Microwave–Hydrothermal Extraction and Degradation of Fucoidan

Article pubs.acs.org/IECR

Microwave−Hydrothermal Extraction and Degradation of Fucoidan from Supercritical Carbon Dioxide Deoiled Undaria pinnatifida Armando T. Quitain,*,† Takahisa Kai,† Mitsuru Sasaki,† and Motonobu Goto‡ †

Graduate School of Science and Technology, Kumamoto University, Kumamoto 860-855, Japan Department of Chemical Engineering, Nagoya University, Nagoya 464-8603, Japan

‡

ABSTRACT: Marine algae, such as Undaria pinnatif ida, commonly known as “wakame” in Japan, contain valuable bioactive organic compounds including lipids and polysaccharides. However, substandard seaweeds that do not meet strict quality standards are normally discarded as wastes or returned to the sea, a situation which is becoming a serious environmental concern. In this work, hydrothermal treatment of the supercritical carbon dioxide deoiled wakame was investigated using microwave and conventional heating to recover and degrade Undaria polysaccharides (i.e., fucoidan) into highly potent low-molecular-weight components of about 5−30 kDa. Results showed advantages of microwave heating compared to conventional heating, obtaining the target molecular weight at a temperature close to 140 °C. Continuous microwave irradiation at constant microwave power in a short irradiation time of 1 min also gave promising results.

1. INTRODUCTION Marine algae of various species contain valuable organic compounds. One of these is Undaria pinnatif ida, a brown alga, commonly known as “wakame” in Japan. This seaweed is edible, is widely consumed, and has been part of the diet in many countries, especially Korea and Japan. This is often used in miso soups, sushi, and salads because of its protein and high mineral content such as calcium, iron, iodine, magnesium, and zinc. It also possesses several functional properties such as antioxidant, anticancer, antiviral, and antiobesity properties.1 Substandard seaweeds are normally discarded as wastes or returned to the sea, and this is becoming a serious environmental concern. Furthermore, this seaweed species has a tendency to rapidly colonize and propagate, and may disturb the balance of marine ecosystem. It also readily disperses and has become rated as “alien and invasive” in Australia, New Zealand, South Africa, and Western Europe.2 These are sometimes considered as pests in these countries, and they are greatly affecting the seashell industry in some regions of New Zealand. In commercial mussel farms, Undaria algae are found growing along with mussels on the long lines supported by floats, causing problems in harvesting the mussels.3 However, this seaweed species contains many useful compounds including fucoidan, a polysaccharide with the chemical structure shown in Figure 1. Fucoidans are highly sulfated cell-wall polysaccharides of brown algae, containing L-fucose as the main sugar unit. The main skeleton of fucoidans consists of α-1,3-linked sulfated Lfucose (I); a repeating sequence of alternating α(1→3) and α(1→4) glycosidic bonds (II) is also possible.4,5 The chemical composition and structure of fucoidans are very diverse and significantly vary depending on the algal source, place of cultivation and habitat, harvesting time, etc.6,7 Biological activity and the medicinal impact of fucoidans depend strongly on their structural properties. These polysaccharides are known to exhibit a wide range of physiological and biological activities, such as anti-inflammatory, antiviral, anticoagulant, antitumor © XXXX American Chemical Society

Figure 1. Chemical structure of fucoidan found in U. pinnatif ida. R = α-L-fucopyranoside, α-D-glucuronic acid, sulfuric base, acetyl base. I = α(1→3); II = α(1→3), α(1→4).

and antimetastatic, and antiangiogenesis activities.8−14 Due to these properties, fucoidans have potential applications in medicine, and thus the production, structures, and properties of these polysaccharides have been intensively investigated. It was also reported that the anticancer activities of fucoidans vary with molecular weight fractions (F) as 18.0−28.5% for F > 30 kDa, 19.2−57.5% for F = 5−30 kDa, and 26.5−36.5% for F < 5 kDa, inhibiting cancer cell growth in a dose-dependent manner.15 Conventional methods for extraction of natural products utilize organic solvent or steam-distillation techniques. However, there are some drawbacks with the use of such methods including the costly posttreatment procedures of separating the extracts from the solvent. Besides, organic solvent remains in the product and may have harmful effects especially if the products are intended for human consumption. Received: February 18, 2013 Revised: May 9, 2013 Accepted: May 20, 2013

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Figure 2. Schematic diagram of apparatus for supercritical carbon dioxide extraction of lipids.

2.3. Supercritical Carbon Dioxide Extraction of Lipids. The work of Watchararuji et al. showed that the yield of proteins and reducing sugars from hydrothermal treatment of rice bran and soybean meal increased with the removal of the oil content.35 The presence of hydrophobic oil in the samples makes them less accessible to the aqueous solvent. Thus, in this work, supercritical carbon dioxide extractions of lipids from dried wakame were performed using the semicontinuous flowtype apparatus shown in Figure 2. CO2 was supplied by an HPLC pump (Jasco PU-2080-100 MPa, Japan) where a chiller (Shibata, Coolman C-560, Japan) was connected to liquefy CO2 at −5 °C. The pressure was adjusted to 40 MPa by a backpressure regulator (AKICO Co., Japan). The CO2 flow rate was set at 4 mL/min. In the 10 mL extractor (Thar Tech, Inc., USA), 5.2 g of dried sample was loaded in the middle of the extractor where the remaining top and bottom sections were trapped with cotton. The extractor was placed in an oven (FC610 ADVANTEC, Japan) to maintain the operating temperature at 40 °C. Extraction was carried out for up to 3 h. The operating conditions were chosen based on our previous studies on extraction of fucoxanthin from the same dried samples, where the highest extraction yield close to 80% was obtained.34 2.4. Conventional Hydrothermal Treatment Methods. An apparatus made by AKICO Co. (Japan) was used for hydrothermal experiments using conventional heating methods. The apparatus consists of a batch-type Inconel reactor (about 8.8 cm3 inner volume) and a heating electric furnace. The apparatus has the capability to monitor and control the temperature inside the furnace using a thermocouple attached to a PID controller. Mechanical stirring of the reactor was provided with a cyclic horizontal swing span of 2 cm fixed at a frequency of 60 cycles/min. About 0.04 g of the freeze-dried sample and 5 mL of water were loaded in an 8.8 mL vessel. The temperature was then set at the desired conditions from 100 to 180 °C. It took about 10−15 min to reach the set temperature as determined by our previous experiments with a thermocouple inserted into the reactor. After treatment, the batch reactor was quenched in a water bath at room temperature for rapid cooling. 2.5. Microwave−Hydrothermal Treatment Methods. Microwave-assisted hydrothermal experiments were performed on supercritical CO2 deoiled samples. Experiments were carried out mostly using a Mars5 IP (CEM Matthews NC Company,

As alternative methods, we have been investigating the application of two environmentally benign pressurized fluid technologies, i.e., supercritical carbon dioxide (SCCO2)16−19 and hydrothermal extraction,20−22 for the isolation of bioactive compounds from various natural products, including seaweeds. Moreover, microwave-assisted techniques have shown significant progress in their application to materials synthesis because of rapid heating and the resulting dramatic increase in reaction rate.23−25 There are many examples of nanoparticles being prepared by hydrothermal methods.26 The combination of both routes is known as microwave−hydrothermal synthesis,27 which has recently been used to prepare nanomaterials.28−30 This technique has also been applied to the extraction of bioactive compounds from henna leaves31,32 and Sophora roots,33 obtaining promising results even in short extraction times and at relatively mild temperatures. In our previous study, SCCO2 extraction was carried out to recover fucoxanthin-containing lipid,34 and was found to be promising even without any cosolvent. Experiments were carried out at extraction temperatures of 40−60 °C and in the pressure range 20−40 MPa, at a liquid carbon dioxide flow rate of 1.0−4.0 mL/min. Results showed that fucoxanthin recovery close to 80% could be obtained at 40 °C and 40 MPa in an extraction time of 180 min. In this study, SCCO2 deoiled U. pinnatif ida algae were treated under hydrothermal conditions heated by microwave irradiation in the temperature range 110−200 °C and treatment time of 5−120 min to recover fucoidan or to degrade it into more valuable low-molecular-weight products of around 5−30 kDa.15 The effects of the operating conditions and pretreatment with EtOH were also investigated.

2. MATERIALS AND METHODS 2.1. Materials. U. pinnatif ida was provided by Isekonbu Co., Ltd. (Mie, Japan). Fucoidan was purchased from SigmaAldrich (United Kingdom). Ethanol and acetonitrile of analytical grade were purchased from Wako Chemicals (Japan). 2.2. Sample Preparation. A dry U. pinnatif ida sample was milled using an IKA-Werke mill (MF 10B S1, Germany) and then sieved using a mesh size of 60. The sample was stored in a refrigerator at 4 °C if the experiment could not be carried out immediately. No other pretreatment method was performed unless otherwise specified. B

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repeated twice while the supernatant was collected. The supernatant was then freeze-dried and analyzed for total nitrogen content by elemental analysis and for the molecular weight of the obtained fucoidan. The total nitrogen was multiplied by the commonly used standard factor of 6.25 to estimate the protein composition of the sample.36

USA) microwave extraction device at a set maximum microwave power of 600 W. This apparatus has the ability to measure and control the temperature and pressure inside the reactor vessel using a fiber optic thermocouple and pressure sensor, respectively. In a typical run, about 0.4 g of sample and 50 mL of water were loaded in a 100-mL Teflon vessel. The prescribed treatment time of 5−120 min and temperature of 110−200 °C were then set with the controller, performing extractions in batch mode. With microwave heating, it took only about 1−3 min to reach the above-mentioned desired temperatures. After each experimental run, the aqueous mixture containing the extracts was separated from the solid residues by centrifugation and then freeze-dried to measure the amounts of extracts. Quantitative and component analyses were then subsequently performed on the extracts. 2.6. Analysis of Molecular Weight Distributions of Fucoidan. Molecular weight distributions of fucoidan obtained after microwave−hydrothermal treatment of wakame residues were determined using an HPLC equipped with an RI detector (RI-930, Jasco, Japan). The fucoidan standard and the sample extracts were diluted with 0.1 N aqueous NaCl solution before injection into a 20-μL sample loop. The column for separation was a Shodex OHpak SB-805HQ (8.0 × 300 mm, 13 μm) (Showa Denko, Japan). The temperature of the column was 35 °C. Component elution was performed in isocratic mode at an eluent flow rate of 1.0 mL/min with a mobile phase consisting of 0.1 N aqueous NaCl solution. Pullulan having molecular weights in the range 5.9−788 kDa (Shodex standard P-82, Showa Denko, Japan) was used as the standard. 2.7. Pretreatment Procedure for Removal of Proteins and Other Components Such as Pigments. The pretreatment procedure for removal of proteins and some other unwanted components is shown in the flowchart in Figure 3.

3. RESULTS AND DISCUSSION 3.1. Preliminary Experiments on MW−Hydrothermal Extraction. Extraction using the microwave−hydrothermal method was performed directly on SCCO2 deoiled samples in 30 min, obtaining an average yield of crude extracts of about 55%. The yield of the target low-molecular-weight fucoidan between 5 and 30 kDa could not be measured due to the unavailability of standard compounds necessary for accurate quantitative HPLC analysis. Thus, in this work only the decomposition and extraction behavior were discussed based on the chromatographic results of HPLC analyses. Figure 4 shows the comparison of (a) microwave and (b) conventional heating on molecular weight distributions of the

Figure 4. Comparison of (a) conventional and (b) microwave heating on hydrothermal extraction and degradation of fucoidan (MW power = 600 W, t = 30 min). Figure 3. EtOH pretreatment procedures of deoiled U. pinnatif ida for removal of proteins.

products obtained after treatment. Results show that microwave heating could extract and degrade fucoidan faster than the conventional heating, as evident from the peak shift toward lower molecular weights. Using microwave heating, lowmolecular-weight fucoidan of about 13 kDa could be obtained at 140 °C, while the fucoidan fraction that was obtained at 110 °C has a higher molecular weight of around 240 kDa. Above an extraction temperature of 160 °C, fucoidan was not detected,

EtOH (99% purity) was added to the sample and was stirred at a rate of 500 rpm, for 12 h at a temperature of 25 °C. The supernatant was separated by centrifugation, and EtOH extraction on the residues was repeated twice. Water was added in the residues and heated to a temperature of 65 °C, and then stirred at 500 rpm for 2 h. This procedure was also C

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such as pigments were performed by ethanol and hot water extraction based on the methods discussed in section 2.7. As a result, this procedure reduced the protein contents of the samples by almost 80% from 20.9 to 4.3 wt %. Other than proteins and pigments, alginates are also present in brown seaweed samples; however, these could not be readily removed by water extraction unless they are converted into sodium form with the addition of sodium carbonate.37 The same tedious procedure was applied by Fenoradosoa et al. to extract and characterize alginates from a brown seaweed species.38 Thus, it was presumed that no alginates were present in the extracts obtained under hydrothermal conditions investigated in this study. Microwave−hydrothermal experiments were then carried out on EtOH-pretreated samples. Results in Figure 6 for the effect

and it was likely that only fucoidan decomposition products were present. Decomposition of fucoidan into monosaccharides such as fucose, mannose, and galactose was promoted at higher temperatures, and it was difficult to obtain fucoidan having the target molecular weight between 5 and 30 kDa. Thus, it is thought that extraction carried out at a temperature of around 140 °C was most suitable for obtaining low-molecular-weight fucoidan in 30 min. The relationships of the treatment time and molecular weight at the extraction temperature of 140 °C were then investigated. 3.2. Effect of Treatment Time. Results of the effect of treatment time on molecular weight at the temperature of 140 °C are shown in Figure 5b. At 140 °C, fucoidan having the

Figure 6. Time-dependent behavior of molecular weight distributions of products obtained by MW−hydrothermal treatment of EtOHpretreated deoiled U. pinnatif ida at 140 °C.

of treatment time on the degradation at 140 °C showed a peak shift to the left of the chromatogram, indicating the presence of higher molecular weight compounds compared to those in Figure 5b, which was obtained without pretreatment. It was also likely that the low-molecular-weight compounds obtained in Figure 5b also contained protein degradation products. 3.4. Comparison of MW and Conventional Heating on Hydrothermal Degradation of EtOH-Pretreated Samples. Figure 7 shows comparison of MW and conventional heating on the average molecular weight distributions of fucoidan at various temperatures. As shown in Figure 7, even at a temperature of 120 °C, MW irradiation could easily hydrolyze fucoidan into the target low-molecular-weight compounds better than the conventional heating method. Also, the relationship of the average molecular weight with the temperature follows a linear trend for conventional methods, but a second-order relation was observed using microwave heating, indicating a possible localized superheating effect other than thermal on the degradation of the polysaccharides.25 3.5. Degradation of Fucoidan at Continuous Microwave Irradiation in Short Time. Continuous microwave irradiation at various powers from 300 to 900 W was applied to the sample in a short time of 1 min. In this range of supplied MW power, the temperature of the mixture reached 62−152 °C. The results for the obtained molecular weight distributions are shown in Figure 8. At 300 W, fucoidan could hardly decompose, and a molecular weight higher than 3000 kDa was obtained. Compounds of various sizes were obtained as the

Figure 5. Comparison of behavior of molecular weight distributions of the products obtained by MW−hydrothermal treatment of seaweeds at (a) 130 and (b) 140 °C.

target molecular weight fractions of 5−30 kDa can be obtained in treatment times between 15 and 60 min. Reducing the temperature by 10 °C, at 130 °C, would require a longer treatment time, which was almost twice, to obtain the same average molecular weight obtained at 140 °C as shown in Figure 5a. 3.3. Behavior of Decomposition of Protein-less Samples. Physical appearances of the products showed browning especially at temperatures above 160 °C, which could be due to a Maillard reaction occurring between the proteins and sugars present in the sample. The presence of proteins could have an effect on the degradation of fucoidan; thus, to be able to study more accurately the degradation of fucoidan under microwave−hydrothermal conditions, pretreatment of samples to remove the proteins and other components D

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4. SPECULATED SYNERGISTIC EFFECT OF COUPLING MICROWAVE AND HYDROTHERMAL TREATMENTS FOR HYDROLYSIS OF FUCOIDAN Fucoidan undergoes degradation under hydrothermal conditions by the cleavage of the glycosidic bond linking the sugar units as shown in Figure 9. This happens even in the absence of

Figure 9. Speculated mechanism of hydrothermal hydrolysis of fucoidan by the cleavage of glycosidic bond.

acid catalysts due to the high ion product of water at elevated temperatures and pressures. The ions produced from the dissociation of water serve as the catalysts for the hydrolysis of fucoidan. In conventional hydrothermal treatment, degradation is attributed to the action of heat and suitable physical properties of water at elevated temperatures. Coupling the hydrothermal process with microwave irradiation can enhance the degradation rate of fucoidan, due to some reasons other than the thermal effects such as “hot spots” or localized heating, molecular agitation, and improved transport properties.

Figure 7. Comparison of microwave and conventional hydrothermal treatments on the average molecular weight distributions of fucoidan at various temperatures.

power was increased to 600 and 750 W, obtaining a molecular weight of less than 100 kDa at a microwave power of 900 W. This treatment with continuous microwave irradiation is costeffective compared to temperature-controlled treatment considering the energy required to obtain the target molecular weight. However, energy analysis of the process should be evaluated to come up with a valid comparison of the two proposed methods.

5. CONCLUSION Supercritical CO2 deoiled U. pinnatif ida algae were treated under hydrothermal conditions using microwave and conventional heating to recover polysaccharides including fucoidan of more valuable low-molecular-weight products of around 5−30 kDa. Hydrothermal treatment at 140 °C could recover the target low-molecular-weight fucoidan. Results also showed the

Figure 8. Molecular weight distributions of the products after continuous MW irradiation treatment in 1 min at various MW powers. E

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(12) Choi, J. I.; Raghavendran, H. R.; Sung, N. Y.; Kim, J. H.; Chun, B. S.; Ahn, D. H. Effect of Fucoidan on Aspirin-Induced Stomach Ulceration in Rats. Chem.-Biol. Interact. 2010, 183, 249−254. (13) Cumashi, A.; Ushakova, N. A.; Preobrazhenskaya, M. E.; D’Incecco, A.; Piccoli, A.; Totani, L. A Comparative Study of the AntiInflammatory, Anticoagulant, Antiangiogenic, and Antiadhesive Activities of Nine Different Fucoidans from Brown Seaweeds. Glycobiology 2007, 17, 541−552. (14) Hahnenberger, R.; Jakobson, A. M. Antiangiogenic Effect of Sulphated and Non-Sulphated Glycosaminoglycans and Polysaccharides in the Chick-Embryo Chorioallantoic Membrane. Glycoconjugate J. 1991, 8, 350−353. (15) You, S.-G.; Yang, C.; Lee, H.-Y.; Lee, B.-Y. Molecular Characteristics of Partially Hydrolyzed Fucoidans from Sporophyll of Undaria Pinnatif ida and Their In Vitro Anticancer Activity. Food Chem. 2010, 119, 554−559. (16) Quitain, A. T.; Moriyoshi, T.; Goto, M. Coupling MicrowaveAssisted Drying and Supercritical Carbon Dioxide Extraction for Coconut Oil Processing. Chem. Eng. Sci. 2013, 1, 12−16. (17) Nerome, H.; Machmudah, S.; Quitain, A. T.; Sasaki, M.; Goto, M. Supercritical Carbon Dioxide Extraction of Carotenoids from Momordica cochinchinesis. J. Sci. Technol. 2011, 49, 177−183. (18) Quitain, A. T.; Oro, K.; Katoh, S.; Moriyoshi, T. Recovery of Oil Components of Okara by Ethanol-Modified Supercritical Carbon Dioxide Extraction. Bioresour. Technol. 2006, 97, 1509−1514. (19) Quitain, A. T.; Katoh, S.; Moriyoshi, T. Isolation of Antimicrobials and Antioxidants from Moso-Bamboo (Phyllostachys heterocycla) by Supercritical CO2 Extraction and Subsequent Hydrothermal Treatment of the Residues. Ind. Eng. Chem. Res. 2004, 43, 1056−1060. (20) Matsunaga, Y.; Wahyudiono; Machmudah, S.; Askin, R.; Quitain, A. T.; Sasaki, M.; Goto, M. Hydrothermal Extraction and Micronization of Polysaccharides from Ganoderma lucidum in a OneStep Process. BioResources 2013, 8, 461−471. (21) Ruen-ngam, D.; Quitain, A. T.; Sasaki, M.; Goto, M. Hydrothermal Hydrolysis of Hesperidin Into More Valuable Compounds Under Supercritical Carbon Dioxide Conditions. Ind. Eng. Chem. Res. 2012, 51, 13545−13551. (22) Quitain, A. T.; Faisal, M.; Kang, K.; Daimon, H.; Fujie, K. LowMolecular-Weight Carboxylic Acids from Hydrothermal Treatment of Organic Wastes. J. Hazard. Mater. 2002, B93, 209−220. (23) Harpeness, R.; Gedanken, A. Microwave Synthesis of Core-Shell Gold/Palladium Bimetallic Nanoparticles. Langmuir 2004, 20, 3431− 3434. (24) Pastoriza-Santos, I.; Liz-Marzan, L. M. Formation of PVP Protected Metal Nanoparticles in DMF. Langmuir 2002, 18, 2888− 2894. (25) Komarneni, S.; Li, D.; Newalkar, B.; Katsuki, H.; Bhalla, A. S. Microwave-Polyol Process for Pt and Ag Nanoparticles. Langmuir 2002, 18, 5959−5962. (26) Cushing, B. L.; Kolesnichenko, V. L.; O’Connor, C. J. Recent Advances in the Liquid-Phase Syntheses of Inorganic Nanoparticles. Chem. Rev. 2004, 104, 3893−3946. (27) Komarneni, S.; Roy, R.; Li, Q. H. Microwave-Hydrothermal Synthesis of Ceramic Powders. Mater. Res. Bull. 1992, 27, 1393−1405. (28) Baldassari, S.; Komarneni, S.; Mariani, E.; Villa, C. MicrowaveHydrothermal Process for the Synthesis of Rutile. Mater. Res. Bull. 2005, 40, 2014−2020. (29) Wilson, G. J.; Will, G. D.; Frost, R. L.; Montgomery, S. A. Efficient Microwave Hydrothermal Preparation of Nanocrystalline Anatase TiO2 Colloids. J. Mater. Chem. 2002, 12, 1787−1791. (30) Murugan, A. V.; Muraliganth, T.; Manthiram, A. Rapid, Facile Microwave-Solvothermal Synthesis of Graphene Nanosheets and Their Polyaniline Nanocomposites for Energy Storage. Chem. Mater. 2009, 21, 5004−5006. (31) Zohourian, T. H.; Quitain, A. T.; Sasaki, M.; Goto, M. Extraction of Bioactive Compounds from Leaves of Lawsonia inermis by Green Pressurized Fluids. Sep. Sci. Technol. 2012, 47, 1006−1013.

advantages of using microwave heating for the degradation of fucoidan compared to conventional heating. Application of continuous microwave irradiation in a short time gave promising results with regard to the degradation of fucoidan into the target highly potent low-molecular-weight components. Pretreatment of samples with EtOH to remove unwanted pigments and proteins to minimize their effects on the hydrothermal degradation of fucoidan was also investigated, obtaining target molecular weights at a rather longer time compared to the nonpretreated samples.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +81-96-342-3665. Fax: +81-96-342-3679. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Kumamoto University Global COE Program “Global Initiative Center for Pulsed Power Engineering”.

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REFERENCES

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