Improving the Efficiency of Organoarsenic Extraction from Seaweeds

Abstract

Seaweeds are a source of arsenosugars (AsSug). AsSug are mainly metabolized to dimethylarsenic species, but their relative health risks have not yet been fully evaluated. Therefore, it is essential to assess the risk of AsSug intake. Although AsSug are water-soluble arsenics, the extraction efficiency of arsenic compounds from wakame seaweed specimens (Undaria pinnatifida) ranges only between 4 and 49%. To develop a high recovery-rate extraction method without altering the chemical structure of the arsenic compounds, we examined the efficacy of a combined enzymatic treatment and methanol (MeOH) extraction method. After treatment with cellulase and alginate lyase and extraction with 100% MeOH, we extracted 88.8% of the arsenic compounds in wakame without arsenic species alteration. Four arsenic peaks were detected using high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS), two of which were identified as 3-[5′-deoxy-5′-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropylene glycol and 3-[5′-deoxy-5′-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropyl-2,3-hydroxypropyl phosphate by using HPLC-electrospray ionization-quadrupole–time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS). Further, the HPLC-ESI-Q-TOF-MS analyses suggested that, of the remaining two peaks, one corresponded to arsenic-hydrocarbon 388 (C₂₁H₄₅OAs) and arsenic-phospholipid 1012 (C₄₉H₉₄O₁₄PAs). Thus, our method can be used to extract arsenic compounds from brown algae with strong cell walls.

1. Introduction

Seaweeds contain large amounts of soluble organic arsenic compounds such as arsenosugars (AsSug) and arsenophospholipids^1,2,3,4). AsSug are mainly metabolized to dimethylarsenic species^5,6). Raml et al.⁶⁾ detected several dimethylarsenic species such as dimethylarsinic acid (DMA), thio-dimethylarsenoethanol (thio-DMAE), and thio-dimethylarsenoacetic acid (thio-DMAA) as major metabolites in urine. In addition, traces of thio-DMA were also detected in urine samples from volunteers following the ingestion of 3-[5′-deoxy-5′-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropylene glycol (AsSug 328). Leffers et al.⁷⁾ demonstrated the cytotoxicity and genotoxicity of DMA and thio-DMA in cultured human bladder cells; thio-DMA, in particular, exhibited high toxicity. Consequently, these substances cannot be considered as nontoxic for humans, and the risk of AsSug ingestion to human health should be considered.

AsSug are water-soluble; therefore, the main method used until date to extract arsenic compounds from seaweed involves the use of a water-methanol (MeOH) solution^8,9). However, this method is not particularly efficient in extracting arsenic compounds from wakame (Undaria pinnatifida)^8,9), one of the most popular edible seaweeds in Japan and Asia. The critical first step of arsenic speciation analysis in seaweed is the development of an efficient extraction method. Here, we hypothesized that polysaccharide compounds in the cell walls, whose function is to protect the cells, are the main structural barriers that prevent efficient arsenic extraction in this type of alga. Thus, we propose the development of an extraction method that is able to achieve high recovery rates under mild conditions without altering the chemical structure of the arsenic compounds.

In this study, we assessed the efficacy of a pre-treatment enzymatic method able to break down the structural polysaccharide compounds of the cell wall, and examined the optimal extractant to use on the enzyme-treated wakame.

2. Materials and Methods

2-1. Chemicals

Sodium arsenite (AsIII), sodium arsenate (AsV), monomethylarsonic acid (MMA), and arsenobetaine (AsBe) were purchased from Wako Pure Chemical Industries (Osaka, Japan). DMA was obtained from Tri Chemical Laboratory (Yamanashi, Japan). The oxo-arsenosugar AsSug 328 was synthesized as described by Traar and Francesconi¹⁰⁾. Ultrapur-100 nitric acid and Ultrapur hydrogen peroxide (Kanto Chemical, Tokyo, Japan) were used for acid digestion. Sodium dihydrogen phosphate dihydrate (NaH₂PO₄·2H₂O; Kanto Chemical, Japan) and sodium dihydrogen phosphate dodecahydrate (NaH₂PO₄·12H₂O; Kanto Chemical, Japan) were used for the buffer solution in the enzyme treatment. For the high performance liquid chromatography (HPLC) mobile phase, we used acetic acid (CH₃COOH for liquid chromatography–mass spectrometry [LC/MS] analysis; Wako Pure Chemical Industries, Japan), ammonium acetate (CH₃CO₂NH₄ for HPLC analysis [99%]; Sigma-Aldrich Japan, Tokyo, Japan), ammonium hydrogen carbonate (NH₄HCO₃ for LC/MS analysis; Sigma-Aldrich, Japan), and MeOH (CH₃OH for LC/MS analysis; Wako Pure Chemical Industries, Japan). Germanium standard solution for the atomic absorption spectrophotometer (Kanto Chemical, Japan) was used as the internal standard for inductively coupled plasma mass spectrometry (ICP-MS) detection. Ultrapure water was prepared using a MilliQ-ICP/MS Ultrapure Water Purification System (Millipore, Tokyo, Japan). Certified reference material, NMIJ CRM 7405-a (Hijiki), from the National Institute of Advanced Industrial Science and Technology in Japan was used to validate the analytical procedure.

2-2. Test Sample

Commercial dry wakame seaweed (harvested from the Sanriku Coast in Japan) was purchased from a retail market in Choshi City, Chiba Prefecture, and was used in the experiment. According to the manufacturer, to obtain the final commercially available dry wakame, the seaweed undergoes four processes after harvest: parboiling, preservation with salt, desalination, and drying. The purchased dried wakame sample was finely ground to powder prior to experimental use.

2-3. Total Arsenic Content Determination

The powdered wakame was subjected to microwave acid digestion by using a closed vessel system (P-25 series, Sanai Science, Aichi, Japan). Subsequently, total arsenic (T-As) content in the samples was analyzed using ICP-MS. For the acid digestion, 100 mg of the powdered samples were introduced into each vessel along with 2.5 mL of acid mixture (HNO₃:H₂O₂ = 5:2, v/v); this mixture was maintained at room temperature for 6 hours prior to digestion in a microwave oven for 8 minutes at 170 W. After cooling the vessels, the solutions were adjusted to 30 mL by adding 5% HNO₃. T-As concentrations were determined using Elan DRCII ICP-MS (PerkinElmer SCIEX, Concord, Ontario, Canada). The ICP-MS assay was conducted under the following conditions: RF power, 1300 W; plasma argon gas flow rate, 15 L/min; auxiliary argon gas flow rate, 1.2 L/min; and nebulizer argon gas flow rate, 1.0 L/min. In addition, we used a coaxial-type nebulizer and skimmer, and the sample cones were made of platinum. Validation was performed through the analysis of NMIJ CRM 7405-a (Hijiki). For NMIJ CRM 7405-a, the certified T-As concentration was 35.8 ± 0.9 mg/kg, and we detected 35.5 ± 0.4 mg/kg of T-As (mean ± S.D., n = 3), validating the T-As determination method.

2-4. Enzymatic Treatment Condition

Alginate lyase (EC No. 4.2.2.3 from Flavobacterium sp.; Sigma-Aldrich, Japan) and cellulase (EC 3.2.1.4; Cellulase Onozuka R-10, from Trichoderma viride, Merck Millipore, MA, USA) were added into 0.1 M phosphate buffer (pH 6.0) at 0.01% final concentration. These enzymes were combined into four different solutions: alginate lyase and cellulase, A(+)C(+); alginate lyase only, A(+)C(−); cellulase only, A(−)C(+); and no enzyme, A(−)C(−). The enzyme solutions were mixed immediately before use and adding 50 mg/mL of gentamicin (Gibco, Life Technologies Japan, Tokyo, Japan) to prevent bacterial proliferation, with the final concentration being 50 ng/μL. The dry wakame (100 mg) was mixed with the enzymatic solution (10 mL) and incubated at 37 °C for 6 hours. The solution was centrifuged at 8000 rpm for 5 minutes, and the T-As content in the supernatant was determined using ICP-MS. The structural conditions of wakame at the cellular level, following enzymatic treatment, were assessed using an optical microscope.

2-5. Concentration of MeOH as the Extractant

We examined the effects of MeOH solutions at 0, 50, and 100% on the extraction of AsSug from wakame following enzymatic treatment. The enzyme treated wakame A(+)C(+) was centrifuged at 8000 rpm for 5 minutes, and after separating the supernatant, the precipitate was subjected to extraction with 10 mL of each extractant. The precipitate and extractant were mixed using a VORTEX-GENIE 2 (Scientific Industries, NY, USA) for 15 minutes and centrifuged at 8000 rpm for 5 minutes. This procedure was repeated three times, and all supernatants were combined in a single tube. Subsequently, the MeOH was allowed to evaporate at 50 °C for 20 minutes. Afterward, 10 mL of MilliQ-water was added, and the sample was filtered through a 0.2-μm PTFE membrane (Millex-LG, Millipore Corp., MA, USA); the filtrate was used for arsenic analysis. A similar extraction procedure was performed for the control samples of A(−)C(−). T-As was determined using ICP-MS, and an arsenic speciation analysis was performed using HPLC with inductively coupled plasma (HPLC-ICP-MS) and HPLC-electrospray ionization-quadrupole–time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS) under the below-described analytical conditions.

2-6. HPLC-ICP-MS Analysis Conditions

Arsenic speciation analysis was performed using two different instrumental systems. The arsenic compounds were separated using cation and anion exchange columns. A PU712 pump, CO631A column oven (GL Science, Tokyo, Japan), and Midas autosampler (Spark, Emmen, Netherlands) were used to separate the different arsenic species, and the Elan DRCII ICP-MS was used to detect the presence of arsenic compounds. We used ⁷²Ge as the internal standard.

A Shodex RSpak NN-614 cation exchange column (150 × 6.0 mm inner diameter; Showa Denko, Tokyo, Japan) under the following analytical conditions: mobile phase, 10 mM ammonium acetate-acetic acid buffer at pH 4.2:MeOH (90:10); flow rate, 0.7 mL/min; column temperature, 40 °C; and injection volume, 50 µL. We used a Hamilton PRP-X100 anion exchange column (250 × 2.1 mm inner diameter; Hamilton, NV, USA) under the following analytical conditions: mobile phase, 20 mM NH₄HCO₃ at pH 9.0 (adjusted with ammonia solution):MeOH (90:10); flow rate, 0.2 mL/min; column temperature, 40 °C; and injection volume, 10 µL.

2-7. HPLC-ESI-Q-TOF-MS Analysis Conditions

For identification of the arsenic species in the wakame extract, we ascertained the accurate ion mass number and MS/MS fragment ion pattern using ESI-Q-TOF-MS. The arsenic species that we targeted are listed in Table 1. The chemical structures of the arsenic species described in Table 1 were determined from previous studies^6,11,12), and the accurate mass number was calculated based on these chemical formulas.

Table 1.List of arsenic compounds used for high performance liquid chromatography-electrospray ionization-quadrupole–time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS).

Name	Abbreviation	Rational formula	Reference
Monomethylarsonic acid	MMA	CH5AsO3	6
Dimethylarsinic acid	DMA	C2H7AsO2
Thio-dimethylarsinic acid	thio-DMA	C2H7AsOS
Oxo-dimethylarsenoacetic acid	oxo-DMAA	C4H9AsO3
Thio-dimethylarsenoacetic acid	thio-DMAA	C4H9AsO2S
Oxo-dimethylarsenoethanol	oxo-DMAE	C4H11AsO2
Thio-dimethylarsenoethanol	thio-DMAE	C4H11AsOS
Trimethylarsine oxide	TMAO	C3H9AsO
Trimethylarsine sulfide	TMAS	C3H9AsS
Oxo-analogue of the unknown arsenic metabolite	oxo-U	C6H15AsO3
Thio-analogue of the unknown arsenic metabolite	thio-U	C6H15AsO2S
Thio-arsenosugar	thio-Gly	C10H21AsO6S
Oxo-arsenosugars	AsSug 254	C7H15AsO5	11
	AsSug 328	C10H21AsO7
	AsSug 391	C10H22AsO8SN
	AsSug 392	C10H21AsO9S
	AsSug 408	C10H21AsO10S
	AsSug 482	C13H28AsO12P
Arsenosugar-phospholipids	As-PL 930	C43H84O14PAs	12
	As-PL 944	C44H86O14PAs
	As-PL 956	C45H86O14PAs
	As-PL 958	C45H88O14PAs
	As-PL 982	C47H88O14PAs
	As-PL 984	C47H90O14PAs
	As-PL 986	C47H92O14PAs
	As-PL 1012	C49H94O14PAs
	As-PL 1014	C49H96O14PAs
	As-PL 1042	C51H100O14PAs
	As-PL 1070	C53H104O14PAs
Arsenic-hydrocarbons	As-HC 332	C17H36OAs
	As-HC 360	C19H40OAs
	As-HC 388	C21H45OAs

We used Synapt G2-S (Waters, MA, USA) and maXis 4G (Bruker Daltonics, MA, USA) for ESI-Q-TOF-MS. The following instrumental conditions were applied for the Synapt G2-S ESI-Q-TOF-MS analysis: capillary voltage, 1.5 kV (negative mode), 2.0 kV (positive mode); ion source temperature, 120 °C; desolvation nitrogen gas temperature, 600 °C; and desolvation gas flow, 20.0 L/min. The following instrumental conditions were applied for the maXis 4G ESI-Q-TOF-MS analysis: capillary voltage, 5.0 kV (positive mode); ion source temperature, 200 °C; desolvation nitrogen gas temperature, 200 °C; and desolvation gas flow, 8.0 L/min. The former was connected to an Acquity ultra performance liquid chromatography (UPLC) system (Waters, USA) while the latter was connected to a Nexera ultra-HPLC (UHPLC) system (Shimadzu, Kyoto, Japan). We injected 10 µL of each sample into the Acquity UPLC system by using a NN-614 cation exchange column (150 × 6.0 mm inner diameter; Showa Denko, Japan) under the same conditions mentioned above. We also injected 10 µL of sample into a Nexera UHPLC system using a PRP-X100 anion exchange column (250 × 2.1 mm inner diameter, Hamilton, USA) under the same conditions mentioned above.

3. Results

3-1. Total Arsenic Concentration in Wakame Seaweed

T-As concentration in wakame seaweed was 25.50 ± 1.05 mg/kg dry weight (mean ± S.D., n = 3).

3-2. Enzymatic Treatment Condition

The efficacy of enzymatic treatment of wakame prior to arsenic compound extraction was verified both through direct observation and by using an optical microscope with 400 × magnification (Fig. 1). After enzymatic treatment with the A(+)C(+) solution, all the cells observed under the microscope were in suspension, and no mesophyll aggregations were left unaltered. The treatment, including only the enzyme alginate lyase A(+)C(−), showed only partially disintegrated cell walls. On the other hand, cell wall digestion was not achieved in samples treated only with cellulase A(−)C(+) as well as in the control samples A(−)C(−).

Fig. 1.

Wakame seaweed after enzymatic treatment observed directly (i) and under an optical microscope (ii).

Table 2 shows the different elution rates used to extract the arsenic compounds from the supernatant following the different enzymatic treatments. Compared to the control treatment A(−)C(−), the amount of arsenic compounds eluted following A(+)C(+), A(+)C(−), and A(−)C(+) treatments were 2.6, 1.8, and 1.3 times higher, respectively, than that in the control. The most effective treatment was A(+)C(+), which achieved an elution of 45% of arsenic from wakame seaweed. The A(+)C(+) treatment was subsequently used to determine the optimal concentration of extractant required, by using the A(−)C(−) solution as the control treatment.

Table 2.Elution rates of arsenic compounds following enzymatic treatment

Enzymatic treatment	Elution rate (%, n = 5, Mean ± S.D.)
A(−)C(−)	17.2 ± 3.7
A(−)C(+)	22.6 ± 0.6
A(+)C(−)	31.2 ± 0.7
A(+)C(+)	45.5 ± 2.2

A: Alginate lyase, C: Cellulase

The elution rates were calculated based on the total arsenic concentration obtained following acid digestion (25.50 mg/kg dry wakame).

3-3. Concentration of MeOH as the Extractant

Table 3 summarizes the extraction rates of arsenic compounds using different MeOH concentrations. The rate of extraction of arsenic compounds increased concomitant with the increase in MeOH concentration in the A(+)C(+) sample, reaching 88.8% for 100% MeOH. In the A(−)C(−) control sample, 100% MeOH showed a significantly higher extraction rate than that obtained at lower concentrations. Therefore, 100% MeOH concentration was sufficient to extract arsenic from wakame seaweed even in the absence of enzymatic extraction.

Table 3.Extraction rates of arsenic compounds in different methanol (MeOH) concentrations

MeOH concentration	Extraction rate (%, n = 3, Mean ± S.D.)
	A(−)C(−)	A(+)C(+)
0%	22.5 ± 1.1	48.3 ± 0.5
50%	21.8 ± 0.9	58.7 ± 4.1
100%	28.1 ± 0.2	88.8 ± 8.4

A: Alginate lyase, C: Cellulase

The extraction rates were calculated based on the total arsenic concentration obtained following acid digestion (25.50 mg/kg dry wakame).

3-4. Arsenic Speciation Analysis by Using HPLC-ICP-MS and HPLC-ESI-Q-TOF-MS

Figure 2 shows the HPLC-ICP-MS and HPLC-ESI-Q-TOF-MS chromatograms of the different arsenic species extracted from the wakame samples by using enzymatic treatment (A(+)C(+)) and 100% MeOH. Four arsenic peaks were detected in the extractant by HPLC-ICP-MS analysis, one of which was eluted at the retention time of an authentic AsSug 328 sample, representing 79.8% of the total fraction of arsenic compounds in the wakame seaweed sample.

Fig. 2.

High performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) (i) and HPLC-electrospray ionization-quadrupole–time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS) (ii) chromatograms of the different arsenic species detected in wakame extract by using a cation exchange column.

We also determined the arsenic species that were present in the extractant from the list of arsenic compounds included in Table 1. The HPLC-ESI-Q-TOF-MS analysis performed using a cation exchange column detected m/z 389.273 and 1013.5648 in the positive mode near the peak (a) on the chromatogram (Fig. 2); these peaks were estimated to correspond to arsenic-hydrocarbon (As-HC) 388 (C₂₁H₄₆OAs) and As-phospholipid (As-PL) 1012 (C₄₉H₉₅O₁₄PAs), respectively (Fig. 3). Near the peak (b), m/z 483.0609 was detected in the positive mode and estimated to correspond to 3-[5′-deoxy-5′-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropyl-2,3-hydroxypropyl phosphate (AsSug 482 (C₁₃H₂₈O₁₂PAs)) (Fig. 3). In addition, m/z 481.0449 was detected under the negative mode, and estimated to correspond to AsSug 482. Based on MS/MS analysis of the m/z 483.0609 peak, this compound was identified as AsSug 482. No ionic forms of the arsenic species listed in Table 1 were detected near the peak (c). The peak corresponding to AsSug 328 was confirmed by the detection of m/z 329.0575 under the positive mode. In addition, AsSug 328 and AsSug 482 were identified using the anion exchange column (data not shown). We, therefore, can confirm the presence of AsSug 328 and AsSug 482 in the samples.

Fig. 3.

Structures of the four arsenic species detected in wakame extract using high performance liquid chromatography-electrospray ionization-quadrupole–time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS).

Table 4 shows that the peaks (a), (b, AsSug 482), (c), and AsSug 328 were detected in all extractants. The extraction rate of peak (a) increased both with the use of enzymatic treatment and with the increase in MeOH concentration during extraction, reaching the maximum at 100% MeOH. The extraction rate of peak (b), corresponding to AsSug 482, increased only with the enzymatic treatment. On the other hand, neither enzymatic treatment nor 100% MeOH extraction influenced the extraction rates for peak (c) and AsSug 328.

Table 4.Arsenic species content in wakame extracts analyzed using high performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) with a cation exchange column

Extraction conditions		Peak position in Figure 2				Sum
		a	b (AsSug 482)	c	AsSug 328
A(−)C(−)	0% MeOH	0.68	1.32	0.57	1.51	4.07
	100% MeOH	4.53	1.38	0.63	1.43	7.97
A(+)C(+)	0% MeOH	5.09	5.94	0.61	1.57	13.22
	100% MeOH	10.46	5.48	0.61	1.49	18.04

(mg/kg dry wakame)

4. Discussion

We provide evidence that treating wakame seaweed samples with either alginate lyase or cellulase can increase the extraction rate of arsenic compounds from it; in addition, the combination of both these enzymes can further increase the extraction efficiency (Table 2). Enzymatic treatment using both enzymes led to complete denaturation of the cell walls, releasing the cell contents into the suspension. Thus, we can suggest that this enzymatic treatment breaks down the cell walls, enhancing arsenic compound extraction. In this study, we detected two types of AsSug: AsSug 328 and 482. Morita et al.¹⁾ reported that AsSug 328, 392 and 482 are detected in wakame and the major AsSug was AsSug 392; their wakame sample was collected in May in Choshi City, Chiba Prefecture Japan. Mirandes et al.⁸⁾ detected AsSug 328 and 482 but not 392 in wakame sampled from the Galicia seashore in Spain. Lai et al.¹³⁾ reported that AsSug content in Fucus gardneri fluctuates by a season. In addition, Shimoda et al.¹⁴⁾ reported that contents and types of AsSugs in Hizikia fusiforme varied according to the harvested area. Our sample was harvested from February to April in Sanriku Coast Japan. Therefore, the AsSug contents in our wakame sample may depend on the harvested time and place. Moreover, Almera et al.⁹⁾ analyzed two different commercially available wakame products purchased from a health food store in Valencia (Spain) and obtained very different results: they detected AsSug 328, 392, and 482, with AsSug 482 being the major component in one product, but neither AsSug 392 nor 482 was detected in the other product. They speculate that the difference was the result of the substantial difference in the extraction efficiency obtained for each product (5 and 49%). Their speculation was consistent with our observations. The extraction rates of AsSug 328 and the peak (c) did not change with the enzymatic treatment; however, those of AsSug 482 and the peak (a) increased following treatment with both enzymes (Table 4). These differences might relate to the differences in the location and distribution of these arsenic compounds within seaweed cells. Therefore, distribution of AsSug in wakame may depend on not only the harvested time and area but also the extraction efficiency.

In the absence of enzymatic treatment, the extraction rate of arsenic compounds from wakame seaweed has been previously shown to be between 5 and 49% by using 50% MeOH extraction⁹⁾; however, previous studies have also shown that seaweed mainly contains water-soluble AsSug^8,9,15,16). After extracting the arsenic compounds present in seaweed samples by mixing with H₂O or 50% MeOH solution, we obtained T-As recovery rates of 22.5% or 21.8%, respectively (Table 3), suggesting that AsSug are mainly located within the cells in wakame seaweed.

The development of convenient enzymatic and/or chemical methods to break down the cellulose in the cell walls in seaweed might help to easily reach the intracellular space¹⁷⁾. Quantitative extraction of AsSug from dietary samples has been shown to often require chemically aggressive extraction conditions to achieve extraction efficiencies greater than 60%¹⁸⁾. AsSug are known to easily decompose under acidic or basic conditions^11,18); for example, AsSug 328 and 482 do not degrade to DMA, although they degrade into 3-[5′-deoxy-5′-(dimethylarsinoyl)-β-ribofuranosyl] (AsSug 254), under simulated gastric juice treatment¹⁸⁾. Under basic conditions, AsSug 328 is stable; however, AsSug 482 is easily degraded into AsSug 328, and the major degradation product from 3-[5′-deoxy-5′-(dimethylarsinoyl)-β-ribofuranosyloxy]-2-hydroxypropyl hydrogen sulfate (AsSug 408) is DMA¹¹⁾. Considering the above background, we attempted the enzymatic treatment under mild conditions. We used cellulase and alginate lyase, because cell walls in brown algae contain a viscous polysaccharide layer composed of alginic acid that is packed within a cellulose frame, offering strong structural protection¹⁹⁾. Neither AsSug 254 nor DMA were detected in this study, and the amount of AsSug 482 detected was higher than that of AsSug 328 after enzymatic treatment (Table 4), suggesting that our enzymatic method did not change the composition of arsenic compounds in wakame. Therefore, this method is suitable for the extraction of arsenic compounds from brown algae with strong cell walls.

In addition, the extraction rate reached 88.8% when using 100% MeOH (Table 3); however, the only arsenic compound whose extraction rate increased using this method was that corresponding to the peak (a) (Table 4), which is estimated to be As-HC 388 or As-PL 1012 based on the HPLC-ESI-Q-TOF-MS analysis. These arsenics are fat-soluble compounds and have been reported to be present in wakame seaweeds¹²⁾. Our analytical conditions differed from those in previous studies, and therefore, As-HC 388 and As-PL 1012 may be co-eluted at the peak (a) position.

In conclusion, our method achieved an approximately 90% extraction rate of the arsenic compounds in wakame seaweed, by using a combination enzymatic treatment of alginate lyase and cellulase with extraction by using 100% MeOH. A summary of the procedure is shown in Figure 4. In addition, our method ensures that the different arsenic species remain unaltered throughout the extraction process. To our knowledge, this represents the most efficient method proposed thus far for the extraction of different arsenic compounds from wakame seaweeds, which may be applied to any species of brown algae with strong cell walls.

Fig. 4.

Summary of the procedure developed in this study, to extract arsenic compounds from wakame seaweed.

Acknowledgments

This study was supported by a grant from the Food Safety Commission, Cabinet Office, Government of Japan (Research Program for Risk Assessment Study on Food Safety, No. 1102) and a Grant-in-Aid for Young Scientists (B) (26860442) from the Japan Society for the Promotion of Science.

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