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Article
Monthly Variation and Ultraviolet Stability of
Mycosporine-like Amino Acids from Red Alga Dulse
Palmaria palmata in Japan
Yuki Nishida 1, Yoshikatsu Miyabe 1,2, Hideki Kishimura 3and Yuya Kumagai 3,*
Citation: Nishida, Y.; Miyabe, Y.;
Kishimura, H.; Kumagai, Y. Monthly
Variation and Ultraviolet Stability of
Mycosporine-like Amino Acids from
Red Alga Dulse Palmaria palmata in
Japan. Phycology 2021,1, 119–128.
https://doi.org/10.3390/
phycology1020009
Academic Editor: Peer Schenk
Received: 31 October 2021
Accepted: 22 November 2021
Published: 25 November 2021
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1Marine Chemical Resource Development, Graduate School of Fisheries Sciences, Hokkaido University,
Hakodate 041-8611, Hokkaido, Japan; karakuchi@eis.hokudai.ac.jp (Y.N.);
yoshikatsu_miyabe@aomori-itc.or.jp (Y.M.)
2Food Research Institute, Aomori Prefectural Industrial Technology Research Center, 2-10 Chikkogai,
Hachinohe-shi 031-0831, Aomori-ken, Japan
3Marine Chemical Resource Development, Faculty of Fisheries Sciences, Hokkaido University,
Hakodate 041-8611, Hokkaido, Japan; i-dulse@fish.hokudai.ac.jp
*Correspondence: yuyakumagai@fish.hokudai.ac.jp; Tel.: +81-138-40-5560
Abstract:
Mycosporine-like amino acids (MAAs) are the natural ultraviolet (UV)-absorbing com-
pounds from micro- and macro-algae. The MAAs in algae change with the environmental conditions
and seasons. We previously determined an efficient extraction method of MAAs from red alga dulse
in Usujiri (Hokkaido, Japan) and revealed monthly variation of MAA in 2019. Dulse samples in
2019 for MAA preparation were suitable from late February to April. In this study, to confirm the
suitable timings to extract MAAs from Usujiri dulse, we also investigated the monthly (from January
to May) variation of MAA content in 2020. There were the most MAAs in the sample on 18 March
(6.696
µ
mol g
−1
dry weight) among the samples from January to May 2020. From two years of
investigation, we deduce that samples of Usujiri dulse from late February to early April were suitable
for MAA preparation. The UV stability of the two major purified MAAs in Usujiri dulse—palythine
and porphyra-334—was tested. The two MAAs and 2-hydroxy-4-methoxybenzophenone were stable
for up to 12 h under a 312 nm lamp at 200
µ
W cm
−2
, but 2-ethylhexyl-4-methoxycinnamate formed
a cis/trans-mixture in a short time. The data in this study show the suitable sampling period for
Usujiri dulse and the possible application for UV protection from food and cosmetics.
Keywords:
red alga; dulse; mycosporine-like amino acids; monthly variation; ultraviolet stability;
ultraviolet absorption; Usujiri Hokkaido
1. Introduction
Although sunlight is essential for photosynthesis in plants, it also contains harmful
ultraviolet (UV) for organisms. UV radiation is classified into UVA (315–400 nm), UVB
(280–315 nm), and UVC (200–280 nm) in terms of their spectra. UVC does not reach Earth
since it is absorbed in the ozone layer. UVA and UVB cause defects of metabolic func-
tion, oxidation of DNA and RNA, and production of reactive oxygen species (ROS) [
1
,
2
].
While UV has beneficial effects for humans, such as the innate immunity response and
biosynthesis of vitamin D [
3
], it also induces skin disorders in humans, e.g., erythema, pig-
mentation, edema, formation of sunburn, phototoxicity, and photoaging [
4
–
7
]. Therefore,
UV protection agents such as UV absorption agents and UV scattering agents are used for
the protection of our skin. However, the use of these agents has raised safety concerns for
the human body and environment. Some agents may cause oxygen radical production,
cancer, and photoallergic skin inflammation [
8
–
10
], while some cause coral bleaching even
in low concentrations [
11
]. Alternative compounds such as polyphenols and flavonoids
from natural products have been used as UV protection agents [
12
–
14
], and natural UV
protection products with a high molar extinction coefficient are now in demand.
Phycology 2021,1, 119–128. https://doi.org/10.3390/phycology1020009 https://www.mdpi.com/journal/phycology
Phycology 2021,1120
Red alga dulse (Palmaria palmata in Japan), which is distributed in the Northern Iwate
prefecture and northward, is an underused marine resource. Dulse has been removed from
Kombu rope in Hakodate, Hokkaido, since Kombu is an important resource for the local
fishermen. When dulse is utilized as a novel local food, we have studied its components
and functions for human health, such as antioxidant activity, peptides for the inhibition
of angiotensin I-converting enzyme activity, prebiotics from xylooligosaccharides [
15
–
18
],
and its mitochondrial and chloroplast genome and phycobiliprotein structures [
19
–
21
]. In
addition, dulse contains UV absorption compounds such as mycosporine like amino acids
(MAAs) [
22
]. MAAs, secondary metabolites of nitrogen products with cyclohexanone or
cyclohexenimine structures, are water soluble low-molecular compounds synthesized by
the shikimate and pentose phosphate pathways [
23
,
24
]. MAAs are used as UV protection
compounds in marine organisms [
25
–
27
]. More than 30 MAA structures with absorption
spectra from 310 to 360 nm have been reported to have a high molar extinction coefficient
(
ε
= 28,000–50,000) [
28
–
30
]. MAAs release heat from UV radiation instead of producing
ROS [
31
–
33
]. However, a problem with utilizing MAAs for UV protection materials is their
low content in seaweeds. The maximum MAA content to have been reported is 14 mg/1 g
dry weight [
34
,
35
]. Therefore, we investigated an effective MAA extraction method using
underused Usujiri dulse [
22
], involving six hours of water extraction to form dulse powder.
We found that dulse from 25 February 2019 contained a high quantity of MAAs when
compared with samples from earlier (to January) and later (to May) in 2019.
In this study, we investigated the monthly variation of MAAs in Usujiri, Hokkaido,
from January to May 2020 to clarify the suitable dulse sampling periods. To demonstrate
the suitable application of MAAs as UV protection materials, we evaluated the UV stability
of two purified MAAs, palythine and porphyra-334, in strong UV conditions.
2. Materials and Methods
2.1. Algal Samples
All dulse samples were collected at 1 m depth in Usujiri, Hakodate, Japan, from
January to May 2020. After collection, the thalli were washed with tap-water to remove sea
salt and epibionts. Soon after, they were frozen and lyophilized. Dried algal samples were
ground into a fine powder by a Wonder Blender WB-1 (Osaka Chemical Co., Osaka, Japan)
and stored in the dark at room temperature until analysis.
2.2. Extraction of Crude MAAs from Dulse
The optimum extraction condition of MAAs from dulse was determined in a previous
study [
22
]. Accordingly, the fine powder samples were soaked in 20 volumes (v/w) of
distilled water at 4
◦
C for 6 h, and the water extracts were collected by centrifugation
at 4
◦
C, 27,200
×
g, for 10 min. After centrifugation, supernatants were lyophilized and
soaked in 20 volumes (volume/powdered sample weight) of methanol at 4
◦
C for 2 h.
The methanol extracts containing MAAs were centrifuged at 4
◦
C, 27,200
×
g, for 15 min.
The supernatants were evaporated, re-dissolved in water, and lyophilized. Then, the solid
samples were designated as crude MAAs and used in the following experiments.
2.3. Spectrophotometric Analysis of MAAs
Crude MAAs solutions were analyzed by the UV-visible ray absorption spectrum
using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan).
2.4. Separation of MAAs by High Performance Liquid Chromatography (HPLC)
The crude MAAs were dissolved in water containing 0.1% trifluoroacetic acid (TFA)
and the solution underwent sequential filtration by a Millex-GV (diameter: 25
Φ
mm, pore
size: 0.22
µ
m) (Merck Millipore, Billerica, MA, USA) and Millex-LG (diameter: 4
Φ
mm,
pore size: 0.20
µ
m) (Merck Millipore, Burlington, MA, USA). The filtrated MAAs were
isolated by reversed-phase HPLC with a Mightysil RP-18GP column (5
µ
m, 10
×
250 mm)
(Kanto Kagaku, Tokyo, Japan) using isocratic elution of ultra-pure water containing 0.1%
Phycology 2021,1121
TFA for 7 min and a linear gradient of acetonitrile (0–70%) containing 0.1% TFA for 13 min
at a flow rate of 4.73 mL/min. The column oven temperature was set at 40
◦
C. The detection
wavelength was set at 330 nm. The peaks of 330 nm were fractionated and evaporated.
Then, the purified MAAs were dissolved in an appropriate quantity of distilled water.
2.5. Ultraviolet Stability
Palythine and porphyra-334 were prepared by HPLC from dulse powder on
25 February 2019
.
The purified palythine and porphyra-334 were dissolved in 0.1 M sodium phosphate buffer
(pH 7.4) at a concentration of 2.2
×
10
−5
M. Then,
2-Hydroxy-4-methoxybenzophenone
and
2-ethylhexyl-4-methoxycinnamate
(Tokyo Kasei Kogyo, Tokyo, Japan) were used as pos-
itive and negative standards, respectively. UV-B radiation (312 nm) was generated by
CSF-15BF (Cosmo Bio Co., Ltd., Tokyo, Japan), and the strength of radiation at 200
µ
W/cm
2
was set by the distance from the UV-B generator. After UV-B exposure, the spectra were
measured for up to 12 h by a spectrophotometer.
2.6. Phycoerythrin (PE) Content
The PE of each sample was prepared from fine powders. Namely, 10 mg of each powder was
dissolved in 1 mL distilled water and extracted at 4
◦
C for 12 h. After centrifugation at 12,000
×
g
for 5 min, the spectra of supernatants were measured. The quantity of PE was determined by
the following equation: PE (mg/mL) = [(A564 −A592)−(A455 −A592)×0.2] ×0.12 [35].
2.7. Abiotic Data in Hakodate
The monthly mean daily maximum ultraviolet index (UVI) was obtained from the
Japan Meteorological Agency (JMA: accessed on 28 January 2021, https://www.data.jma.
go.jp/gmd/env/uvhp/info_uv.html). According to the method of the JMA, the erythemal
UV intensity (mW/m
2
) was calculated by multiplying the UVI by 25 times. Data on the
near-surface chlorophyll concentration (mg/m
3
) were obtained from NASA’s Ocean Color
WEB (accessed on 15 December 2020 https://oceancolor.gsfc.nasa.gov). All data were
recorded in 2020.
2.8. Statistical Analysis
Data are expressed as the mean
±
standard error. All values are the means of triplicate
analysis. Statistical analysis was carried out using Tukey–Kramer’s multiple comparisons
test. All statistical analyses were performed using Statcel 3 software (version 3, OMS
Publisher, Tokorozawa, Japan).
3. Results
3.1. Monthly Variation of Usujiri Dulse MAAs in 2020
We previously determined the extraction condition of MAAs from dulse [
22
]. In
addition, the sampling period affected the MAA contents, with samples from late Febru-
ary to early April found to be suitable MAA sources in 2019. To confirm the sampling
periods in 2020, we prepared crude MAAs and determined the quantities of MAAs from
January to May in 2020. First, we confirmed the MAA components in 2020 by HPLC
(Figure 1)
. The chromatogram showed six peaks (peaks a–f: shinorine, palythine, asterina-
330, porphyra-334, usujirene, and palythene, respectively), which corresponded to the
sample in 2019. We then determined the quantity of each MAA by HPLC (Table 1).
The quantities of MAAs in 1 g of dry-weight dulse increased from January to March and
decreased in April and May 2020. The 18 March sample showed the highest MAA content
(6.696
µ
mol/g dry weight), while that of 27 May was the lowest (1.041
µ
mol/g dry weight).
Phycology 2021,1122
Phycology 2021, 1, FOR PEER REVIEW 4
The chromatogram showed six peaks (peaks a–f: shinorine, palythine, asterina-330,
porphyra-334, usujirene, and palythene, respectively), which corresponded to the sample
in 2019. We then determined the quantity of each MAA by HPLC (Table 1). The quantities
of MAAs in 1 g of dry-weight dulse increased from January to March and decreased in
April and May 2020. The 18 March sample showed the highest MAA content (6.696
µmol/g dry weight), while that of 27 May was the lowest (1.041 µmol/g dry weight).
Figure 1. High Performance Liquid Chromatography (HPLC) chromatogram of crude dulse myco-
sporine-like amino acids (MAA) solution on 14 January 2020. MAAs were eluted by a linear gradient
of acetonitrile from 0% at 7 min to 70% at 20 min. The data represent the typical peaks at retention
times of 4.68 min (a, shinorine), 5.25 min (b, palythine), 5.97 min (c, asterina-330), 9.08 min (d,
porphyra-334), and 13.44 min (e, usujirene, and f, palythene).
Table 1. MAA content in Usujiri dulse.
MAAs Collection Date (2020)
14 January 13 February 18 March 27 April 27 May
Shinorine 0.167 ± 0.002
c
0.226 ± 0.001
a
0.215 ± 0.001
b
0.079 ± 0.003
d
0.031 ± 0.001
e
Palythine 2.444 ± 0.021
b
2.367 ± 0.019
b
2.625 ± 0.006
a
0.588 ± 0.019
c
0.493 ± 0.019
d
Asterina-330 0.080 ± 0.001
c
0.098 ± 0.001
b
0.108 ± 0.001
a
0.039 ± 0.001
d
0.019 ± 0.001
e
Porphyra-334 1.795 ± 0.015
c
1.940 ± 0.019
b
2.526 ± 0.024
a
0.640 ± 0.021
d
0.311 ± 0.015
e
Usujirene +
Palythene 0.402 ± 0.003
c
0.750 ± 0.006
b
1.203 ± 0.006
a
0.623 ± 0.014
d
0.187 ± 0.008
e
Total 4.888 ± 0.035
c
5.380 ± 0.043
b
6.696 ± 0.002
a
1.968 ± 0.057
d
1.041 ± 0.044
e
MAA content is expressed as µmol/g dry weight. Data show mean values ± SE, n = 3. Different
letters for each MAA indicate significant differences in mean value (Tukey–Kramer’s multiple
comparisons test,
a–e
< 0.05).
3.2. Monthly Variation of MAA Contents
The molar percentage (mol%) contents in each MAA in 2020 were compared (Figure
2). The mol% values of shinorine (from 3.2 mol% on 18 March to 4.2 mol% on 13 February)
and asterina-330 (from 1.6 mol% on 18 March to 2.0 mol% on 13 February) were stable at
low values similar to the samples in 2019. The mol% of palythine decreased from 14 Jan-
uary (50 mol%) to 27 April (30 mol%), and then increased to 47 mol% on 27 May.
Porphyra-334 was stable at approximately 37 mol% from 14 January to 18 May, and then
Figure 1.
High Performance Liquid Chromatography (HPLC) chromatogram of crude dulse
mycosporine-like amino acids (MAA) solution on 14 January 2020. MAAs were eluted by a linear
gradient of acetonitrile from 0% at 7 min to 70% at 20 min. The data represent the typical peaks at
retention times of 4.68 min (
a
, shinorine), 5.25 min (
b
, palythine), 5.97 min (
c
, asterina-330), 9.08 min
(d, porphyra-334), and 13.44 min (e, usujirene, and f, palythene).
Table 1. MAA content in Usujiri dulse.
MAAs Collection Date (2020)
14 January 13 February 18 March 27 April 27 May
Shinorine 0.167 ±0.002 c0.226 ±0.001 a0.215 ±0.001 b0.079 ±0.003 d0.031 ±0.001 e
Palythine 2.444 ±0.021 b2.367 ±0.019 b2.625 ±0.006 a0.588 ±0.019 c0.493 ±0.019 d
Asterina-330 0.080 ±0.001 c0.098 ±0.001 b0.108 ±0.001 a0.039 ±0.001 d0.019 ±0.001 e
Porphyra-334 1.795 ±0.015 c1.940 ±0.019 b2.526 ±0.024 a0.640 ±0.021 d0.311 ±0.015 e
Usujirene +
Palythene 0.402 ±0.003 c0.750 ±0.006 b1.203 ±0.006 a0.623 ±0.014 d0.187 ±0.008 e
Total 4.888 ±0.035 c5.380 ±0.043 b6.696 ±0.002 a1.968 ±0.057 d1.041 ±0.044 e
MAA content is expressed as
µ
mol/g dry weight. Data show mean values
±
SE, n= 3. Different letters for each MAA indicate significant
differences in mean value (Tukey–Kramer’s multiple comparisons test, a–e < 0.05).
3.2. Monthly Variation of MAA Contents
The molar percentage (mol%) contents in each MAA in 2020 were compared (
Figure 2
).
The mol% values of shinorine (from 3.2 mol% on 18 March to 4.2 mol% on 13 February)
and asterina-330 (from 1.6 mol% on 18 March to 2.0 mol% on 13 February) were stable
at low values similar to the samples in 2019. The mol% of palythine decreased from
14 January (
50 mol%
) to 27 April (30 mol%), and then increased to 47 mol% on 27 May.
Porphyra-334 was
stable at approximately 37 mol% from 14 January to 18 May, and then
gradually decreased to 30 mol% on 18 May. On the other hand, usujirene + palythene had
a low mol% on 18 January (8.2 mol%), which increased up to 32 mol% on 27 April and then
dropped to 18 mol% on 18 May. Among the five MAAs, the mol% of
usujirene + palythene
varied most greatly, ranging four-fold from 8.2 to 32 mol%, in a similar change to that
observed in the dulse sample of 2019.
Phycology 2021,1123
Phycology 2021, 1, FOR PEER REVIEW 5
gradually decreased to 30 mol% on 18 May. On the other hand, usujirene + palythene had
a low mol% on 18 January (8.2 mol%), which increased up to 32 mol% on 27 April and
then dropped to 18 mol% on 18 May. Among the five MAAs, the mol% of usujirene +
palythene varied most greatly, ranging four-fold from 8.2 to 32 mol%, in a similar change
to that observed in the dulse sample of 2019.
Figure 2. Molar percentages of MAAs in 2020. The quantities of six MAAs—shinorine, palythine,
asterina-330, porphyra-334, and usujirene + palythene—were compared in dulse collected on 14 Jan-
uary, 13 February, 18 March, 27 April, and 27 May. The data show mean values, n = 3.
3.3. Change of MAAs, PE and Erythemal UV Intensity
To understand the variation of MAA content, data on the PE content and erythemal
UV intensity were prepared (Figure 3). The PE contents were stable at 46 µg/mg dry
weight on 14 January and 13 February, and then gradually decreased from 13 February
(47 µg/mg dry weight) to 27 May (19 µg/mg dry weight). Meanwhile, the erythemal UV
intensity increased from January (25 mW/m
2
) to May (115 mW/m
2
). The total MAAs con-
tent correlated with the erythemal UV intensity from January to March, but it did not
correlate with the PE content or erythemal UV intensity from April to May.
Figure 2.
Molar percentages of MAAs in 2020. The quantities of six MAAs—shinorine, paly-
thine, asterina-330, porphyra-334, and usujirene + palythene—were compared in dulse collected on
14 January, 13 February, 18 March, 27 April, and 27 May. The data show mean values, n= 3.
3.3. Change of MAAs, PE and Erythemal UV Intensity
To understand the variation of MAA content, data on the PE content and erythemal
UV intensity were prepared (Figure 3). The PE contents were stable at 46
µ
g/mg dry
weight on 14 January and 13 February, and then gradually decreased from 13 February
(47
µ
g/mg dry weight) to 27 May (19
µ
g/mg dry weight). Meanwhile, the erythemal UV
intensity increased from January (25 mW/m
2
) to May (115 mW/m
2
). The total MAAs
content correlated with the erythemal UV intensity from January to March, but it did not
correlate with the PE content or erythemal UV intensity from April to May.
Phycology 2021, 1, FOR PEER REVIEW 5
gradually decreased to 30 mol% on 18 May. On the other hand, usujirene + palythene had
a low mol% on 18 January (8.2 mol%), which increased up to 32 mol% on 27 April and
then dropped to 18 mol% on 18 May. Among the five MAAs, the mol% of usujirene +
palythene varied most greatly, ranging four-fold from 8.2 to 32 mol%, in a similar change
to that observed in the dulse sample of 2019.
Figure 2. Molar percentages of MAAs in 2020. The quantities of six MAAs—shinorine, palythine,
asterina-330, porphyra-334, and usujirene + palythene—were compared in dulse collected on 14 Jan-
uary, 13 February, 18 March, 27 April, and 27 May. The data show mean values, n = 3.
3.3. Change of MAAs, PE and Erythemal UV Intensity
To understand the variation of MAA content, data on the PE content and erythemal
UV intensity were prepared (Figure 3). The PE contents were stable at 46 µg/mg dry
weight on 14 January and 13 February, and then gradually decreased from 13 February
(47 µg/mg dry weight) to 27 May (19 µg/mg dry weight). Meanwhile, the erythemal UV
intensity increased from January (25 mW/m
2
) to May (115 mW/m
2
). The total MAAs con-
tent correlated with the erythemal UV intensity from January to March, but it did not
correlate with the PE content or erythemal UV intensity from April to May.
Figure 3.
Monthly variation of MAAs and PE from dulse in 2020. The MAA quantity reflects the
total of all MAAs. The quantity of PE from the dry weight was determined using the Beer and Eshel
equation [36]. Erythemal UV intensity was obtained from the Japan Meteorological Agency.
3.4. Stability of Ultraviolet
One of the main functions of MAAs is absorption of UV radiation. Although studies on
porphyra-334 and shinorine have been reported [
31
,
37
–
39
], there are few on palythine. We
Phycology 2021,1124
prepared palythine and porphyra-334 and evaluated their UV stability. The strength of the
UV radiation was set to 200
µ
W cm
−2
. We used 2-hydroxy-4-methoxybenzophenone and
2-ethylhexyl-4-methoxycinnamate
as the non-sensitive and sensitive agent, respectively. The spec-
tra of the two MAAs, palythine and porphyra-334, and
2-hydroxy-4-methoxybenzophenone
were stable (Figure 4). However, 2-ethylhexyl-4-methoxycinnamate immediately changed
from the trans-form to cis/trans-mixture forms [
40
]. This result showed that MAAs are
stable UV-absorption-compounds.
Phycology 2021, 1, FOR PEER REVIEW 6
Figure 3. Monthly variation of MAAs and PE from dulse in 2020. The MAA quantity reflects the
total of all MAAs. The quantity of PE from the dry weight was determined using the Beer and Eshel
equation [36]. Erythemal UV intensity was obtained from the Japan Meteorological Agency.
3.4. Stability of Ultraviolet
One of the main functions of MAAs is absorption of UV radiation. Although studies
on porphyra-334 and shinorine have been reported [31,37–39], there are few on palythine.
We prepared palythine and porphyra-334 and evaluated their UV stability. The strength
of the UV radiation was set to 200 µW cm
−2
. We used 2-hydroxy-4-methoxybenzophenone
and 2-ethylhexyl-4-methoxycinnamate as the non-sensitive and sensitive agent, respec-
tively. The spectra of the two MAAs, palythine and porphyra-334, and 2-hydroxy-4-meth-
oxybenzophenone were stable (Figure 4). However, 2-ethylhexyl-4-methoxycinnamate
immediately changed from the trans-form to cis/trans-mixture forms [40]. This result
showed that MAAs are stable UV-absorption-compounds.
Figure 4. Spectra of UV-B exposure samples. Palythine and porphyra-334 were dissolved in 0.1 M
sodium phosphate buffer (pH 7.4) at the concentration of 2.2 × 10
−5
M. In the experiment, 2-hydroxy-
4-methoxybenzophenone was dissolved in ethanol at the concentration of 2.4 × 10
−5
M, while 2-
ethylhexyl-4-methoxycinnamate was diluted with ethanol at the concentration of 3.3 × 10
−6
M. UV-
B (312 nm) was set at the radiation of 200 µW/cm
2
, and the spectra of samples were measured by a
spectrophotometer. Black lines, 0 h; blue line, 1 h; red dotted lines, 6 h; green dashed lines, 12 h.
4. Discussion
Our previous study showed that dulse from late February to early April in 2019 was
suitable for MAA preparation [22] since dulse then disappeared in summer and reap-
peared in winter. To confirm the period, here, we investigated the suitable period of MAA
preparation from dulse samples in 2020. The maximum quantities of MAAs in 2019 and
2020 were almost equal at 6.930 µmol/g dry weight on 25 February 2019 and 6.696 µmol/g
dry weight on 18 March 2020 (Figure 5). Among the samples from January to May 2020,
the MAA content was high in the samples from February to mid-March. The accumulation
and loss of MAAs in 2020 differed from that of the 2019 samples. Specifically, the total
MAAs on 14 January 2020 (4.888 µmol/g) were higher than on 23 January 2019 (2.649
µmol/g) [22]. The loss of MAAs on 27 April 2020 (1.968 µmol/g) was faster than that on 17
Figure 4.
Spectra of UV-B exposure samples. Palythine and porphyra-334 were dissolved
in
0.1 M
sodium phosphate buffer (pH 7.4) at the concentration of 2.2
×
10
−5
M. In the ex-
periment,
2-hydroxy-4-methoxybenzophenone
was dissolved in ethanol at the concentration of
2.4 ×10−5M
, while 2-ethylhexyl-4-methoxycinnamate was diluted with ethanol at the concentration
of
3.3 ×10−6M
. UV-B (312 nm) was set at the radiation of 200
µ
W/cm
2
, and the spectra of samples
were measured by a spectrophotometer. Black lines, 0 h; blue line, 1 h; red dotted lines, 6 h; green
dashed lines, 12 h.
4. Discussion
Our previous study showed that dulse from late February to early April in 2019 was
suitable for MAA preparation [
22
] since dulse then disappeared in summer and reappeared
in winter. To confirm the period, here, we investigated the suitable period of MAA
preparation from dulse samples in 2020. The maximum quantities of MAAs in 2019 and
2020 were almost equal at
6.930 µmol/g
dry weight on 25 February 2019 and
6.696 µmol/g
dry weight on 18 March 2020 (Figure 5). Among the samples from
January to May 2020
, the
MAA content was high in the samples from February to mid-March. The accumulation and
loss of MAAs in 2020 differed from that of the 2019 samples. Specifically, the total MAAs on
14 January 2020 (4.888
µ
mol/g) were higher than on
23 January 2019
(
2.649 µmol/g
) [
22
].
The loss of MAAs on 27 April 2020 (1.968
µ
mol/g) was faster than that on 17 May 2019
(4.972
µ
mol/g). The difference between 2020 and 2019 was due to the quantity of palythine.
In 2019 samples, this increased from January (1.739
µ
mol/g) to May (3.255
µ
mol/g);
however, in 2020, it was stable from January (2.444
µ
mol/g) to March (2.625
µ
mol/g), and
then drastically decreased in April (0.588 µmol/g).
Phycology 2021,1125
Phycology 2021, 1, FOR PEER REVIEW 7
May 2019 (4.972 µmol/g). The difference between 2020 and 2019 was due to the quantity
of palythine. In 2019 samples, this increased from January (1.739 µmol/g) to May (3.255
µmol/g); however, in 2020, it was stable from January (2.444 µmol/g) to March (2.625
µmol/g), and then drastically decreased in April (0.588 µmol/g).
We previously concluded that the decrease of MAAs was related to the increase of
chlorophyll concentration around the Usujiri area, leading to a shortage of nitrogen
sources for phytoplankton production. Although the quantities of total MAAs decreased
in May 2020 versus 2019, the difference of palythine content may have occurred inde-
pendently of the phytoplankton production. In this study, we employed the parameters
of PE content, erythemal UV intensity, and chlorophyll concentration (data not shown:
data from NASA’s Ocean Color WEB). The PE content is one of the factors reflecting the
quantity of nitrogen compounds in red algae [41–43], suggesting that the loss of PE in
dulse was due to a reduction of nitrogen compounds in the sea, along with the increase in
the chlorophyll concentration in the Usujiri area. Erythemal UV intensity increases the
quantity of MAAs in seaweeds [44], which present as nitrogen compounds in seaweeds.
The decrease of MAAs from March to April was slow compared to that of PE from Feb-
ruary to March, indicating that MAAs are essential compounds in red algae. However,
the MAA contents did not increase with erythemal UV intensity. To understand each
MAA’s content involves taking account of the many reasons for MAAs’ accumulation
such as nutrients, stress from water temperature, and desiccation [45–48]. From these two-
year results, we propose that a suitable sampling period for Usujiri dulse for MAA prep-
aration extends from mid-February to early April.
Although MAAs were unstable in conditions that were acidic (lower than pH 3.0) or
alkaline (higher than pH 10.5), as well as at hot temperatures (higher than 60 °C) [10],
MAAs were stable at moderate conditions of pH 4.0 to 8.5 and temperatures up to 50 °C
[10]. Reports on the UV stability of MAAs are rare. We demonstrated the UV stability of
MAAs for up to 12 h in the expected conditions in the summer season of Wellington, New
Zealand, which is one of the most high-strength locations of radiation on Earth. The MAAs
in this study released heat from UV radiation and were unstable above 60 °C, but we pro-
pose that the experimental conditions (UV irradiation and MAA concentration) represent
a tolerant range of heating for the sample solvent.
Not only do marine photosynthetic organisms use MAAs for UV protection, but also
non-MAA producers such as fish and invertebrates incorporate and use MAAs for UV
protection [25,26,49,50]. Many UV protection agents in products are lipid-soluble materi-
als. MAAs are the amphiphilic compounds. These results show that MAAs have the po-
tential for use as natural UV protection compounds.
Figure 5. Monthly mean of MAA content, daily maximum erythemal UV intensity, and chlorophyll
concentration in 2019 and 2020. (a) MAA content and erythemal UV intensity. Orange bar and line
show MAA content and erythemal UV intensity in 2019; black bar and line show MAA content and
erythemal UV intensity in 2020. (b) Chlorophyll concentration. These data were obtained from JMA
and NASA’s Ocean Color WEB. All data were recorded in 2019 and 2020.
Figure 5.
Monthly mean of MAA content, daily maximum erythemal UV intensity, and chlorophyll
concentration in 2019 and 2020. (
a
) MAA content and erythemal UV intensity. Orange bar and line
show MAA content and erythemal UV intensity in 2019; black bar and line show MAA content and
erythemal UV intensity in 2020. (
b
) Chlorophyll concentration. These data were obtained from JMA
and NASA’s Ocean Color WEB. All data were recorded in 2019 and 2020.
We previously concluded that the decrease of MAAs was related to the increase
of chlorophyll concentration around the Usujiri area, leading to a shortage of nitrogen
sources for phytoplankton production. Although the quantities of total MAAs decreased
in
May 2020
versus 2019, the difference of palythine content may have occurred inde-
pendently of the phytoplankton production. In this study, we employed the parameters
of PE content, erythemal UV intensity, and chlorophyll concentration (data not shown:
data from NASA’s Ocean Color WEB). The PE content is one of the factors reflecting the
quantity of nitrogen compounds in red algae [
41
–
43
], suggesting that the loss of PE in
dulse was due to a reduction of nitrogen compounds in the sea, along with the increase
in the chlorophyll concentration in the Usujiri area. Erythemal UV intensity increases the
quantity of MAAs in seaweeds [
44
], which present as nitrogen compounds in seaweeds.
The decrease of MAAs from March to April was slow compared to that of PE from February
to March, indicating that MAAs are essential compounds in red algae. However, the MAA
contents did not increase with erythemal UV intensity. To understand each MAA’s content
involves taking account of the many reasons for MAAs’ accumulation such as nutrients,
stress from water temperature, and desiccation [
45
–
48
]. From these two-year results, we
propose that a suitable sampling period for Usujiri dulse for MAA preparation extends
from mid-February to early April.
Although MAAs were unstable in conditions that were acidic (lower than pH 3.0)
or alkaline (higher than pH 10.5), as well as at hot temperatures (higher than 60
◦
C) [
10
],
MAAs were stable at moderate conditions of pH 4.0 to 8.5 and temperatures up to
50 ◦C
[
10
].
Reports on the UV stability of MAAs are rare. We demonstrated the UV stability of MAAs
for up to 12 h in the expected conditions in the summer season of Wellington, New Zealand,
which is one of the most high-strength locations of radiation on Earth. The MAAs in this
study released heat from UV radiation and were unstable above 60
◦
C, but we propose that
the experimental conditions (UV irradiation and MAA concentration) represent a tolerant
range of heating for the sample solvent.
Not only do marine photosynthetic organisms use MAAs for UV protection, but also
non-MAA producers such as fish and invertebrates incorporate and use MAAs for UV
protection [
25
,
26
,
49
,
50
]. Many UV protection agents in products are lipid-soluble materials.
MAAs are the amphiphilic compounds. These results show that MAAs have the potential
for use as natural UV protection compounds.
5. Conclusions
We clarified the seasonal variation of Usujiri dulse MAA contents in 2020. Comparing
these with the MAA variation in 2019, we conclude that MAA preparation of samples
Phycology 2021,1126
from Usujiri dulse may be carried out from mid-February to early April. However, the
composition of MAAs differed between the two years. We, therefore, need to investigate
MAAs’ contents in the long term. In addition, we showed the UV stability of MAAs under
pH-controlled conditions. This information will be helpful for the application of MAAs to
produce eco-friendly materials.
Author Contributions:
H.K. and Y.K. conceived and designed the research; Y.N. performed the
experiments; Y.N. and Y.M. analyzed the data; Y.K. and H.K. contributed to writing and editing the
manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data is contained within the article.
Acknowledgments:
We gratefully acknowledge sampling assistance with Palmaria palmata in Japan
at Usujiri from Hiroyuki Munehara, Atsuya Miyajima and Chikara Kawagoe. We also acknowledge
research assistance with MAA preparation from Kanami Sugiyama.
Conflicts of Interest: The authors declare no conflict of interest.
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