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Cite this: DOI: 10.1039/d0fo01750g
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Biological and chemical insight into Gaultheria
procumbens fruits: a rich source of antiinflammatory and antioxidant salicylate glycosides
and procyanidins for food and functional
application†
Piotr Michel, *a Sebastian Granica, b Karolina Rosińska,a Jarosław Rojek,a
Łukasz Poraja and Monika Anna Olszewska a
The fruits of Gaultheria procumbens are traditionally used for culinary and healing purposes as antiinflammatory agents. In the present work, the active components of the fruits were identified
(UHPLC-PDA-ESI-MS3, preparative HPLC isolation, and NMR structural studies), and their biological
capacity was evaluated in vitro in cell-based and non-cellular models. The fruits were revealed to be the
richest known dietary source of salicylates (38.5 mg per g fruit dw). They are also rich in procyanidins
(28.5 mg per g fruit dw). Among five tested solvents, acetone was the most efficient in concentrating the
phenolic matrix (39 identified compounds; 191.3 mg g−1, 121.7 mg g−1, and 50.9 mg g−1 dry extract for
total phenolics, salicylates, and procyanidins, respectively). In comparison to positive controls (dexamethasone, indomethacin, and quercetin), the extract (AE) and pure salicylates exhibited strong inhibitory
activity towards pro-inflammatory enzymes (cyclooxygenase-2 and hyaluronidase). The analytes were
found to be non-cytotoxic (flow cytometry) towards human neutrophils ex vivo. Moreover, they significantly, in a dose-dependent manner, downregulated the release of ROS, TNF-α, IL-1β, and elastase-2 and
Received 6th July 2020,
Accepted 10th August 2020
slightly inhibited the secretion of IL-8 and metalloproteinase-9 in the cells. The observed effects might
support the usage of G. procumbens fruits as functional components of an anti-inflammatory diet and
DOI: 10.1039/d0fo01750g
indicate the potential of AE for use in adjuvant treatment of inflammatory disorders cross-linked with oxi-
rsc.li/food-function
dative stress and associated with the excessive production of TNF-α, IL-1β, and elastase-2.
1.
Introduction
Berries, and fruits in general, are valuable sources of nutrients
and phytochemicals, and their increased intake is associated
with reduced risk of developing a variety of civilization diseases related to chronic inflammation and oxidative stress.1
The beneficial health effects of dietary fruits are usually connected with their antioxidant constituents, especially polyphenols.2 Polyphenols may represent various molecular structures,
among which salicylates exhibit exceptionally strong antiinflammatory activity. Epidemiological and intervention
studies suggest that dietary salicylates may contribute to the
a
Department of Pharmacognosy, Faculty of Pharmacy, Medical University of Lodz,
Muszynskiego 1 St., 90-151 Lodz, Poland. E-mail: piotr.michel@umed.lodz.pl
b
Department of Pharmacognosy and Molecular Basis of Phytotherapy, Faculty of
Pharmacy, Warsaw Medical University, 1 Banacha St., Warsaw 02-097, Poland
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0fo01750g
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beneficial effects of a vegetarian diet in inflammation-associated chronic diseases.3,4 On the other hand, plant salicylates
are relatively rare natural compounds and only a few plant
taxons are able to biosynthesize them at the levels sufficient to
exert disease preventive or therapeutic activity.5 Most of these
are medicinal plants of low dietary value.6 An exception might
be an ericaceous genus Gaultheria L. that produces both
herbal medicines and edible berry-like fruits appreciated for
their taste and aroma resembling those of mint. The aroma of
Gaultheria fruits results from the presence of a methyl salicylate-rich essential oil (wintergreen oil) used to flavour beverages, sweets, and chewing gums.7–9 The fruits of Gaultheria
also contain substantial amounts of pectin,10 ascorbic
acid,11,12 and minerals.13 As dietary products, they are eaten
raw, cooked, preserved, used in pies, or made into jams,
jellies, syrups, and wines, which suggests their significant
potential for functional applications.7,14
Not only the wintergreen oil but also leaves and aerial parts
(leaves with stems and sometimes fruits) of numerous
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Gaultheria species are traditional anti-inflammatory, antipyretic and analgesic herbal medicines used in the treatment of
inflammation-related ailments, including osteoarthritis, rheumatic diseases, influenza, the common cold, pharyngitis,
fever, muscular pain, and some skin and periodontal
problems.15,16 Methyl salicylate, the primary component of
wintergreen oil, is largely formed during distillation, which
suggests that the main native and active components of the
plant materials, including fruits, might be glycosidic salicylates.9 Indeed, five methyl salicylate glycosides have been isolated to date from aerial parts of Gaultheria plants, specifically
from G. yunnanensis (Franch.) Rehder,15 and their anti-inflammatory effects have been confirmed in vitro in a model of
murine macrophages,17–20 and in vivo in animal models.20–22
The suppressed activation of the MAPK/NF-κB signalling
pathway and downregulated release of reactive oxygen species
(ROS), tumour necrosis factor (TNF-α), and pro-inflammatory
interleukins (IL-1β and IL-6) have been suggested as their
main mechanism of action.19,20 However, there is no information available neither on the salicylate profiles of the fruits
of any Gaultheria species nor on their anti-inflammatory
effects.
G. procumbens L. (American wintergreen, eastern teaberry)
is a small, low-growing shrub with evergreen leaves and red
fruits similar in shape and size to huckleberries, cranberries,
lingonberries, and other ericaceous fruits. It is native to northern North America, where it has been used for dietary7,8 and
medicinal purposes.15,16 Due to its decorative and culinary
values, the plant is also widely cultivated in other regions with
temperate climates. G. procumbens is the richest known source
of wintergreen oil, and its fruits yield up to 0.86% (v/w) of the
oil from the fresh mass and over 97% of methyl salicylate in
the oil.23 As for other Gaultheria fruits, there is no direct proof
on the presence of glycosidic salicylates in teaberries, but the
total levels of salicylic acid in hydrolysed fruit extracts have
been reported to several times exceed the content of the free
form, which suggests that most of the methyl salicylate in the
plant is conjugated.9 Previously, the presence of gaultherin
(GT) was confirmed in the stems of the plant by LC-MS/MS.24
The aim of the present study was to characterize the chemical profile and biological activity of the fruits of G. procumbens
with special emphasis on the content and composition of salicylates and their influence on the activity of the fruits. Five
different solvents were used for extraction to select extracts
most suitable for the concentration of active compounds and
specialized functional applications. UHPLC-PDA-ESI-MS3
studies, preparative HPLC isolation, and spectroscopic experiments (1D and 2D) were used for thorough chemical profiling.
In the first stage of activity studies, the extracts were analysed
by in vitro non-cellular tests for inhibition of cyclooxygenase-2
(COX-2), hyaluronidase (HYAL), and lipoxygenase (LOX), as
well as for direct ROS scavenging. In the second step, the most
active extract and the isolated salicylates were investigated for
their effects on viability and proinflammatory and pro-oxidant
functions of human neutrophils ex vivo, including the release
of ROS, IL-1β, IL-8, TNF-α, matrix metalloproteinase 9
Food Funct.
Food & Function
(MMP-9), and elastase-2 (ELA-2). Based on the results, the perspectives of the fruits and the selected extract for functional
food and medicinal applications were discussed.
2. Materials and methods
2.1.
Plant material
Fruits of G. procumbens L. were collected in October 2018 in
the gardening centre of Ericaceae plants, Gospodarstwo
Szkolkarskie Jan Cieplucha (54°44′N, 19°18′E), Konstantynow
Lodzki (Poland), where the plants grew in an open area. The
origin of seeds and their authentication was described previously.24
The
voucher
specimen
(KFG/HB/
18001-GPRO-FRUITS) was deposited in the Medicinal Plant
Garden, Medical University of Lodz (Poland). Samples of the
plant material were air-dried at 35 °C, powdered with an electric grinder, and sieved through a ∅ 0.315 mm sieve.
2.2.
Preparation of extracts
Five samples of the powdered fruits (100 g each) were refluxed
independently with different solvents, i.e. methanol–water
(75 : 25, v/v), ethyl acetate, n-butanol, acetone, and water (3
times, 300 mL × 2 h each time). The combined extracts of each
type were evaporated at 40 °C (in vacuo) to give the dry methanol–water extract (ME), ethyl acetate extract (EAE), n-butanol
extract (BE), acetone extract (AE), and water extract (WE),
respectively. The residual water was removed from ME and WE
by lyophilization using an Alpha 1–2/LD Plus freeze dryer
(Christ, Osterode am Harz, Germany). The extraction procedure was repeated thrice to establish the extraction yield for
each extract. The extraction yields were calculated per dry
weight (dw) of the plant material. All other quantitative results
obtained during the study were calculated per dry weight (dw)
of the extracts.
2.3. Qualitative LC-MS/MS analysis and isolation of
salicylates
The qualitative UHPLC-PDA-ESI-MS3 analysis was carried out
according to Michel et al. (2014)25 using an UHPLC-3000 RS
system (Dionex, Lohmar, Germany) equipped with dual lowpressure gradient pump, thermostated autosampler and
column compartments, a PDA detector, and an Amazon SL ion
trap mass spectrometer with an ESI interface (Bruker Daltonik,
Bremen, Germany). Separations were carried out on a Kinetex
XB-C18 column (1.7 μm, 150 mm × 2.1 mm i.d.; Phenomenex,
Torrance, CA, USA) at the conditions described previously.25
Three salicylate glycosides PH, TG and GT were isolated
from n-butanol extract of G. procumbens (BE, 3.2 g; ESI
Fig. S1†) by preparative HPLC-PDA using a LC-20AP system
(Shimadzu, Kyoto, Japan) equipped with a preparative pump, a
PDA detector, thermostated autosampler and column oven,
and a XB-C18 Kinetex column (5 µm, 150 mm × 22.1 mm i.d.,
Phenomenex, Torrance, CA, USA). A linear gradient system was
applied for preparative elution with the following profile:
0–25 min, 2%–20% B (v/v), 25–30 min, 20%–2% B (v/v), where
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the mobile phase (A) was water-formic acid (100 : 0.1, v/v), and
(B) was acetonitrile-formic acid (100 : 0.1, v/v). Separation was
performed at 25 °C, the flow rate was 20 mL min−1, and the
injection volume was 200 µL. Prior to the isolation, samples of
BE were dissolved in DMSO (4 mL) and filtered through a
PTFE syringe filter (25 mm, 0.2 µm, Ahlstrom, Helsinki,
Finland). The fraction collection was triggered automatically
by signal from UV-Vis detector at λ = 285 nm (methyl salicylate
glycosides). The separation was repeated 20 times, and fractions containing the target analytes were combined and purified by gel permeation over Sephadex LH-20 to afford three
compounds: PH (19.1 mg; isolation yield 90.1%), TG (78.9 mg;
88.7%), and GT (89.2 mg; 94.9%). The HPLC purity of the isolates was 98.3%, 99.2%, and 97.8%, respectively. The HPLC
pure solvents for the analysis were from Avantor (Gliwice,
Poland).
2.4.
Structure elucidation
The acid hydrolysis was performed according to Olszewska
and Roj (2011).26 Free aglycone was identified with an authentic standard by GC-MS according to Magiera et al. (2019).23
The identity and absolute configuration of the sugars from the
aqueous layer were determined after conversion to their 1-[(S)N-acetyl-α-methylbenzylamino]-1-deoxy-alditol
pentaacetate
derivatives, as described earlier.26 As the result, D-glucose was
identified in the hydrolysates of PH, TG and GT; and D-xylose
in the hydrolysates of TG and GT.
1
H NMR, 13C NMR, COSY, HMQC, and HMBC spectra were
recorded at 25 °C on a Bruker Avance III 600 spectrometer
(Bruker BioSpin Co., Billerica, MA, USA) in methanol-d4
(600 MHz for 1H and 150.9 MHz for 13C), with TMS as the
internal standard. HR-ESI-MS spectra were recorded at 25 °C
on a Q-TOF SYNAPT G2-Si spectrometer (Waters, Milford, MA,
USA) coupled with ACQUITY UPLC system (Waters, Milford,
MA, USA). LC-ESI-MS3 spectra were recorded as described in
the previous paper (Michel et al., 2014)25 and the spectral profiles were presented in ESI Table S1.†
Compound PH; methyl salicylate 2-O-(2′-O-β-D-glucopyranosyl)-β-D-glucopyranoside ( physanguloside A). Pale pink amorphous solid: UV (methanol) λmax nm: 285; LC-ESI-MS m/z (relative intensity): [M + HCOO]− 521 (100); MS2: [M − H]− 475
(100), [M − H-32]− 443 (4). HR-ESI-MS (negative mode): found
m/z 521.1512 [M + HCOO]−; calculated for C21H29O15 m/z
521.1506. 1H and 13C NMR data (methanol-d4): see ESI
Table S2.†
Compound TG; methyl salicylate 2-O-(2′-O-β-D-glucopyranosyl-6′-O-β-D-xylopyranosyl)-β-D-glucopyranoside (MSTG-B). Pale
pink amorphous solid: UV (methanol) λmax nm: 285;
LC-ESI-MS m/z (relative intensity): [M + HCOO]− 653 (100);
MS2: [M − H]− 607 (100), [M − H-32]− 575 (13). HR-ESI-MS
(negative mode): found m/z 653.1937 [M + HCOO]−; calculated
for C26H37O19 m/z 653.1929. 1H and 13C NMR data (methanold4): see ESI Table S2.†
Compound GT; methyl salicylate 2-O-(6′-O-β-D-xylopyranosyl)-β-D-glucopyranoside (gaultherin). Pale pink amorphous
solid: UV (methanol) λmax nm: 285; ESI-MS2 m/z (relative inten-
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Paper
sity): [M + HCOO]− 491 (100); MS2: [M − H]− 445 (12), [M −
H-32]− 413 (4), [M − H-152]− 293 (100), [M − H-212]− 233 (2),
[M − H-196]− 149 (4). HR-ESI-MS (negative mode): found m/z
491.1407 [M + HCOO]−; calculated for C20H27O14 m/z 491.1401.
1
H and 13C NMR data (methanol-d4): see ESI Table S2.†
2.5.
Quantitative phytochemical profiling
The total phenolic (TPC) and total proanthocyanidin (TPA)
contents were determined by the Folin–Ciocalteu and
n-butanol-HCl methods, respectively, as described previously.27
Results were expressed as equivalents of gallic acid (GAE) and
cyanidin chloride (CYE), respectively.
The HPLC-PDA analyses were carried out according to
Michel et al. (2019),24 salicylates were used for accurate quantification using an HPLC VWR-Hitachi LaChrom Elite® System
(Hitachi, Tokyo, Japan) equipped with a quaternary pump, a
PDA detector, an autosampler, and a thermostated column
compartment with a C18 Ascentis® Express column (2.7 μm,
75 mm × 4.6 mm i.d.), guarded by a C18 Ascentis® C18
Supelguard column (3 μm, 20 mm × 4 mm i.d.; both from
Supelco, Sigma-Aldrich, St. Louis, MO, USA). Fifteen authentic
standards, including three isolated salicylates, were used for
calibration (five-point statistically significant calibration
equations were established). Apart from the peaks corresponding to the standards, the tentatively identified ones were
quantified as equivalents of the relevant reference compounds,
depending on the PDA spectra. In brief, hydroxybenzoic acids
were estimated as protocatechuic acid or p-hydroxybenzoic
acid; caffeoylquinic acid isomers as chlorogenic acid (5-Ocaffeoylquinic acid); hydroxycinnamic acid derivatives as
p-coumaric acid or caffeic acid; dimeric and trimeric procyanidins as procyanidin B2 (PB2) and procyanidin C1, respectively;
and flavonoid monoglycosides as miquelianin. The standard
set also contained (+)-catechin, (−)-epicatechin, quercetin
(QU), kaempferol, PH, TG and GT. All standards were of HPLC
purity and from Sigma-Aldrich (St. Louis, MO, USA) and
Phytolab (Vestenbergsgreuth, Germany).
2.6.
Antioxidant activity in non-cellular in vitro models
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity
and ferric reducing antioxidant power (FRAP) were determined
as previously described.27 The inhibitory capacity towards
AAPH-induced peroxidation of linoleic acid (LA) was determined according to Matczak et al. (2018)28 with peroxidation
measured by quantitation of thiobarbituric acid-reactive substances (TBARS). The scavenging capacities were evaluated
according to Michel et al. (2014)25 in the case of O2•− (superoxide anion radical) and according to Marchelak et al. (2019)29
in the case of hydroxyl radical (•OH) and hydrogen peroxide
(H2O2). The FRAP values were expressed in µmol of ferrous
ions (Fe2+) produced by 1 g of an analyte. Results of the scavenging and LA-peroxidation tests were expressed in SC50 (scavenging concentration; DPPH, O2•−, •OH, and H2O2) and IC50
(inhibitory concentration; TBARS) values, respectively, which
were defined as concentration levels of analytes required to
produce 50% of the maximal response. Prior to the analyses,
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the analytes were dissolved in methanol–water (75 : 25, v/v) or
PBS and diluted to the final concentrations of 0.8–350.0 µg
mL−1, 0.8–19.5 µg mL−1, 0.5–450.0 µg mL−1, 1.5–1000.0 µg
mL−1, 15.0–1500.0 µg mL−1, and 2.5–1000.0 µg mL−1 for
DPPH, FRAP, TBARS, O2•−, •OH, and H2O2 tests, respectively.
In all tests quercetin (QU) and TX (Trolox®, (±)-6-hydroxy2,2,7,8-tetramethylchroman-2-carbo-xylic acid) were used as
positive controls. All reagents and standards were purchased
from Sigma-Aldrich (St. Louis, MO, USA). All tests were performed using 96-well plates and monitored using a microplate
reader SPECTROstar Nano (BMG Labtech GmbH, Ortenberg,
Germany). The samples were incubated in a constant temperature using a BD 23 incubator (Binder, Tuttlingen, Germany).
2.7. Anti-inflammatory activity in non-cellular in vitro
models
The ability of the analytes to inhibit lipoxygenase (LOX) and
hyaluronidase (HYAL) was examined as described by Matczak
et al. (2018),28 while the inhibitory effect on cyclooxygenase-2
(COX-2) was evaluated by ELISA test following the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA).
All other reagents and standards, including indomethacin
(IND), dexamethasone (DEX) and QU, used as positive controls, were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Prior to the assays, the analytes were dissolved in monosodium
phosphate buffer ( pH = 7.0) with 0.01% BSA, sodium borate
buffer ( pH = 9.0) or ELISA buffer, and diluted to the final concentrations of 50–1200 µg mL−1, 2.5–80.0 µg mL−1, and
25–1000 µg mL−1 for LOX, HYAL, and COX-2 tests, respectively.
The results were expressed as IC50 values. A microplate reader
SPECTROstar Nano and 96-well plates were used in the assays.
2.8. Antioxidant and anti-inflammatory effects in cellular
model
2.8.1. Isolation of human neutrophils and viability studies.
Neutrophils were isolated from buffy coat fractions, a byproduct of blood fractionation for transfusions. The fractions
were obtained from Warsaw Blood Donation Centre, where
they were collected from adult human donors (18–35 years
old). The donors were confirmed to be healthy and routine laboratory tests showed all values within the normal ranges. The
study conformed to the principles of the Declaration of
Helsinki, and as it involved commercially available biological
material, it did not require an approval of a bioethics
committee.
Isolation was carried out with a standard method of
dextran sedimentation prior to hypotonic lysis of erythrocytes
and centrifugation in a Ficoll Hypaque gradient according to
Michel et al. (2019).24 The purity of the neutrophils fraction
was over 97%. After isolation, cells were suspended in (Ca2+)free Hanks’ balanced salt solution (HBSS buffer), (Ca2+)-free
phosphate buffered saline (PBS buffer), or RPMI 1640 culture
medium, and maintained at 4 °C before use. A (Ca2+)-free
phosphate buffered saline (PBS) was purchased from Biomed
(Lublin, Poland), Ficoll Hypaque gradient from PAA
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Food & Function
Laboratories (Pasching, Austria), and all other reagents and
media from Sigma-Aldrich.
The potential cytotoxicity (influence on cell wall integrity)
of the analytes was evaluated according to Michel et al.
(2019).24 The analytes were tested at the levels of 25–150 µg
mL−1 for AE and 25–75 µM for pure compounds. Cell viability
was analysed using propidium iodide (PI) staining and a flow
cytometer (BD FACSCalibur, BD Biosciences, San Jose, CA,
USA) with 10 000 events recorded per sample. Cells that displayed high permeability to PI were expressed as a percentage
of PI(+) cells. Cells treated with Triton X-100 solution were
used as a positive control (98.6% of PI(+) cells).
2.8.2. Evaluation of ROS production by human neutrophils. The ROS levels in neutrophils stimulated by N-formyl-Lmethionyl-L-leucyl-L-phenylalanine ( fMLP) were determined by
luminol-dependent chemiluminescence testing24 using 96-well
plates and monitored using a microplate reader (Synergy 4,
BioTek, Winooski, VT, USA). The concentrations of the analytes
were the same as applied in the viability studies. The response
from the fMLP-stimulated control untreated by the analytes
was assumed as 100% of ROS production. QU (25–75 µM) was
used as a positive control. All reagents and standard were purchased from Sigma-Aldrich.
2.8.3. Evaluation of IL-8, IL-1β, TNF-α and MMP-9 release.
The release of cytokines (IL-8, IL-1β, and TNF-α) and MMP-9 by
lipopolysaccharide (LPS)-stimulated neutrophils was carried
out according to Michel et al. (2019)24 and evaluated by ELISA
tests following the manufacturer’s instructions (BD
Biosciences, San Jose, CA, USA or R&D Systems, Minneapolis,
MN, USA) using 96-well plates and a microplate reader
(Synergy 4). The concentrations of the analytes were the same
as applied in the viability studies. The response from the LPSstimulated control untreated by the analytes was assumed as
100% of the release. DEX (25–75 μM) was used as a positive
control. LPS from Escherichia coli and all other reagents and
media were purchased from Sigma-Aldrich.
2.8.4. Evaluation of ELA-2 release by human neutrophils.
The secretion of ELA-2 by fMLP + cytochalasin B-stimulated
neutrophils was determined using N-succinyl-alanine-alaninevaline p-nitroanilide (SAAVNA) as a substrate according to
Michel et al. (2019).24 The release of p-nitrophenol was
measured at 412 nm over a period of 300 min with 20 min
intervals using 96-well plates and a microplate reader (Synergy
4). The concentrations of the analytes were the same as
applied in the viability studies. The response from the stimulated control untreated by the analytes was assumed as 100%
of the release. QU (25–75 µM) was used as a positive control.
All reagents and media were purchased from Sigma-Aldrich.
2.9.
Statistical and data analysis
The results were expressed as the mean ± standard deviation
(SD) of replicate determinations. The statistical analyses (calculation of SD, one-way analysis of variance, HSD Tukey tests and
linearity studies) were performed using Statistica12Pl software
for Windows (StatSoft Inc., Krakow, Poland), with p values
<0.05 regarded as significant.
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3. Results
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3.1.
Qualitative chemical profile of the fruit extracts
The UHPLC-PDA-ESI-MS3 assay of the extracts obtained with
different solvents revealed their similar qualitative profile and
the presence of 45 phenolic constituents (Fig. 1, UHPLC peaks
1–45), 39 of which were fully or tentatively identified (ESI
Table S1†) based on their chromatographic and spectral
properties.24,25,30–32
Four major groups were distinguished among the analytes,
including: (i) salicylates; (ii) flavan-3-ols and procyanidins; (iii)
flavonoids; and (iiii) phenolic acids. The most structurally
diverse were flavan-3-ols and procyanidins, i.e. procyanidin
A-type dimers (13, 15, 16, 18), B-type dimers (7, 8, 16–18, 25,
29), A-type trimers (24, 26, 34), B-type trimers (15, 27, 28, 31),
(+)-catechin (10), and the dominant (−)-epicatechin (21).
Among flavonoids, two flavonol aglycones, i.e. QU (44) and
kaempferol (45), five quercetin glycosides (35–37, 39, 40) and
one kaempferol glycoside (41) were identified, with the prevailing miquelianin (37). Among phenolic acids, simple hydroxybenzoic and hydroxycinnamic acids (2, 3, 5, 12, 13, 20), as well
as chlorogenic acid isomers (4, 9), were present. The primary
peaks were assigned to glycosidic salicylates, but all three com-
Paper
pounds of this group (11, 14, 22) had to be isolated for full
structural identification. They were isolated from BE (ESI
Fig. S1†), with high purity and yield, and identified after
thorough spectral profiling (LC-ESI-MS, 1H NMR, 13C NMR,
COSY, HMBC, and HMQC) (ESI Table S2†) as methyl salicylate
2-O-(2′-O-β-D-glucopyranosyl)-β-D-glucopyranoside ( physanguloside A, PH), methyl salicylate 2-O-(2′-O-β-D-glucopyranosyl-6′-Oβ-D-xylopyranosyl)-β-D-glucopyranoside (TG), and 2-O-(2′-O-β-Dxylopyranosyl)-β-D-glucopyranoside (GT), respectively.33–37 Their
structures are presented in Fig. 2.
3.2.
Quantitative chemical profile of the fruit extracts
The extracts differed significantly in quantitative profiles and
extraction yields (Table 1). The total phenolic content varied in
the range of 17.3–79.7 mg GAE per g dw (TPC, determined by
the Folin–Ciocalteu method) and 36.3–135.2 mg per g dw
(TPH, determined by HPLC-PDA), with the highest levels
observed in ME and AE, respectively. Except for WE, the TPH
levels of all other extracts were significantly higher than their
TPCs. Methyl salicylate glycosides dominated in the extracts
and their total levels (TSAL, 31.1–121.7 mg per g dw) constituted 86–95% of the TPH values. The main salicylate was GT,
which contents reached up to 89% of the TSAL values. The
Fig. 1 Representative UHPLC-PDA chromatograms of different solvent extracts (ME, AE and WE) of G. procumbens fruits at 280 nm. The peak
numbers refer to those implemented in ESI Table S1.†
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Fig. 2
Table 1
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Structures of the isolated methyl salicylate glycosides PH, GT and TG.
Extraction yield and phenolic profile of G. procumbens fruit dry extracts (mg per g dw)
Compound/fraction
Extraction yield
Phenolic fractions:
TPC
TPH
TSAL
TPA
TLPA
TPHA
TFL
Primary compounds:
GT, gaultherin (22)
TG, methyl salicylate triglycoside (14)
PH, physanguloside A (11)
PB2, procyanidin B2 (18)
(−)-Epicatechin (21)
Procyanidin A-type trimer (26)
Miquelianin (37)
Quercetin (44)
Protocatechuic acid (2)
p-Hydroxybenzoic acid (5)
Chlorogenic acid (9)
Methanol–water (ME)
456.71 ± 25.84
D
Ethyl acetate (EAE)
77.03 ± 3.85
A
n-Butanol (BE)
388.14 ± 20.71
Acetone (AE)
C
118.59 ± 5.93
Water (WE)
B
502.67 ± 27.13 D
79.71 ± 1.15 E
94.49 ± 1.54 C
83.66 ± 0.88 C
62.39 ± 0.80 E
7.41 ± 0.16 B
2.25 ± 0.03 A
1.17 ± 0.06 E
17.26 ± 0.45 A
115.37 ± 1.68 D
109.28 ± 1.76 D
0.58 ± 0.01 A
n.d.
6.03 ± 0.24 E
0.065 ± 0.002 A
23.46 ± 0.38 B
67.23 ± 1.14 B
63.77 ± 0.74 B
2.92 ± 0.17 B
n.d.
2.80 ± 0.06 B
0.66 ± 0.04 C
56.05 ± 0.83 D
135.23 ± 1.78 E
121.67 ± 1.44 E
41.26 ± 1.36 D
9.59 ± 0.26 C
3.23 ± 0.11 C
0.74 ± 0.08 D
46.62 ± 1.41 C
36.26 ± 0.74 A
31.09 ± 0.59 A
30.01 ± 0.31 C
1.24 ± 0.04 A
3.60 ± 0.13 D
0.33 ± 0.03 B
48.89 ± 1.52 C
22.31 ± 0.70 D
12.45 ± 0.41 D
2.86 ± 0.03 C
2.36 ± 0.11 C
0.89 ± 0.03 B
0.95 ± 0.04 C
0.14 ± 0.01 B
0.16 ± 0.03 A
0.08 ± 0.01 A, B
0.37 ± 0.05 A
98.25 ± 1.84 E
2.45 ± 0.16 A
8.58 ± 0.08 C
n.d.
n.d.
n.d.
n.d.
0.031 ± 0.001 B
0.35 ± 0.05 C, D
0.22 ± 0.02 C
0.59 ± 0.03 B
29.35 ± 0.68 B
27.81 ± 0.13 E
6.61 ± 0.08 A
n.d.
n.d.
n.d.
n.d.
0.52 ± 0.03 C
0.43 ± 0.08 D
0.07 ± 0.01 A
0.35 ± 0.02 A
93.63 ± 0.23 D
11.95 ± 0.60 B
16.09 ± 0.68 E
2.28 ± 0.08 B
1.57 ± 0.02 B
3.48 ± 0.14 C
0.52 ± 0.04 B
0.16 ± 0.03 B
0.30 ± 0.03 B, C
0.21 ± 0.02 C
0.76 ± 0.04 C
2.73 ± 0.04 A
20.59 ± 0.73 C
7.77 ± 0.11 B
0.34 ± 0.03 A
0.22 ± 0.02 A
0.68 ± 0.07 A
0.29 ± 0.02 A
0.021 ± 0.001 A
0.27 ± 0.05 B
0.09 ± 0.01 B
0.40 ± 0.03 A
Results are presented as mean values ± SD (n = 3). Means with different superscript capital letters within the same row differ significantly (p <
0.05). Extraction yield calculated per dry weight (dw) of fruits and other results per dw of the extracts. TPC: total phenolic content (FolinCiocalteau assay) in gallic acid equivalents, TPH: total phenolic content (HPLC), TSAL: total salicylates (HPLC), TPA: total proanthocyanidins
(n-butanol/HCl assay) in cyanidin chloride equivalents, TLPA: total proanthocyanidins (HPLC), TPHA: total phenolic acids (HPLC), TFL: total
flavonoids (HPLC). Numbers in parentheses refer to peak numbering in Fig. 1 and ESI Table S1.†
second relevant group of polyphenols was formed by flavan-3ols and procyanidins. Their total contents differed significantly, depending on the extract and the assay protocol. In
ME, AE and WE, the TPA levels (determined by n-butanol/HCl
assay) were up to 24-times higher than the TLPA values (determined by HPLC-PDA), respectively. In both EAE and BE, only
low amounts of TPA could be found. As the RP-HPLC technique is able to detect proanthocyanidin oligomers built from
less than four flavan-3-ol monomers, the difference between
the TPA and TLPA values could be ascribed to the presence of
highly polymerised homologues. Minor components of the
extracts were phenolic acids (TPHA) and flavonoids (TFL).
3.3. Antioxidant and anti-inflammatory activity in noncellular in vitro models
The investigated extracts showed concentration-dependent
ability to: (i) scavenge free radicals, both synthetic (DPPH) and
generated in vivo (O2•−, •OH, and H2O2); (ii) inhibit linoleic
acid peroxidation (TBARS); and (iii) reduce ferric ions (FRAP)
(Table 2). AE revealed the strongest antioxidant capacity in all
Food Funct.
non-cellular assays, but its activity parameters did not differ
significantly ( p > 0.05) from those of ME in the DPPH, FRAP,
and •OH scavenging tests. The activity of AE and ME was significantly weaker than that of standard antioxidants (QU and
TX) and a model procyanidin (PB2), but it surpassed the
capacity of pure salicylates. As regards the anti-inflammatory
potential of the extracts, they revealed significant and concentration-dependent abilities to inhibit COX-2 and HYAL, but
their activity toward LOX was weak (Table 3). AE was the strongest enzyme inhibitor among the extracts. The COX-2 inhibitory effect of AE was significantly stronger ( p < 0.05) than that
observed for positive standards of synthetic anti-inflammatory
drugs (DEX and IND). The activity of AE towards HYAL was
about 2-times weaker than that of DEX and IND, but it was
comparable to that observed for QU, a natural HYAL inhibitor.
Due to the low number of tested extracts (n = 4 data points),
most of the linear correlations between the concentration and
activity parameters were not significant (ESI Table S3†).
However, high correlation coefficients in concert with the
capacity of pure compounds and their concentration in the
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Table 2
models
Paper
Antioxidant activity of G. procumbens fruit extracts (AE, ME, BE and WE) and primary constituents (TG, GT, PH and PB2) in non-cellular
Analyte
DPPH
SC50 a (µg mL−1)
FRAP
mmol Fe2+ per g b
TBARS
IC50 c (µg mL−1)
O2•−
SC50 a (µg mL−1)
•
OH
SC50 a (µg mL−1)
H2O2
SC50 a (µg mL−1)
AE
ME
BE
WE
TG
GT
PH
PB2
QU
TX
44.59 ± 2.01 D
40.40 ± 1.64 D
237.00 ± 6.49 F
75.46 ± 2.12 E
308.02 ± 12.41 H
265.69 ± 11.28 G
274.58 ± 13.24 G
2.37 ± 0.02 B
1.65 ± 0.04 A
4.31 ± 0.06 C
1.75 ± 0.04 C
1.65 ± 0.05 C
0.93 ± 0.03 B
0.99 ± 0.03 B
0.59 ± 0.04 A
0.64 ± 0.04 A
0.63 ± 0.03 A
29.57 ± 0.11 E
47.09 ± 0.61 F
11.89 ± 0.25 D
37.41 ± 1.22 D
56.67 ± 2.47 E
251.68 ± 18.85 G
71.84 ± 0.94 F
312.32 ± 16.12 I
269.40 ± 15.17 G,H
278.57 ± 13.67 H
2.54 ± 0.13 B
1.78 ± 0.06 A
4.68 ± 0.24 C
175.33 ± 8.36 D
333.41 ± 8.55 E
877.63 ± 44.56 H
322.94 ± 10.18 E
519.05 ± 16.46 G
451.76 ± 14.16 F
475.28 ± 12.87 F
3.62 ± 0.05 A
7.58 ± 0.21 B
135.24 ± 1.01 C
863.95 ± 24.88 G
824.04 ± 37.86 G
676.29 ± 40.95 F
1150.67 ± 49.59 H
552.87 ± 18.64 E
488.52 ± 9.69 D
498.84 ± 11.58 D
121.65 ± 3.42 B
42.48 ± 4.07 A
165.45 ± 2.99 C
166.36 ± 7.36 C
332.15 ± 6.33 D
497.95 ± 5.96 F
422.95 ± 4.51 E
657.19 ± 16.43 H
587.86 ± 14.08 G
603.35 ± 13.46 G
15.05 ± 0.56 B
7.52 ± 0.38 A
15.87 ± 0.33 B
SC50: scavenging efficiency in μg of the dry extract or compound per mL of the reaction solution. b Values expressed per g of the dry extract or
standard. c IC50: inhibition concentration in μg of the dry extract or compound per mL of the reaction solution. The positive controls: QU (quercetin) and TX (Trolox®, (±)-6-hydroxy-2,2,7,8-tetramethylchroman-2-carboxylic acid). Results presented as mean values ± SD (n = 3). For each parameter different superscript capital letters indicate significant differences (p < 0.05).
a
Table 3 Inhibitory activity of G. procumbens fruit extracts (AE, ME, BE
and WE) and primary constituents (TG, GT, PH and PB2) towards proinflammatory enzymes
Analyte
COX-2
IC50 a (μg mL−1)
HYAL
IC50 a (µg mL−1)
LOX
IC50 a (µg mL−1)
AE
ME
BE
WE
TG
GT
PH
PB2
IND
DEX
QU
152.89 ± 7.64 A
230.83 ± 11.54 C
224.08 ± 11.20 C
713.36 ± 35.67 H
266.18 ± 10.28 D
346.16 ± 15.31 E
368.25 ± 16.98 E
829.13 ± 39.15 I
178.40 ± 7.92 B
507.63 ± 15.38 G
471.96 ± 15.02 F
28.39 ± 1.16 E
32.72 ± 0.26 E,F
49.30 ± 1.02 H
39.34 ± 0.75 G
24.29 ± 0.68 D
28.58 ± 1.28 E
30.34 ± 0.95 E
21.65 ± 1.03 C
12.77 ± 1.91 A
14.18 ± 1.05 B
30.78 ± 1.84 D
644.79 ± 22.83 F
743.61 ± 33.55 G
850.42 ± 16.86 H
837.96 ± 15.75 H
448.93 ± 11.75 D
561.16 ± 15.93 E
584.32 ± 13.56 E
167.20 ± 8.23 C
92.60 ± 3.71 A
118.14 ± 5.15 B
89.23 ± 7.13 A
IC50: inhibition concentration in μg of the dry extract or compound
per mL of the reaction medium. The positive controls: IND (indomethacin), DEX (dexamethasone) and QU (quercetin). Results are presented as mean values ± SD (n = 3). For each parameter different superscript capital letters indicate significant differences (p < 0.05).
a
extracts might suggest that direct antioxidant effects of the
extracts are mediated by procyanidins and salicylates are primarily responsible for their COX-2 inhibitory capacity, while
both salicylates and procyanidins contributed to their activity
towards HYAL.
3.4. Influence on pro-oxidant and proinflammatory functions
of human neutrophils
3.4.1. Effects on neutrophil viability and ROS production.
Among the extracts, AE was selected for the cell-based study. It
was tested in a concentration range of 25–150 µg mL−1, which
means 6–36 µM of total salicylates (TSAL), and 8.5–50 µM of
the sum of total salicylates and procyanidins (TSP = TSAL +
TLPA + TPA; where molar concentration of TPA was calculated
as PB2). During the viability assay, it was confirmed that
neither the extract nor its pure constituents (PH, TG, GT and
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PB2) were cytotoxic at the concentration range investigated,
which means that no significant reduction ( p > 0.05) in membrane integrity was observed between the control and analytestreated cells (ESI Fig. S2†). For antioxidant activity testing, ROS
were produced by neutrophils after stimulation by fMLP. As
shown in Fig. 3A, AE revealed significant ( p < 0.001) and dosedependent antioxidant effects, downregulating the ROS level
in stimulated neutrophils by up to 65% at 150 µg mL−1 (50 µM
TSP). A similar range of activity was observed for pure compounds with relatively stronger capacity found for PB2 (the
ROS level reduced by 67% at 50 µM) in comparison to salicylates (the ROS release decreased by up to 50% at 50 µM) (ESI
Fig. S3A†). Considering the high molar proportion of salicylates to the fraction (0.72 TSP), both these groups of compounds might be considered responsible for the antioxidant
effect of AE, however, with a prominent role for salicylates. In
comparison to QU, the cellular antioxidant activity of the analytes, especially salicylates, was noticeably stronger than their
relative ability to directly scavenge ROS, as observed in chemical models (Table 2).
3.4.2. Effects on the release of proinflammatory cytokines
and enzymes. AE and its model constituents were able to
modulate the release of proinflammatory cytokines (TNF-α,
IL-1β and IL-8) and tissue remodelling enzymes (ELA-2 and
MMP-9) from neutrophils stimulated by LPS or fMLP + cytochalasin B, depending on the test (Fig. 3B–F). Pure salicylates
exhibited comparable or relatively stronger effects compared to
PB2 (ESI Fig. S3B–F†). In view of the large proportion of TSAL
in AE (Table 1), salicylates were considered as main contributors to the observed capacity of the extract. The effects of all
analytes were dose-dependent and strongest towards the
release of TNF-α, IL-1β and ELA-2. For instance, the extract at
24 µM TSAL downregulated the secretion of TNF-α, IL-1β, and
ELA-2 by 47%, 64%, and 77%, respectively, in comparison to
the stimulated control ( p < 0.001). In contrast, in the same
conditions, only a 14% and 9% decrease was observed in the
release of IL-8 and MMP-9, respectively. Moreover, in the case
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Fig. 3 Effect of acetone fruit extract (AE) at 6, 12, 24, and 36 µM TSAL (25–150 µg mL−1) on: (A) ROS production, and secretion of (B) TNF-α, (C)
IL-1β, (D) IL-8, (E) MMP-9, and (F) ELA-2 by stimulated human neutrophils. The positive controls (QU and DEX) tested at 25 µM (7.5 and 10 µg mL−1,
respectively). Data expressed as means ± SD of three independent experiments performed with cells isolated from five independent donors.
Statistical significance: #p < 0.001 compared to the non-stimulated control; *p < 0.05, **p < 0.01, ***p < 0.001 decreased compared to the stimulated control.
of TNF-α and IL-1β, the extract effects did not differ significantly ( p > 0.05) from those of a reference anti-inflammatory
drug (DEX) at the relevant concentration (25 µM).
Furthermore, the effect of AE towards ELA-2 was significantly
stronger ( p < 0.05) than that of the positive control QU at
25 µM. Interestingly, the activity of pure salicylates and PB2
appeared to be relatively weaker than that of AE. For example,
50–75 µM TG, GT or PH were required to obtain effects comparable to those of 25 µM DEX (secretion of TNF-α and IL-1β),
and 25 µM individual salicylates were necessary to inhibit the
release of ELA-2 in a manner similar to 25 µM QU.
Food Funct.
4.
Discussion
Chronic inflammation and oxidative stress are important etiological factors linked to age-related human disorders, such as
cardiovascular disease, metabolic disorders, rheumatoid
arthritis, and inflammatory bowel disease, which negatively
influence both quality of life and lifespan.38 Apart from
designing new synthetic drugs, the search for dietary intervention strategies, as well as new sources of anti-inflammatory
functional foods, is an ever-increasing area of research.1,39
This work was focused on teaberries, the edible and willingly
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Food & Function
consumed fruits of G. procumbens and a traditional herbal
medicine recommended (as whole aerial parts) for the treatment of inflammatory disorders,7,15 with yet unexplored
chemical composition and biological effects.
For phytochemical investigation, the fruits were extracted
with different polar solvents with relatively high ability to
solvate Gaultheria polyphenols including glycosidic salicylates.
The extracts with methanol–water (75 : 25, v/v) and water
reflected the composition of the overall fruit matrix, some processed food products (liquors and wines), and the most
popular medicinal preparations (tinctures and infusions).
Ethyl acetate, n-butanol and acetone were used for concentration of polyphenolic fraction according to our previous
works on leaves and stems of G. procumbens.24,25 After preliminary experiments, nonpolar solvents such as petroleum ether
and chloroform were excluded from the study due to trace
recovery of polyphenols (results not shown). The extracts
obtained with the selected solvents differed slightly in qualitative composition but significantly in the amounts and relative
proportions of individual components. Among 39 polyphenols,
which were fully or tentatively identified by LC-MS/MS, the
most structurally diversified were flavan-3-ols and procyanidins
(20 analytes), while the highest concentration was observed for
salicylates with only three detected representatives (PH, TG
and GT). Despite the presence of salicylates, which is in
accordance with the phytochemistry of the genus Gaultheria,15
their abundance distinguishes teaberries among fruits of other
Gaultheria species investigated to date, such as G. shallon,14,40
G. mucronata and G. antarctica,12 as well as G. phyllireifolia and
G. poeppiggi,32 where procyanidins or flavonoids prevailed and
salicylates have not been detected.
The salicylates PH, TG and GT were isolated from the fruits
and identified by means of spectroscopic studies, including
1D and 2D NMR experiments. All isolates were glycosides that
confirmed the suggestions of Ribnicky et al. (2003).9 PH was
previously isolated from Physalis angulata whole plant35 and
probably from the leaves of G. procumbens. However, the low
amount and low purity of the isolated compound allowed the
earlier suggestion of the structure based only on the 1H-NMR
spectra.33 TG was obtained for the first time from
G. yunnanensis aerial parts,22,41 and since then, it was found
only in two other plant materials, i.e. in ripe fruits of the
cherry tomato Lycopersicum esculentum var. cerasiforme34 and
aerial parts of G. trichoclada.37 The presence of GT has been
observed in the leaves and stems of several Gaultheria plants,
including G. procumbens,9,24,31,33 but here, it was detected for
the first time in Gaultheria fruits. Moreover, the present work
is the first report providing full NMR data of PH, GT and TG,
especially for their sugar moieties, compared to only partial
assignments reported earlier.33–37
The total levels of polyphenols in plants are routinely determined by the Folin–Ciocalteu method, because the results may
be easily compared with the data in the literature. However, in
the case of methyl salicylate glycosides, the TPC levels may
strongly underestimate the real contents due to the blocked
hydroxyl group and related low reactivity of the aglycone in
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Paper
redox reactions. The levels of low-molecular weight polyphenols (TPH) in teaberries were thus accurately established by
HPLC-PDA, while total procyanidins including the condensed
ones (TPA) were estimated by the n-butanol/HCl assay. The
results indicated that TPH + TPA levels or even TPH contents
alone indeed surpassed the TPC values in most extracts.
Previously, Pliszka et al. (2009, 2016)13,42 have only examined
the TPC contents in fresh fruits extracted with aqueous methanol (1.3 mg GAE per g fw) and with water acidified with citric
acid (1.8–4.9 mg GAE per g fw). These values are in good agreement with the TPC levels obtained by us for the ME and WE
and the fruit fresh weight (4.9–7.7 mg GAE per g fw) (ESI
Table S4†). However, as indicated above, the real total phenolic
content in the extracts is better reflected in the TPH levels or
in the sum of TPH and TPA. Nevertheless, the TPC levels
(extracted with aqueous methanol) are in the range commonly
achieved by willingly consumed ericaceous berries with
acknowledged health-promoting properties, such as salal
berries (G. shallon; 9.7 mg GAE per g fw), blueberries
(Vaccinium myrtillus; 5.8 mg GAE per g fw), and cranberries (V.
oxycoccos; 3.7 mg GAE per g fw).14,43 The levels of salicylates in
teaberries calculated in equivalents of salicylic acid (9.9 mg
per g fruit dw, considering the extraction yield of ME) (ESI
Table S4†) significantly exceed those of spices, such as cumin,
coriander, and vanilla beans (0.1–0.6 mg per g dw), known to
date as the richest sources of dietary salicylates.5
The anti-inflammatory effects of plant salicylates are relatively well documented and connected with their potential to
treat inflammatory disorders, primarily rheumatoid arthritis,
osteoarthritis, swelling, muscular pain, and inflammationrelated skin and periodontal problems.6 This activity profile
corresponds well to the therapeutic application of
G. procumbens in traditional medicine.15,16 To verify the antiinflammatory potential of teaberries and to select the fruit
extract most suitable for functional application, four extracts
(AE, ME, BE and WE) were chosen for biological activity tests
due to the high recovery of total polyphenols and salicylates.
EAE revealed the lowest extraction yield and TPC levels and
was excluded from further research.
Inflammation is closely linked to oxidative stress. Immune
cells release a number of ROS at the site of inflammation,
leading to oxidative stress and tissue injury, which in turn may
trigger cellular signalling and secretion of proinflammatory
mediators, enhancing ROS production.38 Polyphenols may
interrupt the ROS-inflammation cycle by various direct and
indirect mechanisms.39 The current study indicated that the
extracts of teaberries exhibit dose-dependent, but relatively
weak, direct scavenging activity towards both synthetic and
ROS generated in vivo by immune cells (O2•−, •OH, and H2O2).
In consequence, they also revealed a low ability to inhibit ROSdependent lipid peroxidation (TBARS) and low reducing power
(FRAP). Similar to low TPC levels, this fact might be linked to
the high content of salicylates and their low redox reactivity.
Thus, the direct antioxidant capacity of the extracts might be
influenced by other constituents, especially procyanidins, as it
was suggested by the correlation studies and potent activity of
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the model compound (PB2). In contrast, the extracts turned
out to be strong inhibitors of two proinflammatory enzymes,
COX-2 and HYAL. This activity was assigned to salicylates.
COX-2 is an inducible enzyme converting arachidonic acid
into prostaglandins and thromboxane, the chief mediators of
inflammation.44 HYAL is one of the tissue remodelling
enzymes that catalyses the degradation of hyaluronic acid (a
constituent of the extracellular matrix), increases tissue permeability and facilitates the migration of proinflammatory
mediators.45 In clinical practice, both enzymes, especially
COX-2, are targets for anti-inflammatory therapies.
Downregulation of COX-2 activity, either directly or by modulation of proinflammatory signalling pathways, is believed to
be one of the major molecular mechanisms of in vivo effects of
both natural and synthetic salicylates.46 We observed that AE
is a stronger direct inhibitor of COX-2 than the popular antiinflammatory drugs DEX and IND. Interestingly, the activity of
AE surpassed that of pure salicylates. It might suggest some
synergy between its individual salicylate components.
Moreover, the use of extracts instead of pure compounds
might offer benefits coming from procyanidins and their
synergic effects. Due to the relatively high antioxidant capacity
and enzyme inhibitory potential, AE was selected for further
study in a cellular ex vivo model. Moreover, as differences in
chemical composition and activity between AE and ME were
relatively small in most cases, AE might well represent the
overall matrix and traditional medicinal preparations of the
fruits.
Neutrophils are the most abundant immune cells in
human blood and one of the first responders to infection or
tissue injury. After activation, they release a large amount of
ROS and proinflammatory mediators orchestrating the inflammation process. The persistent activation of neutrophils may
induce tissue damage and contribute to the pathogenesis of
chronic inflammatory disorders.47
The study revealed that AE and its main constituents did
not deteriorate the viability of neutrophils but significantly
and in a dose-dependent manner downregulated the levels of
ROS, proinflammatory cytokines, and extracellular matrix
degrading enzymes in cell cultures stimulated by fMLP, LPS, or
fMLP + cytochalasin B, depending on the test. The most spectacular effects were observed towards ROS, TNF-α, IL-1β and
ELA-2.
The factor fMLP is a bacterial derived potent chemoattractant that activates NADPH oxidase, initiating the process of
oxidative burst and rapid release of ROS, including O2•−, •OH,
and H2O2.48 We observed that, in comparison to the positive
control (QU), the cellular antioxidant activity of the analytes,
especially salicylates, was significantly stronger than their relative ability to directly scavenge ROS observed in non-cellular
tests. It suggested that the antioxidant effects of AE and its salicylate constituents in cells are mediated by some indirect
mechanisms. These findings are in accordance with previous
research on both synthetic and natural salicylates. The synthetic ones have been reported to reduce the binding of fMLP
to its receptors in intact neutrophils,49 thus inhibiting both
Food Funct.
Food & Function
the priming and activation of NADPH oxidase.47,48 They were
also proven to downregulate transcription factors, such as NFκB, and several kinases that control the secretion of regulatory
cytokines, including TNF-α,46 which can prime cells for oxidative burst.47,48 Among natural salicylates, three glycosides
from aerial parts of G. yunnanensis, including GT, have been
previously reported to inhibit the production of ROS in
immune cells, but animal macrophages (RAW 267.4) stimulated by LPS were used as a model.18,19 The mechanistic
studies confirmed that the effects of Gaultheria salicylates are
connected with their ability to modulate the MAPK/NF-κB signalling pathway.19,20 In the same studies, the inhibitory effect
on the release of proinflammatory cytokines (TNF-α, IL-1β, and
IL-6) from LPS-stimulated murine cells has been documented
for G. yunnanensis salicylates, which is in accordance with our
results from the G. procumbens and human neutrophil model.
Moreover, during the current study, we observed that AE and
Gaultheria salicylates are strong inhibitors of the secretion of
ELA-2 from immune cells. Salicylates were indeed found to be
chief contributors to the observed activity, but few additive
and/or synergistic effects might be linked to procyanidins,
despite their lower proportion in the extract. At the relevant
molar concentration of salicylates, the inhibitory effect of AE
on the release of TNF-α and IL-1β did not differ significantly
from that of DEX. Furthermore, the effect of AE towards ELA-2
was significantly stronger than that of the positive control QU
at the relevant level.
The proinflammatory factors TNF-α, IL-1β and ELA-2 are
connected with the progression of numerous chronic human
disorders. TNF-α and IL-1β are powerful priming agonists of
neutrophils and pleiotropic master cytokines that orchestrate
the immune response and stimulate gene expression and
release of numerous proinflammatory chemokines and
enzymes, including COX-2. They are also important neutrophil
prosurvival agents and modulators of the recruitment and
adhesion of various immune cells to vascular endothelium,
which is a key step in the development of inflammation.47,50,51
ELA-2 is a tissue remodelling matrix metalloproteinase that
especially degrades elastin and collagen, increases tissue permeability, and stimulates the release of some neutrophil chemoattractants, such as IL-8, thus potentiating inflammation.52
All of these factors are targets for anti-inflammatory therapies
against e.g. rheumatoid arthritis, gout, inflammatory bowel
disease, psoriasis, and chronic obstructive pulmonary
disease.50–52 The potential of the teaberry extract to downregulate the levels of TNF-α, IL-1β, ELA-2, and ROS in stimulated
neutrophils and its relevant capacity to directly inhibit COX-2
and HYAL might thus support the therapeutic use of the plant
in inflammatory disorders, as reported by traditional
medicine.15,16 It might also be an argument for the preventive
value of the fruits as dietary ingredients against chronic
inflammation connected with oxidative stress and for the use
of AE in inflammation-targeted functional food products.
The previous studies on G. yunnanensis suggested that the
significant anti-inflammatory potential of salicylate-rich fractions and pure Gaultheria salicylates in cellular in vitro
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Food & Function
models18–20 stands in correspondence with their anti-inflammatory effects in vivo.20–22,36 In particular, the significant suppression of the levels of TNF-α and IL-1β by methyl salicylate 2O-β-D-lactoside was evidenced both in vitro (RAW 267.4 macrophages) and in vivo (in rats), and the later observations correlated with the alleviated symptoms of inflammation in adjuvant arthritic animals.19,20 This might be a result of the relatively high bioavailability of salicylates and their pharmacokinetics. According to the accumulated knowledge, glycosidic
salicylate esters, such as GT, are cleaved by intestinal microflora to their aglycones, for example, methyl salicylate, which
are fast absorbed and hydrolysed by non-specific esterases in
blood and liver to free salicylate as the main active
metabolite.21,36,53 Both the liberated salicylic acid and trace
methyl salicylate are partly conjugated with glucuronic
acid.53,54 Finally, all of the metabolites present in the intestine
and serum have the same salicylate active core as their parent
glycosides. Moreover, the bioavailability of salicylate glycosides, judged by the example of salicin (the most extensively
studied natural salicylate glycoside), was about 43%, calculated
as total salicylate in serum, which surpasses the values typically observed for non-salicylate polyphenols including procyanidins.54 All these facts correspond with the effectiveness of
natural salicylates as anti-inflammatory agents in vivo. For
instance, significant alleviation in the symptoms of induced
inflammation was revealed in animals treated by either pure
glycosides of salicylic acid esters, including GT,21,36 or the salicylate-enriched fraction from aerial parts of G. yunnanensis,
containing 50% GT.22 The effects observed at doses of 400 mg
per kg and 800 mg per kg body weight, respectively, were equivalent to those of aspirin at 200 mg per kg body weight. Here,
we report on the TSAL and GT levels in G. procumbens fruit
extracts of 121.7 mg per g dw and 93.6 mg per g dw, respectively. Thus, the beneficial in vivo effects might be expected for
AE or the source fruits at relevant oral doses. In the application
context, it is worth emphasizing that GT and other glycosidic
salicylates do not cause side effects typical for the synthetic
ones, such as gastric ulcer toxicity, and are generally regarded
as safe in internal applications,21 except in the case of hypersensitivity to salicylates.
5. Conclusions
This is the first study on the phytochemical profile of
G. procumbens fruits and their anti-inflammatory and antioxidant effects. The results revealed that the fruits accumulate
a diversified fraction of polyphenols at the levels comparable
to those of willingly consumed ericaceous berries. Moreover,
teaberries seem to be the richest dietary source of salicylates
known. They are also procyanidin abundant. Their active polyphenols including salicylates and procyanidins may be
efficiently concentrated by extraction with acetone, and the
obtained dry extract (AE) might be recommended for specialized functional applications. Three methyl salicylate glycosides
dominate in AE and primarily contribute to its biological
This journal is © The Royal Society of Chemistry 2020
Paper
effects in vitro. The extract is able to significantly modulate the
proinflammatory and pro-oxidant functions of human neutrophils, especially to downregulate the release of TNF-α, IL-1β,
ELA-2, and ROS from the stimulated cells. The direct inhibitory effects of AE towards COX-2 and HYAL are also noticeable.
According to the accumulated knowledge on plant salicylates
and their pharmacokinetics, the results obtained during the
present study might support the usage of the fruits as components of an anti-inflammatory diet. However, further and
more intrinsic studies are required to evaluate the in vivo
effects (both health-promoting and potentially toxic) of the
fruits and AE in short- and long-term consumption and to
establish the effective dose that would produce a biological
response necessary for both therapeutic and adjunctive or prophylactic application of the extract and fruits as functional
food products. The further molecular mechanisms that might
be related to the in vitro and in vivo effects should also be
addressed in future research.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgements
Funding: This work was supported by National Science Centre,
Poland (Grant Project: UMO-2015/19/N/NZ7/00959).
References
1 F. Zhu, B. Du and B. Xu, Anti-inflammatory effects of phytochemicals from fruits, vegetables, and food legumes: A
review, Crit. Rev. Food Sci. Nutr., 2018, 58, 1260–1270.
2 T. Hussain, B. Tan, Y. Yin, F. Blachier, M. C. B. Tossou and
N. Rahu, Oxidative stress and inflammation: What polyphenols can do for us?, Oxid. Med. Cell. Longevity, 2016, 2016,
7432797.
3 G. G. Duthie and A. D. Wood, Natural salicylates: foods,
functions and disease prevention, Food Funct., 2011, 2,
515–520.
4 G. Vizzari, M. C. Sommariva, M. D. Cas, S. Bertoli,
S. Vizzuso, G. Radaelli, A. Battezzati, R. Paroni and
E. Verduci, Circulating salicylic acid and metabolic profile
after 1-year nutritional–behavioral intervention in children
with obesity, Nutrients, 2019, 11, 1–11.
5 S. Malakar, P. R. Gibson, J. S. Barrett and J. G. Muir,
Naturally occurring dietary salicylates: A closer look at
common Australian foods, J. Food Compos. Anal., 2017, 57,
31–39.
6 P. Mao, Z. Liu, M. Xie, R. Jiang, W. Liu, X. Wang, S. Meng
and G. She, Naturally occurring methyl salicylate glycosides, Mini-Rev. Med. Chem., 2014, 14, 56–63.
7 S. Facciola, Cornucopia: a source book of edible plants,
Kampong Publications, Vista, USA, 2nd edn, 1998.
Food Funct.
View Article Online
Published on 19 August 2020. Downloaded by Cornell University Library on 8/20/2020 7:26:43 AM.
Paper
8 D. E. Moerman, Native American food plants: an ethnobotanical dictionary, Timber Press, Portland, USA, 2010.
9 D. M. Ribnicky, A. Poulev and I. Raskin, The determination
of salicylates in Gaultheria procumbens for use as a natural
aspirin alternative, J. Nutraceuticals, Funct. Med. Foods,
2003, 4, 39–52.
10 E. Villagra, C. Campos-Hernandez, P. Cáceres, G. Cabrera,
Y. Bernardo, A. Arencibia, B. Carrasco, P. D. S. Caligari,
J. Pico and R. García-Gonzales, Morphometric and phytochemical characterization of chaura fruits (Gaultheria
pumila): a native Chilean berry with commercial potential,
Biol. Res., 2014, 47, 1–8.
11 S. Karuppusamy, G. Muthuraja and K. M. Rajasekaran,
Antioxidant activity of selected lesser known edible fruits
from Western Ghats of India, Indian J. Nat. Prod. Resour.,
2011, 2, 174–178.
12 A. Ruiz, I. Hermosín-Gutiérrez, C. Vergara, D. von Baer,
M. Zapata, A. Hitschfeld, L. Obando and C. Mardones,
Anthocyanin profiles in south Patagonian wild berries by
HPLC-DAD-ESI-MS/MS, Food Res. Int., 2013, 51, 706–713.
13 B. Pliszka, J. Waźbińska and G. Huszcza-Ciołkowska,
Polyphenolic compounds and bioelements in fruits of
eastern teaberry (G. procumbens L.) harvested in different
fruit maturity phases, J. Elem., 2009, 14, 341–348.
14 G. J. Mcdougall, C. Austin, E. Van Schayk and P. Martin,
Salal (Gaultheria shallon) and aronia (Aronia melanocarpa)
fruits from Orkney: phenolic content, composition and
effect of wine-making, Food Chem., 2016, 205, 239–247.
15 W. R. Liu, W. L. Qiao, Z. Z. Liu, X. H. Wang, R. Jiang,
S. Y. Li, R. B. Shi and G. M. She, Gaultheria: Phytochemical
and pharmacological characteristics, Molecules, 2013, 18,
12071–12108.
16 B. Luo, R. Gu, E. J. Kennelly and C. Long, Gaultheria, ethnobotany and bioactivity: blueberry relatives with antiinflammatory, antioxidant, and anticancer constituents,
Curr. Med. Chem., 2017, 25, 5168–5176.
17 W. Xin, C. Huang, X. Zhang, G. Zhang, X. Ma, L. Sun,
C. Wang, D. Zhang, T. Zhang and G. Du, Evaluation of the
new anti-inflammatory compound ethyl salicylate 2-O-β-Dglucoside and its possible mechanism of action, Int.
Immunopharmacol., 2013, 15, 303–308.
18 D. Zhang, R. Liu, L. Sun, C. Huang, C. Wang, D. M. Zhang,
T. T. Zhang and G. H. Du, Anti-inflammatory activity of
methyl salicylate glycosides isolated from Gaultheria yunnanensis (Franch.) Rehder, Molecules, 2011, 16, 3875–3884.
19 T. Zhang, L. Sun, R. Liu, D. Zhang, X. Lan, C. Huang,
W. Xin, C. Wang, D. Zhang and G. Du, A novel naturally
occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-kappaB activity in
RAW264.7 macrophages, Mol. Pharm., 2012, 9, 671–677.
20 X. Zhang, J. Sun, W. Xin, Y. Li, L. Ni, X. Ma, D. Zhang,
D. Zhang, T. Zhang and G. Du, Anti-inflammation effect of
methyl salicylate 2-O-β-D-lactoside on adjuvant inducedarthritis rats and lipopolysaccharide (LPS)-treated murine
macrophages RAW264.7 cells, Int. Immunopharmacol., 2015,
25, 88–95.
Food Funct.
Food & Function
21 B. Zhang, X. L. He, Y. Ding and G. H. Du, Gaultherin, a
natural salicylate derivative from Gaultheria yunnanensis:
Towards a better non-steroidal anti-inflammatory drug,
Eur. J. Pharmacol., 2006, 530, 166–171.
22 B. Zhang, J. B. Li, D. M. Zhang, Y. Ding and G. H. Du,
Analgesic and anti-inflammatory activities of a fraction
rich in gaultherin isolated from Gaultheria yunnanensis,
(Franch.) Rehder, Biol. Pharm. Bull., 2007, 30, 465–469.
23 A. Magiera, M. Sienkiewicz, M. A. Olszewska, A. Kicel and
P. Michel, Chemical profile and antibacterial activity of
essential oils from leaves and fruits of Gaultheria procumbens L. cultivated in Poland, Acta Pol. Pharm. – Drug Res.,
2019, 76, 93–102.
24 P. Michel, S. Granica, A. Magiera, K. Rosińska, M. Jurek,
Ł. Poraj and M. A. Olszewska, Salicylate and procyanidinrich stem extracts of Gaultheria procumbens L. inhibit proinflammatory enzymes and suppress pro-inflammatory and
pro-oxidant functions of human neutrophils ex vivo,
Int. J. Mol. Sci., 2019, 20, 1–17.
25 P. Michel, A. Dobrowolska, A. Kicel, A. Owczarek,
A. Bazylko, S. Granica, J. P. Piwowarski and
M. A. Olszewska, Polyphenolic profile, antioxidant and antiinflammatory activity of eastern teaberry (Gaultheria procumbens L.) leaf extracts, Molecules, 2014, 19, 20498–20520.
26 M. A. Olszewska and J. M. Roj, Phenolic constituents of the
inflorescences of Sorbus torminalis (L.) Crantz, Phytochem.
Lett., 2011, 4, 151–157.
27 M. A. Olszewska, A. Presler and P. Michel, Profiling of phenolic compounds and antioxidant activity of dry extracts
from the selected Sorbus species, Molecules, 2012, 17, 3093–
3113.
28 M. Matczak, A. Marchelak, P. Michel, A. Owczarek,
A. Piszczan, J. Kolodziejczyk-Czepas, P. Nowak and
M. A. Olszewska, Sorbus domestica L. leaf extracts as functional products: phytochemical profiling, cellular safety,
pro-inflammatory enzymes inhibition and protective effects
against oxidative stress in vitro, J. Funct. Foods, 2018, 40,
207–218.
29 A. Marchelak, A. Owczarek, M. Rutkowska, P. Michel,
J. Kolodziejczyk-Czepas, P. Nowak and M. A. Olszewska,
New insight into antioxidant activity of Prunus spinosa
flowers: extracts, model polyphenols and their phenolic
metabolites in plasma towards multiple in vivo-relevant
oxidants, Phytochem. Lett., 2019, 30, 288–295.
30 J. Katanić, E. M. Pferschy-Wenzig, V. Mihailović, T. Boroja,
S. P. Pan, S. Nikles, N. Kretschmer, G. Rosić, D. Selaković,
J. Joksimović and R. Bauer, Phytochemical analysis and
anti-inflammatory effects of Filipendula vulgaris Moench
extracts, Food Chem. Toxicol., 2018, 122, 151–162.
31 P. Michel, A. Owczarek, M. Kosno, D. Gontarek,
M. Matczak and M. A. Olszewska, Variation in polyphenolic
profile and in vitro antioxidant activity of eastern teaberry,
(Gaultheria procumbens L.) leaves following foliar development, Phytochem. Lett., 2017, 20, 356–364.
32 D. Mieres-Castro, G. Schmeda-Hirschmann, C. Theoduloz,
S. Gómez-Alonso, J. Pérez-Navarro, K. Márquez and
This journal is © The Royal Society of Chemistry 2020
View Article Online
Food & Function
33
Published on 19 August 2020. Downloaded by Cornell University Library on 8/20/2020 7:26:43 AM.
34
35
36
37
38
39
40
41
42
F. Jiménez-Aspee, Antioxidant activity and the isolation of
polyphenols and new iridoids from Chilean Gaultheria phillyreifolia and G. poeppigii berries, Food Chem., 2019, 291,
167–179.
S. Carlin, D. Masuero, G. Guella, U. Vrhovsek and
F. Mattivi, Methyl salicylate glycosides in some Italian varietal wines, Molecules, 2019, 24, 1–15.
M. Ono, Y. Shiono, T. Tanaka, C. Masuoka, S. Yasuda,
T. Ikeda, M. Okawa, J. Kinjo, H. Yoshimitsu and T. Nohara,
Three new aromatic glycosides from the ripe fruit of cherry
tomato, J. Nat. Med., 2010, 64, 500–505.
C. P. Sun, X. F. Nie, N. Kang, F. Zhao, L. X. Chen and
F. Qiu, A new phenol glycoside from Physalis angulata, Nat.
Prod. Res., 2017, 31, 1059–1065.
C. Wang, T. T. Zhang, G. H. Du and D. M. Zhang, Synthesis
and anti-nociceptive and anti-inflammatory effects of
gaultherin and its analogs, J. Asian Nat. Prod. Res., 2011,
13, 817–825.
G. L. Xu, Z. Z. Liu, M. Xie, X. Zhang, Y. Yang, C. Yan,
W. L. Qiao, W. R. Liu, R. Jiang, X. H. Wang and G. M. She,
Salicylic acid derivatives and other components from
Gaultheria trichoclada, Chem. Nat. Compd., 2016, 52, 301–
303.
L. Zuo, E. R. Prather, M. Stetskiv, D. E. Garrison,
J. R. Meade, T. I. Peace and T. Zhou, Inflammaging and oxidative stress in human diseases: from molecular mechanisms to novel treatments, Int. J. Mol. Sci., 2019, 20, 4472.
N. Yahfoufi, N. Alsadi, M. Jambi and C. Matar, The immunomodulatory and anti-inflammatory role of polyphenols,
Nutrients, 2018, 10, 1–23.
A. Ferguson, E. Carvalho, G. Gourlay, V. Walker, S. Martens,
J. P. Salminen and C. P. Constabel, Phytochemical analysis
of salal berry (Gaultheria shallon Pursh.), a traditionallyconsumed fruit from western North America with exceptionally high proanthocyanidin content, Phytochemistry,
2018, 147, 203–210.
Z. Liu, R. Jiang, M. Xie, G. Xu, W. Liu, X. Wang, H. Lin,
J. Lu and G. She, A rapid new approach for the quality
evaluation of the folk medicine Dianbaizhu based on chemometrics, Chem. Pharm. Bull., 2014, 62, 1083–1091.
B. Pliszka, G. Huszcza-Ciołkowska and E. Wierzbicka,
Effects of solvents and extraction methods on the content
and antiradical activity of polyphenols from fruit Actinidia
arguta, Crataegus monogyna, Gaultheria procumbens and
Schisandra chinensis, Acta Sci. Pol., Technol. Aliment., 2016,
15, 57–63.
This journal is © The Royal Society of Chemistry 2020
Paper
43 G. I. Hidalgo and M. P. Almajano, Red fruits: extraction of
antioxidants, phenolic content, and radical scavenging
determination: a review, Antioxidants, 2017, 6, 7.
44 M. D. Ferrer, C. Busquets-Cortés, X. Capó, S. Tejada,
J. A. Tur, A. Pons and A. Sureda, Cyclooxygenase-2 inhibitors as a therapeutic target in inflammatory diseases, Curr.
Med. Chem., 2018, 26, 3225–3241.
45 K. Girish, K. Kemparaju, S. Nagaraju and B. Vishwanath,
Hyaluronidase inhibitors: a biological and therapeutic perspective, Curr. Med. Chem., 2009, 16, 2261–2288.
46 M. T. Baltazar, R. J. Dinis-Oliveira, J. A. Duarte,
M. L. Bastos and F. Carvalho, Antioxidant properties and
associated mechanisms of salicylates, Curr. Med. Chem.,
2011, 18, 3252–3264.
47 H. L. Wright, R. J. Moots, R. C. Bucknall and
S. W. Edwards, Neutrophil function in inflammation and
inflammatory diseases, Rheumatology, 2010, 49, 1618–1631.
48 G. T. Nguyen, E. R. Green and J. Mecsas, Neutrophils to the
ROScue: Mechanisms of NADPH oxidase activation and bacterial resistance, Front. Cell. Infect. Microbiol., 2017, 25, 373.
49 S. B. Abramson, B. Cherksey, D. Gude, J. LeszczynskaPiziak, M. R. Philips, L. Blau and G. Weissmann,
Nonsteroidal antiinflammatory drugs exert differential
effects on neutrophil function and plasma membrane viscosity. Studies in human neutrophils and liposomes,
Inflammation, 1990, 14, 11–30.
50 H. M. Hoffman and A. A. Wanderer, Inflammasome and
IL-1β-mediated disorders, Curr. Allergy Asthma Rep., 2010,
10, 229–235.
51 G. D. Kalliolias and L. B. Ivashkiv, TNF biology, pathogenic
mechanisms and emerging therapeutic strategies, Nat. Rev.
Rheumatol., 2016, 12, 49–62.
52 P. A. Henriksen, The potential of neutrophil elastase
inhibitors as anti-inflammatory therapies, Curr. Opin.
Hematol., 2014, 21, 23–28.
53 [CIR] Cosmetic Ingredient Review, Safety assessment of salicylic acid, butyloctyl salicylate, calcium salicylate, C12-15
alkyl salicylate, capryloyl salicylic acid, hexyldodecyl salicylate, isocetyl salicylate, isodecyl salicylate, magnesium salicylate, MEA-salicylate, ethylhexyl salicylate, potassium salicylate, methyl salicylate, myristyl salicylate, sodium salicylate, TEA- salicylate, and tridecyl salicylate, Int. J. Toxicol.,
2003, 22(Suppl 3), 108.
54 B. Schmid, I. Kötter and L. Heide, Pharmacokinetics of
salicin after oral administration of a standardized willow
bark extract, Eur. J. Clin. Pharmacol., 2001, 57, 387–391.
Food Funct.