Radical-induced oxidative damage to body cells is associated with the causation and progression of inflammatory diseases and other chronic diseases [1,2].

On the other hand, various vegetables and fruits have been considered as preventive foods against these diseases, because the antioxidative components in these foods have radical scavenging activities against the oxygen radical attack on the important biomolecules such as proteins, nucleic acids and lipids in human body [3,4].

In Asian countries such as China, Korea and Japan, besides these edible plants, various edible algae have been traditionally consumed as healthy food stuffs. Some of the constituents in edible algae showed strong radical scavenging activities. For example, several brown algae contain significant amounts of antioxidant phlorotannin [5]. Antioxidant phenolic compounds such as epigallocatechin and cathechol were found in specific green algae, Halimeda species [6]. Furthermore, strong antioxidant activities were detected in the specific low molecular weight fractions of the sulfated polysaccharides extracted from a brown alga Laminaria japonica (Ma-konbu) by acid hydrolysis [7] or radical process degradation [8].

On the other hand, in Japan, an edible red alga, Porphyra tenera (P. tenera, Asakusa-nori) has been consumed as a traditional popular food stuff. Usually, P. tenera is dried and roasted under high temperature condition, which is called “Yaki-nori”. Generally, it has been believed that the purpose of roasting treatment of P. tenera is the improvement of its taste and flavor until present. Previously, we found the remarkable enhancing effect of roasting treatment on the radical?scavenging activity in the extract of P. tenera by using DPPH radical assay [9]. Furthermore, we also detected the roasting effect on the radical scavenging activity in Laminaria japonica (Ma-konbu), which was analyzed by using several radical generating systems and the active principles responsible for the enhancing effect on the radical?scavenging activity were analyzed [10].

 In our present study, we also found the strong enhancing effect of the high temperature roasting treatment on the radical scavenging activity in the extract of another brown alga Hijikia fusiformis (Hijiki), and the possible active principles responsible for the enhancing effect on the radical?scavenging activity were analyzed. Especially, the role of the specific polysaccharide fraction in Hijiki extract for the enhancing effect on the radical scavenging activity was analyzed. The significance of the enhancing effect of roasting treatment on the radical scavenging activity of Hijiki is discussed from the viewpoint of medical food science.

Chemicals and Reagents

Aluminum chloride, caffeic acid, soluble starch, tannic acid, Vanillin and 8-amino-5-chloro-7-phenylpirido[3,4-d]pyridazine-1,4-[2H,3H]dione (L-012) were purchased from Wako Pure Chemicals (Osaka, Japan). Linoleic acid and o-phenanthroline were purchased from Kanto Chemicals (Tokyo, Japan). Bovine serum albumin, diphenylpicrylhydrazyl (DPPH), Folin-Denis reagent, DEAE cellulose and yeast zymosan were purchased from Sigma-Aldorich Japan (Tokyo, Japan).

Preparation of the Extract of the Roasted Hijikia Fusiformis (Hijiki)

Hijiki was harvested at Ise-shima district of Mie prefecture in Japan. The dried fronds of Hijiki were minced extensively by an electric milling blender (Type MC100R, San-Ei Co. Tokyo, Japan), and the Hijiki powder was roasted at 100ºC, 130ºC, 150ºC, 180ºC or 200ºC for 30 min in an electric oven (Type NE-A251, Panasonic Co, Osaka, Japan). One gram of the roasted Hijiki powder was suspended with 49 ml of distilled water and the suspension was agitated by a stirring apparatus at 4ºC for 3 hr. After each suspension was centrifuged at 1,500 x g for 15 min, the supernatant was stored at -20ºC in a freezer as the Hijiki extract for further experiments.

Separation of the Specific Polysaccharide Fraction of Hijiki Extract

The specific polysaccharide fraction of Hijiki was separated by the modification of the previous method [11]. Ten gram of the unheated Hijiki powder mentioned above was added and mixed with 500 ml of hot water (95ºC) and the mixture was mildly agitated by a stirring apparatus for 3 hr at 4ºC. After the suspention of the Hijiki powder was centrifuged at 1,500 x g for 15 min, the supernatant fraction was mixed with three volumes of cold ethanol with 1/10 volume of 3M NaCl solution and kept for 3 hr at -20ºC. The polysaccharide fraction of the mixture was separated by a centrifugation at 3,000 x g for 15 min as a precipitate fraction, which was dissolved with 20 ml of distilled water and dialized against 2 L of cold distilled water by using a dialysis membrane (cut size : 3.5 kdaltons, Spectrum Medical Industries, Los Angels, USA) at 4ºC for one overnight. The dializate containing polysaccharides was applied on the DEAE cellulose column chromatography. After the column was washed mildly with 25 mM phosphate buffer (pH 6.8), the bound polysaccharides were eluted by the same buffer containing 0.5 M NaCl and recovered. The recovered polysaccharide fraction was dialized against distilled water at 4ºC and dried by a rotary evaporator at 60ºC, and the dried powder was used as a polysaccharide fraction for further experiments. As our preliminary physicochemical analysis, this polysaccharide fraction contains significant amount of fucose, galactose, mannose and uronic acid as the polysaccharide component. However, the polysaccharide fraction in the present study did not contain alginic acid, because we could not detect D-mannuronate and L-gluronate as alginic acid component in this polysaccharide fraction. Generally, the extraction of alginic acid from brown algae is carried out by strong alkaline pH condition. Furthermore, significant amount of proteins or amino acids were not detected in this preparation. Possibly, this polysaccharide fraction seems to correspond to fucoidans.

Roasting Experiment of the Polysaccharide Fraction of Hijiki Extract

The polysaccharide fraction of Hijiki mentioned above was dissolved with distilled water at the concentration of 50 mg/ml in each glass test tube and heated to 100ºC, 130ºC, 150ºC , 180ºC or 200ºC for 30 min in an electric oven (Type NE-A251, Panasonic Co, Osaka, Japan). The dried powder of each roasted polysaccharide fraction was dissolved with distilled water at the concentration of 5 mg/ml, and its radical scavenging activities were measured by various in vitro assay systems.

Measurement of Total Saccharide Concentration in Test Solution

The saccharide concentration in test solution was measured by the modification of phenol/H₂SO₄ method in the previous report [12] by using soluble starch as standard saccharide.

Measurement of Polyphenol Concentration in Hijiki Extract

Polyphenol concentration in Hijiki extract was measured by the modification of Folin-Denis method in the previous report [10]. In brief, after 200 µl Hijiki extract was diluted with the same volume of distilled water, the mixture was added with 2 ml of Folin-Denis reagent and 2 ml of saturated sodium carbonate solution. After keeping the mixture solution for 20 min at room temperature, it was centrifuged at 1,500 x g for 15 min. The photometric absorbance of the supernatant of the mixture was measured at 700 nm by a Shimadzu UV-265 spectrophotometer. The same experiment was carried out by using successively diluted caffeic acid solution as standard polyphenol.

Measurement of Tannin Concentration in Hijiki Extract

Tannin concentration in Hijiki extract was measured by the modification of vanillin/HCl method in the previous report [11]. 500 µl of Hijiki extract was mixed with 2 ml of vanillin/HCl reagent. After keeping the mixture at room temperature for 15 min, it was centrifuged at 1,500 x g for 15 min. The photometric absorbance of the supernatant fraction of the mixture solution was measured at 525 nm by a Shimadzu UV-265 spectrophotometer. The same experiment was carried out by using successively diluted tannic acid solution as standard tannin.

Assay for DPPH Radical Scavenging Activity of Test Solution

The radical?scavenging activity in Hijiki extract or test solution was analyzed by using diphenylpicrylhydrazyl (DPPH) radical according to the modification of the previous method [13]. Namely, 200 µl of Hijiki extract or test solution was mixed with 3.8 mL of 0.2 mM DPPH in ethanol. A negative control experiment was performed with distilled water in place of test sample. The photometric absorbance of the reaction mixture (At) was measured at 30 min after the initiation of the assay reaction by a Shimadzu UV-265 spectrophotometer at 520 nm. The background absorbance (Ab) of Hijiki extract itself was subtracted from the apparent value of absorbance in the assay reaction mixture, and its value was called At. The radical scavenging activity (RSA) was calculated by the following equation: RSA (%) = ((Ao – At)/ (Ao – Ab)) x 100%, where Ao is the absorbance at 520 nm at the start of the assay reaction. As a standard radical scavenging substance, ascorbic acid was successively diluted with deionized water, and the DPPH radical scavenging activity of ascorbic acid solution was assayed as described above.

Assay for Hydroperoxide Radical Generated By Linoleic Acid Peroxidation

The amount of hydroperoxide radical generated by linoleic acid peroxidation was measured by the slight modification of previous aluminum chloride method [14]. One ml of Hijiki extract was mixed with 1 ml of linoleic acid in ethanol and 2 ml of sterile 50 mM phosphate buffer (PB, pH 7.5) in a glass test tube and stood for 2 weeks at 35ºC. As a negative control experiment, 1 ml of sterile distilled water was mixed in place of test solution. After the peroxidation reaction of linoleic acid, the reaction mixture mentioned above (200 µl), 250 µl of 0.2% KI solution in ethanol, 250 µl of 2% aluminum chloride solution and 1 ml of hexane were mixed in a glass test tube and incubated for 5 min at 37 ºC, then 250 µl of starch solution and 7.5 ml of 10 mM HCl were mixed and shaken vigorously. After the mixture was centrifuged at 1,500 x g for 10 min, the absorbance of the lower layer fraction of the mixture was measured at 560 nm by using Shimadzu UV-265 spectrophotometer.

Assay for Oxygen Radical Generation in Human Blood Neutrophils

Isolation of human neutrophils and assay for oxygen radical generation in human neutrophils were carried out by the previous method [15]. Human venous blood obtained from a healthy nonsmoking male donor was recovered in the presence of 3.8% citrate and centrifuged using Monopoly Resolving Medium (Dainihon Pharmatheutical Co. Osaka, Japan) and Krebs Ringer phosphate solution (KRP, pH 7.4). The neutrophils in the cell layer were recovered and washed two times with KRP by centrifugation and kept in an ice box until using for the experiment. Isolation of the blood human neutrophils and other associated experimental procedures were carried out according to the rules of the experimental moral and hazard committee in Osaka Prefecture University. Human neutrophils (1 x 10⁵ cells) were incubated with 50 mM phosphate buffered saline (PBS pH 7.4) in the presence of 400 µM L-012 (chemiluminescence reagent) at a final volume of 500 µl. After the preincubation of the cell suspension mixture including Hijiki extract or test solution at 37ºC for 3 min, the reaction was started by the addition of opsonized yeast zymosan (Opz, 1 µg/ml) and further incubated another 30 min. The chemiluminescence (CHL) intensity of the cell suspension mixture was recorded continuously during the incubation at 37ºC by using a CHL detector apparatus (Type BLR-102, Aloka Co., Tokyo, Japan).

Statistical Analysis of Experimental Results

The experimental result was expressed as the mean and SD (standard deviation) of triplicate assays. The statistical comparison of the experimental results between the control (RT: room temperature) and different roasting temperature conditions was analyzed by using Student’s t test. A P value less than 0.05 was considered to be significantly different.

Enhancing Effect on the Radical Scavenging Activity of the Hijiki Extract against DPPH Radical by Roasting Treatment

First, we analyzed the radical scavenging activity of the water extract of Hijiki?treated?under different temperature roasting conditions by using a stable radical compound diphenylpicrylhydrazyl (DPPH). As shown in Fig1, the water extract of unheated Hijiki kept at room temperature (RT: 22 ºC) exhibited a weak but significant DPPH radical scavenging activity (14.1% inhibition). When roasted at 100ºC, 130ºC or 150ºC for 30 min, the Hijiki extracts showed the weak but significant increase in the radical scavenging activities (16.5%, 18.2% and 19.5% inhibition, P <0.05 ) as compared with the control activity at room temperature. Furthermore, when roasted at 180ºC or 200ºC for 30 min, the Hijiki extracts exhibited the very strong radical scavenging activities (74.1% and 90.8% inhibition, P <0.001). This experimental result indicates that the Hijiki extracts roasted under higher temperature conditions (180ºC and 200ºC) have the strong radical scavenging activities against DPPH radical.

Enhancing Effect of Roasting Treatment on the Radical Scavenging Activity of Hijiki Extract against Hydroperoxide Radical Generation by Linoleic Acid Peroxidation

Second, we analyzed the radical scavenging activity of the water extract of Hijiki treated under different temperature conditions by using hydroperoxide radical generating system in linoleic acid peroxidation. As shown in Fig.2, the water extract of unheated Hijiki kept at room temperature (RT) exhibited a weak radical scavenging activity against hydroperoxide radical generation in linoleic acid peroxidation as compared with the control experiment of water (15.9% inhibition). When Hijiki was roasted at 100ºC for 30 min, the significant increase in the radical scavenging activity could not detected as compared with the extract of non-roasted Hijiki, but when roasted at 130ºC or 150ºC for 30 min, the extracts showed moderate increase in the radical scavenging activities (21.0% and 24.8% inhibition, P <0.05) as compared with the control activity at room temperature. Furthermore, when roasted at 180ºC or 200ºC for 30 min, the Hijiki extracts caused the strong radical scavenging activities (58.4% and 74.6% inhibition, P <0.01 and P <0.001). This experimental result suggests that Hijiki extracts roasted under higher temperature conditions (180ºC and 200ºC) have the strong radical scavenging activities against hydroperoxide radical generation in linoleic acid peroxidation.

Suppressive Effect of the Extract of Roasted Hijiki on Opz-Stimulated Oxygen Radical Generation in Human Blood Neutrophils

In addition to the cell-free experiments mentioned above, we analyzed the effects of the Hijiki extracts on the oxygen radical generation in opsonized zymosan (Opz)-stimulated human blood neutrophils as a typical oxygen radical-producing cell system. As shown in Fig.3, opsonized zymosan (Opz) strongly enhanced oxygen radical generation in human blood neutrophils which showed the very strong L-012-dependent chemiluminescence (CHL) generation. When the extract of unheated Hijiki kept at room temperature (RT) was added before the Opz stimulation, a weak suppressive activity against Opz-induced oxygen radical generation (12.6% inhibition) was observed. When Hijiki was roasted at 100ºC, the extract did not show the

significant increase in the radical scavenging activity as compared with the control experiment of unheated Hijiki extract kept at room temperature (RT). However, the roasting treatment at 130ºC or 150ºC for 30 min exhibited the mild but significant increase in the radical scavenging activities (18.0% and 23.8% inhibition, P <0.05) as compared with the control activity at room temperature (RT). Furthermore, the Hijiki extracts roasted at 180ºC and 200ºC showed the strong suppressive effects on Opz-induced oxygen radical generation (66.0% and 80.3% inhibition, P <0.001). This experimental result indicates that Hijiki extracts roasted under higher temperature conditions (180ºC or 200ºC) cause the strong radical scavenging activities against oxygen radical generation in Opz-stimulated human blood neutrophils.

Possible Active Components Responsible For the Roasting-Induced Enhancement of the Radical Scavenging Activity of the Hijiki Extract

 To analyze the active principles responsible for the roasting-induced enhancement of the radical scavenging activity in the Hijiki extract, we measured the concentrations of possible active components in the Hijiki extracts under different temperature roasting conditions.

First, as a possible active component, we measured the concentration of polyphenol in the Hijiki extract according to the previous report [6]. As shown in Table 1, although the polyphenol concentration in Hijiki extract kept at room temperature was relatively low (18.4 µg/ml), the polyphenol concentrations in Hijiki extracts roasted at 100ºC, 130ºC or 150ºC increased significantly (27.0, 30.6 and 38.2 µg/ml). Furthermore, the polyphenol concentrations in the Hijiki extracts roasted at 180ºC and 200ºC remarkably increased (183.7 and 290.3 µg/ml, P <0.001). This experimental result suggests that the radical scavenging activity in the extract of roasted Hijiki is highly correlated with the polyphenol concentration in the extract.

Next, we measured the tannin concentration in the extracts of roasted Hijiki according to the previous study [5]. As indicated in Table 2, although the tannin concentration in the extract of Hijiki kept at room temperature was 4.5 mg/ml, the tannin concentrations in Hijiki extracts roasted at 100ºC, 130ºC or 150ºC increased moderately (5.7, 6.3 and 7.9 mg/ml). Furthermore. the tannin concentrations in the extracts roasted at 180ºC or 200ºC were relatively high (19.3 and 30.7 mg /ml, P <0.01) as compared with the tannin concentrations under lower temperature conditions. This result indicates a possibility that the radical scavenging activity in the extract of roasted Hijiki is associated with the tannin concentration in the Hijiki extract under different roasting temperature conditions.

Furthermore, we measured the saccharide concentration in the Hijiki extracts under different temperature conditions according to the previous studies [7,8]. As shown in Table 3, although the saccharide concentration in the extract of Hijiki kept at room temperature was 16.3 mg/ml, the saccharide concentrations increased moderately at 100ºC, 130ºC and 150ºC (22.6, 25.1 and 28.3 mg/ml, P <0.05). Furthermore, the saccharide concentrations in the extracts roasted at 180ºC or 200ºC were relatively high (56.8 and 78.2 mg/ml, P <0.01) as compared with those under lower temperature conditions. These experimental results suggest a possibility that the radical scavenging activity in the extract of roasted Hijiki is correlated with the saccharide concentration in the extract.

The Role of the Specific Polysaccharide Fraction for the Enhancing Effect on the Radical Scavenging Activity in the Roasted Hijiki

As a possible active component for the enhancing effect on the radical scavenging activity in the roasted Hijiki, the role of the specific polysaccharide fraction in Hijiki extract was analyzed.

We separated the specific polysaccharide fraction from the non-roasted Hijiki extract, which seems to correspond to fucoidans as explained in Materials and Methods. The radical scavenging activities of this polysaccharide fraction under different temperature roasting conditions were analyzed by several in vitro assay systems.

First, we analyzed the effect of roasting treatment of the polysaccharide fraction on the radical scavenging activity against DPPH radical. As shown in Table 4, the non-roasted polysaccharide fraction did not exhibit significant DPPH radical scavenging activity. When the polysaccharide fraction was roasted at 100ºC or 130ºC for 30 min, the weak but significant radical scavenging activities were observed (6.8% and 9.5% inhibition, P <0.05). When roasted at 150ºC for 30 min, the polysaccharide fraction showed the significant radical scavenging activity (23.8% inhibition, P <0.01). Furthermore, the polysaccharide fractions roasted under higher temperature conditions (180ºC and 200ºC) caused the strong radical scavenging activities against DPPH radical (56.3% and 78.2% inhibition, P <0.001).

Second, we analyzed the effect of roasting treatment the polysaccharide fraction of Hijiki on the radical scavenging activity against hydroperoxide generation in linoleic acid peroxidation. As shown in Table 5, the non-roasted polysaccharide fraction did not exhibit significant radical scavenging activity in the control experiment (room temperature). When the polysaccharide fraction of Hijiki roasted at 100ºC or 130ºC for 30 min, they showed weak but significant radical scavenging activities (8.3%, 11.6% inhibition, P <0.05). Furthermore, when roasted at 150ºC for 30 min, the polysaccharide fraction exhibited the significant radical scavenging activity (28.0% inhibition, P <0.01). When the polysaccharide fraction was roasted at 180ºC for 30 min, it showed the considerable radical scavenging activity against hydroperoxide radical generation (46.8% inhibition, P <0.002). Furthermore, when roasted at 200ºC for 30 min, the polysaccharide fraction caused the strong radical scavenging activity (65.9% inhibition, P <0.001). This experimental result suggests that the polysaccharide fraction roasted under higher temperature conditions (180ºC or 200ºC) exhibited the relatively high radical scavenging activities against hydroperoxide radical generation in linoleic acid peroxidation.

Next, we analyzed the effect of roasting treatment of the polysaccharide fraction of Hijiki on the radical scavenging activity against oxygen radical generation in Opz-stimulated human blood neutrophils. As shown in Table 6, the non-roasted polysaccharide fraction (room temperature) did not exhibit significant radical scavenging activity. When the polysaccharide fraction was roasted at 100ºC and 130 ºC, they showed weak but significant radical scavenging activities (10.4% and 16.2% inhibition, P <0.05). However, the roasting treatment at 150ºC for 30 min exhibited the considerable radical scavenging activity (36.8% inhibition, P <0.01). Furthermore, the polysaccharide fraction roasted at 180ºC or 200ºC for 30 min showed the strong suppressive effects (65.0% and 84.7% inhibition, P <0.001). This experimental result indicates that the specific polysaccharide fraction of Hijiki roasted under higher temperature roasting conditions (180ºC or 200ºC) cause the strong radical scavenging activities against oxygen radical generation in Opz-stimulated human blood neutrophils.

In the present study, we found the strong enhancing effect of the high temperature roasting treatment on the radical scavenging activity in the extract of a Japanese brown alga, Hijikia fusiformis (Hijiki), which was estimated by using several radical generating systems such as (i) a stable radical compound DPPH, (ii) hydroperoxide radical generating system in linoleic acid peroxidation and (iii) opsonized zymosan (Opz)-induced oxygen radical generating system in human blood neutrophils (Fig.1, 2 and 3).?

Generally, it has been believed that the purpose of roasting edible algae is the improvement of their tastes and flavors until present. However, the significance of the roasting treatment of edible algae has been poorly elucidated from the viewpoint of antioxidant or radical scavenging activity.

For example, in Japan, two types of commercially available Porphyra?tenera (P. tenera,?Asakusa-nori) have been known as traditional popular food stuffs. One type is dried P. tenera , which is called “Hoshi-nori”, and the other type is roasted P. tenera, which is called “Yaki-nori”. In our previous study, the strong enhancing effect of roasting treatment on the radical scavenging activity of P. tenera (Yaki-nori) extract against DPPH radical was observed in very short roasting time (10-20 sec) at 180ºC [9]. However, the significant enhancing effect of Hijiki on radical scavenging activity was not detected by the same short roasting treatment at 180ºC. The different roasting effects between P. tenera and Hijiki seem to be responsible for their different body structures. Apparently, P. tenera has thin and labile fronds, but Hijiki has thick and tough body structures. We considered that Hijiki may exhibit the similar roasting effect on the radical scavenging activity by more strong roasting treatment. As shown in the present study, Hijiki extract showed the strong radical scavenging activities by much stronger roasting treatments (180ºC and 200ºC for 30 min) (Fig. 1, 2 and 3).

Figure 1: Enhancing effect of roasting treatment on the radical scavenging activity of Hijiki extract against DPPH radical.

The column and bar in figure show the mean and SD of DPPH radical scavenging activities of the Hijiki extract in triplicate assays. The radical scavenging activity of the Hijiki extract against DPPH radical was calculated as described in Materials and Methods. The statistical difference of the results between the control (RT: room temperature) and different roasting temperature conditions is analyzed by Student’s t test. (* : P <0.05, ** : P <0.001 ) SD: standard deviation, DPPH: diphenylpicrylhydrazyl

Figure 2: Enhancing effect of roasting treatment on the radical scavenging activity of Hijiki extract against hydroperoxide radical generation from linoleic acid peroxidation.

The amount of hydroperoxide radical generated from linoleic acid peroxidation in the absence and presence of Hijiki extract was measured by aluminum chloride method as described in Materials and Methods. The column and bar in figure show the mean and SD of the radical scavenging activities of the Hijiki extract against hydroperoxide radical generation from linoleic acid peroxidation in triplicate assays. The statistical difference of the results between the control (RT: room temperature) and different roasting temperature conditions is analyzed by Student’s t test. (* : P <0.05, ** : P <0.01, *** : P <0.001 )

Figure 3: Suppressive effect of the extract of roasted Hijiki on the Opz-induced CHL generation in blood neutrophils.

The oxygen radical generating activity in Opz-induced human blood neutrophils was measured by L-012-dependent CHL generation as described in Materials and Methods. The column and bar in figure show the mean and SD of CHL generating activities of triplicate assays. The statistical difference of the results between the control (RT : room temperature) and different roasting temperature conditions is analyzed by Student’s t test. (* : P<0.05, ** : P<0.001) Opz: Opsonized zymosan, CHL: Chemiluminescence, L-012: 8-amino-5-chloro-7-phenylpirido[3,4-d]pyridazine-1,4-[2H,3H]dione (Chemiluminescence reagent), PBS: Phosphate buffered saline, ND: Not detected

Furthermore, we assumed that heterogenous radical-scavenging substances might be associated with the roasting-induced enhancing effect on the radical scavenging activity of the Hijiki extract, and there may be possible reasons for the increase in radical scavenging activity in the extract of Hijiki by roasting treatment.

One possibility is the increase in the extractability of radical scavenging substances by effective roasting treatment. For example, the concentrations of polyphenol, tannin and saccharide in the Hijiki extract were correlated with the radical scavenging activities of the extracts under different roasting conditions (Figure 1, 2 and 3, Table 1, 2 and 3).

Table 1: Polyphenol concentrations in the Hijiki extracts under different temperature conditions.

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