Food Chemistry 131 (2012) 318327

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Direct characterisation of phenolic antioxidants in infusions from four

Mapuche medicinal plants by liquid chromatography with diode array
detection (HPLC-DAD) and electrospray ionisation tandem mass spectrometry
(HPLC-ESIMS)
Mario J. Simirgiotis a,b, Mario Silva c, Jos Becerra c, Guillermo Schmeda-Hirschmann d,
a

Departamento de Ciencias y Tecnologa Vegetal, Universidad de Concepcin, Campus Los ngeles, J. A. Coloma 201, Los ngeles, Chile
Laboratorio de Productos Naturales, Departamento de Qumica, Facultad de Ciencias Bsicas, Universidad de Antofagasta, Camino a Coloso S/N, Antofagasta 02800, Chile
c
Departamento de Botnica. Facultad de Ciencias Naturales y Oceanogrcas. Universidad de Concepcin, Casilla 160, Concepcin, Chile
d
Laboratorio de Qumica de Productos Naturales, Instituto de Qumica de Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chile
b

a r t i c l e

i n f o

Article history:
Received 22 July 2010
Received in revised form 3 March 2011
Accepted 31 July 2011
Available online 9 August 2011
Keywords:
Adesmia emarginata
Escallonia illinita
Linum chamissonis
Quinchamalium chilensis
Mapuche medicinal plants
Antioxidant
Phenolics
High performance liquid chromatography
Electrospray ionisationmass spectrometry

a b s t r a c t
A simple, fast and direct method was developed for the qualitative analysis of phenolic constituents from
infusions of Mapuche medicinal plants. Teas made of Linum chamissonis Schiede, Quinchamalium chilensis
Mol., Adesmia emarginata Clos. and Escallonia illinita K. Presl. were analysed by high-performance liquid
chromatography with diode array detector (HPLC-DAD) and electrospray mass spectrometry (ESIMS).
This technique allowed for the rst time the tentative identication of 16 phenolic compounds in E. illinita,
27 in Q. chilensis, 10 in L. chamissonis and 19 in A. emarginata. The compounds were mainly phenolic acids,
avonoid glycosides, anthocyanins and tannins. The total phenolic and avonoid content of the infusions
was assessed as well as the free radical scavenging capacity measured by the bleaching of a solution of the
2,2-diphenyl-1-picrylhydrazyl (DPPH ) radical. From the four species, Q. chilensis exhibited the strongest
antioxidant activity with highest total phenolic and avonoid content.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Endemic plants have played an important role in the life of local
people and amerindians in the southern part of South America.
Indeed, Mapuche communities have maintained a long standing
plant gathering tradition which has provided food and medicine
to these indigenous and local populations particularly in Southern
Chile and Argentina (Ladio, Lozada, & Weigandt, 2007). Chemical
composition analysis of native South American plants is very
important for the exploitation of local species in food industry as
well as for the further development of medicinal plants as crop
plants. In the last decades the presence of phenolic compounds in
fruits, legumes and medicinal plants has been suggested to be
responsible for many human health benets and stimulated the
development of methods and application of several analytical techniques for their identication (Robards, 2003). Indeed, high performance liquid chromatography with UVVis diode array detection
(HPLC-DAD) (Chen, Zuo, & Deng, 2001), gasliquid chromatography
Corresponding author. Tel.: +56 71 200288/201573.
E-mail address: schmeda@utalca.cl (G. Schmeda-Hirschmann).
0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.07.118

with ame ionisation (GC-FID) chromatography coupled with mass

spectrometry (GCMS) (Chen & Zuo, 2007; Vallejo, TomsBarbern, & Ferreres, 2004) and capillary electrophoresis coupled
to electrospray ionisation time-of-ight mass spectrometry
(CEESI-TOF-MS) (Gmez-Caravaca, Verardo, Segura-Carretero,
Caboni, & Fernndez-Gutirrez, 2008) were used to detect, characterise and quantify phenolic compounds present in different food
and medicinal plants. Particularly, HPLC methods hyphenated with
mass spectrometric ionisation at atmospheric pressure (API) like
electrospray ionisation (ESI) enable direct, fast and easy analysis
of complex polar mixtures in liquid solutions by mass spectrometry, since different structures, sizes and types of polar molecules
such as phenolic acids, branched avonoid glycosides and tannins
can be separated by HPLC and ionised by ESI for MS investigation.
Within this scenario we have developed screening methods using
LC/DAD and ESI/MS for the identication of phenolics in Chilean
medicinal and food plants. So far the native edible white strawberry
Fragaria chiloensis (Simirgiotis, Theoduloz, Caligari, & SchmedaHirschmann, 2009) the highland papaya Vasconcellea pubescens
(Simirgiotis, Caligari, & Schmeda-Hirschmann, 2009) and the
worlwide known medicinal plant Peumus boldus (Simirgiotis &

M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

Schmeda-Hirschmann, 2010) were analysed, using this fast, direct

and precise analytical technique.
Following our studies on antioxidant phenolic compounds in
South American plants, we have selected four medicinal plants,
widely used in southern Chile in the treatment of hepatobiliary
ailments, as anti-inammatory and to relieve gastrointestinal
disorders (Montes & Wilkomirsky, 1985).
The plant infusions investigated comprises some of the most
commonly used crude drugs from the former Mapuche culture.
According to Murillo (1889), Escallonia illinita (Saxifragaceae) was
recommended to treat liver diseases while quinchamali (Quinchamalium chilensis) (Santalacae) was reported to be used in decoction
of the plant sweetened with bee honey as an effective treatment for
liver abscesses as well as an anti-inammatory crude drug. The
preparation was to be taken early in the morning. Houghton and
Manby (1985) report the use of an infusion of the aerial parts
(leaves and stem) of E. illinita K. Presl. to relieve liver diseases, rheumatism and kidney pain. Linum chamissonis Schiede (Linaceae),
known under the trivial name retamilla in northern Chile or
nanco or nanco-lahuen (eagles herb) in the southern part of
the country, was used as a digestive and febrifuge (Murillo, 1889).
Quinchamali and nanco are bitter tasting crude drugs and as such,
regarded as having effect as digestive and to treat liver diseases.
Msbach (1991) describe the use of quinchamal (Quinchamalium ericoides, Quinchamalium majus and Quinchamalium gracile) in
decoction to treat colds and ue and as blood depuratives. As the
taxonomy of the species seems to need revision, the crude drug
probably include several close related species that have the same
or similar taste and properties. The same author describes E. illinita
as a very unpleasant smelling shrub and no information is given on
a possible use. As the study of Msbach is posterior to the recopilation of Murillo, the use of this species probably diminished in the
last decades. L. chamissonis is regarded as an effective remedy
against dyspepsia and as a digestive (Msbach, 1991). Montes
and Wilkomirsky (1985) report the use of E. illinita leaves to treat
liver complaints, L. chamissonis aerial parts as a laxative and carminative and infusions or decoctions of dry leaves of Adesmia emarginata as aphrodisiac.
Since no studies were reported on the online qualitative analysis of these important medicinal Mapuche plants using HPLCDADMS/MS, the main objective of this study was to compare
the phenolic and avonoid content in infusions of the four species
and provide HPLC ngerprints for fast and direct analysis of the
crude drugs. Free radical scavenging effect was assessed in order
to relate the phenolic contents with this activity.
2. Materials and methods
2.1. Plant material
Dried aerial parts, including owers of L. chamissonis Schiede
(local name: anco) collected in Mulchn-Collipulli, A. emarginata Clos. (local name: paramela) and Quinchamalium chilense Mol.
(local name: quinchamali) collected at the slopes of Volcn Antuco as well as E. illinita K. Presl (local name: barraco) gathered at
the Laguna del Laja in January 2008, were purchased at the local
market of Los Angeles, VIII Region, Bio-Bio Province, Chile. Voucher
specimens are deposited at the Herbarium of the Department of
Sciences and Plant Technology, Concepcin University, Los Angeles,
Chile with the numbers 206, 207, 209 and 210, respectively.
2.2. Chemicals
FolinCiocalteu phenol reagent, 1,1-diphenyl-2-picrylhydrazyl
radical, AlCl3, NaNO2, NaOH, gallic acid and quercetin, were

319

purchased from Sigma Chemical Co. (St. Louis, MO, USA). HPLCgrade acetonitrile, MeOH, and HPLC-grade water, were obtained
from J.T. Baker (Phillipsburg, NJ, USA), and formic acid from Merck
(Darmstadt, Germany). The purity of the chemicals used was as
follows: AlCl3 p.a. P 99%, NaOH reagent grade P 98%, NaNO2
ACS reagent P97.0%, gallic acid (purity P99%), quercetin (purity
P97%), FolinCiocalteu phenol reagent (2 M, respect to acid,
Sigma), 1,1-diphenyl-2-picrylhydrazyl radical (purity = 95%
SigmaAldrich), and formic acid (P 99%).
2.3. Instrumentation
The HPLC system used for DAD analysis of extracts was an HPLC
system consisting of a Shimadzu LC-10AD (Kyoto, Japan) equipment with a Shimadzu SPD-M10A VP diode array detector with
an SCL-10A VP control system. Shimadzu LC-Solution software
was used for graphic visualisation and data acquisition. The HPLC
was equipped with a 250  4.60 mm i.d., 5 lm C18-RP Luna column (Phenomenex, Torrance, CA, USA) maintained at 25 C. The
compounds were monitored at 280 nm, and UV spectra from 200
to 600 nm were recorded for peak characterisation. For anthocyanins, monitoring was at 520 nm. The HPLC analysis were performed using a linear gradient solvent system consisting of 1%
aqueous formic acid (A) and acetonitrile (B) as follows: 9075% A
over 30 min; followed by 7540% A from 30 to 45 min at a ow
rate of 1 mL/min. The injected volume was 20 lL. LCMSMS
was conducted using the same column with an Agilent 1100 HPLC
system connected through a split to an Esquire 4000 Ion Trap LC/
MS System (Bruker Daltoniks, Germany). An UV-160 Shimadzu
spectrophotometer was used for DPPH, total phenol (TP) and total
avonoid (TF) assays.
2.4. ESI mass spectrometric conditions
Full scan ESI mass spectra were measured between m/z 150 and
2000 U in negative ion mode for all compounds. Nitrogen was used
as nebuliser gas at 172.36 kPa, 350 C and at a ow rate of 8 L/min.
The mass spectrometric conditions were as follows: electrospray
needle, 4000 V; end plate offset, 500 V; skimmer 1, 56.0 V;
skimmer 2, 6.0 V capillary exit offset, 84.6 V; collision induced
dissociation (CID) spectra were obtained with a fragmentation
amplitude of 1.00 V (ms/ms) using helium as the collision gas.
2.5. Extraction and sample preparation
The sample solutions for HPLC comparison purposes were prepared as previously reported (Simirgiotis et al., 2009), with some
modications. The aerial parts were air-dried and ground in a mill.
Infusions were immediately prepared by adding 5 g of powdered
aerial parts (corresponding to about 2 tea bags) to 250 mL (1 cup)
of hot (90 C) deionised water (Milli-Q) and left to stand for 5 min,
ltered through a 0.45 lm PTFE lter (Waters) and the solution
loaded onto an reverse phase solid phase extraction (SPE) column
(C-18 Strata, Phenomenex, CA). The volume of SPE column was 6 mL.
The column was rinsed with 25 mL distilled water and eluted with
25 mL MeOHformic acid (99:1, v/v). The eluate was evaporated to
dryness under reduced pressure below 40 C to afford the extracts
as follows: Q. chilensis (205.2 mg), E. illinita (219.3 mg), A. emarginata
(158.5 mg) and L. chamissonis (120.2 mg), respectively.
2.6. HPLC analysis
A portion of the extracts obtained as explained above was dissolved in MeOH: formic acid, (99:1 v/v) to prepare standard solutions of 1 mg per mL, ltered through a 0.45 lm lter and
directly submitted to DAD and HPLCMS analysis.

320

M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

2.7. Total phenolic and total avonoid content

The total phenolic content (TPs) was determined by the Folin
Ciocalteu method as previously described (Simirgiotis et al.,
2009; Simirgiotis, Theoduloz, et al., 2009). All samples and gallic
acid were dissolved in 50% (v/v) aqueous methanol. Samples
(50 lL) were placed into test tubes and 250 lL FolinCiocalteu
reagent were added. The mixture was left to stand for 5 min, and
750 lL of 20% sodium carbonate (20%, v/v, in water) and 5 mL distilled water was added. After 30 min of incubation at room temperature (20 C) the resulting absorbance was measured at 765 nm.
The calibration curve was performed with gallic acid (concentrations ranging from 31.3 to 500.0 lg/mL) and the results were
expressed as mg of gallic acid equivalents per 100 g of dry plant
material. The total avonoid (TF) content of the extracts was determined as follows. Briey, 250 lL of the sample (1 mg of the dry
extract per mL) was diluted with 1.25 mL of water. Then 75 lL of
5% NaNO2 solution were added to the mixture. After 5 min,
150 lL of 10% AlCl3 6H2O were added and the mixture was allowed
to stand for 5 min. Then 500 lL of 1 M NaOH solution and 275 lL
of distilled water were added to make a total of 2.5 mL. The absorbance was measured immediately against the prepared blank at
510 nm. The results were expressed as mg quercetin equivalents
per 100 g of dry material.
2.8. Scavenging of DPPH radicals
The scavenging of DPPH radicals was assayed as previously
reported with some modications (Simirgiotis, Theoduloz, et al.,
2009). All extracts were dissolved in 50% (v/v) aqueous methanol
to prepare stock solutions of 1 mg/mL. These stock solutions were
serially diluted with methanol, mixed with an equal volume of
DPPH solution (60 lM) and shaken vigorously. The mixture was
incubated at room temperature for 30 min before the absorbance
at 517 nm was read. Solutions of quercetin were used as a positive
control. The scavenging activity was determined by comparing the
absorbance with that of the blank (100%) that contained only DPPH
and solvent. Antiradical DPPH- bleaching activity is expressed as
IC50 in lg/mL which denoted the concentration of sample required
to scavenge 50% of DPPH free radicals. The lower the IC50 value, the
higher the antiradical activity. Values lower than 50 lg/mL are
considered high, while values between 50 and 100 lg/mL are considered moderate.
3. Results and discussion
3.1. Phenolic and avonoid content and free radical scavenging
activity of the extracts
In this study, all extracts presented free radical scavenging
effect, measured by the bleaching of the DPPH free radical
(Table 1). HPLC qualitative analysis (Table 2, Fig. 1) showed 16
main phenolic compounds in E. illinita, 27 in Q. chilensis, 10 in L.

chamissonis and 19 in A. emarginata. Q. chilensis presented the highest TP and TF values (1591.5 mg gallic acid and 982.2 mg quercetin/100 g dry weight, respectively) followed by
E. illinita
(554.1 mg gallic acid and 200.5 mg quercetin/100 g dry weight,
respectively). Three out of the four extracts presented good antioxidant activity, including Q. chilensis, A. emarginata and E. illinita
infusions with IC50 values of 32.2, 37.9 and 43.2 lg/mL, respectively. Under our assays conditions, the IC50 of quercetin was
29.3 lg/mL. The effects of the three extracts was similar to that
of the reference compound and suggest a relevant antioxidant
effect of the infusions.
3.2. HPLC with DAD and ESIMS/MS analysis
The phenolic constituents of four Andean medicinal plant infusions were analysed by HPLC/DAD and electrospray ion trap tandem mass spectrometry (HPLC/ESIMSMS, Table 2). Those
compounds contained in the infusions were separated on a Luna
C18 column and identied or tentatively characterised based on
UV spectra and MS fragmentation behavior in comparison with
previously published data (Fig. 1 and Table 2). Phenolic compounds
show different UV absorption characteristics. Flavonols and their
glycosides present two absorptions maxima at 330370 nm and
250270 nm, due to their B-ring (cinnamoyl structure) and A-ring
(benzoyl structure), respectively. The most common glycosylation
pattern observed in plant avonols is at the 3-O or 7-O position.
In this study, the band I appears at about 355 nm and its intensity
is somewhat lower than that of band II (250270 nm), which characterise most of them as avonol-3-O-glycosides. When ring B has
two free OH as in quercetin derivatives, band II appears at 255 nm,
and if ring B has only one free OH, the band I present a maximum
at 265 nm, as for kaempferol derivatives. Flavan-3-ols are readily
distinguished from other avonoids because they typically exhibit
only one intense band (band II at 270280 nm). Caffeoylquinic
acids show absorption maxima at 310330 nm and a weaker band
at 240 nm, while anthocyanidins absorb at 520 and 505 nm for
cyanidin and pelargonidin glucoside, respectively.
In this study negative and positive modes of ESI mass detection
were employed. However, in positive mode only small [M + H]+
ions and some product ions were observed in MSMS experiments,
excepting the anthocyanidin compounds detected in Q. chilensis. In
the negative mode, most of the [M H] ions were abundant
enough to be subjected to tandem mass analysis and provided better structural information. In total, 71 compounds were detected
and 64 compounds were tentatively characterised or identied,
including avonoids, phenolic acids, anthocyanidins and quinic
acid derivatives. Peaks 9 and 14 occurring in Q. chilensis are the
only anthocyanidins found and were identied as cyanidin and
pelargonidin glucose, respectively (Simirgiotis, Theoduloz, et al.,
2009).
Peaks 7, 1012, 27 and 30 were identied as chlorogenic acid
derivatives, while peaks 15, 16, 21, 45, 47, 52, 58, 60 and 70 are
quinic acid conjugates. Among the compounds with a quinic acid

Table 1
Total phenolic content (TP), total avonoid content (TF), scavenging of the free radical DPPH and percent w/w extraction yield of water infusions of Mapuche medicinal plants on
the basis of dry starting material.
Species and common name

TPa

TFa

DPPHa

w/w extraction yield (%)

Adesmia emarginata (paramela)

Escallonia illinita (barraco)
Linum chamissonis (anco)
Quinchamalium chilensis (quinchamal)
Quercetin

190.1 0.4
554.1 6.8
101.0 2.7
1591.5 9.1

18.1 0.4
200.5 0.8
46.5 0.2
982 11

37.9 0.6
43.7 1.5
73.8 3.1
32.2 0.3
29.3 2.3

6.34
8.77
4.83
8.24

a
All measurements are expressed as mean S.D. (n = 3). TP expressed as mg gallic acid equivalents/100 g dry weight. TF expressed as mg quercetin equivalents/100 g dry
weight. Antiradical DPPH bleaching activity is expressed as IC50 in lg/mL. Yield expressed as g/100 g dry weight.

M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

321

Table 2
Tentative identication of phenolic compounds in Mapuche medicinal plant infusions by LC-DAD, LCMS and MS/MS data.
Peak

Rt (min)

kmax (nm)

[M H]

MS/MS ions

Tentative identication

Species

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71

2.0
2.4
2.5
3.0
5.0
5.5
6.5
7.0
7.5
7.2
7.5
7.8
8.0
8.5
9.3
9.5
10.0
9.0
11.0
10.5
11.5
10.5
11.2
11.0
12.0
13.0
12.5
12.8
12.0
13.5
14.8
13.5
14.8
15.0
14.8
15.0
15.0
15.3
16.4
17.0
16.0
17.0
17.2
18.0
19.0
19.8
20.4
21.3
21.5
22.2
22.3
23.0
23.2
23.4
23.6
23.8
24.0
24.8
25.5
26.2
26.8
27.2
27.5
28.3
28.3
28.6
28.8
29.0
31.8
32.1
33.8

272
364, 264
365, 266
322, 244
355, 255
332, 295sh, 240
355, 255
520, 265
332, 295sh, 240
332, 295sh, 240
328, 295sh, 240
364, 295sh, 262
505, 264
305
326, 295sh, 240
355, 255
320sh, 284
280
326, 295sh, 240
328, 295sh, 240
355, 255
279
355, 255
279
355, 253
332, 295sh, 242
364, 264
364, 320sh 264
332, 295sh, 240
354, 254
364, 260
364, 264
355, 254
279
280
355, 255
355, 254
355, 254
356, 300sh, 267

665
331
901
913
537
589
353
589
449
551
551
551
593
433
367
609
629
579
745
291
515
593
561
839
561
651
551
577
739
551
713
739
577
565
745
273
609
417
463
585
401
463
623
549
515
461
515
971
567
565
433
777
567
565
565
689
285
689
735
763
719
591
723
721
595
471
939
709
599
369
571

605,
271,
739,
621,
353,
439,
191
439,
287
389,
389,
389,
561,
271
343,
577,
373,
417,
569,
247,
483,
419,
435,
593,
543,
609,
389,
533,
593,
515,
521,
593,
457,
433,
535,
217,
301,
301,
301,
453,
255,
301,
477,
417,
483,
285
483,
941,
403,
547,
300,
565,
433,
433,
433,
561,
239,
561,
589,
719,
301
283,
693,
621,
287,
387,
909,
611,
255,
311,
283,

Hydroxyl-piperitol-demethoxy-dihexoside
Monogalloyl glucose
K-di-rutinoside
K-hexosyl-rhamnosyl conjugate
Salvianolic acid H
Unknown Q-derivative
CA
Unknown Q-derivative
Cyanidin glucoside
CA-isomer conjugate
CA-isomer conjugate
CA-isomer conjugate
K-coumaroyl-hexoside
Pelargonidin glucoside
Feruloyl-quinic acid
Caffeoyl-quinic acid derivative
Q-sorbyl-hexoside
Eriodictyol 7-O-neohesperidoside
Gallocatechin-catechin gallate or isomer
Unknown quinic acid derivative
Di-caffeoyl-quinic acid
Q-deoxyhexosyl-deoxyhexoside
Afzelechincatechin dimer
Q-rhamnosyl-rhamnoside derivative
Afzelechincatechin dimer
Q-acetyl-rutinoside
CA-isomer conjugate
K-di-rhamnoside
K-di-rhamnosyl-hexoside
CA-isomer conjugate
Unknown Q derivative
K-di-rhamnosyl-hexoside
Isorhoifolin (Apigenin hexosyl-rhamnoside)
Q-pentosyl-pentoside
Gallocatechin catechin gallate or isomer
Afzelechin
Rutin
Q-derivative
Q-hexoside
Q-galloyl pentoside
Ferulic acid glucoside
Q-hexoside (Hyperoside)
Isorhamnetin glucosyl-rhamnoside
K-di-pentoside
Di- caffeoyl-quinic acid
K-glucuronide
Di caffeoyl quinic acid
Unknown K-derivative
K-hydroxy-benzoyl-hexoside isomer
Q-pentosyl-pentoside
Q-pentoside
Tri-caffeoyl succinoyl quinic acid
K-hydroxy-benzoyl-hexoside isomer
Q-pentosyl-pentoside
Q-pentosyl-pentoside
Unknown
K
Unknown caffeoyl derivative
Unknown Q-conjugate
1,4,5-tri-caffeoyl-3-methoxy-oxaloyl-quinic acid
Unknown Q-derivative
Acacetin-rutinoside
Sinapoyl-feruloylgentiobioside
Q-di-hexoside
Naringenin-7-O-rutinoside
Methyl-epigallocatechin gallate
Pentagalloyl glucose ester
Unknown
Unknown Q-conjugate
Trihydroxy-cinnamoyl-quinic acid
Unknown Q-conjugate

E.i.
Q.c.
A.e.
Q.c.
E.i.
Q.c.
E.i.
Q.c.
Q.c.
E.i.
E.i.
E.i.
Q.c.
Q.c.
E.i.
Q.c.
E.i.
A.e.
E.i.
Q.c.
E.i.
A.e.
Q.c.
A.e.
Q.c.
E.i.
L.c.
Q.c.
A.e.
E.i.
E.i.
A.e.
Q.c.
E.i.
L.c.
Q.c.
A.e.
A.e.
Q.c.
E.i.
A.e.
Q.c.
E.i.
Q.c.
L.c.
A.e.
L.c.
A.e.
L.c.
L.c., Q.c.
A.e.
L.c.
Q.c.
L.c.
L.c.
Q.c.
A.e.
Q.c.
Q.c.
Q.c.
Q.c.
A.e.
Q.c.
Q.c.
A.e.
L.c.
A.e.
Q.c.
A.e.
A.e.
A.e.

354, 254
354, 254
364, 264
326, 295sh,
348, 266
328, 295sh,
364, 262
364, 300sh,
370, 271
355, 255
326, 295sh,
364, 300sh,
355, 255
355, 255
260
364, 262
320
354, 254
330, 300sh,
354, 254
364, 254
325
355, 255
327sh, 280
254, 354
272
255
355, 255
326, 295sh,
355, 255

240
240
266

240
266

242

240

575,
211,
593,
593,
191,
301

503, 341, 323

191, 109
285
285
165

301
195,
195,
195,
307,

163
163
163
285

335,
489,
343,
255,
277
191
353,
300,
273,
300,
435,
351,
195
407,
431,
351,
207
431,
337,
301,
373,
189
255,
255,
179,
301,
193,
300,
387,
285
353,

181, 109
353, 191
221, 161
137

195,
137
207
179,
407,
301,

135

151, 137
289, 273, 207
191, 135

285
285
301, 191, 135
285
271, 269, 155
207, 179, 151
343
179,
179,
151
287
119
271,
315,

151
151, 119

179, 151
207

191

353,
809,
253,
433,
239,
403,
313,
403,
301,
435
199,
435
301,
601

191
639,
208
403,
179,
191,
253,
301,
387,

268,
625,
301
253
311
867,
431,
179,
268,
255,

211
529, 435, 257

343, 285, 253

301, 179, 151
151
179
169
179, 151
207, 179, 151

119
179, 151

769, 617, 401

257
151
195, 191
179, 151

Compounds abbreviations: CA: Chlorogenic acid; K: Kaempferol; Q: Quercetin; Plant species abbreviations: A.e.: Adesmia emarginata (paramela); E.i.: Escallonia illinita
(barraco); L.c.: Linum chamissonis (anco); Q.c.: Quinchamalium chilensis (quinchamal).

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M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

Fig. 1. HPLC-DAD chromatograms at 280 nm of Mapuche medicinal plants infusions. (A) Escallonia illinita, (B) Linum chamissonis, (C) Quinchamaliun chilensis (280 nm), (D)
Quinchamaliun chilensis (520 nm), and (E) Adesmia emarginata.

moiety, seven constituents (peaks 7, 1012, 15, 21 and 30) occurs

in E. illinita, three compounds (peaks 27, 45 and 47) in L. chamissonis, three in Q. chilensis (peaks 52, 58 and 60) and one in A. emarginata (peak 70). Peaks 19, 23, 25, 35, 36 and 66 were identied as
avanol derivatives. Peaks 3, 4, 13, 17, 18, 22, 24, 26, 28, 29, 3134,
3740, 42, 43, 44, 46, 4851, 5355, 57,59, 61, 62, 64 and 65 are
tentatively identied as avonol derivatives. Among them, peaks
6, 8, 17, 22, 24, 26, 34, 3740, 42, 50, 51, 54, 55, 59, 61, 64 and
69 are in agreement with quercetin derivatives. Peaks 3, 4, 13,
28, 29, 32, 44, 46, 49, 53 and 57 are kaempferol derivatives, peak
33 is tentatively assigned to apigenin rutinoside (or neohesperidoside), while peak 62 can be assigned as acacetin rutinoside and
peak 43 as isorhamnetin rutinoside. Peaks 18 and 65 are in agreement with avanones, peak 18 was tentatively identied as eriodictyol neohesperidoside and peak 65 as a naringenin rutinoside.
Peak 2 in Q. chilensis and peak 67 in A. emarginata were identied as monogalloyl and pentagalloyl glucose, respectively. Peaks 6,
8, 20, 31, 48, 56, 59, 61, 68, 69 and 71 remain unidentied. However, compounds 6, 8, 31, 48, 59, 61, 69 and 71 showed UV spectra
characteristic of avonols while the UV trace of peak 20 is characteristic of a chlorogenic acid. Selected ESI mass spectra and proposed structures of avonoids are presented in Fig 2a for
compounds 18, 26, 28, 32 and 34 and Fig 2b for compounds 33,
37, 39, 43 and 65, respectively. Additional full scan ESI mass spectra and proposed structures are showed in Fig 3a (compounds 2, 7,
9, 14 and 67) and Fig 3b (compounds 19, 25 and 36). More details
on the identication of all phenolic compounds are described
below.

3.3. Identication of phenolic compounds

Peak 1 showed an deprotonated molecular ion at m/z 665 and a
[M H 162] ion at m/z 503 corresponding to the loss of a dehydrated hexose. MS3 analysis of the ion at m/z 503 produced ions
at m/z 341 and m/z 323. These MS spectra are consistent with data
previously reported for demethoxy-hydroxyl-piperitol-O-diglucoside (Ye, Yan, & Guo, 2005). Peak 2 was identied as a monogalloyl
glucose ester (MW = 332). Peak 3 showed UV max consistent with
a kaempferol derivative, a [M H] ion at m/z 901 and daughter MS
ions at m/z 739 (loss of hexose) and 593 (loss of a rutinose moiety).
The ion at m/z 593 loses another rutinose to produce the kaempferol ion at m/z 285 which is consistent with literature [M 2 rutinose H] (Kite, Stoneham, & Veitch, 2007). Full mass scan analysis
of compound 4 showed a [M H] ion at m/z 913 and also product
MS ions at m/z 739 (loss of 174 U), 593 and 285 (kaempferol aglycon, loss of rutinose from the ion at m/z 593). A compound with
similar MS characteristics has been reported to be a glucosyldi-rhamnosyl kaempferol derivative with an additional unknown
sugar moiety (loss of 174 U). Full MS spectra for peak 5 showed
an deprotonated molecule at m/z 537 with MSMS ions at m/z
353, 191 and 165 and was identied as an caffeic acid oligomer
or depside with a MW of 538 U (salvianolic acid H) as previously
reported (Li, Song, Zheng, Liu, & Liu, 2008). Peaks 6 and 8 both with
prominent signals at m/z 589 in the full MS spectra and MS3 peaks
at m/z 301 were identied as quercetin derivatives (kmax 254 and
354, MS3 at m/z 179 and 151) conjugated with an unknown sugar
moiety. Peak 9 and 14 detected at 520 nm were present only in Q.

M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

323

Fig. 2. Full scan negative ESI mass spectra and proposed structures of avonoids identied with peaks 18, 26, 28, 32, 33, 34, 37, 39, 43 and 65, occurring in Mapuche
medicinal plant infusions. (a): Compounds 18, 26, 28, 32 and 34; (b): Compounds 33, 37, 39, 43 and 65.

chilensis (Fig. 1) and were identied as proanthocyanidin hexosides

of cyanidin and pelargonidin, respectively. Peak 13 with a [M H]
ion at m/z 593 and a MS ion at m/z 285 was identied as a kaempferolcoumaroylhexoside as previously reported (Simirgiotis, Theoduloz, et al., 2009). Peak 7 was identied as one of the known
chlorogenic acids, caffeoylquinic acid (molecular anion at m/z
353, MS2 at m/z 191 and 173 (191H2O) by comparison with
authentic sample, while peak 15 showed a signal at m/z 367 and
MS ions at m/z 343, 191, 181, and 109 and was tentatively identied as feruloyl-quinic acid. Peak 16 was identied as a caffeoylquinic acid derivative with a MW of 610 due to MS2 fragment at m/z
353 (chlorogenic acid) and 191. Peak 21 and 45 with a [M H]
at m/z 515 (MS fragments at m/z 353, 195, 173 and 135) were identied as above as 1,3,3,4- and 1,4-di-O-caffeoyl quinic acid isomers

(Lin & Harnly, 2008). Peak 1012, 27 and 30 were isomers of a

structure with a pseudomolecular ion of 551, which fragmented
to a base peak MS2 ion at m/z 389 (loss of hexose) and MS3 ions
at m/z 353 (chlorogenic acid) 195 and 163. Thus, the compounds
are suggested to be isomers of a chlorogenic acid derivative. Peak
18 with UV max at 284, 320 nm showed a negative molecular
ion at m/z 579 [M H] and MS fragments at m/z 459 [M H 120] ,
313 [M H 146 120] , and 271 [M H 308] . The compound
was tentatively identied as the avanone eriodictyol 7-O-neohesperidoside according to data previously published (Abad-Garca,
Berrueta, Garmn-Lobato, Gallo, & Vicente, 2009).
Peak 17 had a [M H] ion at m/z 627 in negative ion mode and
[M + H]+ ion of 629 U in positive ion mode, while the fragments m/z
153, 229 and 257 allowed the assignment of this compound as a

324

M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

Fig. 2 (continued)

quercetinsorbylhexoside as previously reported (Termentzi, Kefalas, & Kokkalou, 2008). Peak 22 with a [M H] ion at m/z 593 and
a pseudoquercetin MS2 ion at m/z 300 (593-di-sugar-H) was tentatively assigned as a quercetin deoxyhexosyl-deoxyhexoside. Peak
19 present in E. illinita and peak 35 present in L. chamissonis
showed very different retention times but the same deprotonated
molecule in their full MS spectrum (at m/z 745) and were identied
as isomers of an gallocatechin catechin gallate (Lin, Chen, & Harnly,
2008). Peak 26 had a molecular anion at m/z 651 and MS ions at m/
z 609 (quercetin rutinoside, loss of CH2CO moiety) and m/z 301
(quercetin ion, through loss of rutinose moiety). The compound
could be tentatively assigned as a quercetin acetyl rutinoside by
comparison with previously reported data (Djoukeng, Arbona,
Argamasilla, & Gomez-Cadenas, 2008). Peaks 23 and 25 detected
in Q. chilensis both showed a pseudomolecular ion at m/z 561
and were identied as tannin dimer isomers composed of afzelechin and catechin. In a similar way, peak 36 was identied in the

same species as the monomer afzelechin (Lin et al., 2008). Peak

24 showed a molecular anion at m/z 839 and was identied as a
quercetin glycoside derivative(kmax 254 and 354) containing a
deoxyhexosyl-deoxyhexoside moiety (MS2 peak at m/z 593, MS3
peaks at m/z 300, 179 and 151) while peaks 29 and 32 present in
the same species (A. emarginata) showed an [M H] ion at m/z
739 and MS ions at m/z 593, 431, 285 were identied as isomers
of a di-rhamnosyl-hexosyl kaempferol. Peak 28 showed MS characteristics of a kaempferol derivative with a MW of 578 and was tentatively assigned as a kaempferol di-rhamnoside (Ferreres et al.,
2008). Full scan MS spectrum of peak 31 showed a prominent peak
at m/z 713 U and MS2 peak at m/z 521, and 207 and remains as an
unknown avonoid (kmax 254 and 354). Peak 33 (kmax 364 and
264) showing a deprotonated molecule at m/z 577 afforded MS2
ions at m/z 271, 269 and 155 and was identied as apigenin
hexosyl-rhamnoside. The data matched with that of the apigenin
rutinoside isorhoifolin reported previously (Sanchez-Rabaneda

M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

325

Fig. 3. Full scan ESI mass spectra and proposed structures of chlorogenic acid (peak 7) anthocyanins (peaks 9 and 14, detected in positive mode), galloyl and pentagalloyl
glucose esters (peaks 2 and 67), and avanols (peaks 19, 25 and 36) identied in four Mapuche medicinal plant infusions. (a): Compounds 2, 7, 9, 14 and 67; (b): Compounds
19, 25 and 36.

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M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

et al., 2003). Peak 37 was identied as the quercetin rutinoside

rutin, (Simirgiotis et al., 2009) identity conrmed with spiking
experiments using authentic standard. Peak 38 with a pseudomolecular ion at m/z 417, UV spectrum (kmax 254 and 354) and MS
ions at m/z 301, 255, 179 and 151 was identied as a quercetin
derivative. Peaks 39 and 42 both showing a [M H] ion at m/z
463 (mainly MS2 ions at 301, 179 and 151) could be identied as
quercetin-hexoside isomers (Castillo-Muoz et al., 2009) while
peak 40 was identied as quercetin galloyl pentoside (Michodjehoun-Mestres et al., 2009). Peak 41 is in agreement with ferulic
acid hexoside. Peak 44 with a MW of 550 could be differentiated
from dipentosyl luteolin (Figueirinha, Antonio, Perez-Alonso,
Santos-Buelga, & Batista, 2008) by the shape of UV spectra and
MS fragmentations (MS2 at 417 and 285) in agreement with dipentosyl kaempferol. Peak 46 present in A. emarginata was identied
as kaempferol glucuronide (Mertz, Cheynier, Gnata, & Brat,
2007). Peak 52 showed characteristic pattern of a caffeoyl-quinic
acid derivative with MS2 ions at m/z 191 (quinic acid) and m/z
179 (caffeic acid) and was identied as a tricaffeoyl succinoyl quinic acid with a MW of 778 (Lin et al., 2008). Full MS spectra of
peaks 34, 50, 54 and 55 showed an [M H] ion at m/z 565. Further
fragmentation of the [M H] ion yielded ions at m/z: 433 (M-132,
loss of a pentosyl moiety), and 301 (433132, loss of a second
pentosyl moiety). The m/z 301 ion produced major ions at m/z
179 and 151 which matches the fragmentation pattern of quercetin. Thus, those compounds were identied as quercetin pentosyl
pentoside isomers with a difference in the position of attachment
of the sugar moieties (Cai, Xing, Sun, Zhan, & Corke, 2005). Similarly, peak 51 with a [M H] ion at m/z 433 and an artifact peak
[2M H] ion at m/z 867 was identied as a quercetin pentoside
(Fig 1). Peak 48 with a pseudomolecular ion at m/z 971 was identied as a kaempferol derivative (kmax at 254 and 364, MS3 ion at
m/z 285) with unknown structure. Peaks 49 and 53 are identied
as isomers of an acylated avonol, UV and MS data is coincident
with that reported for kaempferol hydroxy-benzoyl-glucoside
(Ye et al., 2005). Peak 43 was attributted to an isorhamnetin hexosyl-rhamnoside, with a [M H] ion at m/z 623 that gave rise to the
fragments at m/z 477 and 315 U, with a neutral loss of 308 U in
agreement with a rutinose moiety. Peak 60 could be identied as
a glycoside of the avonol anhydroicaritin identied in Epimedium
plants (Zhao et al., 2008) according to MS (MS ion at m/z 763).
However, it was identied as another caffeoyl derivative, namely
1,4,5-tri-caffeoyl-3-methoxy-oxaloyl-quinic acid due to UV data
and MS fragmentations (Lin et al., 2008). Peak 61 was tentatively
identied as a quercetin derivative but the structure remains
unknown. Peak 38 was identied as a avonoid rhamno-hexoside,
in agreement with acacetin rutinoside as suggested by the deprotonated molecule at 591 which gave through loss of a rutinose
moiety an MS2 peak at 283 (acacetin, MS3 268, 211) (Parejo
et al., 2004). Peak 63 matched the MS data reported for sinapoylferuloyl-gentiobioside detected in Brasica rapa (Francisco et al.,
2009). Peak 65 was identied as the avanone naringenin-7-Orutinoside (Abad-Garca et al., 2009), while MS spectra of peak
66 is in agreement with that reported for methyl epigallocatechin
gallate. Peak 67 was identied as pentagalloyl glucose, while peak
69 and peak 71 were tentatively identied as quercetin glycosides
(MS3 peaks at 179 and 151) (Cai et al., 2005). Peak 70 had a
[M H] ion at m/z 369 and provided three fragments units at m/
z 195, 191, and 151 in MS/MS and thus was identied as tri-hydroxy-cinnamoyl-quinic acid (Fang, Yu, & Prior, 2002).

MS. Some 71 main phenolics were detected and 60 compounds

were directly characterised based on their UV spectra and MS fragmentation patterns. Sixteen phenolics were tentatively identied
by LC-UV and LCMSMS analysis in E. illinita, 27 in Q. chilense,
10 in L. chamissonis and 19 in A. emarginata. In the infusions investigated, 34 avonoids were identied. Their occurrence is as follows: four avonoids in L. chamissonis (peaks 49, 50, 54 and 55)
6 in E. illinita (peaks 17, 26, 31, 34, 40 and 43), 16 in A. emarginata
(peaks 3, 18, 22, 24, 29, 32, 37, 38, 46, 48, 51, 57, 62, 65, 69 and 71)
and 14 in Q. chilensis (peaks 4, 6, 8, 13, 28, 33, 39, 42, 44, 50, 53, 59,
61 and 64). In addition, several compounds possessing the quinic
acid moiety such as feruloyl quinic acid (peak 15) chlorogenic acid
(peak 7) and several chlorogenic acid derivatives (peaks 1012 and
30) were detected in E. illinita, 4 quinic acid derivatives were found
in Q. chilensis (peaks 16, 20, 58 and 60), 1 in A. emarginata (peak 70)
and 4 in L. chamissonis (peaks 27, 45, 47 and 52). HPLC-DAD screening at 520 nm showed 2 anthocyanidins (peaks 9 and 14) detected
only in Q. chilensis. Tannins were also detected, including gallocatechin catechin gallate and its isomer (peaks 19 and 35) in E. illinita
and L. chamissonis, respectively. Isomers of the dimer catechin-afzelechin (peaks 23 and 25) were found in Q. chilensis, as well as the
monomer afzelechin (peak 36) in Q. chilensis and methyl-epigallocatechin gallate (peak 66) in L. chamissonis.
Little is know on the chemistry of Adesmia species. The occurrence of quercetin, isovitexin, isorhamnetin-3-O-rutinoside, pinitol
and chlorogenic acid from Adesmia aegiceras was reported by
Agnese, Prez, & Cabrera (2001). Kaempferol, kaempferol-3,7-O-dirhamnoside and kaempferol-7-neohesperidoside were reported
from Argentinian Adesmia species (Agnese & Cabrera, 1996). The
infusion of Adesmia emarginata contained mainly avonol and dihydroavone glycosides bearing one or two hydroxyl groups in the
B-ring and phenylpropanoids as well as hydrolysable and condensed
tannins. The occurrence of quercetin and kaempferol glycosides in
this plant is in general agreement with the trend observed for the
genus Adesmia. Iridoid glycosides have been described for Escallonia
species as well as the avonoid 5-hydroxy-3,7-dimethoxyavone
(Buskingham, 2010). From the 16 compounds detected/tentatively
identied in E. illinita infusion, seven correspond to chlorogenic/caffeoylquinic acid derivatives and six are avonoids based on quercetin, kaempferol or isorhamnetin. The infusion of L. chamissonis was
characterised by the occurrence of 10 main compounds, including
four chlorogenic acid/caffeoylquinic acid derivatives and four avonoid glycosides, as quercetin and kaempferol derivatives. The most
complex pattern of phenolic compounds was found for the Santalacae Quinchamalium chilensis infusion. At the same time and from the
four selected species, this is the most widely used crude drug. The
avonoids were glycosides from the avonols quercetin, kaempferol
and the avone apigenin (15 out of 27 compounds). Quinic acid and
caffeoylquinic acids were also present as well as hydrolisable
tannins, cyanidin and pelargonidin glycosides.
The reputed medicinal properties of the infusions investigated
in this study can be in part attributed to the phenolic compounds
identied. Assay-guided isolation of the active compounds using
appropriate bioassays is needed to fully disclose the identity of
the active constituents. The HPLC proles and phenolic patterns
reported for these species could be used to characterise and differentiate these medicinal Mapuche plants and serve as a starting
point for new studies on the occurrence and distribution of phenolics in different populations of the plants.
Acknowledgments

4. Conclusions
The phenolic compounds composition of infusions from four
Mapuche medicinal plants was analysed for the rst time by LC/

The authors would like to thank CONICET (Argentina), Bicentennial Programme in Science and Technology of Chile, Project PBCT
Grants ACT-38. Mario J. Simirgiotis is a research member of
CONICET.

M.J. Simirgiotis et al. / Food Chemistry 131 (2012) 318327

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