KR101866906B1 - a extraction method of fucoidan - Google Patents

Abstract

The present invention relates to a method for extracting fucoidan. More specifically, the present invention relates to a method for extracting fucoidan, comprising the following steps: (a) preparing a first mixture by mixing choline chloride and ethylene glycol; (b) adding water to the first mixture and stirring the same to prepare a second mixture; (c) crushing kelp, adding a solvent, and stirring the same to remove impurities; and (d) mixing the second mixture and the kelp from which the impurities have been removed, and then heating the same to obtain fucoidan.

Description

A extraction method of fucoidan}

The present invention relates to a method for extracting fucoidan, and more particularly, to a method for extracting fucoidan, comprising: (a) preparing a first mixture by mixing choline chloride and ethylene glycol; (b) adding water to the first mixture and stirring to prepare a second mixture; (c) crushing kelp, adding a solvent and stirring to remove impurities; And (d) mixing the second mixture and the kelp from which the impurities have been removed, followed by heating and extracting to obtain fucoidan.

Seaweeds have been used for a long time in edible and industrial applications. In Asia, seaweeds are mainly used as food materials, while western countries are using algae to produce new compounds.

Among seaweeds, kelp contains a large amount of polysaccharides such as alginate, laminarin, mannitol, fucoidan, dietary fiber and minerals.

Fucoidan is a polysaccharide mainly containing L-fucose and containing a large number of sulfate groups, and is contained in large amount in brown algae such as kelp, sea mustard, and tortoise. The chemical structure, physiological activity, and molecular weight of fucoidan depend on the type of brown algae, the place of harvest, and the harvest time.

Fucoidan exhibits various physiological activities such as enhancement of immune activity, anticoagulant effect, antioxidant effect, anticancer effect and antibacterial effect. Based on these effects, researches for application to pharmaceuticals, cosmetics, food industry and the like are actively conducted.

In this connection, Korean Patent Laid-Open Nos. 10-2008-0106741, 10-2013-0055926 and 10-2014-0020005 disclose an extraction method of fucoidan.

However, the technology disclosed in the above document uses an acid, a base, an organic solvent, an enzyme, and the like as a solvent for extracting fucoidan, which causes environmental pollution problems, threatens the health of workers, There is a drawback that it takes time.

Korean Patent Laid-Open No. 10-2008-0106741 Korean Patent Publication No. 10-2013-0055926 Korean Patent Publication No. 10-2014-0020005

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above problems of the prior art, and it is an object of the present invention to provide a method for extracting fucoidan by using a eutectic solvent to replace an existing solvent such as acid, base, organic solvent, The present invention has been made in view of the above problems.

It is another object of the present invention to provide a method for extracting fucoidan which is harmless to human body, has low cost, and has high stability because of using no toxicity by using choline chloride and ethylene glycol as a eutectic solvent.

In order to accomplish the above object, the present invention provides a method for preparing a mixture comprising: (a) preparing a first mixture by mixing choline chloride and ethylene glycol; (b) adding water to the first mixture and stirring to prepare a second mixture; (c) crushing kelp, adding a solvent and stirring to remove impurities; And (d) mixing the second mixture and the kelp from which the impurities have been removed, followed by heating extraction to obtain fucoidan.

In one embodiment of the present invention, the step (a) is characterized in that the molar ratio of choline chloride and ethylene glycol is 1: 1-3.

In one embodiment of the present invention, the step (b) is characterized in that the volume ratio of the first mixture and water is 30-60: 40-70.

In one embodiment of the present invention, the step (d) is characterized in that the second mixture and the kelp from which the impurities are removed are mixed and heated and extracted at 80 to 120 ° C for 60 to 180 minutes to obtain fucoidan.

In one embodiment of the present invention, the weight ratio of the second mixture and the sea tangle from which the impurities are removed is 5 to 30: 1.

The present invention can provide an eco-friendly, safe and economical method of extracting fucoidan by using eutectic solvents for extraction of fucoidan, thereby replacing existing solvents such as acids, bases, organic solvents and enzymes.

Further, by using choline chloride and ethylene glycol as the eutectic solvent, the present invention can provide a method for extracting fucoidan which is harmless to the human body, has low toxicity, is inexpensive, and has high stability.

Figure 1 shows the FT-IR spectrum of fucoidans obtained using three eutectic solvents.
Fig. 2 shows the cytotoxicity evaluation of fucoidan prepared from Example 1. Fig.

Hereinafter, the present invention will be described in detail based on examples. It is to be understood that the terminology, examples and the like used in the present invention are merely illustrative of the present invention in order to more clearly explain the present invention and to facilitate understanding of the ordinary artisan, and should not be construed as being limited thereto.

Technical terms and scientific terms used in the present invention mean what the person skilled in the art would normally understand unless otherwise defined.

The present invention relates to a process for preparing a mixture comprising: (a) preparing a first mixture by mixing choline chloride and ethylene glycol; (b) adding water to the first mixture and stirring to prepare a second mixture; (c) crushing kelp, adding a solvent and stirring to remove impurities; And (d) mixing the second mixture and the kelp from which the impurities have been removed, followed by heating and extracting to obtain fucoidan.

The step (a) is a step of preparing a deep eutectic solvent (DES), and the present invention can use choline chloride and ethylene glycol as eutectic solvents.

The eutectic solvent is prepared by mixing and heating two solvents having a low melting point. Since the eutectic solvent has no toxicity, it is harmless to the human body, has low cost, has high stability and low vapor pressure, Solvents, enzymes, etc.) can be substituted.

The present invention relates to a process for the preparation of the eutectic compound of the present invention, which comprises using as the eutectic solvent tetramethylammonium chloride, hydroxymethyltrimethylammonium chloride, hydroxyethyltrimethylammonium chloride (choline chloride), hydroxyethyltetramethylammonium chloride, hydroxyethyltripropylammonium chloride, Hydrogen bond acceptors such as tetrabutyl ammonium chloride and hydroxyethyl tributyl ammonium chloride; And hydrogen bond donors such as ethylene glycol, 1,2-butanediol, phenol and urea. In particular, the present invention can dramatically improve the yield of fucoidan extracted from sea tangle by using choline chloride and ethylene glycol as the eutectic solvent (first mixture).

The molar ratio of the choline chloride to the ethylene glycol is preferably 1: 1 to 3, and when the molar ratio is less than 1: 1 or the molar ratio is more than 1: 3, the yield of the fucoidan to be extracted decreases.

The eutectic solvent may be prepared by mixing choline chloride and ethylene glycol, followed by stirring at 80 to 120 ° C for 10 to 60 minutes.

In the step (b), a solvent is added to the eutectic solvent to prepare a mixed solvent (second mixture). In the present invention, water may be used as a solvent to be added.

Examples of the solvent include water, ethanol, methanol, hexane, chloroform, methylene chloride, ethyl acetate, diethylene glycol monoethyl ether and the like.

In particular, the present invention can dramatically improve the yield of fucoidan extracted from sea tangle by using water as a solvent to be added.

The volume ratio of the eutectic solvent and water is preferably 30 to 60:40 to 70. When the volume ratio is less than 30:70, the amount of fucoidan to be extracted decreases. When the volume ratio is more than 60:40, The yield of fucoidan is reduced.

The step (c) is a step of removing impurities by adding a solvent (water, alcohol, etc.) after shredded tallow, and stirring to remove impurities (salts, coloring matters, residue) before extracting fucoidan from sea tangle. The efficiency can be increased.

For example, 100 parts by weight of distilled water is added to 10 parts by weight of crushed kelp, and the mixture is stirred for 10 to 60 minutes to discard the supernatant. The above procedure is repeated until the salinity becomes 0%, and 100 to 500 parts by weight of 85% ethanol is added, followed by stirring for 5 to 15 hours to remove the pigment. The kelp from which the pigment is removed can be washed with acetone, filtered and then dried to pretreat the kelp.

(D) is a step of mixing the second mixture and the kelp from which the impurities have been removed, followed by heat extraction to obtain fucoidan, wherein the second mixture and the kelp from which the impurities have been removed are mixed and heated at 80 to 120 ° C And then heated and extracted for 60 to 180 minutes to obtain fucoidan.

The weight ratio of the second mixture to the sea tangle from which the impurities are removed is preferably 5 to 30: 1 in terms of the yield of fucoidan.

For example, 60 ml of the second mixture is added to 3 g of kelp from which impurities have been removed, and the mixture is stirred at 100 ° C for 2 hours to obtain an extract. The extract is centrifuged to separate the supernatant, calcium chloride is added to the separated supernatant, and the mixture is stirred at 4 ° C for 12 hours to remove alginate precipitate. Ethanol is added to the supernatant from which alginic acid has been removed and centrifuged to obtain fucoidan, which is then dried to obtain high purity fucoidan.

Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples. The following examples are intended to illustrate the practice of the present invention and are not intended to limit the scope of the present invention.

(Example 1)

The choline chloride and ethylene glycol were mixed in a molar ratio of 1: 2, and then the mixture was stirred at 100 DEG C for 20 minutes to prepare a first mixture.

Water was added to the first mixture and stirred to prepare a second mixture, wherein the volume ratio of the first mixture and water was 50:50 (F-ChCl-EG-50%).

After kelp was crushed, 200 parts by weight of distilled water was added to 10 parts by weight of crushed kelp, and the mixture was stirred for 30 minutes, and the supernatant was removed. The above procedure was repeated until the salinity became 0%. 200 parts by weight of 85% ethanol was added thereto, and the mixture was stirred for 12 hours to remove the coloring matter. The dyed kelp was washed with acetone, filtered, and dried to pretreat the kelp.

60 ml of the second mixture was added to 3 g of the pretreated kelp, and the mixture was stirred at 100 ° C for 2 hours to obtain an extract. The extract was centrifuged to separate the supernatant, calcium chloride was added to the separated supernatant, and the mixture was stirred at 4 ° C for 12 hours to remove alginate precipitate. Ethanol was added to the supernatant from which alginic acid had been removed and centrifuged to obtain fucoidan, which was then dried at 60 DEG C to obtain high purity fucoidan.

(Example 2)

Fucoidan was obtained in the same manner as in Example 1 except that choline chloride and ethylene glycol were mixed in a molar ratio of 1: 0.9.

(Example 3)

Fucoidan was obtained in the same manner as in Example 1 except that choline chloride and ethylene glycol were mixed in a molar ratio of 1: 4.

(Example 4)

Fucoidan was obtained in the same manner as in Example 1, except that the first mixture and water were mixed in a volume ratio of 25:75 (F-ChCl-EG-25%).

(Example 5)

Fucoidan was obtained in the same manner as in Example 1 except that the first mixture and water were mixed in a volume ratio of 65:35.

(Comparative Example 1)

After kelp was crushed, 200 parts by weight of distilled water was added to 10 parts by weight of crushed kelp, and the mixture was stirred for 30 minutes, and the supernatant was removed. The above procedure was repeated until the salinity became 0%. 200 parts by weight of 85% ethanol was added thereto, and the mixture was stirred for 12 hours to remove the coloring matter. The dyed kelp was washed with acetone, filtered, and dried to pretreat the kelp.

To 3 g of the pretreated sea tangle, 60 ml of water was added (F-ChCl-EG-0%), and the mixture was stirred at 100 ° C for 2 hours to obtain an extract. The extract was centrifuged to separate the supernatant, calcium chloride was added to the separated supernatant, and the mixture was stirred at 4 ° C for 12 hours to remove alginate precipitate. Ethanol was added to the supernatant from which alginic acid had been removed and centrifuged to obtain fucoidan, which was then dried at 60 DEG C to obtain high purity fucoidan.

(Comparative Example 2)

Fucoidan was obtained in the same manner as in Example 1 except that tetramethylammonium chloride and 1,2-butanediol were used as eutectic solvents.

The characteristics of fucoidan prepared from the above Examples and Comparative Examples were measured and the results are shown in Table 1 below.

(Yield of fucoidans)

The yield of the obtained fucoidan was calculated according to the following equation.

(Salinity)

After kelp was crushed, 200 parts by weight of distilled water was added to 10 parts by weight of crushed kelp, and the mixture was stirred for 30 minutes, and the salinity was measured using a salinity meter.

(pH)

Fucoidan obtained in distilled water was dissolved at a concentration of 1 mg / ml and then measured using a pH meter.

(Molecular Weight)

The molecular weight of fucoidan was measured by gel filtration chromatography (GFC). The column was an Agilent 1200 Series HPLC system, with a flow rate of 1 ml / min and a bed of 0.02% sodium azide. The calibration curve was obtained from pullulan.

(Sulfuric acid group content)

The sulfate content of fucoidan was measured by Dodgson's barium chloride-gelatin method. Calibration curves were obtained from sodium sulfate.

division Example Comparative Example One 2 3 4 5 One 2 yield(%) 1.38 0.96 0.98 0.93 0.99 0.93 0.91 Salinity (%) 0 0 0 0 0 0 0 pH 6.47 6.75 6.81 6.86 6.69 6.83 6.61 Mn
(× 10 ⁴ g / mol) 1.6 1.8 2.3 3.1 2.1 4.9 2.8 Mw
(× 10 ⁴ g / mol) 9.3 9.7 10.2 13 9.9 15 10.5 PI 5.86 5.62 5.66 4.13 5.29 3.10 5.57 Sulfate content
(%) 26.18 16.31 17.89 13.91 17.05 15.99 16.07

From the results shown in Table 1, Examples 1 to 5 show higher yields (0.96 to 1.38%) than Comparative Examples 1 and 2, and Example 1 shows the highest yield.

The salinity of the sea tangle is completely 0%, and the pH of the seaweed is completely removed (pH 6.47 ~ 6.86). Since it meets the food quality standard, the fucoidan of the present invention can be applied to foods, cosmetics and pharmaceuticals .

The obtained fucoidan had a number average molecular weight of 1.6 × 10 ⁴ to 3.1 × 10 ⁴ g / mol, a weight average molecular weight of 9.3 × 10 ⁴ to 13 × 10 ⁴ g / mol, and a polydispersity index (PI) 4.13 ~ 5.86.

It was also confirmed that as the content of the eutectic solvent was increased, the average molecular weight was lowered and the PI was increased (Example 1, Example 4, Comparative Example 1).

In addition, Examples 1 to 5 show higher sulfuric acid group contents than Comparative Examples 1 and 2.

Therefore, by using choline chloride and ethylene glycol as the eutectic solvent, fucoidan which is harmless to human body, low in cost, and high in stability can be obtained with high yield.

Figure 1 shows the FT-IR spectrum of fucoidans obtained using three eutectic solvents. All three cases exhibit peaks seen in polysaccharides, especially S = O asymmetric stretching peaks at around 1250 to 1257 cm ^-1 .

Fig. 2 shows the cytotoxicity evaluation of fucoidan prepared from Example 1. Fig.

Cytotoxicity was assessed using 3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H- tetrazolium (MTS) HeLa cell lines were treated with 1, 3, and 5 mg / ml, and analyzed for cytotoxicity by MTS reduction assay.

The cytotoxicity level at each concentration was similar to that of the control without drug treatment, so that the obtained fucoidan was found to be non-toxic to HeLa cells.

Claims (5)

(a) preparing a first mixture by mixing choline chloride and ethylene glycol;
(b) adding water to the first mixture and stirring to prepare a second mixture;
(c) crushing kelp, adding a solvent and stirring to remove impurities; And
(d) mixing the second mixture and the kelp from which the impurities have been removed, followed by heating and extracting to obtain fucoidan.
The method according to claim 1,
Wherein the molar ratio of choline chloride to ethylene glycol is 1: 1-3.
3. The method of claim 2,
Wherein the volume ratio of the first mixture and the water is 30 to 60:40 to 70,
The method of claim 3,
Wherein the step (d) comprises mixing the second mixture and the kelp from which the impurities have been removed, heating and extracting the mixture at 80 to 120 ° C for 60 to 180 minutes to obtain fucoidan.
5. The method of claim 4,
Wherein the weight ratio of the second mixture to the kelp from which the impurities are removed is 5 to 30: 1.

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