Ultraviolet radiation (UVR) is a part of the solar electromagnetic spectrum and defined as wavelengths from 200 to 400 nm composed of ultraviolet A (UVA) (315–400 nm), ultraviolet B (UVB) (280–315 nm), and ultraviolet C (UVC) (200–280 nm). UVR reaching on the Earth’s surface is only a small portion of the entire UVR, which is composed of wavelengths above 290 nm (mainly UVA and up to 10% of UVB) [1
]. In the past few decades, the amount of UVR on the Earth’s surface has been increased due to climate change with the decrease in aerosols and cloud [4
], resulting in exposure of UVR to a wide variety of biological systems. In human, exposure of UVR induces diseases such as cancer and skin aging by denature of DNA or proteins (e.g., formation of cyclobutane purine/pyrimidine dimers) [6
]. Therefore, organisms under UVR must take some defense strategies to minimize UV-induced damage [1
Mycosporine-like amino acids (MAAs) are synthesized and accumulated as photoprotective compounds by marine phototrophs (e.g., dinoflagellates, cyanobacteria and macro algae) [1
]. MAAs are nitrogenous secondary metabolites of the low molecular weight (<400 Da) with the maximum absorption ranging from 310 to 360 nm and have the high molar extinction coefficients (28,100 to 50,000 M−1
]. MAAs are composed of a cyclohexanone or cyclohexenimine ring conjugated with amino acids, amino alcohols, or amino groups. MAAs are classified into two types by the core structure of cyclohexanone or cyclohexenimine ring, showing oxo-MAAs or imino-MAAs, respectively. The major oxo-MAAs are mycosporine-glycine and mycosporine-taurine, while major imino-MAAs are shinorine, palythine, asterina-330, porphyra-334, usujirene and palythene [1
]. According to Hoyer et al. [12
], the biosynthesis pattern of MAAs in red alga is classified into three categories: (i) species with no capacity for MAAs biosynthesis; (ii) species with the high content of MAAs permanently; (iii) species changing the content of MAAs with the environmental conditions. While the species of category (i) typically represent red seaweed of lower sublittoral species of category (ii) and (iii) grow from the mid-sublittoral zone to the supralittoral zone [12
]. Biosynthesis of MAAs is expected to the shikimate pathway and the pentose phosphate pathway, and many MAAs are produced in the synthesis steps [1
]. However, the ratio and product patterns of MAAs are still unclear because of the complicated environmental condition and the lack of knowledge for seaweed’s molecular biology [18
MAAs have the physicochemical advantage in the point of high melting point, photostability, solubility in water, and organic solvents, stability in a wide range of pH and temperature [9
]. UV-protective compounds are divided into two types: UV-reflective and UV-absorbable. MAAs are UV-absorbable materials and dissipate the energy as heat without generating reactive oxygen species (ROS) [9
]. Recently, functions of MAAs have been reported as sunscreens, activators of cells proliferation, anti-cancer agents, anti-photoaging molecules, and stimulators of skin renewal. MAAs are nontoxic and biodegradable compounds. Therefore, MAAs have been attracted attention among various biotechnological industries [1
]. Antioxidant capacity is one of the main functions of MAAs [1
]. The in vitro antioxidant capacity has been reported using oxygen radical absorbance capacity (ORAC) assay (measuring hydrogen atom transfer (HAT) reaction) and 2,2′-azino-bis (ABTS) radical scavenging assay (measuring electron transfer (ET) reaction) [10
]. On the other hand, inconsistencies in strength of antioxidant capacity remain an issue [24
]. Although research on relationship between the activity and pH has been reported [10
], antioxidant capacity of MAAs is still controversial.
Among macro algae, dulse (Palmaria palmata
) is known to possess MAAs [18
]. Dulse is a red alga distributed mainly across coastal areas in high-latitude such as Ireland and Atlantic Canada. Dulse also grows well in Hokkaido, Japan. Hakodate, the southern part of Hokkaido, is famous for Kombu farming, and dulse grew there and disturbed growth of Kombu (Laminaria
]. Dulse is underused alga in Japan; therefore, the use of dulse was requested. To utilize dulse in Usujiri, Hakodate, Japan, we clarified genetic character [27
] and showed the nutrient characters (approximately 40 g protein/100 g dried dulse). The major component of proteins is phycoerythrin (PE) [29
], and the thermolysin hydrolysate showed the angiotensin-I-converting enzyme inhibitory activity [26
]. The chromophores from PE showed antioxidant capacity [32
]. In addition, β-(1→3)-xylosyl-xylobiose prepared from dulse xylan exhibited the prebiotic effect on enteric bacterium [33
]. In this way, dulse contains many healthy functional ingredients. On the other hand, seaweeds contain low content of MAAs (up to 14 mg g−1
dry weight) [9
]. Usujiri dulse grew in the depth of 1–2 m, which is expected a large content of MAAs, since the depth of biotope was a key factor of MAAs synthesis [12
]. In order to use MAAs from Usujiri dulse for biotechnological applications, it is necessary to develop the efficient extraction method. However, little has been reported on the efficient extraction from macro algae.
In this study, we first determined the efficient extraction condition to evaluate the content of MAAs of Usujiri dulse and then investigated the monthly variation (January–May 2019). Finally, we evaluated the effects of pH on the antioxidant capacity of MAAs by ABTS radical scavenging and ferrous reducing power assays.
4. Materials and Methods
4.1. Algal Material
All dulse samples were collected at 1 m depth in Usujiri, Hakodate, Japan (41°56′N, 40°56′E) on March 2018 and from January to May 2019. After collection, the thalli were washed with tap-water to remove sea salt and epibionts. Soon after, they were lyophilized. Dried algal samples were ground into a fine powder by Wonder Blender WB-1 (Osaka Chemical Co., Osaka, Japan) and stored in the dark at room temperature until analysis.
4.2. Determination of the Extraction Condition of Dulse Crude MAAs
To determine the optimum extraction conditions of MAAs, water extraction time from dulse powder was evaluated. The powdered samples were soaked in 20 volumes (v/w) of distilled water at 4 °C for 2 to 24 h. The water extracts were collected by centrifugation at 4 °C, 27,200 g for 10 min. After centrifugation, the supernatants were lyophilized and soaked in 20 volumes (volume/powdered sample weight) of methanol at 4 °C for 2 h. The MAAs containing methanol extracts were centrifuged at 4 °C, 27,200× g for 15 min, and designated as dulse crude MAAs solutions. The supernatants were evaporated, re-dissolved in water, and lyophilized. Then, the solid samples were designated as dulse crude MAAs and used following experiments.
4.3. Spectrophotometric Analysis of Dulse Crude MAAs Solutions
Dulse crude MAAs solutions were analyzed by the UV-visible ray absorption spectrum using a spectrophotometer (250–400 nm, UV-1800, Shimadzu, Kyoto, Japan).
4.4. Separation of MAAs by HPLC
The dulse crude MAAs were dissolved in ultra pure water containing 0.1% trifluoroacetic acid (TFA) and applied to sequential filtration by Millex-GV (pore size: 0.22 μm) (Merck Millipore, Billerica, MA, USA) and Millex-LG (pore size: 0.20 μm) (Merck Millipore). The filtrated MAAs were isolated by reversed-phase HPLC with a Mightysil RP-18GP column (5 µm, 10 × 250 mm) (Kanto Kagaku, Tokyo, Japan) using an isocratic elution of ultra pure water containing 0.1% 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 having 330 nm were fractionated and evaporated. Then, the purified MAAs were dissolved in an appropriate amount of distilled water.
4.5. Identification of MAAs by MALDI-TOF/MS
The mass-to-charge ratio of MAAs was determined by the Matrix Assisted Laser Desorption/Ionization Time Of Flight Tandem Mass Spectrometry (MALDI-TOF/MS) method using a 4700 Proteomics Analyzer with Denovo Explorer software (Applied Biosystems, Carlsbad, CA, USA). The fractionated MAAs were lyophilized and dissolved in ultra pure water containing 0.1% TFA. Then, the samples were mixed with 5 mg/mL α-cyano-4-hydroxycinnamic acid matrix and detected by positive-mode.
4.6. Calculation of the Content of MAAs in HPLC
The content of individual MAAs was determined using Lambert–Beer law. Using the purified individual MAAs, the relationship between the content of MAAs and HPLC peak area was determined. The results were expressed as µmol g−1 DW (dry weight).
4.7. ABTS Radical Scavenging Assay
ABTS radical scavenging assay was carried out according to the method of Binsan et al. [66
] with some modifications. The working solution was prepared by mixing with an equal volume of 14.8 mM ABTS and 5.2 mM potassium persulphate and incubated at room temperature in dark conditions for 12 h. The ABTS reagent having the absorbance at 734 nm of 1.00 ± 0.02 was prepared by the dilution of working solution with 0.2 M phosphate buffer (pH 5.8, 6.6, 7.4, and 8.0). The ABTS radical scavenging assay was performed as follows: 50 µL of sample (dulse crude MAAs or the purified MAAs) or distilled water were mixed with 950 µL of the ABTS reagent or the phosphate buffer. Then, the mixture was incubated at room temperature for 2 h in the dark. After the incubation, the solution was centrifuged at 4 °C, 2000× g
for 5 min to remove insoluble, and the supernatant was measured absorbance at 734 nm. ABTS radical scavenging activity (%) was calculated from the equation [1 − (As − Asb)/(Ac − Acb)] × 100, where As is the absorbance of sample mixed with the ABTS reagent, Asb is the absorbance of sample mixed with the phosphate buffer, Ac is the absorbance of distilled water mixed with the ABTS reagent, and Acb is the absorbance of distilled water mixed with the phosphate buffer. Ascorbic acid was used as a standard.
4.8. Ferrous Reducing Power Assay
Ferrous reducing power was determined according to the method of Kuda et al. [67
] with some modifications. In addition, 0.4 mL of sample (dulse crude MAAs or the purified MAAs) or distilled water were mixed with 0.4 mL of 0.2 M phosphate buffer (pH 5.8, 6.6, 7.4, and 8.0) and 0.4 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 20 min. Then, 0.4 mL of 10% trichloroacetic acid was added to the reaction mixture. A half volume of samples were extracted and then mixed with 960 µL of 0.017% ferric chloride and incubated for 10 min at room temperature. Absorbance at 700 nm of solutions was measured. Ferrous reducing power (OD) was calculated from the equation As-Asb, where As is the absorbance of sample and Asb is the absorbance of control. Ascorbic acid was used as a standard.
4.9. Abiotic Data in Hakodate (Usujiri)
The monthly mean of daily maximum ultraviolet index (UVI) was obtained from Japan Meteorological Agency (JMA: https://www.data.jma.go.jp/gmd/env/uvhp/info_uv.html
). According to the method of JMA, erythemal UV intensity (mW/m2
) was calculated by multiplying UVI by 25 times. Data on chlorophyll concentration (mg/m3
) of near-surface were obtained from NASA’s Ocean Color WEB (https://oceancolor.gsfc.nasa.gov
). All data were recorded in 2019.
4.10. Statistical Analysis
Data are expressed as the mean ± standard error. All values are mean 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 No. 3, OMS Publisher, Tokorozawa, Japan).