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Phytochemistry 86 (2013) 8391

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Phytochemistry
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HPLCUVESI-MS analysis of phenolic compounds and antioxidant properties


of Hypericum undulatum shoot cultures and wild-growing plants
Nuno Rainha a, Kamila Koci c, Ana Varela Coelho c, Elisabete Lima a,b, Jos Baptista a,b,
Manuel Fernandes-Ferreira d,e,f,
a

Department of Technological Sciences and Development (DCTD), University of Azores, 9501-801 Ponta Delgada, Portugal
Research Center for Agricultural Technology (CITA-A), University of Azores, 9700-042, Angra do Herosmo, Portugal
Instituto de Tecnologia Qumica e Biolgica, Universidade Nova de Lisboa, Avenida da Repblica, 2780-157 Oeiras, Portugal
d
Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), University of Trs-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal
e
Department of Biology, Faculty of Science, University of Porto, 4169-007 Porto, Portugal
f
Mapprod Lda, Rua Antnio de Mariz, 22, 4715-278 Braga, Portugal
b
c

a r t i c l e

i n f o

Article history:
Received 7 June 2012
Received in revised form 7 October 2012
Available online 7 November 2012
Keywords:
Wavy St. Johns Wort
Free radical scavenging activity
Reducing power
Total phenolics
Electrospray ionization mass spectrometry

a b s t r a c t
LCUV and LCMS analysis were used to study the phenolic composition of water extracts of Hypericum
undulatum (HU) shoot cultures and wild-growing (WG) plants. Total phenolic content (TPC), determined
using the FolinCiocalteu assay, and the antioxidant activity measured by two complementary methods
were also performed for each sample. Mass spectrometry revealed several phenolics acids with quinic
acid moieties, avonols, mostly quercetin, luteolin and apigenin glycosides, avan-3-ols (catechin and
epicatechin) and the xanthonoid mangiferin. Differences in phenolic composition prole and TPC were
found between the samples. The major phenolic in HU culture-growing (CG) samples is chlorogenic acid,
followed by epicatechin, quercitrin and isoquercitrin. The WG plants presents hyperoside as the main
phenolic, followed by isoquercitrin, chlorogenic acid and quercetin. The TPC and antioxidant activity
were higher in samples from WG plants.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Hypericum (Guttiferae) is a large genus of herbs or shrubs (ca
450 species) used for centuries as traditional medicinal plants at
several parts of the world (Robson, 2003). Hypericum perforatum
L., also known as St. Johns Wort (SJW), is the best-documented
and most studied species due to its many therapeutic applications,
including neuralgic, vulnerary, diuretic conditions, sciatica and
poisonous reptile bites (Bilia et al., 2002). The success and economic relevance of H. perforatum as a natural alternative for
mild-to-moderate depression therapy was observed, for instance,
in Germany, in which the prescription of Hypericum-based products was approximately 20 times higher than uoxetine hydrochloride (Prozac), one of the most prescribed antidepressants
(Greeson et al., 2001).
Studies of etnopharmacology in Portugal report the use of several Hypericum species for therapeutic purposes (Novais et al.,
2004; Rivera and Obn, 1995). Hypericum undulatum Schousb. ex.
Willd (HU), also known as Wavy St. Johns Wort, is one of the three
Corresponding author at: Centre for the Research and Technology of AgroEnvironmental and Biological Sciences (CITAB), University of Trs-os-Montes e Alto
Douro, 5001-801 Vila Real, Portugal. Tel.: +351 220 402 732.
E-mail address: manuel.ferreira@fc.up.pt (M. Fernandes-Ferreira).
0031-9422/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.phytochem.2012.10.006

Hypericum species most used in Portuguese folk medicine along


with H. androsaemum and H. perforatum (Nogueira, 2000). HU is reported to be very similar, both morphological and chemically to
SJW and its main applications are for the treatment of migraine
and for bladder and gall bladder ailments. It is also reported to
be hepatic protector, renal antispasmodic and intestinal-inammatory (Ferreira et al., 2006). According to some authors, HU can also
prevent the development of Alzheimers disease, once its ethanolic
extracts and decoctions showed acetylcholinesterase inhibitory
activity (Ferreira et al., 2006; Hernandez et al., 2010). Such type
of extracts are mainly composed of phenolic compounds, namely,
quercetin and its glycoside derivatives, hyperoside, isoquercitrin
and quercitrin, and the phenylpropanoid chlorogenic acid as well
demonstrated by Hernandez et al. (2010) and in our previous research on a water extract from HU aerial parts (Rainha et al.,
2011). The presence of hypericin was also reported, along with
quercetin sulfate, rutin and mangiferin, in alcoholic extracts of
leaves and aerial parts of HU (Seabra et al., 1991, 1992).
Culture-growing (CG) plants, in sterile and standardized conditions, is an attractive tool for mass multiplication and production
of bioactive secondary metabolites from Hypericum species
(Guedes, 2009). Many studies have been performed to establish
and to enhance the production of naphthodianthrones (hypericin
and pseudohypericin) and the phloroglucinol hyperforin on SJW

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N. Rainha et al. / Phytochemistry 86 (2013) 8391

and other Hypericum species. It is well known that Hypericum


species have been widely researched due to: the growing demand
of standardized phytopharmaceutical products, the limited area of
occurrence of the important species, seasonal harvesting, loss of
biodiversity, variability in quality, and also potential cross-contamination (Gadzovska et al., 2005; Karppinen et al., 2007; Pavlk et al.,
2007).
Studies on the secondary metabolite production from CG plants
of HU are scarce and the recent reports in literature only revealed
the characterization of essential oil extracts (Guedes, 2009) and the
quantication of the plant naphthodianthrone content (Rainha
et al., 2012). The aim of the present research was to study and to
compare the chemical prole of HU wild-growing (WG) and CG
plants in order to support the cultivation and the production of
bioactive molecules from the latter plants. We present the qualitative (LCUV and LCMS) and quantitative (LCUV) analysis of the
major phenolic compounds present in water extracts of HU CG
plants in comparison with that WG plants. The total phenolic content (TPC) and the in vitro antioxidant potential were also determined. This latter activity was evaluated by two different
methods, the free radical scavenging activity (FRSA) and the reducing power.

2. Results and discussion


2.1. Qualitative analysis of phenolic compounds from H. undulatum
water extracts
A qualitative study of the phenolic compounds present in HU
samples was performed by LCUVESI-MS method (k = 280 nm).
Before the LCMS analysis a direct infusion of chlorogenic acid
(1) was performed in order to tune ESI-MS conditions to achieve
the best sensitivity for HU compounds. The typical HPLC chromatograms of water extracts for HU WG and CG (sample HU03) plants
are presented in Figs. 1A and B, respectively. The identied peaks
are labeled with numbers. For identication see Table 1.
Table 1 summarizes the mass spectrometry data acquired for
HU WG and CG samples. Mass spectrometry was used to conrm
the identication of 5-O-caffeoylquinic acid (peak 2), quercetin
(peak 23) only in HU WG and its glycosides hyperoside (peak
15), isoquercitrin (peak 16) and quercitrin (peak 20). Compounds
were identied by comparing RT values with standards analyzed
under the same conditions and by comparing the m/z values together with MS/MS fragmentation patterns (when available) with
the molecular structure and MS/MS data reported in literature
(see Table 1) (Exarchou et al., 2006; Gioti et al., 2009). The referred
compounds were previously described as the major phenolic compounds present in water extracts of HU WG samples (Hernandez
et al., 2010; Rainha et al., 2011).
Three caffeoylquinic acid isomers (X-CQA) were identied
based on [M+H]+, [MH] and MS/MS information (see Table 1).
4-CQA (peak 4, RT 16.4 min), also known as crypto-chlorogenic
acid, was identied in both HU WG and CG samples based on the
MS/MS fragments 173 and 179, which has a distinctive MS/MS
behavior for a caffeoylquinic acid isomer previously reported by
Clifford et al. (2003) and Fang et al. (2002). Fig. 2 shows the molecular mass of 3-CQA and 4-CQA and their different fragmentation
patterns. 3-CQA (peak 1) was located on the chromatogram and
identied as the major caffeoylquinic acid by comparison with a
commercial standard and consequently the peak assigned at
13.2 min was identied as the isomer 5-O-caffeoylquinic acid,
based on the retention behavior of caffeoylquinic acids in reverse
phase chromatography (Clifford et al., 2003). The previous was
only detected at HU WG samples revealing a distinct chemical prole. It was also detected a signal [M+H]+ at m/z 355 and at 32.1 min

Fig. 1. Representative RP-HPLC/UVESIMS chromatograms of water extracts from


(A) wild-growing H. undulatum and (B) culture-growing H. undulatum (sample
HU3), detected at 280 nm. Experimental conditions as mentioned in Section 4.4.3.
Peak numbers refer to Table 1. b, blank peaks;  unknown peaks.

(for both HU WG and CG plants) that could be assigned to di-CQA,


that fragmented before the mass analyzer after the loss of one caffeyol moiety. Such compounds are more difcult to identify because the detected ions do not correspond to the expected
molecular ions but to the fragment ions (Mauri and Pietta, 2000).
[MH] ions corresponding to p-coumaroylquinic acid derivatives (m/z 337) were detected in both HU CG and WG samples.
The molecular ion [MH] of feruloylquinic acid derivative (m/z
367) was also detected in HU CG samples. The identication of this
substances was based in their chromatographic behavior and
molecular ions consulted in the literature (Plazonic et al., 2009).
One of the main achievements was the identication of epicatechin (peak 10, RT 26.1 min) (see Table 1) as one of the major phenolic compounds of HU CG water extracts. The compound was
identied by its precursor ion [MH] at m/z 289, and its MS/MS
fragment at m/z 245, which results from the loss of a CO2 or CH2CHOH group (Bravo et al., 2006). A similar precursor ion was detected at m/z 289 (peak 7, RT 19.4 min), for both CG and WG HU
samples, indicating the existence of catechin. The report of catechin derivatives was never described for HU extracts. Nevertheless,
catechin, epicatechin and their dimers (proanthocyanidins) were
previously identied in SJW with a molecular mass of 578 Da
(Ploss et al., 2001). The [MH]and [M+H]+ ions at m/z 577 and
579, respectively were recorded in the mass spectrum at RT
24.2 min. Gallic acid derivatives of catechin and epicatechin were
also searched in HU samples, but the correspondent m/z were
not found.
Xanthones are a very typical class of metabolites, present in
plants from the Guttiferae family. These substances are usually
found in the roots but trace amounts can also be found in the aerial
parts of the plants (Erdelmeier et al., 2000). The ion [MH] detected at 20.7 min with m/z 421 (peak 8) seems to correspond to
mangiferin, a C-glucosylxanthone previously reported in ethanolic
extracts of aerial parts of H. undulatum (Seabra et al., 1992). Dias

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N. Rainha et al. / Phytochemistry 86 (2013) 8391


Table 1
Compounds identied in water extracts of H. undulatum wild growing (WG) and culture growing (CG) samples by mass spectrometry.
Name

3-O-caffeoylquinic acid (3-CQA)


5-O-caffeoylquinic acid (5-CQA)
3-p-Coumaroylquinic acid
4-O-caffeoylquinic acid (4-CQA)
Unknown
o-Feruloylquinic acid
Catechin
Mangiferin
Catechin or epicatechin dimer
Epicatechin
Rutin
Unknown (X-CQA)
Quercetin sulfate
Miquelianin
Hyperoside
Isoquercitrin
Luteolin hexoside
Quercetin-pentoside
Luteolin hexoside
Quercitrin
Apigenin-7-O-glucoside
Flavonoid aglycon
Quercetin
a

Compound

H. undulatum
a

Peak number

RT (min)

[M+H] m/z

[MH] m/z

Fragments for negative ions

CG samples

WG sample

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23

9.1
13.2
13.8
16.4
17.1
17.8
19.4
20.7
24.2
26.1
27.7
32.1
33.0
33.6
35.1
35.4
36.8
37.0
37.5
37.6
39.5
41.1
42.1

355
355

355

353
353
338
353
415, 355
367
289
421
577
289
609

381
477
463
463
447
433
447
447
432
263
301

191, 179, 135


191, 179

173, 179

x
x
x
x
x
x
x
x
x
x
x
x
x
x
x

x
x
x

x
x
x
x

422
579
291

355

479
551, 465
465
449
435
449
449;

303

245, 205

245, 205

301
301
284
301
284
301

179, 151

x
x
x
x
x
x
x
x
x
x
x

Retention in total ion chromatograms.

et al. (2000, 2001) reported the production of several xanthones in


calli and suspended cells of H. androsaemum and H. perforatum
when produced under CG conditions. Curiously, mangiferin was
only detected at HU CG revealing the possibility for the existence
of several other substances from this class.
Table 1 also reports the molecular mass information of several
avonols. Peak 11 detected at RT 27.7 min for HU CG corresponds
to the molecular ion [MH] of rutin, an usual avonol found in
SJW (Rainha et al., 2011). Miquelianin (peak 14, RT 33.6 min;
[MH] m/z 477) was detected in HU WG plants and quercetin sulfate (peak 13, RT 33.0 min; [MH] m/z 381) at HU CG plants. The
peaks 17 and 19 assigned with RT 36.8 and 37.5 min, respectively,
with precursor ion [MH] m/z 447 and MS/MS fragment at 284
resulting by the loss of the hexose moiety, correspond to a luteolin
hexoside. Peak 18 assign at RT 37.0 min corresponds to a quercetin
pentoside, probably guaijaverin (quercetin-3-O-arabinoside)
([MH] m/z 433, [M+H]+ m/z 435 and MS/MS fragment in negative mode at m/z 301 corresponding to the aglycone quercetin).
Guaijaverin is a common quercetin glycoside found in Hypericum
species, but in smaller amounts compared to rutin, hyperoside or
isorquecitrin, which was already reported in SJW (Jrgenlienmk
and Nahrstedt, 2002) and eleven Hypericum species from the Trigynobrathys section (Nunes et al., 2010). The molecular ion [MH]
at m/z 432 corresponding to the molecular mass of apigenin glucoside (peak 21) was also detected at 39.5 min.
Qualitative variations of the phenolic prole of both HU CG and
WG were detected. The major difference is the absence of quercetin in HU CG samples. Other relevant chromatographic differences
were detected at RT 32.1, 35.9, 41.1 and 43.3 min for HU CG, but
the acquired m/z values and the literature reports are not sufcient
for their condent identication.
2.2. Quantitative analysis of the major phenolic compounds from H.
undulatum water extracts
HPLC has been widely applied for the analysis of the secondary
metabolites of Hypericum species. Usually, HPLC analyses are carried out with C18 columns. However several methods reported
are not economical in terms of time and solvent usage (Pellati

et al., 2005). Since, the method developed by us and used in previous studies of the chemical composition of Hypericum species
(Rainha et al., 2011) using a reversed phase C12 column was adequate to study the chemical composition of these HU samples separating all the relevant peaks in less than 45 min.
The quantitative variation of the major phenolic compounds of
HU water extracts are shown in Table 2. Chlorogenic acid (1) was
found to be the major phenolic in HU CG samples with an average
amount of 13.04 1.56 mg g1 dE which is slightly lower than the
amount determined for HU WG plants (15.09 0.77 mg g1 dE).
However samples HU10 and HU12 presented higher amounts of
(1) as compared to HU WG samples. These results are in accordance with those published elsewhere reporting that (1) and its
positional isomers are the major phenolic acids present in Hypericum species.
Hypericum species are widely known for its richness in avonoids especially by the avonols derived from quercetin glycosilation. As expected for HU WG plants, the major avonols found
were hyperoside (15), isoquercitrin (16) and quercetin (23) with
32.97, 15.38 and 6.61 mg g1 dE, respectively. Low amounts of
quercitrin (20) were also detected but not quantied since the
peak was not completely separated from the neighbor peaks. In
comparison with HU CG plants the avonoid distribution was completely different from that found for HU WG plants. The amounts of
(15) varied between 0.50 and 0.88 mg g1 dE which is two orders
of magnitude lower than the amount found in HU WG samples.
Compound (16) was also found in lower levels (average value of
2.10 0.46 mg g1 dE) than those of HU WG plants. The major avonol of HU CG samples was (20) with an average amount of
4.76 0.75 mg g1 dE but with some signicant variations between the HU CG group. Other avonols such as the identied
guaijaverin and luteolin hexoside were also detected, but their
amounts are lower than those of the referred compounds.
The second main phenolic detected in HU CG samples is epicatechin (10) a compound from the avonoid class avan-3-ols or
sometimes referred to as avanols. Epicatechin was determined
in an average amount of 5.03 0.80 mg g1 dE also with signicant
differences between the HU CG group. Epicatechin was also
detected in HU WG samples by LC/MS/ESI, although its levels are

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N. Rainha et al. / Phytochemistry 86 (2013) 8391


353.08

[M-H]-

2500

(A) 3-O-Caffeyolquinic acid

m/z = 353.08
Intensity

2000
1500

[M-Na]-

m/z = 354.08

m/z = 375.07

[M-K]m/z = 391.03

1000
354.08

500
332.93 334.94
336.91 339.04 340.96 3 4 2 .9 5 344.93

335

340

345

375.07

355.07

3 4 8 .9 3 351.02

350

391.03

373.02
3 56 .2 4 3 59 .0 0 360.90

355

360

365

376.09

368.92 370.93

364.90366.91

370

m/z

379.93

375

392.98

382.96 384.91 386.95 388.94

380

385

390

396.90 398.91

395

401.01

400

191.05

700

MS2

m/z = 191.05

600
500

Intensity

[M]

m/z = 179.04

400

179.04

m/z = 135.03

300
200
100

135.03
105.98

0
100

110

120

130

172.99

155.00 161.05

134.11 137.23

140

150

160

170

188.90 191.67 203.02

180

190

200

236.84247.15

210

220

230

240

254.82

250

m/z

270

280

290

300

353.08

900

321.21 328.64 335.02

320

330

340

350

360

370

380

390

400

m/z = 353.08

800

Intensity

310

(B) 4-O-Caffeyolquinic acid

[M-H]-

1000

308.78

270.91 278.80 285.04 293.91

260

[M]

700

[M-Na]-

[M-K]-

m/z = 375.02

m/z = 390.96

m/z = 354.11

600
500
400
300
200
100
0

317.03
314.97

302.92
306.97

305

310

332.88

321.00

311.04

315

373.06

354.11

318.90

320

325.01

325

330.93

330

375.02
334.92

335

340.97

340

346.94

345

348.91

350

355.05

355

m/z

358.97 360.96

360

365

370

390.96
379.96

366.91 368.94376.94

375

392.93

384.98 386.91

380

385

390

398.92 400.98

395

402.91

400

408.93

405

410

173.06

120

MS2

m/z = 179.04

m/z = 173.06

Intensity

100
80

m/z = 191.05

179.06

m/z = 135.05

60
40

191.05

20
135.05

151.02 154.96

127.06

110

120

130

140

150

160

188.95

170

180

190

199.47

200

2 2 0 .8 8

m/z

210

220

230

240

2 4 9 .12 252.94

259.96

250

260

269.15

270

276.83

280

284.86

293.93

290

300

Fig. 2. Full mass spectrum of (A) 3-O-Caffeyolquinic acid and (B) 4-O-Caffeyolquinic acid and their respective MS/MS spectrum in negative mode from precursor ion m/z 353.

below the limits of quantication. Its epimer catechin was also detected in HU water extracts but also below the limits of quantication by HPLC/UV.
Person correlation matrix was applied to identify possible
chemical composition correlations within the HU CG samples. It
was determined a correlation between chlorogenic acid and
quercitrin (R2 = 0.421, P < 0.05). A strong correlation was observed
between avonols hyperoside and isoquercitrin (R2 = 0.740,
P < 0.01), and hyperoside and quercitrin (R2 = 0.763, P < 0.01). Isoquercitrin and quercitrin were also correlated (R2 = 0.395,
P < 0.05). Such correlations were also observed by Kusari et al.
(2009) for different Hypericum species from Slovakia.

2.3. Determination of total phenolic content (TPC) in H. undulatum


water extracts
Plant phenolics constitute one of the major groups of compounds acting as primary antioxidants or free radical terminators.
Their total amount can be a useful tool to distinguish plants and
extracts as potential resources to be explored as commercial products. The content of phenolic compounds (mg g1 dE) in HU water
extracts, determined from regression equation of calibration curve
(y = 1.0204x0.0397, R2 = 0.996) and expressed in gallic acid equivalents (GAE), varied between 119.90 and 191.77 mg GAE/g dE.
Table 3 summarizes the TPC results for each individual HU sample.

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N. Rainha et al. / Phytochemistry 86 (2013) 8391

Table 2
HPLC quantitative variations of the major phenolic compounds identied in water extracts of H. undulatum (HU) wild growing (WG) and culture growing (CG, referred as HU1 to
HU12) samplesa.
H. undulatum

Compound (mg g1 dry extract)


Chlorogenic acid

HU1
HU2
HU3
HU4
HU5
HU6
HU7
HU8
HU9
HU10
HU11
HU12
WG HU

13.29 0.88
12.39 0.43
11.90 0.48
12.66 0.30
10.39 0.15
13.55 0.37
12.92 0.45
12.33 1.23
12.32 0.34
15.39 0.71
12.93 0.11
16.37 0.20
15.09 0.77

Epicatechin
abc
ad
ad
ad
d
abc
ab
ad
ad
ce
ab
e
bce

4.41 0.26
4.42 0.24
5.34 0.33
6.36 0.07
3.93 0.38
4.53 0.46
6.31 0.39
5.50 0.10
5.64 0.08
4.23 0.54
4.85 0.30
4.80 0.27
tr

Hyperoside
abc
abc
bcde
e
a
abcd
e
cde
de
ab
abcd
abcd
f

0.64 0.02
0.50 0.07
0.87 0.07
0.66 0.03
0.71 0.01
0.67 0.01
0.82 0.04
0.80 0.03
0.87 0.06
0.88 0.03
0.65 0.02
0.80 0.02
32.97 1.05

Isoquercitrin
ab
a
e
b
bce
bc
de
cde
e
e
b
cde
f

1.96 0.13
1.53 0.01
3.20 0.07
2.13 0.13
1.99 0.01
1.89 0.10
1.95 0.06
2.06 0.09
2.65 0.09
2.31 0.24
1.51 0.05
1.97 0.08
15.38 0.67

Quercitrin
ab
cd
e
ab
ab
d
ab
ab
f
bf
c
ab
g

4.54 0.24
3.94 0.14
4.86 0.41
3.50 015
4.47 0.05
4.23 0.09
4.67 0.29
5.64 0.18
5.54 0.22
5.72 0.05
4.34 0.12
5.66 0.04
tr

Quercetin
ab
ac
b
c
ab
ab
b
d
d
d
ab
d
e

n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
6.61 0.53

Values marked with different letters within a column are signicantly different (P < 0.05). Data are mean SD, n = 3. tr, trace; n.d., not determined.
a
The HPLC analytical conditions as referred in Section 4.4.2.

The highest amount was found in HU WG samples. The variation


within the CG samples was narrow with an average TPC of
145.78 7.21 mg GAE/g dE. To test the precision of both HPLC
quantication of phenolic compounds and their total amount measured by the FolinCiocalteu reagent, the total area of the chromatograms recorded at 280 nm was correlated with the TPC
values for all HU samples. The Pearson coefcient (r-value) obtained between the total phenolic content and the total area recorded at 280 nm was r = 0.641 with P < 0.01 which reveals a
linear relationship between the results measured by the two methods, although there is some dispersion of the results, especially for
HU CG samples.
2.4. Evaluation of potential antioxidant activity of H. undulatum water
extracts
2.4.1. Free radical scavenging activity (FRSA)
Nature has allowed living systems with numerous antioxidant
molecules. These natural antioxidants are known to minimize the
adverse effects of free radicals in living system. Assessments of
antioxidant properties of natural compounds are very important
due to their applications in medicine, food and cosmetic (Mishra

et al., 2012). The use of DPPH assay is routinely practiced for FRSA
assessment of extracts and it is considered as one of the standard
and easy colorimetric methods for the evaluation of antioxidant
properties. The results of the FRSA for HU samples are summarised
in Table 3. The average IC50 for HU CG samples was
24.51 3.42 lg ml1 with the IC50 varying from 19.35 to
29.60 lg ml1. The majority of FRSA values are not statistically different (P < 0.05) from that found for HU WG samples
(IC50 = 20.81 lg ml1).
An extract is considered to have high antioxidant activity based
on the comparison, under the same assay conditions, with natural
or synthetic antioxidant standards such as ascorbic acid
(IC50 = 5.85 lg ml1) or BHT (butylhydroxytoluene, IC50 = 19.4 lg ml1) (Mishra et al., 2012). As any of these compounds reect
the chemical composition of the HU extracts, four of the major
phenolics (compounds 1, 15, 16, and 20) identied on HU extracts
were assayed on the same conditions as the extracts to verify if
they were responsible for such activity. The results are also summarised in Table 3. The FRSA was more prominent in hyperoside
(15) followed by isoquercitrin (16), chlorogenic acid (1) and
quercitrin (20). All the four standards tested presented the FRSA
higher than all the HU extract in study, revealing that these

Table 3
Free radical scavenging activity (IC50), reducing power and total phenolic content of water extracts of H. undulatum (HU) WG (wild growing) and CG (culture growing) (referred as
HU1 to HU12) samples. Comparison of the antioxidant activities with pure standards.
Plant material and standards

DPPH IC50 (lg ml1)


(Condence Limits)

Reducing power (lg ml1)


(Condence Limits)

Total phenolic content


(mg GAE/g dry extract)a

HU1
HU2
HU3
HU4
HU5
HU6
HU7
HU8
HU9
HU10
HU11
HU12
WG HU
Chlorogenic acid
Hyperoside
Isoquercitrin
Quercitrin

26.13
20.92
22.86
26.58
29.60
25.33
28.25
24.98
26.48
n.d.
19.35
19.61
20.81
3.90
2.77
3.10
6.10

66.66
69.38
68.30
72.96
70.11
74.19
59.45
68.19
64.44
69.55
73.42
73.68
53.84
9.70
12.31
18.43
15.82

119.90 6.86
148.81 6.02
159.10 8.86
146.69 12.23
140.97 2.14
138.20 9.99
151.43 11.76
161.06 0.98
147.34 4.49
147.18 4.71
145.71 5.57
142.93 3.06
191.77 6.87

(23.0129.96)
(17.7324.49)
(19.5326.85)
(23.0230.80)
(25.6034.81)
(21.9729.32)
(24.5332.71)
(21.3629.42)
(24.1929.17)
n.d.
(16.4223.06)
(17.1422.79)
(18.0324.01)
(3.404.60)
(2.413.21)
(2.713.69)
(5.407.15)

Data are mean SD, n = 3. n.d., not determined; GAE, gallic acid equivalents.
a
Values marked with different letters are signicantly different (P < 0.05).

(58.3271.94)
(63.7874.45)
(65.2171.32)
(69.6675.25)
(65.8874.97)
(70.8677.03)
(57.1863.29)
(65.2372.18)
(61.0267.38)
(66.1473.53)
(70.2676.31)
(68.4979.12)
(48.7155.89)
(8.2010.31)
(11.5212.98)
(17.5019.63)
(13.8517.96)

a
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
c

88

N. Rainha et al. / Phytochemistry 86 (2013) 8391

compounds are some of the responsible for the FRSA detected in


HU extracts. These results are comparable to those referred by Hernandez et al. (2010) in which the authors determined an IC50 of the
same order of magnitude for the reference compounds: (1)
3.9 lg ml1, (16) 1.3 lg ml1 and (20) 1.3 lg ml1. The same
authors determined an IC50 = 17.0 lg ml1 for HU WG samples.
The FRSA of HU WG and CG samples can be explained by the presence of the identied compounds. A Pearson correlation matrix between the FRSA and the chemical composition for all HU samples
reveal that chlorogenic acid (1) is the only substance in which
the quantitative variation is correlated with the FRSA activity
(R2 = 0.572, P < 0.05). However, as observed (1) is not the only compound present in the extract that produces a FRSA effect. There was
no correlation observed between the total phenolic content and
the FRSA, which is usually observed in this type of studies (Bocco
et al., 1998; Khknen et al., 1999).
2.4.2. Reducing Power
The reducing capacity of a compound may serve as a signicant
indicator of its potential antioxidant activity (Meir et al., 1995).
Table 3 shows the reducing power of the extracts obtained as
EC50 value (lg ml1) by interpolation from linear regression analysis of concentration vs absorbance at 700 nm. The reducing power
was dose-dependent for all samples analyzed. EC50 value varied
between 59.45 and 74.19 lg ml1 for HU CG water extracts.
According to the results presented in Table 3 the reducing power
effect is not comparable to that determined for HU WG water
extract (EC50 = 53.84 lg ml1). The reducing power effect was also
determined for compounds (1), (15), (16), and (20). The results
(Table 3) show that chlorogenic acid (1) possesses the highest
reducing power effect followed by hyperoside (15), quercitrin
(20) and isoquercitrin (16). These reference compounds identied
in HU water extracts exhibit higher reducing power in comparison
to the extracts. The high amounts of (15) and (16) determined for
HU WG plants should justify the higher reducing power of this
sample. A correlation of the individual amount of the compounds
with the reducing power reveals that (15) and (16) are correlated
with the reducing power found for HU WG plants, but the quantitative variations of the compounds are not correlated with the HU
CG reducing power activity. In the latter case the combined activity
of several compounds should reect the determined activity. The
reducing power activity was correlated (R2 = 0.641, P < 0.05) with
the total phenolic content.
3. Conclusions
In this research study, LCMS and LCUV techniques were used
to identify and quantify several phenolic acids and avonoids from
Hypericum undulatum CG plants in comparison to WG plants.
Quantitative differences were demonstrated in the WG plants
showing high levels of the avonols hyperoside, isoquercitrin and
quercetin. On the other hand the major avonol in HU CG plants
was quercitrin. Phenolic acids with quinic acid moieties were
shown to be present in similar amounts in both WG and CG samples. WG plants exhibit higher antioxidant activities (compared to
the majority of the CG plants) and higher total phenolic content.
These differences between HU WG and CG plants could be explained by the qualitative and quantitative differences in the phenolic composition.
In spite of the differences between CG and WG plants in terms
of their antioxidant potential and phytochemical content, HU CG
plants could be consider as an alternative for the standardization
of HU phytotherapeutic products. More research will be necessary
to improve the chemical composition of these shoot cultures and
assess their health benets.

4. Experimental
4.1. Plant material
Wild-growing (in vivo) plants of Hypericum undulatum Schousb.
ex Willd (HU) and shoot cultures (twelve samples, referred as HU1
to HU12) were supplied by a Portuguese medicinal and aromatic
plant company, MAPPROD Lda., which multiplied and kept them
growing in land plots located near Braga (Northern Portugal).
HU1 to HU12 correspond to 12 accessions (aseptic in vitro plants
growing together in each one of 12 culture asks) of the same
micropropagated clone of H. undulatum. Micropropagation started
from one only nodal stem segment of one plant from the same in
nature WG H. undulatum population used in this study. The clone
was kept in vitro over four years, through subcultures, with three
months interval, on MS (Murashige and Skoog, 1962) medium,
containing sucrose (2%) and devoid of growth regulators. The pH
of the medium was adjusted to 5.7 and it was solidied with
0.8% agar prior to autoclaving at 15 psi for 20 min at 121 C. Sterile
primary explants were incubated at 25 2 C under a photoperiod
16 h light/8 h dark. Illumination was supplied by cool white uorescent tubes with a light intensity of 52 lmol m2s1. The present
culture conditions were reported by Guedes (2009) in which all the
plantlets grown and acclimatized in the described conditions survived, showing higher physiological development in comparison
to other different culture mediums. All HU1 to HU12 CG plant
accessions were harvested at the end of 85 days since the last subculture and dried in shady environmental conditions before processing for extraction. Voucher specimens were deposited at the
Department of Technological Sciences and Development, University of Azores (voucher numbers UAc-DCTD 165 and UAc-DCTD
166177, for HU and HU1 to HU12, respectively).
4.2. Chemicals
Butylated
hydroxyltoluene
(BHT),
2,2-diphenyl-1-picrylhydrazyl (DPPH), trichloroacetic acid, potassium ferricyanide,
ferric chloride, gallic acid, sodium carbonate, FolinCiocalteu reagent (2 N), the HPLC grade and LCMS grade solvents (methanol
and acetonitrile) and the standard compounds chlorogenic acid
(1), epicatechin (10), quercitrin (20) and quercetin (23) were obtained from SigmaAldrich Chemical Co. (St. Louis, MO, USA).
Hyperoside (15) was acquired from Extrasynthese (Lyon, France)
and isoquercitrin (16) was obtained from Fluka Chemicals (Buchs,
Switzerland). Ammonium acetate, sodium dihydrogen phosphate,
glacial acetic acid were purchased from E. Merck (Darmstad, Germany). Deionised water with 0.050 lS cm1 conductivity was obtained with an in-house Milli-Q water purication system
(Millipore, Molsheim, France).
4.3. Sample preparation
The infusions were prepared by boiling 200 mg of nely powdered dry plant material in 25 ml of water for 5 min with manual
agitation. After cooling for 10 min at room temperature, the extracts were ltered through a Whatman No. 1 lter, concentrated
under reduced pressure and then lyophilized. After quantication,
the residue was stored (under N2) in dark at 4 C until further analysis. The extraction procedure was performed as far as possible under daylight protection. Prior to HPLC analysis, 2 mg of each
sample was dissolved in 1 ml of deionised water using an ultrasonic bath for a few minutes. Then, they were submitted to centrifugation (10g for 15 min) and ltered through a type HA 0.45 lm
membrane lter (Millipore, Molsheim, France) before injected into
the HPLC system.

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N. Rainha et al. / Phytochemistry 86 (2013) 8391

4.4. HPLC analysis of phenolics compounds from H. undulatum water


extracts
4.4.1. Standard calibration curves
The reference standards, namely chlorogenic acid (1), epicatechin (10), hyperoside (15), isoquercitrin (16), quercitrin (20) and
quercetin (23), were accurately weighed and dissolved in water
or methanol using a mixer when necessary, to facilitate the dissolution. These stock solutions were kept at 46 C and brought to
room temperature before use. A series of working standard solutions of six additional calibration levels were prepared by serial
dilution of the stock solutions with water or methanol, in appropriate quantities, to fulll the requirements for the construction of the
calibration standard curves. The curves were constructed using the
external standard method by plotting the peak-area of each standard versus the concentration. Each standard solution was injected
in triplicate and averaged. Table 4 shows the calibration data acquired for each standard compound.
4.4.2. HPLCUV conditions for the quantication and identication of
the major phenolic compounds
The HPLC system consisted of a Waters 626 pump and Waters
600S controller coupled to a Waters 486 Tunable Absorbance Detector. The detection was performed at 270 nm for all HU phenolic
components. The analysis was carried on a Phenomenex Synergi
MAX-RP C12 column (80 , 150  4.6 mm i.d., 4 lm) which was protected by Security Guard Cartridge, and maintained at a constant
temperature of 40 C. The mobile phase consisted of: eluent A
10 mM ammonium acetate buffer equilibrated to pH 4.0 with glacial
acetic acid, eluent B MeCN:MeOH:H2O (3:1:1 v/v/v), and eluent C
methanol. A multilinear gradient was applied from 87:13:0
(A:B:C) to 70:30:0 in 25 min, then changed to 40:2:58 in 10 min, nally changed to 2:2:96 in 15 min, and held at this composition for
more 10 min. The total run time was 60 min and the equilibration
time between runs was 15 min. The ow rate was kept at
0.7 ml min1, and the injection volume was 20 ll. The average of
triplicate measurements was used to calculate the amount of each
compound. The chromatographic data of the quantitative determination was achieved by the external standard method using the
PeakSimple Chromatography Software version 3.93 for Windows.
Peak identity of the individual peaks was assigned by retention
time (RT) values, based on comparison with the standards, spike of
authentic standard to the sample and also conrmed by chromatographic pattern, superimposing the spectrum of each peak with the
corresponding standard spectrum. Liquid chromatography coupled
to electrospray ionization mass spectrometry was also used to conrm the identity of several compounds.
4.4.3. Liquid chromatographymass spectrometry (LCMS) analysis
An HPLC system (Surveyor, ThermoFinnigan) equipped with a
diode array detector set at k = 280 nm operated by the XCalibur

software was used. The HPLC separation was performed at a ow


rate of 0.2 ml min1 on a reverse phase column (Synergi Max-RP
80 , 150  2.0 mm i.d., 4.0 lm; Phenomenex) with a security
guard column (4  2.0 mm; Phenomenex) heated at 40 C using
gradient elution (eluent A 10 mM ammonium acetate pH 4, eluent B MeCN:MeOH:H2O, 3:1:1 v/v/v). Eight lL of the sample extract was injected into the LC system and separated according to
the following program: 025 min (9680% A), 2535 min (80
60% A), 3550 min (602% A), 5055 (2% A) and 5566 (96% A).
Acquisition of MS data was performed with a linear ion trap
mass spectrometer (LTQ, ThermoFinnigan) equipped with an electrospray ionization (ESI) source operated in both ion modes, positive and negative. MS data were acquired under optimized ESI-MS
conditions (source = 5 kV and 3,5 kV respectively; capillary voltage = 25 V and 49 V respectively; tube lens offset = 75 V and
97 V respectively; capillary temperature = 300 C) in full scan
mode (m/z 502000, scan time = 3 micro scans and maximum inject time = 50 ms) in the form of TIC (Total Ion Current) chromatogram. Fragmentation was done by collision induced dissociation
using helium as a collision gas, normalized collision energy = 30%,
activation time = 30 ms and radio frequency (activation Q control) = 0.250. Nitrogen was used as sheath and auxiliary gas with
ow rates of 50 and 20 arbitrary units, respectively. Compound
identication was assigned by RT values, m/z value of the molecular ion and the fragmentation pattern, when available.
4.5. Determination of total phenolics content (TPC) in H. undulatum
water extracts
TPC in plant extracts was determined by using FolinCiocalteu
colorimetric methodology according to Waterhouse (2001). Determinations were carried out in triplicate averaged and calculated
from a calibration standard curve of gallic acid. An aliquot of
20 lL of each sample solution (2 mg ml1) and each calibration
solution or blank was pipetted into separated cuvettes and
1.58 ml water plus 100 lL of 2 N FolinCiocalteu reagent were
added and well mixed. Additionally, 300 lL of 20% (w/v) Na2CO3
were added within 30 s to 8 min interval and shake to mix. The
sample was incubated for 2 h at room temperature. The absorbance was measured at 765 nm and was plotted vs concentration
using methanol as a blank. TPC in plant extracts was expressed
in gallic acid equivalents (GAE).
4.6. Determination of antioxidant activity in H. undulatum water
extracts
4.6.1. DPPH free radical scavenging activity (FRSA) assay
Antioxidant activity was assayed by the DPPH radical scavenging assay according to Blois (1958). Serial dilutions of plant extracts dissolved in methanol or reference compounds chlorogenic
acid, hyperoside, isoquercitrin and quercitrin were carried out in

Table 4
Calibration data for the standard compounds found in H. undulatum water extracts, including regression equation, linearity range, average retention time and relative standard
deviationa (SD).
Compound

y = ax + bb

Regression coefcient (R2)

Linearity range (lg ml1)

Average RT (min)

RSD of RT (%)

0.9995
0.9993
0.9993
0.9996
0.9987
0.9973

3.930125.750
1.563306.000
1.09449.500
1.18847.000
1.75040.000
3.37550.000

3.779 0.042
10.669 0.109
19.782 0.239
20.492 0.295
24.575 0.340
37.055 0.309

1.11
1.02
1.21
1.44
1.38
0.83

Slope a
Chlorogenic acid (1)
Epicatechin (10)
Hyperoside (15)
Isoquercitrin (16)
Quercitrin (20)
Quercetin (23)

28.262
15.693
45.380
49.286
57.812
44.523

The P value was <0.001 for all calibration curves.


a
The HPLC analytical conditions as referred in Section 4.4.2. RT, retention time (mean of triplicate samples); RSD, relative standard deviation.
b
For each curve the equation is y = ax + b, where y is the peak area, x the concentration of the analyte (lg ml1), and the value of the intercept b for all the analytes was zero.

90

N. Rainha et al. / Phytochemistry 86 (2013) 8391

96-well microplates, added to a methanolic DPPH solution and the


absorbance at 517 nm was measured after 30 min in the dark. In
each assay, a control was prepared, in which the sample or standard was substituted by the same amount of solvent. Percentage
of antioxidant activity (% AA) was calculated as:

% AA 1001  Acontrol  Asample =Acontrol 

where Acontrol is the absorbance of the control and Asample is the


absorbance of the extract or standard. All assays were carried out
in triplicate and results expressed as IC50 values (50% inhibitory
concentration).
4.6.2. Reducing power assay
The reducing power effect of the extracts was determined
according to the method of Oyaizu (1986). Each extract (concentration range 31.25500 lg ml1) in water (0.4 ml) was mixed with
0.4 ml of 300 mM of phosphate buffer (pH 6.6) and 0.4 ml of potassium ferricyanide (1% w/v), in a 2 ml eppendorf, and the mixture
was incubated at 50 C for 20 min. After 0.4 ml of trichloroacetic
acid (10% w/v) was added, the mixture was centrifuged at 10g for
5 min. The upper layer (1 ml) was mixed with 1 ml of deionised
water and 0.2 ml of FeCl3 (0.1% w/v), and the absorbance was measured at 700 nm against a blank. The blank solution containing
water instead of plant extract solution was used. A higher absorbance indicates a higher reducing power. EC50 value (lg extract/
ml) is the effective concentration at which the absorbance was
0.5 for reducing power and was obtained by interpolation from linear regression analysis. Each set of points were repeated three
times. Chlorogenic acid (1), hyperoside (15), isoquercitrin (16)
and quercitrin (20) were used for comparison.
4.7. Statistical analysis
Data of the antioxidant and total phenolic assays are expressed
as the means standard deviation (SD) of three independent measurements. Statistical analysis of the calibration curves of the reference compounds (1, 10, 15, 16, 20 and 23) was acquired by
Statistical Package for the Social Sciences (SPSS) software package
for Windows, version 17.0. Pearson correlation was performed between the results to verify possible correlations between the measurements. Mean differences were achieved by Tukey test.
Acknowledgments
This study was nancially supported by funds from Science and
Technology Foundation (FCT-Portugal) project PTDC/AGR-AAM/
70418/2006 (Hypericum Biotech), through grant # PEst-OE/EQB/
LA0004/2011 and through the National Re-equipment Program
for Rede Nacional de Espectrometria de Massa - RNEM (REDE/
1504/RNEM/2005).
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