups: (i) compounds whose quantity increased scan mode showed the deprotonated molecules [M–H]– (xanthones X6, X7, X10, X12, X13, X14, X15, X17, X19, of catechin and epicatechin at m/z 289 (compounds F3, X24, X28), (ii) compounds whose quantity decreased F7), with characteristic MS2 ions at m/z 245 and 205 (xanthone X11), (iii) compounds whose quantity was not and UV maximum at 278 nm. Compounds F5, F8 and significantly modified (xanthones X1, X9, X18, X22), (iv)

1017 Xanthone production in Hypericum perforatum hairy roots

compounds that were not detectable (xanthones X8, X20, of 1,3,5,6-tetraoxygenated xanthone. A distinct shoulder X26), and (v) compounds that were de novo synthesized at 365 nm revealed conjugation with a pyran ring. MSn (xanthones X2, X3, X4, X5, X16, X21, X23, X25, X27). and UV spectra were in complete agreement with The compound X1 was putatively identified as mangiferin. those of toxyloxanthone, previously reported by Dias HPLC–MS/MS analysis of this compound gave a et al. [32]. Xanthones X14 and X17 were identified molecular ion m/z [M–H]– of 421 and major –MS2 fragments as 1,3,7-trihydroxy-6-methoxy-8-prenyl xanthone at m/z 331 [M–H–90]– and 301 [M–H–120]–, thus proving and 1,3,6-trihydroxy-7-methoxy-8-prenyl xanthone that this compound loses the characteristics of C-hexosyl (molecular ions [M–H]– at m/z 341) using previously compounds [26]. Compounds X4, X6, and X11 showed published data [27,31,32]. Compounds X16 and X18 UV spectral characteristics of the 1,3,5,6 oxygenated were putatively identified as isomers of γ-mangostin xanthones, with band IV reduced to shoulder while most (1,3,6,7-tetrahydroxyxanthone-C-bis-prenyl), since they of the other identified xanthones had UV spectra similar have a similar molecular ion [M–H]– of 395 but different to mangiferin typical of the 1,3,6,7 oxygenation pattern UV spectra and retention times. Compound X19 had a with a very well-defined band IV [30]. Compounds X6 similar fragmentation pattern to compound X14, thus and X7 were identified as 1,3,5,6-tetrahydroxyxanthone indicating that compound X19 is similar in nature to and 1,3,6,7-tetrahydroxyxanthone aglycones, compound X14. We can tentatively term compound X19 respectively (single intense molecular ion [M–H]– at as trihydroxy-1-metohy-C-prenyl xanthone. Comparisons m/z 259). Compounds X4 and X5 gave molecular to previously published data for UV and MS spectra ions [M–H]– at m/z 517. Major –MS2 fragments at indicate that compound X21 is γ-mangostin (molecular m/z 365 and 257, respectively, characterized them ion [M–H]– at m/z 395). Compounds X22, X24, X26 and as dimers of 1,3,5,6-tetrahydroxyxanthone and X27 gave deprotonated molecular ions [M–H]– at m/z 1,3,6,7-tetrahydroxyxanthone. Compound X9 was 461, 463, 477 and 413, respectively. Their MS2 spectra

putatively identified as mangiferin-C-prenyl isomer. were generated by the loss of a prenyl residue C4H8 HPLC–MS/MS analysis of this compound gave molecular (56 amu) and two prenyl residues (112 amu). So, ions [M–H]– at m/z 489 and major MS2 fragments at compounds X22, X24, X26 and X27 were identified as m/z 399 [M–H–90]–, 369 [M–H–120]– with loss of the banaxanthone D, garcinone E, banaxanthone E and characteristics of C-hexosyl compounds [28] and 327 garcinone C, respectively. Several other peaks (X2, X3, as a base peak (1,3,6,7-tetrahydroxyxanthone-C-prenyl X20, X23, X25 and X28) were categorized as xanthone residue). Compounds X10 and X15 had UV spectra derivatives, but were not fully identified. 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-C- 4. Discussion prenyl isomers. It is commonly argued in literature that in some Hypericum species the C-prenyl moiety 4.1 Establishment of hairy roots can be in position 2 or 8 [31]. They can be tentatively In the present study, we have successfully described termed 1,3,6,7-tetrahydroxy-8-prenyl xanthone and a method for an A. rhizogenes A4 mediated 1,3,6,7-tetrahydroxy-2-prenyl xanthone. Compound transformation of H. perforatum. The results showed X11 had the same fragmentation pattern as X10 that root segments, as primary explants, displayed and X15 but different UV spectra, characteristic of susceptibility to an A. rhizogenes infection, which 1,3,5,6-tetrahydroxyxanthone, and was therefore termed resulted in the development of HR cultures. Namely, 1,3,5,6-tetrahydroxy-8-prenyl xanthone [32]. Compound HR formation with pRiA4 occurred at a transformation X12 gave molecular ion [M–H]– at m/z 327, but showed frequency of about 33%. Recent studies on different a different fragmentation pattern in comparison with primary explants infected with A. rhizogenes reported the other compounds with the same mass. In the MS2 lower HR transformation rates. Efficient transformation – it exhibited a loss of a hydroxyl group [M–H2O] to give with A. rhizogenes A4M70GUS was observed in 21% the base peak at m/z 309, indicating that the OH group of infected shoots [10]. Di Guardo et al. [9] showed that is not linked to the xanthone aglycone, but to the prenyl 25% of explants and only 13% of root segments had group. In the next MS3 step, after the loss of the prenyl been successfully transformed with A. rhizogenes ATCC moiety, a base peak at m/z 257 was detected. In line with 15834. These authors suggested that the transformation this behaviour and literature data, it is evident that this of leaf segments was more troublesome and occurred compound is 1,3,7-trihydroxy-2-(2-hydroxy-3-methyl-3- only on a medium supplemented with indole-3-acetic butenyl)-xanthone [33]. Compound X13 gave a [M–H]– acid and zeatin. Phytohormones promote cell division peak at m/z 325. The UV spectrum was characteristic of the host target tissue and it is reasonable that

1018 O. Tusevski et al.

wound sites associated with actively dividing cells are rapid growth and capability for enhanced production capable of undergoing a successful transformation of secondary metabolites [37]. So far, phenolic profile [9]. As presently established, efficient Agrobacterium- of H. perforatum HR cultures has not been the subject mediated transformation occurred when H. perforatum of extended research. Therefore, in the present study root segments were maintained on a hormone-free we used HPLC/DAD/ESI-MSn method to thoroughly medium. Therefore, it is possible to consider that root analyse HR extracts for the production of various segments are promising explants and better target sites groups of phenolics. The results revealed the presence for a higher transformation rate. of phenolic acids, flavonol glycosides, flavonoid Present results confirmed that transformed roots of aglycones, flavan-3-ols, and xanthones in root extracts. H. perforatum had characteristic traits of HR previously The HPLC profiles obtained in the course of this work described by Tepfer [34]. Namely, H. perforatum HR clearly evidenced a distinct phenolic production between phenotype includes a high degree of lateral branching, control roots and HR cultures. plagiotropism, and an exponential growth pattern As established, while HR did not exhibit a superior on hormone-free medium. A slow growth of HR was potential for the accumulation of various phenolic recorded in the first week of culture, followed by a gradual acids, it is noteworthy to mention in this study that increase of biomass in the next 3 weeks. Thereafter, the they did exhibit the potential to accumulate quinic retarded growth phase began and it reached stationary/ acid. Quinic acid is the most important component declining trend on 5th week, when HR started to senesce as a key intermediate in the biosynthesis of aromatic due to the nutrient depletion. In addition, H. perforatum compounds. The condensation between quinic acid and HR lines showed a homogeneous morphology and caffeic acid leads to the formation of chlorogenic acid similar growth patterns among individual root clones. in the shikimic acid pathway. Chlorogenic acid is an The uniformity of HR phenotypes obtained in this study important antioxidative compound recently produced by is curious, because the HR morphological traits depend H. perforatum adventitious roots cultivated in bioreactor of particular expression levels of various rol genes [38], shoot cultures [39] and transgenic plantlets [13]. within the clones, differences in length or copy number With regard to the class of flavonol glycosides, our of inserted T-DNA, positional effects or by an epigenetic results showed that HR have the capability to produce control [7]. quercetin derivatives such as quercetin 6-C-glucoside, quercetin 3-O-rutinoside (rutin) and isorhamnetin 4.2 Molecular analysis O-hexoside. However, there is no available study for T-DNA of agropine type of Agrobacterium Ri plasmid the potential of H. perforatum root cultures to produce consists of TL-DNA and TR-DNA which is separated flavonol derivatives. Several differences can be pointed by 16-18 kb non-transferred DNA sequence [35]. Both out when comparing the composition of flavonol

TL-DNA and TR-DNA are transferred and integrated glycosides in HR extracts with those of H. perforatum in independently into the host plant genome, but the vitro cultures. In our previous work [22,23], we indicated transfer of TL-DNA is essential for HR formation. White that H. perforatum cells, calli and shoots demonstrate et al. [35] identified the rol loci on TL-DNA to be the a considerable potential for producing quercetin, most important virulent factors and indicated that rolB isoquercitrin and quercitrin upon elicitation with jasmonic gene has a main role in pathogenicity. In our study, acid and salicylic acid. The LC-MS screening of twelve the integration of TL-DNA region in H. perforatum HR H. perforatum HR transgenic plant lines showed a large genome was confirmed by showing the presence of variability in the content of rutin, hyperoside, quercetrin rolB gene segment. In other studies, the transgenic and quercetin [13]. Moreover, the abovementioned nature of H. perforatum HR cultures was verified by flavonol glycosides had been identified inH. perforatum the amplification of rolC gene [9], while transgenosis of regenerated plantlets [40] and H. undulatum shoot H. tetrapterum and H. tomentosum was confirmed by cultures [41]. the presence of rolABCD genes [11]. Considering that HPLC-MS analysis of flavonoid aglycones in HR the rol genes are essential genetic determinants, it is cultures resulted in the identification of kaempferol but reasonable to assume that these gene loci have a large the aglycone quercetin was not detected. Kaempferol impact on secondary metabolism in transformed plant and quercetin are typical flavonoid aglycones in H. cells [36]. perforatum wild plants, which are considered to have strong antioxidant properties and neuroprotective 4.3 Production of phenolic compounds action [42]. The absence of aglycone quercetin in HR The main advantage of using HR lies in their extracts represents a potentially interesting finding; differentiated nature, genetic and biochemical stability, since it is well known that quercetin is a biologically

1019 Xanthone production in Hypericum perforatum hairy roots

active flavonoid that interacts synergistically with other enzymes that influence xanthone accumulation in bioactive substances [43]. calli and suspended cells of H. perforatum and H. One of the main achievements in this study was androsaemum [47]. The presence of xanthones was the identification of flavan-3-ols (catechins) as the also confirmed in H. perforatum undifferentiated calli major flavonoid fraction in root extracts. Namely, HR [40,48]. However, callus cultures are not a valid choice cultures were better producers of both catechin and for large-scale production due to the lack of available epicatechin than control roots. Furthermore, catechin technology and due to their low productivities [47]. To this and epicatechin play important role as antioxidants view, H. perforatum root cultures elicited with chitosan and can exert marked medicinal effects [44]. and supplemented with indol-3-butiryc acid represent a H. perforatum in vitro cultures had never been reported valuable tool for obtaining extracts with stable quantities to posses catechin derivatives. Nevertheless, catechin, of xanthones [2,49]. These authors suggested that epicatechin and proanthocyanidin dimers had been root cultures grow continuously on nutrient media previously identified in shoots and calli of H. erectum supplemented with auxins, but sometimes repetitive [45] and H. undulatum shoot cultures [41]. subcultures may induce loss of morphogenetic potential, Our data demonstrated that xanthones correspond resulting in poor or negligible secondary metabolite to the major peaks recorded in the chromatograms production. On the other hand, our results showed that of H. perforatum root extracts. It is worth noting that H. perforatum HR successfully grow on hormone-free transformed roots synthesized and stored significant media and represent a continuous source for high- quantities of xanthones compared to control roots. level secondary metabolite production. Therefore, we Among the twenty-eight detected xanthones, eleven can consider that H. perforatum HR cultures are a were up-regulated in HR cultures. Moreover, four promising biotechnological system for mass-production xanthones identified as 1,3,5,6-tetrahydroxyxanthone, of xanthones. 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 5. Conclusions could be related to a stress-induced response due to the infection with A. rhizogenes A4. The possible importance In conclusion, we have developed an efficient of xanthones as defence compounds is also reported transformation system for H. perforatum, which leads to in H. perforatum cells elicited with Colletotrichum the formation of HR cultures producing various groups of gloeosporioides [27], A. tumefaciens [31] and chitosan phenolic compounds. A distinct phenolic profile between [46]. Taken together, these compelling results support control and HR cultures was shown as detailed for the the hypothesis that xanthones belong to the chemical first time. HR cultures showed biosynthetic potential defence arsenal employed by H. perforatum to combat for the production of specific secondary metabolites biological stress factors due to the transformation such as quinic acid, quercetin 6-C-glucoside, quercetin process. Recent studies showed that Hypericum in vitro 3-O-rutinoside (rutin), isorhamnetin O-hexoside, cultures have the potential to accumulate xanthones and kaempferol, catechin and epicatechin. More importantly, their production could be manipulated by the hormonal HR cultures synthesized and stored significant quantities supplementation [47] or/and by the culture type [40]. of xanthones. Therefore, H. perforatum HR cultures It is probable that phytohormones either facilitate or represent a promising experimental system for studying hamper the expression and activity of specific xanthone the regulation of xanthone biosynthesis.

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