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Antioxidant and ACE Inhibitory Activity of Cultivated and Wild
Angelica gigas
Nakai Extracts Prepared Using Different Extraction
Conditions
Bo-Young Noh, Hye-Jin Lee, Jeong-Ryong Do, and Hyun-Ku Kim
Korea Food Research Institute, Gyeonggi 463-746, Korea
Prev. Nutr. Food Sci. 2014;19(4):274-280
http://dx.doi.org/10.3746/pnf.2014.19.4.274
pISSN 2287-1098ㆍeISSN 2287-8602
Received 11 September 2014; Accepted 10 October 2014; Published online 31 December 2014
Correspondence to Hyun-Ku Kim, Tel: +82-31-780-9134, E-mail: hyunku@kfri.re.kr
Copyright © 2014 by The Korean Society of Food Science and Nutrition. All rights Reserved.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT: The purpose of this study was to investigate the biological activities of cultivated Angelica gigas Nakai (CAG)
and wild Angelica gigas Nakai (WAG) extracts prepared by extraction with water, 30% ethanol, 60% ethanol, or 90%
ethanol. The electron donating ability of the WAG extracts was higher than that of the CAG extracts and 0.1% and 1.0%
solutions of the comparative substance, L-ascorbic acid. The superoxide dismutase-like activity of the CAG extracts was
higher than that of WAG extracts. Superoxide dismutase-like activity was highest (33.95%) in the CAG water extract.
The total polyphenol content was highest in the 60% ethanol extracts of WAG. The nitrite scavenging ability of the CAG
and WAG extracts was highest at a pH of 1.2. The tyrosinase inhibitory effect was highest (43.72%) in the water extract
of WAG. The angiotensin converting enzyme inhibitory activity was highest (83.84%) in the 60% ethanol extract of
WAG. The results of the present study will be useful for understanding the antioxidant and angiotensin-converting en-
zyme inhibitory activities of Angelica gigas Nakai extracts.
Keywords: antioxidant, cultivated Angelica gigas Nakai, wild Angelica gigas Nakai, extraction condition
INTRODUCTION
Dang-gwi (Angelica gigas Nakai; AG), a perennial plant in
the Apiaceae family, can be found throughout Korea,
China, and Japan. Based on its area of distribution, Angelica
gigas is classified into different cultivars: AG, Angelica si-
nensis (Oliv.) Diels, and Angelica acutiloba Kitagawa. AG
grows and is cultivated in the alpine region of Korea and
is called Cham-dang-gwi in Korean (1). The main com-
ponents of AG (i.e., decursin, decursinol, nodakenin,
-pinene, limonene, -eudesmol, and elemol) have vari-
ous pharmacologic effects. Over the years, AG has been
widely used for its various medicinal effects, including
its anti-bacterial effect, sedative action, analgesic effect,
and diuretic effect and its effects on uterine function,
blood circulation, anemia, and hemostasis. Since the
pharmacologic effects of AG became widely known, re-
searchers have studied the extraction, chemical compo-
nents (1), isolation and purification (2,3), anti-anemia
effect (4), cytotoxic effect (5), and immune-enhancing
effects (6) of AG. In addition, AG has been shown to
have positive effects on leukemia, kidney toxicity, dia-
betic hypertension, and cancer (7). The biological activ-
ity and antioxidant effects of AG have also been studied
(8-10). In addition, there have been several food patent
development studies exploring the incorporation of AG
into bread (11), fish cakes (12), kimchi (13), syrup, sik-
hye, and functional drinks. However, the study of the
development of natural functional foods containing AG
remains insufficient.
The present study will examined differences in the bi-
ological activities of cultivated Angelica gigas Nakai
(CAG) and wild Angelica gigas Nakai (WAG). Of partic-
ular interest are the effects of the growing environment
on the electron donating ability (EDA), superoxide dis-
mutase (SOD)-like activity, total polyphenol content, ty-
rosinase inhibitory effect, nitrite-scavenging effect, and
angiotensin-converting enzyme (ACE) inhibitory activity
of AG. This information will provide fundamental data for
the development natural functional foods containing AG.
MATERIALS AND METHODS
Materials and extraction
Two kinds of AG were used in this study: cultivated and
Antioxidant Activity of Angelica gigas Nakai Extracts 275
wild. CAG was purchased from the markets in Jeongseon,
Gangwon and WAG was taken from Mt. Sobaek
(Yeongju, Korea). After freeze-drying (FD 5512, Ilshin
Lab Co., Ltd., Seoul, Korea), the AG were ground and
stored frozen in a sealed 0.2 mm polyethylene film.
Afterwards, they were extracted in 25 mL of water (i.e.,
0% ethanol), 30% ethanol, 60% ethanol, or 90% ethanol
with microwave assisted extraction (MAE; Soxwave-100,
Prolabo, Paris, France) at 60 W for 5 min.
Electron donating ability
The reducing power of each extract was determined by
measuring the EDA according to a modified method by
Blois (14). Briefly, 0.2 mL of each extract was mixed
with 0.8 mL of 4×10
-4
M 1,1-diphenyl-2-picrylhydrazyl
(DPPH) and dissolved in 2 mL of 99.9% ethanol to yield
a total volume of 3 mL. After vortexing the mixtures for
10 s and incubating them at room temperature for 10
min, the absorbance of each mixture was measured at
525 nm using a SpectraMax M2 (Molecular Devices,
LLC., Sunnyvale, CA, USA). Ascorbic acid was used as
the positive control.
Superoxide dismutase (SOD)-like activity
To control the oxidation rate of the pyrogallol, which is
oxidized by superoxide, the SOD-like activity of each ex-
tract was measured by the method of Marklund and
Marklund (15). The pH of each sample was adjusted to
8.5 using a tris-HCl buffer [50 mM tris(hydroxymethyl)
aminomethane, 10 mM EDTA]. Three milliliters of the
tris-HCl buffer and 0.2 mL of 7.2 mM pyrogallol were
added to 0.2 mL of each sample. The mixtures were in-
cubated at 25
o
C for 10 min and then 0.2 mL of 1 N HCl
was added to stop the reaction. The absorbance of the
resulting mixtures was measured at 420 nm using a
UV/VIS spectrometer (SpectraMax M2, Molecular Devi-
ces, LLC.). Ascorbic acid was used as the positive con-
trol.
Total polyphenol content
The total polyphenol content of each extract was meas-
ured by the method of Folin-Denis (16). Briefly, 0.5 mL
of 1 N Folin-Ciocalteu reagent was added to 0.5 mL of
each sample. The mixture was allowed to incubate for 3
min and then 10 mL of 2% Na
2
CO
3
solution was added.
The resulting solution was mixed, incubated for 1 h, and
then the absorbance was measured at 750 nm using a
UV/VIS spectrometer (V-570, Jasco Inc., Hachioji, Japan).
The total polyphenol content was determined from a
tannic acid standard curve.
Tyrosinase inhibitory effect
The tyrosinase inhibitory effect was measured by the
method of Wong et al. (17). A crude tyrosinase solution
was prepared by dissolving mushroom tyrosinase in 50
mM sodium phosphate buffer (pH 7.0). Subsequently,
0.2 mL of the crude tyrosinase solution and 0.1 mL of
extract were added to 2.8 mL of 10 mM catechol
solution. For the control sample, 50 mL of sodium phos-
phate buffer (pH 7.0) was added to the mixture instead
the crude tyrosinase solution. The absorbance of each
reaction mixture was measured at 420 nm by a UV/VIS
spectrometer (V-570, Jasco Inc.). Ascorbic acid was used
as the positive control.
Nitrites cavenging effect
The method described by Gray and Dugan (18) was
used to measure the nitrite scavenging effect of each
extract. Briefly, 0.1 mL of 1 mM NaNO
2
solution was
added to 0.2 mL of each extract, and the pH values of
the resulting mixtures were adjusted to 1.2 (0.1 N HCl),
3.0, 4.2, or 6.0 using a 0.2 N of buffer solution. The final
volume of each sample was adjusted to 1 mL. The sam-
ples were allowed to react at 37
o
C for 1 h, mixed thor-
oughly with 5 mL of 2% acetic acid and 0.4 mL of Griess
reagent, and then incubated at room temperature for 15
min. The nitrite content of each mixture was determined
by measuring the absorbance at 520 nm (SpectraMax M2,
Molecular Devices, LLC.). The control was processed
with the same method, but was prepared with 0.4 mL of
distilled water instead of 0.4 mL Griess reagent. Ascor-
bic acid was used as the positive control.
ACE inhibitory activity
ACE inhibitory activity was measured by the modified
method of Cushman (19). ACE crude enzyme extract
was prepared by adding rabbit lung acetone powder
(Sigma, St. Louis, MO, USA) to 300 mM NaCl-100 mM
sodium borate buffer (pH 8.3) at a ratio of 0.2 g/10 mL
(w/v). The mixture was incubated at 4
o
C for 24 h and
then centrifuged (4
o
C, 8,000 rpm, 70 min) to obtain
ACE crude enzyme extract.
To measure ACE inhibitory activity, 100 L of 450
mM NaCl-100 mM sodium borate buffer (pH 8.3) and
50 L of 5 mM hippuryl-histidyl-leucine were added to
50 L of each AG extract, and the mixtures were
pre-cultivated at 4
o
C for 10 min. Next, 50 L of the ACE
crude enzyme extract was added to each mixture and the
mixtures were incubated at 37
o
C for 30 min. Then 100
L of 1.75 N HCl and 1.5 mL of ethyl acetone were add-
ed to each mixture and 1 mL of supernatant was
obtained. The supernatant was dried at 100
o
C for 1 h.
Then 1 mL of distilled water was added to dissolve the
residue, and the absorbance of the resulting solution
was measured at 228 nm using a UV/VIS spectrometer
(V-570, Jasco Inc.). The control was processed with the
same method, but was prepared with 50 L of distilled
water instead of the AG extract.
276 Noh et al.
Fig. 1. Electron donating ability of microwaved
Angelica giga
s
Nakai extracts. □: cultivated
Angelica gigas
Nakai, ■: wild
Angelica gigas
Nakai. Microwave-assisted extraction (60 W, 5
min) was performed on a mixture composed of 2 g of sample
and 50 mL of solvent.
1)
Means with the same letter are not sig-
nificantly different (
P
＜0.05).
Fig. 2. SOD-like activity of microwaved
Angelica gigas
Nakai
extracts.
□: cultivated
Angelica gigas
Nakai, ■: wild
Angelic
a
gigas
Nakai. Microwave-assisted extraction (60 W, 5 min) was
performed on a mixture composed of 2 g of sample and 50 mL
of solvent.
1)
Means with the same letter are not significantly dif-
ferent (
P
＜0.05).
Statistical analysis
Duncan’s multiple range tests were used to examine the
significance of the differences among the experimental
groups (SAS version 8.0, SAS Institute Inc., Cary, NC,
USA).
RESULTS AND DISCUSSION
Electron donating ability
Fig. 1 shows the DPPH radical scavenging activity of cul-
tivated and wild AG extracts. For all extraction methods,
the EDA of WAG was higher than the EDA of CAG
(WAG: 88.05∼96.03%, CAG: 68.02∼93.94%). The EDA
of the ethanol extracts was higher than the EDA of the
water extracts; EDA increased as the ethanol content of
the extracts increased. Among the extracts tested, the
EDA of the 90% ethanol extract of WAG was the high-
est (96.03%). The EDA of the 90% ethanol extract of
CAG and WAG was 93.94% and 96.03%, respectively,
while the EDA of 0.1% L-ascorbic acid and 1% L-ascor-
bic acid, the comparative substance, was 86.97% and
90.83%, respectively.
According to the research of Chang et al. (20), the
EDA of an AG extract by reflux condensing added to the
water with 1:20 ratio (v/w) is 94.37%, which is higher
than the EDA value measured in the AG water extracts
in the present study. However, other authors have re-
ported AG water extract EDA values that are lower than
those measured in the present study. For example, Kang
et al. (8) reported that the EDA of a water extract of AG
was 66.8% and Ahn et al. (9) reported that the EDA of a
water extract of AG was 27.89%. These differences in
EDA may be due to differences in the extract to solvent
ratio and differences in the extraction methods used.
The EDA values reported in the present study are
greater than those reported for other medicinal plants.
For example, Lee et al. (21) found that the EDA of
Angelica genuflexa Nutt, another medicinal plant of the
Apiaceae family, is between 0.51% and 49.79%, depend-
ing on the extract contents and solvents. According to
Jeong et al. (22), the EDA of most of the medicinal plants
that are available at the market is lower than the EDA of
AG. For example, an 80% methanol (MeOH) extract of
peony has an EDA of 57.1%, an 80% MeOH extract of
ginger has an EDA of 48.3%, and an 80% MeOH extract
of microphyll has an EDA 37.2%. In a study of Angelica
utilis Makino, the EDA of red Angelica utilis Makino was
higher than that of green Angelica utilis Makino, depend-
ing on kinds of Angelica sp. and extract solvents showed
the highest (23) as AG’s activity changes. Although the
EDA of AG was different on kinds, extract methods, ex-
tract solvents, and extract ratio, the results of the cur-
rent study indicate that the EDA of AG is greater than
the EDA of other medicinal plants.
SOD-like activity
The results of the SOD-like activity determinations are
shown in Fig. 2. The SOD-like activity of CAG and WAG
was under 34%, while the activity of 0.1% L-ascorbic
acid and 1% of L-ascorbic acid was 60.65% and 75.93%,
respectively. Overall, the SOD-like activity of CAG was
higher than that of WAG, regardless of the solvent used.
While SOD-like activity was higher in the water-ex-
tracted AG samples than in the ethanol-extracted AG
samples, SOD-like activity was not affected by the con-
centration of ethanol in the extraction solvent.
Kim et al. (24) reported that the SOD-like activity of
AG is 11.6%, which is lower than the SOD-like activity
of AG measured in this study. This difference may be
due to differences in the extraction method used and the
contents of the extracts. The SOD-like activity measured
Antioxidant Activity of Angelica gigas Nakai Extracts 277
Fig. 3. Total polyphenol contents (mg%) of microwaved
Angelic
a
gigas
Nakai extracts. □: cultivated
Angelica gigas
Nakai, ■:
wild
Angelica gigas
Nakai. Microwave-assisted extraction (60 W,
5 min) was performed on a mixture composed of 2 g of sample
and 50 mL of solvent.
1)
Means with the same letter are not sig-
nificantly different (
P
＜0.05).
Fig. 4. Tyrosinase inhibitory effects of microwaved
Angelic
a
gigas
Nakai extracts. □: cultivated
Angelica gigas
Nakai, ■:
wild
Angelica gigas
Nakai. Microwave-assisted extraction (60 W,
5 min) was performed on a mixture composed of 2 g of sample
and 50 mL of solvent.
1)
Means with the same letter are not sig-
nificantly different (
P
＜0.05).
in the CAG and WAG extracts of the current study was
lower than the SOD-like activity of extracts from other
medicinal plants, such as Epimedium koreanum Nakai
(42.4%), Acanthopanax (24.2%), and Phlomis root
(21.3%), that were extracted under the same conditions
as those used in the current study (7). The antioxidant
activity of water extracts from elm tree and Hemiptelea
davidii is 14.51∼98.24% and the antioxidant activity of
ethanol extracts from elm tree and Hemiptelea davidii is
0.34∼80.00%. Antioxidant activity increased with the
extracted content (25).
Previous work indicates that the SOD-like activity of
water-extracted Vitex seed is higher than the SOD-like
activity of ethanol-extracted Vitex seeds and upon the
density of extracts (26). Similarly, it is expected that the
SOD-like activity of AG will increase as the content of
the extract increases. The SOD-like activity of an ethanol
extract of Angelica dahurica root was higher than that of a
water extract of Angelica dahurica root (23.24% vs.
15.86%, respectively). In addition, the SOD-like activity
of the reflux extract of Angelica dahurica root was higher
than that of the MAE extract of Angelica dahurica root
(27). Together, these results indicate that the SOD-like
activity of AG could be affected by the extract method
and the extract contents.
Total polyphenol content
The total polyphenol content of each extract (in mg%) is
shown in Fig. 3. The total polyphenol content for WAG
was 114.97∼146.41 mg% and 133.05∼178.02 mg%.
The total polyphenol content of all of the extracts was
higher than 100 mg%, but at least two times lower than
the activity of the comparative substance, L-ascorbic
acid. The 60% ethanol extracts of CAG and WAG had
the highest total polyphenol content (144.88 mg% and
178.02 mg%, respectively), while the 90% ethanol ex-
tracts of CAG and WAG had the lowest total polyphenol
content (114.97 mg% and 133.05 mg%, respectively).
The rank order (highest to lowest) of the total poly-
phenol content of the extracts tested in this study is:
60% ethanol extracts＞ 30% ethanol extracts＞ water
extracts＞ 90% ethanol extracts.
Chang et al. (20) reported that the total polyphenol
content of a hot water extract of AG is 5.77 mg/g and
the total polyphenol content of a hot water extract treat-
ed with cold ethanol and partly refined is 6.16 mg/g.
Similar to the results of the present study, the total poly-
phenol content of a 60% ethanol extract of rosemary is
greater than the total polyphenol content of a water ex-
tract of rosemary (25.7 mg/g vs. 24.3 mg/g, respectively)
(28). Likewise, the total polyphenol content of an 80%
ethanol extract of Morus bombycis Koidzumi is greater
than that of a water extract of Morus bombycis Koidzumi
(29). The rank order of the total polyphenol content of
fresh Pleurotus eryngii is as follows: hot water extract＞
50% ethanol extract＞ 100% ethanol extract (30). We hy-
pothesize that these differences in polyphenol content
occur because polyphenol components exist in various
forms and kinds in plants and therefore elute differently
under different extraction conditions (31). Lee et al.
(25) and Kroyer et al. (32) report that DPPH radical
scavenging activity increases with total polyphenol
content. However, this study was to estimate the correla-
tion between the electron donating ability and poly-
phenols.
Tyrosinase inhibitory effect
The tyrosinase inhibitory effect of AG extracts is shown
in Fig. 4. The tyrosinase inhibitory effect of WAG ex-
tracts increased from 26.58% to 37.56% as the ethanol
concentration of the extraction solvent increased. In
contrast, the tyrosinase inhibitory effect of CAG extracts
278 Noh et al.
Table 1. Nitrite scavenging effect of microwaved
Angelica giga
s
Nakai extracts
Solvent
Nitrite scavenging ability (%)
pH 1.2 pH 3.0 pH 4.2 pH 6.0
Cultivated
Angelica gigas
Nakai
Wild
Angelica gigas
Nakai
0.1% L-ascorbic acid
1% L-ascorbic acid
Water
30% ethanol
60% ethanol
90% ethanol
Water
30% ethanol
60% ethanol
90% ethanol
97.50±0.35
a1)
98.63±0.18
a
97.60±0.09
a
92.04±0.22
a
86.35±0.35
a
101.20±0.83
a
101.07±1.64
a
93.55±1.08
a
84.38±0.14
a
84.93±0.24
a
66.71±0.52
b
82.66±0.41
b
70.22±0.26
b
62.33±0.15
b
50.64±0.53
b
88.10±0.30
b
88.08±5.06
b
71.82±1.56
b
50.48±1.87
b
85.01±0.05
a
25.59±1.56
c
28.80±2.01
c
25.91±1.57
c
21.37±1.47
c
20.04±0.76
c
32.34±9.33
c
33.53±1.88
c
25.32±1.49
c
30.68±1.37
c
84.34±0.23
b
13.44±2.67
d
11.33±2.12
d
13.51±0.67
d
7.45±3.26
d
12.42±0.42
d
6.73±1.21
d
15.60±2.41
d
15.69±1.63
d
14.78±1.36
d
76.49±0.46
c
1)
Within each row, means with the same letters are not significantly different (
P
＜0.05).
decreased from 43.72% to 1.84% as the ethanol concen-
tration of the extraction solvent increased. The highest
tyrosinase inhibitory effect (43.72%) was two times low-
er than the tyrosinase inhibitory effect of the com-
parative solutions, 0.1% L-ascorbic acid (96.71%) and
1% L-ascorbic acid (100.47%).
Jung et al. (33) measured the tyrosinase inhibitory ef-
fect of medicinal plant extracts (2 g dry weight extracted
with 100 mL water). They found that the tyrosinase in-
hibitory effect of AG is 39%, which is slightly higher
than the maximum tyrosinase inhibitory effect measured
in the 1:25 ratio (v/w) WAG extracts of the present
study. The tyrosinase inhibitory effect of a snake beard
water extract is 22.80%, which is lower than that of the
AG measured in the present study (34). Previous reports
indicate that the tyrosinase inhibitory effect of plum is
88.0%∼100%, which is similar to that of the comparative
substance, L-ascorbic acid, used in the present study (35).
Song et al. (23) reports that the whitening effect of ty-
rosinase observed in Angelica utilis Makino is affected by
the total polyphenol content. Likewise, previous work
indicates that the highest tyrosinase inhibitory effect is
observed in the white sandalwood extracts with the
highest total polyphenol content (36). However, the re-
sults of the present study did not reveal a correlative re-
lationship between the tyrosinase inhibitory effect and
the total polyphenol content of AG.
Nitrite scavenging effect
As shown in Table 1, the nitrite scavenging effect of the
AG extracts was affected by pH. The nitrite-scavenging
effect of CAG and WAG was highest at a pH of 1.2 and
decreased as pH increased, indicating that the nitrite
scavenging effect was higher in more acidic conditions.
In addition, at a pH of 1.2, the scavenging effect of all of
the extracts tested was higher than that of the com-
parative solutions, 0.1% L-ascorbic acid and 1% of L-as-
corbic acid (84.34% and 84.93%, respectively). There
was no distinct difference in the nitrite scavenging ef-
fects of CAG and WAG in the present study. The highest
scavenging ability was observed in the 30% ethanol ex-
tracts of CAG and WAG. It demonstrated a higher ni-
trite scavenging effect at water extract of CAG and etha-
nol extract of WAG.
The present study provides support for previous reports
that AG has a superior nitrite scavenging effect. Earlier
studies report that AG water extracts and methanol ex-
tracts have a very high nitrite scavenging effect at a pH of
1.2 (92.87∼96.51% and 93.00∼98.86%, respectively)
(10). The results of the current study also support pre-
vious reports that the nitrite scavenging effect of medici-
nal herbs varies with pH, and that there is no difference
in the nitrite scavenging effect of water and ethanol AG
extracts (37). However in the previous study, the nitrite
scavenging effect of water and ethanol AG extracts with
a pH of 1.2 was 37% and 35%, respectively. Previous re-
ports indicate that the nitrite scavenging effect of
Korean mountain ginseng, Rubus coreanus, and Angelica
dahurica leaves decreases with increasing pH and that the
nitrite scavenging effect of these leaves is at least two
times greater than that of AG (38-40).
ACE inhibitory activity
Angiotensin II increases blood pressure by inducing
blood vessel contraction and increases the secretion of
aldosterone into body fluids. Angiotensin II is produced
by ACE when rennin, an enzyme, acts on angiotensi-
nogen to produce angiotensin I (41). To prevent blood
pressure, it is required to inhibition of ACE. The ACE
inhibitory activity of CAG extracts was measured in this
study. As shown in Fig. 5, the rank order of the ACE in-
hibitory activity of CAG extracts was: 30% ethanol ex-
tract (79.13%)＞ water extract (73.58%)＞ 60% ethanol
extract (73.28%)＞ 90% ethanol extract (56.17%). In
contrast, the rank order of the ACE inhibitory activity of
WAG extracts was: 60% ethanol extract (83.84%)＞
90% ethanol extract (64.67%)＞ water extract (38.76%)＞
30% ethanol extract (36.29%).
Previously, Do et al. (42) reported that the ACE in-
hibitory activity of 10 mg/mL Psoralea corylifolia L. is
Antioxidant Activity of Angelica gigas Nakai Extracts 279
Fig. 5. ACE inhibitory ability of microwaved
Angelica gigas
Nakai
extracts.
□: cultivated
Angelica gigas
Nakai, ■: wild
Angelic
a
gigas
Nakai. Microwave-assisted extraction (60 W, 5 min) was
performed on a mixture composed of 2 g of sample and 50 mL
of solvent.
1)
Means with the same letter are not significantly dif-
ferent (
P
＜0.05).
65.2%, and Kang et al. (43) noted that the ACE in-
hibitory activity of pine needles and a hot water extract
of mugwort are 61.0% and 75.1%, respectively. In addi-
tion, the ACE inhibitory activity of Phellinus ribis is rela-
tively low (12.0%) (44). Therefore, the results of the
present study indicate that AG has notable ACE in-
hibitory activity.
AUTHOR DISCLOSURE STATEMENT
The authors declare no conflict of interest.
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