TW201929884A - Polysaccharides of brown algae, method of producing the same and application thereof - Google Patents

Abstract

The present invention relates to polysaccharides of brown algae, a method of producing the same and applications thereof. The yield of polysaccharides of brown algae purified by hot water extraction facilitated by microwave-assisted and/or ultrasonication-assisted treatments can be increased. The low-molecular-weight polysaccharides of the brown algae, which significantly inhibit lipid biosynthesis and tumor necrotic factor-[alpha] (TNF-[alpha]) production, can be applied to a medicinal composition for inhibiting lipid biosynthesis and proinflammatory factors.

Description

Brown algae polysaccharide, its manufacturing method and application

The invention relates to a polysaccharide and a preparation method thereof, in particular to a brown algae polysaccharide having the effects of inhibiting lipid synthesis and inhibiting proinflammatory factors, a preparation method thereof and application thereof.

Feed additives refer to non-nutritive substances that have been announced by the central competent authority to improve the efficiency of feed, maintain feed quality, promote the development of livestock, poultry, and aquatic animals, maintain their health or other uses, and add to feed without chemicals. Feed additives include microorganisms, enzymes, preservatives and antioxidants, iodinated proteins, acidity regulators, quality enhancers (emulsifiers, granulating agents, colorants, flavoring agents, anti-caking agents and mycotoxin adsorbents), Animal/aquatic technology additives, drugs, etc., including drug feed additives generally include antibiotic products, non-antibiotic products, and Carbadox products.

In actual use, up to 27% of feed additives use antibiotics, but bacteria are resistant to antibiotics. In recent years, various animal diseases have emerged, indicating that it is necessary to delete the drug-containing feed additives. As for other chemical feed additives, although there are restrictions and specifications in various countries, the raw materials are heavy. Metal contamination and chemical toxicity, and chemical feed additives themselves have problems such as environmental or human residual or cumulative toxicity. It is necessary to develop feed additives for natural ingredients.

Taiwan is surrounded by the sea and the algae resources are very rich. According to the characteristics of pigments and storage substances in the algae, the seaweed can be divided into four major categories: Cyanophyte, Chlorophyte, Phaeophyte, and Red Algae. Recently, the global warming, part of the large seaweed growing in the intertidal zone has been multiplied, which not only interferes with the fishermen's operations, but also causes troubles in the sea and fishing port operations. These algae species are also less converted into ingredients, which are rarely used as feed. Or it is waste.

There are various ways to extract brown algae polysaccharides. Some extraction methods are: after the high temperature and high pressure treatment (burst cake machine), after extracting with hot water of 70-100 ° C for 1 to 3 hours, the obtained fucoidan (ie, brown algae polysaccharide) has DPPH free radical scavenging and No algae smell. In other ways, after extracting Sargasso from about 1 to 3 hours (5% (w/w)) with hot water of about 100 °C, it is treated with enzyme, acidification and chemical modification (addition of sulfate) to obtain sulfation. Sargassum polysaccharide has anti-enteric virus type 71 effect.

However, the brown algae polysaccharide obtained in the above manner still contains a pigment, an algal protein, a brown alginic acid, etc., and the brown algae polysaccharide having a large molecular weight is not easily absorbed after the gastrointestinal tract, thereby not only reducing the bioavailability but also affecting the efficacy of the brown algae polysaccharide. In view of this, it is urgent to provide a new process for the brown algae polysaccharide, to improve the above-mentioned shortcomings of the traditional brown algae polysaccharide, and to expand the application surface of the large seaweed.

Therefore, one aspect of the present invention provides a method for producing a brown algae polysaccharide which can improve the purification amount of fucoidan by a hot water extraction step and a microwave assisted extraction treatment and/or an ultrasonic assisted extraction treatment.

Another aspect of the present invention provides a fucoidan polysaccharide which is obtained by the above method to produce a low molecular weight fucoidan.

A further aspect of the invention provides a composition comprising an effective amount of the above-described fucoidan.

Still another aspect of the present invention provides a use of a fucoidan polysaccharide for the preparation of a pharmaceutical composition for inhibiting lipid synthesis and inhibiting proinflammatory cytokines, for inhibiting cell lipid synthesis reaction and inhibiting tumor necrosis factor (TNF)-α produce.

According to the above aspect of the invention, a method for producing a brown algae polysaccharide is proposed. In one embodiment, the method for producing the fucoidan may include performing at least one pretreatment on the dried algal material to obtain a first crude extract. Next, the first crude extract is subjected to a hot water extraction step to obtain a second crude extract, wherein the hot water extraction step is to immerse the first crude extract in water at 100 ° C for microwave assisted extraction and/or ultrasonic assist In the extraction treatment, the liquid-solid ratio (mL/g) of the first crude extract to water is 10 to 20, and the hot water extraction step is carried out for no more than 1 hour. Thereafter, the second crude extract is post-treated to remove the protein, alginate of the second crude extract and obtain a brown algae polysaccharide, wherein the molecular weight of the fucoidan is not more than 3.2 kilo-dalton (kDa).

In an embodiment of the present invention, the dried seaweed material may include, but is not limited to, Sargassum siliquosum , S. hemiphyllum , and S. polycystum .

In an embodiment of the invention, the pretreatment comprises selectively performing a high temperature and high pressure step on the dried algal material. After the high temperature and high pressure step, the seaweed powder is selectively subjected to a crude extraction step and a solvent removal step to obtain a crude raw material.

In an embodiment of the invention, the post-treatment described above includes a step of removing the protein, a step of removing the alginate, a chromatography, and/or a partial hydrolysis.

According to another aspect of the present invention, a brown algae polysaccharide is provided which is obtained by the above method, wherein the molecular weight of the fucoidan is not more than 3.2 kDa.

According to still another aspect of the present invention, a composition comprising an effective amount of fucoidan is provided, wherein the fucoidan has a molecular weight of not more than 3.2 kDa.

According to still another aspect of the present invention, a use of a brown algae polysaccharide for preparing a pharmaceutical composition for inhibiting lipid synthesis and inhibiting proinflammatory cytokines is proposed, wherein the molecular weight of the fucoidan polysaccharide is not more than 3.2 kDa to inhibit cell lipid synthesis reaction. And the production of tumor necrosis factor (TNF)-α.

The brown algae polysaccharide of the present invention and the method for producing the same, which utilize the hot water extraction step and the microwave assisted extraction treatment and/or the ultrasonic assisted extraction treatment, not only improve the purification amount of the brown algae polysaccharide, but also obtain the low molecular weight fucoidan having the cell inhibiting effect. The lipid synthesis reaction and the effect of tumor necrosis factor (TNF)-α production can be applied to the preparation of a pharmaceutical composition for inhibiting lipid synthesis and inhibiting pro-inflammatory factors.

The above and other objects, features, advantages and embodiments of the present invention will become more apparent and understood. 1A to 1I are fluorescent micrographs of cells treated in vitro using different concentrations of the fucoidan of Preparation Example 1 and the second crude extract of Comparative Example 1 according to an embodiment of the present invention.

2A to 2L are fluorescent micrographs of cells treated with fucoidan of Preparation Examples 1 to 4 at different concentrations according to an embodiment of the present invention.

[Fig. 3] is a graph showing the relative absorbance of cells of Fig. 2A to Fig. 2L calculated by flow cytometry.

[Fig. 4] shows that the cells in the LPS-induced inflammatory reaction in vitro have a long TNF-α content after treatment with different concentrations of the fucoidan of Preparation Example 1 and the second crude extract of Comparative Example 1 according to an embodiment of the present invention. Bar chart.

As described above, the present invention provides a brown algae polysaccharide and a method for producing the same, which utilizes a hot water extraction step and a microwave assisted extraction treatment and/or an ultrasonic assisted extraction treatment to improve not only the purification amount of the brown algae polysaccharide but also the low molecular weight fucoidan obtained. It has the effect of inhibiting cell lipid synthesis reaction and tumor necrosis factor (TNF)-α production.

The fucoidan polysaccharide referred to herein as a product refers to a product obtained by extracting seaweed raw material by water. In one embodiment, the above-mentioned fucoidan is low molecular weight and low sulfate content, and the molecular weight is not more than 3.2 thousand Daltons. (kilo-dalton; kDa) and fucoidan having a sulfate content of less than 20% are preferred, and fucoidan having a molecular weight of not more than 3.2 kDa and a sulfate content of less than 19% is preferred.

The above brown algae polysaccharide can be obtained by the following method. In one embodiment, first, the dried seaweed material is pretreated to obtain a crude material. The seaweed raw material may be optionally washed with water to remove surface salt, and then dried by various conventional methods, for example, drying at a temperature lower than 100 ° C for several hours to several tens of hours, thereby obtaining a dried seaweed raw material. In the above embodiments, the dried seaweed material may include, but is not limited to, Sargassum siliquosum , S. hemiphyllum , and S. polycystum . In another example, the dried seaweed material can be, for example, S.

In the above embodiments, suitable pre-processing may include including a first pre-processing. In an example, the first pretreatment may include performing high temperature and high pressure treatment on the dried seaweed material at a temperature of 140 ° C to 250 ° C and a pressure of 1.5 kg / cm ² to 19 kg / cm ² for 4 seconds to 10 seconds. To obtain the raw material of the expanded seaweed. In an example, the high temperature and high pressure treatment can be carried out using commercially available equipment (for example, a high temperature and high pressure blasting machine) to obtain a swelled seaweed material.

In some examples, after the first pre-treatment, the pre-treatment may selectively perform a second pretreatment on the expanded algae material to obtain a first crude extract.

In the above examples, the second pretreatment comprises a crude extraction step and a solvent removal step. In the above examples, the above-mentioned expanded seaweed material may be directly subjected to a crude extraction step. In other examples, the above-mentioned expanded seaweed raw material is optional After the pulverization into the seaweed powder by a conventional pulverization method (for example, grinding or the like), the seaweed powder is subjected to a crude extraction step.

In the above crude extraction step, the swelled seaweed material may be immersed in a polar solution (for example, a 95% ethanol solution), and subjected to crude extraction at a liquid-solid ratio of, for example, 10 mL/g for 4 hours to 24 hours to obtain a crude extract. . The crude extraction step also removes lipids, mannitol, pigments and partial salts contained in the expanded seaweed material. Next, the crude extract may be selectively subjected to a solid-liquid separation step (for example, centrifugation), whereby a first crude extract is obtained.

In one embodiment, after the first crude extract is obtained, the first crude extract is then subjected to a hot water extraction step to obtain a second crude extract. In this embodiment, the hot water extraction step may immerse the first crude extract in water at 100 ° C and use microwave assisted extraction and/or ultrasonic assisted extraction. In the above embodiment, the liquid-solid ratio (mL/g) of the first crude extract to water may be, for example, 10 to 25. In the above examples, the hot water extraction step is carried out for no more than one hour.

In the above embodiment, the hot water extraction step and the microwave assisted extraction treatment and/or the ultrasonic assisted extraction treatment can effectively increase the purification amount of the brown algae polysaccharide by more than 30%. In other embodiments, the hot water extraction step and microwave assisted extraction treatment can effectively increase the yield of fucoidan from 35% to 45%.

In the above embodiment, the microwave-assisted extraction treatment may treat the first crude extract, for example, with a microwave power of 750 W, wherein the liquid-solid ratio (mL/g) of the first crude extract to water is 15 to 25 treatments for 5 minutes to 20 minutes is appropriate, but liquid to solid ratio (mL / g) 15 to 20 treatment for 10 minutes to 20 minutes for comparison Preferably, it is more preferably treated with a liquid-solid ratio (mL/g) of 15 to 20 for 10 minutes to 15 minutes. In one embodiment, the hot water extraction step and microwave assisted extraction treatment increase the amount of fucoidan purified by about 45%.

In the above embodiment, the ultrasonic assisted extraction treatment may treat the first crude extract, for example, with an ultrasonic energy of 50 to 200 watts. When the liquid-solid ratio (mL/g) of the first crude extract to water is 15 to 25, it is preferably treated with ultrasonic energy of 50 to 200 watts for 10 minutes to 20 minutes; however, when the first crude extract is used When the liquid-solid ratio (mL/g) to water is 15 to 20, it is preferably treated with ultrasonic energy of 50 to 150 watts for 10 minutes to 15 minutes; and, when the liquid ratio of the first crude extract to water is When (mL/g) is 15, it is more preferably treated with ultrasonic energy of 100 watts for about 10 minutes. If the first crude extract is treated with more than 200 watts of ultrasonic energy for more than 20 minutes, the fucoidan contained in the second extract will be excessively degraded, and the expected purified amount of fucoidan cannot be obtained.

In one embodiment, after the second crude extract is obtained, the second crude extract can be selectively dialyzed for hours to tens of hours to remove components having a molecular weight below 10 kDa. In some illustrations, a second solid extract after dialysis may be selectively subjected to a conventional solid-liquid separation step (eg, filtration) to remove solids from the second crude extract.

In one embodiment, after the hot water extraction step, the second crude extract may be post-treated to remove the protein and alginate of the second crude extract and obtain a brown algae polysaccharide. In some illustrations, the foregoing post-treatment may include, but is not limited to, a protein removal step, an alginate removal step, a chromatography method, and/or a partial hydrolysis method.

In one example, the step of removing protein may comprise isoelectric precipitation for several hours in an acidic (eg, pH 3 to pH 3.5) room temperature environment to remove proteins contained in the second crude extract. In the above exemplification, the second crude extract can be subjected to isoelectric precipitation for about 4 hours at room temperature of pH 3 to remove about 83% of the protein in the second crude extract.

In an example, the alginate removal step may add a final concentration of 20 g/L to 40 g/L of calcium chloride (CaCl ₂ ) to the second crude extract, and perform static at 0 ° C to 10 ° C. After a few hours, the alginate contained is removed by solid-liquid separation (for example, centrifugation). In the above illustration, the second crude extract may be treated with calcium chloride having a final concentration of 20 g/L to remove about 74% of the alginate.

In one example, the chromatographic method can comprise chromatographing a second crude extract with a 1M to 4M sodium chloride solution as a rinse using an anion exchange column. In the above exemplification, the second crude extract can be chromatographed using a commercially available anion exchange column and a 2M sodium chloride solution as a extract to obtain a brown algae polysaccharide having a high recovery rate.

In one example, the partial hydrolysis method may include treating the obtained fucoidan polysaccharide with a hydrogen peroxide solution to obtain a low molecular weight fucoidan. The low molecular weight fucoidan as referred to herein means a molecular weight of not more than 3.2 kilodaltons (kda) and a sulfate content of less than 19%, and a molecular weight of not more than 3.2 kDa and a sulfate content lower than that. 19% are preferred. In this illustration, the obtained fucoidan polysaccharide is further treated with a 0.025 M to 0.1 M hydrogen peroxide solution for 30 minutes to 120 minutes, and the yield of the low molecular weight fucoidan can be increased, wherein 0.05 M is utilized. It is preferred to treat the 0.1 M hydrogen peroxide solution for about 60 minutes to 120 minutes, and more preferably for about 60 minutes with a 0.1 M hydrogen peroxide solution. After the above treatment, the conventional method (for example, nitrogen blowing, 95% ethanol solution washing, etc.) may be selectively used to further remove the residual hydrogen peroxide of the fucoidan, which is well known to those of ordinary skill in the art to which the present invention pertains. Therefore, I will not repeat them.

The low molecular weight fucoidan obtained above has been proved to have the effect of inhibiting cell lipid synthesis reaction and tumor necrosis factor (TNF)-α production by in vitro cell experiments, and can be applied to pharmaceutical compositions. The following examples are used to illustrate the application of the present invention, and are not intended to limit the present invention. Those skilled in the art can make various changes without departing from the spirit and scope of the present invention. Retouching.

Example 1: Production of low molecular weight fucoidan

This example uses a S. cerevisiae derived from Lake Biwa to produce a low molecular weight fucoidan. S. cerevisiae mainly contains 78.26% of total sugar, 19.48% of sulphate, 6.09% of protein, 5.52% of alginate, 4.41% of phenolic compounds, and the like. This embodiment utilizes pretreatment, hot water extraction steps, and post treatment to increase the yield of low molecular weight fucoidan.

Preparation Example 1

Preparation Example 1 was carried out under the conditions shown in Table 1. Briefly, in Preparation Example 1, S. cerevisiae was selected, and the epiphytes and surface salts were removed by washing with water, and then dried at 60 ° C for 48 hours to obtain dried algae raw materials. Next, using a commercially available high temperature and high pressure blasting machine, the dried seaweed material is subjected to high temperature and high pressure treatment at a temperature of 140 ° C to 250 ° C and a pressure of 1.5 kg / cm ² to 19 kg / cm ² for 4 seconds to 10 seconds. To obtain the raw material of the expanded seaweed.

Then, the above-mentioned expanded seaweed material is ground by a conventional grinding apparatus to be pulverized into seaweed powder. Thereafter, the seaweed powder (1 g) was immersed in a 95% ethanol solution at a liquid-solid ratio of 10 mL/g, and allowed to stand at room temperature (crude extraction) for 4 hours to remove the contained lipid, mannitol, pigment, and part of the salt. This gave the first crude extract.

Next, the first crude extract is immersed in water at 100 ° C for a hot water extraction step, and subjected to microwave-assisted extraction treatment and/or ultrasonic-assisted extraction treatment to obtain a second crude extract, wherein the first crude extract and water The liquid-solid ratio (mL/g) was 15, and it was treated at a microwave power of 750 W for 10 minutes.

The second crude extract obtained above was subjected to dialysis for 24 hours using a commercially available dialysis membrane having a molecular weight cut off (MWCO) of 10 kDa to remove a component having a molecular weight of less than 10 kDa in the second crude extract. Then, the second crude extract was filtered using a 0.22 μm filter device to obtain a supernatant of the second crude extract.

Thereafter, the second crude extract obtained above was subjected to isoelectric precipitation for 4 hours at room temperature of pH 3, and after centrifugation at 6000 rpm for 3 minutes, 83% of the protein was removed. The second crude extract after removing the protein has a protein concentration reduced to 3.18 g/L, and the total sugar concentration is increased to 21.18 g/L and 42.44% (based on the total amount of the second crude extract not subjected to post-treatment is 100). %), the sugar recovery was 92.65% (based on 100% of the total amount of the second crude extract not subjected to post-treatment).

Then, calcium chloride (CaCl ₂ ) having a final concentration of 20 g/L was added to the second crude extract after the protein removal, and after standing at 4 ° C for 4 hours, it was centrifuged at 6000 rpm for 3 minutes. The precipitated alginate is removed. The second crude extract after removing the alginate has a total sugar concentration of 20.57 g/L and 54.85% (based on 100% of the total amount of the crude extract), and the sugar recovery rate is 89.95% (based on the crude extract). The total amount is 100%).

The second crude extract (300 mg) obtained above is dialyzed, filtered, and lyophilized, and then subjected to a layer using a commercially available anion exchange column (for example, DEAE Sephadex A-25) as a solution of 4 M sodium chloride solution. Analysis to obtain a second crude extract containing fucoidan. The second crude extract obtained by the above chromatography is used to detect the optical density value at a wavelength of 490 nm by a phenol-sulfuric acid coloring method, and the sugar content is calculated, wherein the total sugar concentration of the second crude extract after chromatography is increased to 21.18 g. /L and 78.65% (based on the total amount of the crude extract is 100%), the sugar recovery rate is 78.26% (based on the total amount of the crude extract is 100%).

After the second crude extract obtained above was treated with a 0.025 M to 0.1 M hydrogen peroxide solution for 60 minutes, the average molecular weight of the fucoidan contained was 3.2 kDa, and was subjected to subsequent evaluation.

Preparation Examples 2 to 14 and Comparative Examples 1 to 3

Preparation Examples 2 to 11 and Comparative Examples 1 to 3 were carried out in the same manner as in Preparation Example 1, except that the process conditions of Preparation Examples 2 to 11 and Comparative Examples 1 to 3 were different, as shown in Table 1.

Example 2, evaluation of the efficacy of low molecular weight brown algae polysaccharide

1. Structure of brown algae polysaccharide

In this example, the structural analysis of the brown algae polysaccharide of Preparation Example 1 was carried out by electrospray ionization-collision induced dissociation-mass spectrometry (ESI-CID-MS/MS). According to the ESI-CID-MS/MS results (not shown), the main structure of the fucoidan of Preparation Example 1 is L-fucose bonded with α- (1,3)-glycosidic bonds, and most of the sulfuric acid The base is at the position of carbon No. 2 and No. 4 carbon. Secondly, the fucoidan obtained in Preparation Example 1 has many branches, mainly D-galactose bonded by (1,3)-glycosidic bond, and the branching point is located on the No. 4 carbon of fucose, and D-half The sulfate groups on the lactose are mainly located on the No. 4 and No. 6 carbons, and a few are distributed on the No. 2 carbon. From the above results, it is understood that the structure of the fucoidan of Preparation Example 1 is as shown in the formula (I), wherein Gal represents D-galactose bonded by a (1,3)-glycosidic bond, and Fuc represents fucose:

2. In vitro cell assay of brown algae polysaccharide (I) - anti-lipid biosynthesis activity

This example was subjected to an in vitro cell test using the fucoidan of Preparation Example 1 and the second crude extract of Comparative Example 1 to evaluate the anti-fat of fucoidan The effect of quality synthesis.

First, the human hepatoma cell line HepG2 cells (purchased from the FIRDI Bioresource Conservation and Research Center (BCRC), accession number: RM60025; and another ATCC number: HB-8065) were cultured at 37 °C. In a 5% CO ₂ environment, the cell culture medium contains 90% (Minimum essential medium; MEM, Eagle), containing 2 mM L-glutamine, 10% fetal bovine serum (FBS), and 1.5 g/L sodium bicarbonate adjusted with Earle's balance salt solution (Earle's balance salt solution; Earl's BSS), 0.1 mM non-essential amino acids and 1.0 mM Sodium pyruvate.

At the time of evaluation, HepG2 cells (1×10 ⁵ cells) were added to the HepG2 cell culture medium or the different concentrations (20-80 μg/mL) of the fucoidan of Preparation Example 1 or the second crude extract of Comparative Example 1 After an hour, it was co-cultured with 1 mM free fatty acid (FFA; oleic acid and palmitic acid at a molar ratio of 2:1) for 24 hours. Thereafter, the phenomenon of lipid accumulation (presenting red) in HepG2 cells was observed by oil red staining, and the results are shown in FIGS. 1A to 1I.

1A to FIG. 1I are diagrams showing microscopy of human hepatoma cell line HepG2 cells treated with different concentrations of the fucoidan of Preparation Example 1 and the second crude extract of Comparative Example 1 according to an embodiment of the present invention. Photo (magnification 40 times). Figure 1A shows the control group (unprocessed, Con) The appearance of HepG2 cells, FIG. 1B is an image of lipid accumulation in HepG2 cells co-cultured with FFA, and FIG. 1C shows lipid accumulation in HepG2 cells co-cultured with FFA and co-treated with pioglitazone (Pioglitazon; Pio; a diabetic drug). Image; FIG. 1D to FIG. 1F are respectively treated with the second crude extract of Comparative Example 1 co-cultured with FFA and treated with 20 μg/mL (FIG. 1D), 40 μg/mL (FIG. 1E), and 80 μg/mL (FIG. 1F). Images of lipid accumulation in HepG2 cells; Figures 1G to 1H are preparations of Preparation Example 1 co-cultured with FFA and utilized 20 μg/mL (Fig. 1G), 40 μg/mL (Fig. 1H), and 80 μg/mL (Fig. 1I), respectively. Image of lipid accumulation in HepG2 cells after treatment with fucoidan.

The results from Fig. 1A to Fig. 1C show that, compared with the control group cells (as shown in Fig. 1A), HepG2 cells co-culture with FFA did produce free fatty acid-induced lipid accumulation (as shown in Fig. 1B), using pioglitazone. After treatment, hepatic cell de novo lipogenesis can be inhibited, thereby reducing the amount of intracellular lipid accumulation (as shown in Figure 1C).

From the results of FIG. 1G to FIG. 1I, the HepG2 cells were treated with different concentrations of the fucoidan of Preparation Example 1 and co-cultured with FFA, and the amount of intracellular lipid accumulation decreased as the amount of the second crude extract was increased. The fucoidan representative of Preparation Example 1 does have an effect of inhibiting fat regeneration in vitro.

In contrast, when HepG2 cells were treated with different concentrations of the second crude extract of Comparative Example 1 and co-cultured with FFA, the amount of intracellular lipid accumulation did not decrease as the amount of the second crude extract increased. 1D to Figure 1F.

Next, please refer to FIG. 2A to FIG. 2L, which are illustrated according to the present invention. MODE FOR CARRYING OUT THE INVENTION A micrograph (magnification of 40 times) of cells treated in vitro with different concentrations of the fucoidan of Preparation Examples 1 to 4 was used. 2A to 2C are respectively treated with FFA and treated with 20 μg/mL (Fig. 2A), 40 μg/mL (Fig. 2B), and 80 μg/mL (Fig. 2C) of the fucoidan of Preparation Example 4 (average molecular weight 107.3 kDa). HepG2 cell image (referred to as control group, Con). 2D to 2F are respectively treated with FFA and treated with 20 μg/mL (Fig. 2D), 40 μg/mL (Fig. 2E), and 80 μg/mL (Fig. 2F) of the fucoidan of Preparation 3 (average molecular weight 68.5 kDa). HepG2 cell image (abbreviated as L1). 2G to 2I are respectively co-cultured with FFA and treated with 20 μg/mL (Fig. 2G), 40 μg/mL (Fig. 2H), 80 μg/mL (Fig. 2I), preparation of fucoidan (average molecular weight 31.5 kDa) HepG2 cell image (abbreviated as L2). 2J to 2L are respectively co-cultured with FFA and treated with 20 μg/mL (Fig. 2J), 40 μg/mL (Fig. 2K), and 80 μg/mL (Fig. 2L) of the fucoidan (average molecular weight 3.2 kDa) of Preparation Example 1 HepG2 cell image (abbreviated as L3). Fig. 2A to Fig. 2L show the phenomenon of accumulation of lipids (presenting red) in HepG2 cells by oil red staining.

Please refer to FIG. 3 together, which shows that the cells of FIG. 2A to FIG. 2L are tested by the enzyme immunoassay tester for the absorption at a wavelength of 490 nm (showing the amount of oil red staining in the cells, and the oil red staining amount representing the lipid content). The relative absorbance bar graph (100% in the control group), wherein the vertical axis is the relative absorbance of the cells detected at a wavelength of 490 nm, and the horizontal axis is the respective treatment groups. The figure number "*" in Fig. 3 represents that the mean value ± standard deviation has a statistically significant difference ( P < 0.05) compared to the control group, and the figure number "**" represents that the average value has statistics compared to the control group. Significant differences ( P < 0.01), the figure number "***" represents that the mean is statistically significant ( P < 0.005) compared to the control group (three replicates per group).

The results of the 490 nm absorbance measured by the results of FIG. 2A to FIG. 2L and the enzyme immunoassay analyzer of FIG. 3 show that the smaller the average molecular weight of the brown algae polysaccharide, the better the effect of inhibiting fat regeneration. Taking 80 μg/mL of fucoidan as an example, the ratio of the brown algae polysaccharides of Preparation Examples 1 to 4 inhibiting the fat regeneration of HepG2 cells was 71.1% (Fig. 2L), 31.6% (Fig. 2I), 28.9% (Fig. 2F). 28.9% (Fig. 2C), the effect of the brown algae polysaccharide (i.e., Preparation Example 1) showing an average molecular weight of 3.2 kDa for inhibiting fat regeneration was significantly increased. Secondly, the results of the results of FIG. 2J to FIG. 2L and the enzyme immunoassay tester of FIG. 3 show that the effect of the fucoidan of the preparation example 1 on suppressing fat regeneration is also superior to that of pioglitazone (as shown in FIG. 3). The brown algae polysaccharide (ie, Preparation Example 1) having an average molecular weight of 3.2 kDa has potential as a drug candidate for the treatment of obesity.

In contrast, the brown algae polysaccharide having an average molecular weight of 31.5 kDa to 107.3 kDa (i.e., Preparation Example 2 to Preparation Example 4) has little effect in inhibiting fat regeneration.

3. In vitro cell assay of brown algae polysaccharides (II) - anti-inflammatory activity

In this example, an in vitro cell test was carried out using the fucoidan of Preparation Example 1 and the second crude extract of Comparative Example 1, and the anti-inflammatory effect of fucoidan was evaluated by detecting changes in the content of tumor necrosis factor (TNF)-α.

At the time of evaluation, Bone marrow-derived dendritic cells (BMDC; 1×10 ⁶ cells) were first treated with the drug or different concentrations (0.25 μg/mL to 1.0 μg/mL) of the brown algae of Preparation Example 1. The polysaccharide or the second crude extract of Comparative Example 1 was pretreated for 1 hour, and an inflammatory reaction was induced with 50 μM of lipopolysaccharide (LPS) for 24 hours. Thereafter, the content of TNF-α was measured by an enzyme-linked immunosorbent assay (ELISA), and the results are shown in FIG. 4 .

Please refer to FIG. 4 , which illustrates TNF-α treated by the LPS-induced inflammatory reaction in vitro according to an embodiment of the present invention, using different concentrations of the fucoidan of Preparation Example 1 and the second crude extract of Comparative Example 1. Content bar chart. In Fig. 4, the vertical axis represents the content of TNF-α (ng/mL), and the horizontal axis represents each treatment group, including the control group (untreated, Con), the LPS treatment group (LPS), and the drug treatment group (quercetin). , quercetin; Q), the second crude extract treatment group of Comparative Example 1 (abbreviated as LPS+Crude extract) and the fucoidan treatment group of Preparation Example 1 (abbreviated as LPS+Fucoidan).

The results of Fig. 4 show that the BMDC cells induced by the LPS inflammatory reaction did not inhibit the TNF-α content after treatment with the second crude extract of Comparative Example 1, but the second of Comparative Example 1 was used at 1.0 μg/mL. After treatment with the crude extract, the TNF-α content increased significantly. However, after treatment with the fucoidan of Preparation Example 1, it has an effect of inhibiting the TNF-α content. For example, compared to the result of using the second crude extract of Comparative Example 1 at 0.5 μg/mL, the treatment with 0.5 μg/mL of the fucoidan of Preparation Example 1 reduced the TNF-α content by 13.3%. Similarly, the brown of Preparation Example 1 was used at 0.25 μg/mL as compared with the result of using the second crude extract of Comparative Example 1 at 0.25 μg/mL. After treatment with algal polysaccharides, the TNF-α content of 6.7% can be reduced, and it has anti-inflammatory activity.

In summary, although the present invention is exemplified by a specific type of brown algae, a specific process, or a specific evaluation method, the brown algae polysaccharide of the present invention and a method for producing the same are described, but any one of ordinary skill in the art to which the present invention pertains can be known. The invention is not limited thereto, and the brown algae polysaccharide of the present invention and the method for producing the same may be carried out using other types of brown algae, other processes, or other evaluation methods without departing from the spirit and scope of the invention. For example, the fucoidan polysaccharide of the present invention can be applied to other types of compositions, for example, to prepare a pharmaceutical composition for inhibiting lipid synthesis and inhibiting pro-inflammatory factors, a drug candidate for obesity treatment, etc., to effectively process the traditional fucoidan and Improve the application of brown algae polysaccharides.

It can be seen from the above examples that the nucleated brown algae polysaccharide of the present invention and the method for producing the same have the advantages that the hot water extraction step and the microwave assisted extraction treatment and/or the ultrasonic assisted extraction treatment not only improve the purification amount of the brown algae polysaccharide, but also the post treatment. The obtained low molecular weight fucoidan has the effects of inhibiting cell lipid synthesis reaction and tumor necrosis factor (TNF)-α production, and can be applied to prepare a pharmaceutical composition for inhibiting lipid synthesis and inhibiting proinflammatory factors.

While the invention has been described above in terms of several embodiments, it is not intended to limit the scope of the invention, and the invention may be practiced in various embodiments without departing from the spirit and scope of the invention. The scope of protection of the present invention is defined by the scope of the appended claims.

Claims (14)

A method for producing a brown algae polysaccharide, comprising: performing at least one pretreatment on a dry seaweed material to obtain a first crude extract; performing a hot water extraction step on the first crude extract to obtain a second crude extract The hot water extraction step is performed by immersing the first crude extract in water at 100 ° C and treating it with a microwave-assisted extraction treatment and/or an ultrasonic assisted extraction treatment, the first crude extract and one of the water being liquid-solid. The ratio (mL/g) is 10 to 25, and the hot water extraction step is carried out for no more than 1 hour; and the second crude extract is subjected to a post-treatment to remove the protein, alginate of the second crude extract And obtaining the brown algae polysaccharide, wherein the fucoidan has a molecular weight of not more than 3.2 kilodaltons (klo). The method for producing a brown algae polysaccharide according to claim 1, wherein the dried algae raw material is selected from the group consisting of Sargassum siliquosum , S. hemiphyllum , and Litchi . S. polycystum ) is a group of people. The method for producing a brown algae polysaccharide according to claim 1, wherein the at least one pretreatment comprises a first pretreatment comprising a temperature of 140 ° C to 250 ° C and 1.5 kg / cm ² The dried seaweed material is subjected to a high temperature and high pressure step at a pressure of 19 kg/cm ² for 4 seconds to 10 seconds to obtain a swelled seaweed material. According to the method for producing a brown algae polysaccharide according to claim 3, after the first pretreatment, the at least one pretreatment further comprises at least a second pretreatment of the expanded algae raw material to obtain the crude raw material. Where the second pretreatment comprises a crude extraction step and a solvent removal Steps to obtain the first crude extract. The method for producing a brown algae polysaccharide according to the fourth aspect of the invention, wherein the crude extraction step is performed by immersing the expanded algae raw material in a 95% ethanol solution or pure ethanol for 4 hours to 24 hours. The method for producing a brown algae polysaccharide according to claim 1, wherein the microwave-assisted extraction treatment treats the first crude extract with a power of 750 W for 5 minutes to 20 minutes, and the first crude extract and the One of the water has a liquid to solid ratio (mL/g) of 15 to 25. The method for producing a brown algae polysaccharide according to claim 1, wherein the post-treatment comprises a step of removing a protein, a step of removing an alginate, a method of chromatography, and/or a partial hydrolysis. The method for producing a brown algae polysaccharide according to the above aspect of the invention, wherein the step of removing the protein is carried out by an isoelectric point precipitation method for treating the second crude extract in an environment of pH 3 to pH 3.5 for 4 hours. The method for producing a brown algae polysaccharide according to the above aspect of the invention, wherein the alginate removing step is treating the second crude extract with a calcium chloride solution of 20 g/L to 40 g/L. The method for producing a brown algae polysaccharide according to the first aspect of the invention, wherein the chromatographic method treats the second crude extract by anion exchange chromatography. The method for producing a brown algae polysaccharide according to the above aspect of the invention, wherein the partial hydrolysis method treats the second crude extract with a hydrogen peroxide solution of 0.025 M to 0.1 M for 30 minutes to 60 minutes. A brown algae polysaccharide obtained by the method according to any one of claims 1 to 11, wherein the fucoidan has a molecular weight of not more than 3.2 kDa. A composition comprising an effective amount of a brown algae polysaccharide, wherein the fucoidan has a molecular weight of no greater than 3.2 kDa. The use of a brown algae polysaccharide for preparing a pharmaceutical composition for inhibiting lipid synthesis and inhibiting proinflammatory factors, wherein the molecular weight of the fucoidan polysaccharide is not more than 3.2 kDa, thereby inhibiting a cell lipid synthesis reaction and tumor necrosis factor (TNF)-α produce.