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: Hypericum 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: Agrobacterium 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]
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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
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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
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
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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
Figure 1.
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.
Control roots (A) and hairy roots (B) of H. perforatum cultivated on solid hormone-free MS/B5 medium.
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Xanthone production in Hypericum perforatum hairy roots
3.2 Molecular analysis
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.
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).
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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.
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
proanthocyanidin dimer
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
proanthocyanidin dimer
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
xanthone derivative 2
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
xanthone derivative 3
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
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.
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Xanthone production in Hypericum perforatum hairy roots
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
1,3,5,6-tetrahydroxyxanthone
8-prenyl xanthone
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
1,3,6,7-tetrahydroxyxanthone
2-prenyl xanthone
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
xanthone derivative 3
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
xanthone derivative 4
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
xanthone derivative 5
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
xanthone derivative 6
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
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.
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
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.
HPLC/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)
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Xanthone production in Hypericum perforatum hairy roots
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
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].
4.2 Molecular analysis
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
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Xanthone production in Hypericum perforatum hairy roots
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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Xanthone production in Hypericum perforatum hairy roots
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