WO2008041799A1 - Bioavailable fucoidan and methods for preparing the same - Google Patents
Bioavailable fucoidan and methods for preparing the same Download PDFInfo
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- WO2008041799A1 WO2008041799A1 PCT/KR2007/004453 KR2007004453W WO2008041799A1 WO 2008041799 A1 WO2008041799 A1 WO 2008041799A1 KR 2007004453 W KR2007004453 W KR 2007004453W WO 2008041799 A1 WO2008041799 A1 WO 2008041799A1
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- fucoidan
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/20—Fish extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/40—Shell-fish
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
- A23L17/60—Edible seaweed
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
- A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
Definitions
- the present invention relates to a bioavailable fucoidan and methods for preparing it.
- Functional polysaccharides which are widely used in the food industry are contained in algae.
- the representative of the functional polysaccharides includes agar, alginic acid, carrageenan, and fucoidan whose physiological activities are proved.
- Fucoidan is a complex sulfated polysaccharide which includes esterifed sulfate group containing L-fucose as a primary constituent sugar and small amount of galactose, xylose and glucuronic acid.
- Fucoidan is a mucous high molecular weight polysaccharide having 40,000- 2,000,000 dalton of weight average MW (molecular weight). It is mainly contained in the mucous of Mollusca such as cuttlefish and abalone or in the brown algae such as undaria seaweeds and sea tangles. The weight average MW of fucoidan extracted from sporophyll of undaria seaweeds is 170,000 dalton.
- the acidic polysaccharide fucoidan derived from brown algae is functional food materials which have been developed in Japan and are in early stage of development in South Korea. Most of fucoidan on the market in South Korea is high molecular weight compound extracted from brown algae with hot water and has high viscosity.
- fucoidan has the physiological activities such as enhancement of immunity, anti-tumor activity, (cancer cell apoptosis), suppression of blood glucose level increase, anticoagulant effects, suppression of triglyceride and cholesterol level increase, anti-oxidative activity, and anti-ulcer effects (Scand. J. Urol. Nephrol., 40(1), 2006; Am. J. Hematol., 78(1), 2005; Journal of the Korean Chemical Society, 46(2); The Monthly Food Industry, November Special Issue, 2005).
- the process of low-molecularization of high molecular weight fucoidan to more fluent fucoidan is key technology for commercialization, which can expand the usefulness and maximize the value added of fucoidan because this process can ensure the uniformity and improve the demerits of physical properties.
- the present inventors have made intensive researches to prepare a low molecular weight bioavailable fucoidan which have improved physical properties and physiological activities, and to provide novel method for preparing the low molecular weight bioavailable fucoidan which overcomes the demerits and limitations of conventional enzymatic method.
- the present inventor has discovered that where the fucoidan polysaccharide is treated with proper organic acids such as pyruvic acid to effectively produce a low molecular weight fucoidan, its molecular weight can be controlled by regulating the concentration and type of organic acids, the hydrolyzed products of fucoidan polysaccharide have structural similarity to high molecular weight fucoidan molecules, and its physiological activities (for example, activity to inhibit the proliferation of cancer cells) has been remarkably improved.
- bioavailable fucoidan characterized in that the bioavailable fucoidan has (a) 20-40% of the content of sulfate group; (b) 1,000-30,000 Da of the weight average molecular weight; (c) 1- 6 of polydispersity; and (d) 0-200 centi-poise of viscosity.
- the present inventors have made intensive researches to prepare a low molecular weight bioavailable fucoidan which have improved physical properties and physiological activities, and to provide novel method for preparing the low molecular weight bioavailable fucoidan which overcomes the demerits and limitations of conventional enzymatic method.
- the present inventor has discovered that where the fucoidan polysaccharide is treated with proper organic acids such as pyruvic acid to effectively produce a low molecular weight fucoidan, its molecular weight can be controlled by regulating the concentration and type of organic acids, the hydrolyzed products of fucoidan polysaccharide have structural similarity to high molecular weight fucoidan molecules, and its physiological activities (for example, activity to inhibit the proliferation of cancer cells) has been remarkably improved.
- the present invention relates to a bioavailable fucoidan which shows improved physical properties and physiological activities.
- bioavailability used herein with reference to fucoidan has been used to express low molecular weight fucoidan which has enhanced absorption rate and water solubility in the body, and shows higher physiological activities than those of naturally occurring fucoidan.
- the bioavailable fucoidan of this invention has very low molecular weight with compared to the conventional high molecular weight fucoidan (more than 170,000 Da) derived from brown algae ⁇ e.g., Undaria pinnatifida).
- the weight average molecular weight of hydrolyzed product of the fucoidan is 1,000-20,000 Da, more preferably, 3,000-10,000 Da, most preferably 1,500-3,000 Da.
- weight average molecular weight means that weight average molecular weight of fucoidan molecules which have various molecular weights and are present in the bioavailable fucoidan of this invention.
- the bioavailable fucoidan of this invention is obtained by treating the high molecular weight fucoidan with organic acids, the treated resulting products contain variable fucoidan molecules having diverse molecular weights.
- the "weight average molecular weight” used herein is the weight average molecular weight of these variable fucoidan molecules included in the treated resulting product.
- the content of sulfate group of the low molecular weight of the instance invention is 20-35%, most preferably, 20-30%.
- the sulfate group is a very pivotal moiety for fucoidan to exert its physiological activities (Biochem Pharmacol, 65, 2003). Accordingly, the physiological activities of the low molecular weight fucoidan of this invention can be greatly improved because of its high content of sulfate group.
- the bioavailable fucoidan of this invention has polydiversity of 1-6, preferably, 1-5, more preferably 1-4, most preferably 1-2. Such a low degree of polydiversity addresses that the size distribution of bioavailable fucoidan of this invention is significantly narrow.
- the bioavailable fucoidan of this invention is the low molecular weight fucoidan having homogeneous physical and physiological properties. Where commercial products are produced using the bioavailable fucoidan of this invention, the equal quality of fucoidan products may become reliable.
- the bioavailable fucoidan of the instant invention (more specifically, aqueous solution of the bioavailable fucoidan) has considerably low viscosity, preferably, 0-100 centi-poise, most preferably 0-50 centi-poise.
- the very low viscosity of the bioavailable fucoidan demonstrates that its solubility to water is almost 100.
- the bioavailable fucoidan of the present invention is obtained by treating high molecular weight fucoidan with pyruvic acid, citric acid, acetic acid or mixtures thereof.
- the bioavailable fucoidan having desirable physical properties and physiological activities can be obtained by controlling the concentration and type of organic acid.
- the preparing method for the bioavailable fucoidan will be described in more detail hereinunder:
- a method for preparing the bioavailable fucoidan having the afore-mentioned characteristics which comprises the steps of: (a) hydrolyzing a high molecular weight fucoidan by treating the high molecular weight fucoidan with pyruvic acid, citric acid, acetic acid or mixtures thereof; and (b) recovering the hydrolyzed product of high molecular weight fucoidan.
- high molecular weight fucoidan used herein means fucoidan of at least 170,000 Da, preferably at least 150,000 Da, more preferably at least 100,000 Da, most preferably at least 40,000 Da.
- the high molecular weight fucoidan is fucoidan molecules which are extracted from seaweeds such as sea mustard (Undaria pinnatifida), sea tangle ⁇ Laminaria), sea weed fusiforme (Hizikia fust ! forme) and sea oak (Eisenia bicyclis), and various ocean organisms such as abalone, cuttlefish and sea star.
- the present invention is the process to prepare bioavailable fuco- oligosaccharides of interest by effectively hydrolyzing the high molecular weight fucoidan.
- the organic acid used to hydrolyze fucoidan is pyruvic acid, citric acid, acetic acid or mixtures thereof.
- the step (a) is carried out by using the mixture of pyruvic acid and acetic acid. More preferably, the volumetric ratio of pyruvic acid to acetic acid in the mixture of pyruvic acid and acetic acid is 1:2-1:20.
- the present inventors have made experimentation for fucoidan hydrolysis with various organic acids and have discovered that edible organic acids such as pyruvic acid, citric acid, acetic acid or mixtures thereof are remarkably effective to hydrolyze fucoidan polysaccharide.
- pyruvic acid and citric acid are used to hydrolyze fucoidan, they have significant advantages in preparing foods by use of products of fucoidan hydrolysis. Furthermore, it has been discovered that pyruvic acid and citric acid make it possible to provide fucoidan molecules having desirable and controlled molecular weights because of their effective hydrolysis activities on high molecular weight fucoidan.
- Acetic acid for hydrolysis can be used in the form of eatable vinegars such as brewed vinegar, synthetic vinegar or alcoholic vinegar.
- the vinegars are edible those containing acetic acid as a major component.
- the alcoholic vinegar comprises citric acid, lactic acid and succinic acid as well as acetic acid.
- the other vinegars are mainly composed of acetic acid.
- the acid may be used in concentrations of typically 0.001- 10 N, preferably 0.01-10 N, more preferably 0.1-10 N, still more preferably 1-8 N, most preferably 2-7 N.
- a solution of high molecular weight fucoidan in concentrations of typically 0.1-2%, preferably 0.4-1.5%, more preferably 0.8-1.2%, most preferably about 1% is used and then the solution is treated with vinegar having 6% acidity in at least 20% (v/v), preferably at least 60% (v/v), more preferably at least 80% (v/v), still more preferably at least 100% (v/v), most preferably 50% (v/v) of 10-fold concentrated solution of 6% acidity vinegar.
- the volumetric ratio of organic acid solution and vinegar solution both of which are in the same concentration is preferably 1:1-1:20, more preferably 1:1- 1:10, most preferably 1:1-1:5.
- High molecular weight fucoidan as starting materials in the hydrolysis step of this invention is used in preferably 0.1-20% (w/v), more preferably 0.1-10 % (w/v), most preferably 0.1-6% (w/v).
- the reaction temperature is preferably 40-120 ° C and the reaction time is preferably 1-10 hours.
- the step of recovering fucoidan hydrolysis product may be performed according to various protocols.
- membrane filters having a constant molecular weight cutoff value ⁇ e.g., 1,000- 50,000 Da of cutoff value are used to filter the hydrolysis product.
- the concentration under reduced pressure may be performed to evaporate water and organic acids and then to concentrate, providing final products in the form of liquid, or further processing such as ethanol washing, decolorizing and drying may be carried out to provide final fucoidan products in the form of powder.
- the weight average molecular weight of fucoidan hydrolysis product is 1,000-30,000 Da, more preferably 1,000-20,000 Da, still more preferably 3,000-10,000 Da, most preferably 1,500-3,000 Da.
- the molecular weight of bioavailable fucoidan prepared by the present method can be controlled by selecting the type or concentration of organic acids used in this invention, which is one of the prominent features of the instant invention.
- the striking feature of this invention is not to provide prepare fucoidan molecules with low molecular weight but to provide fucoidan molecules having molecular weight which exhibit higher bioavailability by treatment with suitable organic acids.
- the fucoidan hydrolysis product has preferably sulfate group content of 20-40%, more preferably 20-35%, most preferably 20- 30%.
- the finally produced bioavailable fucoidan has higher content of sulfate groups compared to high molecular weight fucoidan molecules used as starting materials.
- the sulfate group content in the fucoidan hydrolysis product is increased by 1-20%, more preferably 2-15%, most preferably 5-10% compared to high molecular weight fucoidan.
- the sulfate group is a very important moiety for fucoidan to exert physiological activity (Biochem Pharmacol, 65, 2003). Accordingly, the present bioavailable fucoidan molecules showing high sulfate group content have enhanced physiological activities ⁇ e.g., anti-tumor activity).
- the fucoidan hydrolysis product has polydispersity of preferably 1-6, more preferably 1-4, most preferably 1-2.
- This low degree of polydiversity shows that size distribution of the present bioavailable fucoidan is very narrow. Therefore, according to this method, the bioavailable fucoidan having uniform size (molecular weight) can be obtained, and as a result, the low molecular weight fucoidan having homogeneous physical and physiological properties can be prepared.
- the equal quality of products can be accomplished.
- the bioavailable fucoidan of the instant invention (more specifically, aqueous solution of the bioavailable fucoidan) has very low viscosity.
- the present method can highly reduce the viscosity of high molecular weight fucoidan used as starting material.
- the bioavailable fucoidan shows viscosity preferably of 0-300 centi-poise, more preferably of 0-200 centi-poise, more and more preferably of 0-100 centi-poise, most preferably of 0-50 centi- poise.
- the very low viscosity of the bioavailable fucoidan means that its solubility to water is almost 100. From this view, the method of this invention is also expressed as 'method for regulating the solubility of fucoidan to water'.
- physiological activity of the prepared bioavailable fucoidan has been improved compared to that of high molecular weight fucoidan used as starting material (see the example 7).
- the method of the present invention it is possible not only to prepare bioavailable fucoidan with improved efficiency, convenience and economical efficacy compared to the conventional enzymatic method but also to regulate the molecular weight of bioavailable fucoidan.
- the method of this invention does not affect the basic construction of fucoidan (see the example 3) so that it does not make any damage to native physiological activities.
- differential activities of fucoidan owing to the variable molecular weights are expected.
- High molecular weight fucoidan has various physiological activities. However, in spite of these broad functionalities, high molecular weight fucoidan has problems of high molecular weight and viscosity, and low absorption rate in the body because of the low solubility, limitation of commercialization owing to the inequality of product quality according to variability of starting materials and preparing process.
- the present invention overcomes successfully shortcomings associated with high molecular weighted fucoidan molecules. Since the low molecular weighted fucoidan of the present invention has higher content of sulfate groups, low weight average molecular weight of less than 30,000 Da, low polydispersity and viscosity, and high water-solubility, its absorption rate in the body is greatly increased resulting in high improvement in its bioavailability. Accordingly, it would be appreciated that the present low molecular weight fucoidan sufficiently exerts its unique physiological activities in the body (inhibition of cancer cell proliferation, anti-oxidative activities, suppression of blood glucose level, anticoagulant effects, suppression of triglyceride and cholesterol level, and improvement in the therapeutic effect to gastric ulcer). In addition, the improved physical properties of bioavailable fucoidans of this invention ensure to overcome problems owing to non-homogeneous quality in conventional fucoidan products. BRIEF DESCRIPTION OF THE DRAWINGS
- Fig. 1 shows results of gel-chromatography analysis of molecular weight of bioavailable fucoidan molecule prepared according to the method of this invention.
- Rg. 2 shows results of Fourier transformed FT-IR Spectrophotometer analysis for verifying the structural properties of bioavailable fucoidan molecule of the present invention.
- Fig. 3 shows results of analysis of the content of sulfate group in bioavailable fucoidan molecule of the instant invention.
- Fig 4a and 4b show results of water-solubility and viscosity analysis of bioavailable fucoidan molecule of this invention.
- Fig 5 shows the anti-proliferation activities of the present bioavailable fucoidan molecule in Squamous Cell Carcinoma compared to those of high molecular weight fucoidan molecule.
- 1% (w/v) of high molecular weight fucoidan of weight average molecular weight of about 170,000 Da was added to (i) edible organic acids of 2N of pyruvic acid, citric acid, and acetic acid; (ii) 100% (v/v) brewed vinegar, synthetic vinegar, and alcoholic vinegar having 6% acidity; (iii) mixture (50/50, v/v) of 2N pyruvic acid and 100% vinegar. Afterwards, it was hydrolyzed at 100 ° C for 4 hours.
- the molecular weight of hydrolyzed products with organic acid of example 1 was analyzed by employing gel-permeation chromatography (Waters Co. Ltd.) equipped with column (PL aquagel,OH30*2, Waters Co. Ltd.), RI detector (waters 410 Differential Refractometer, Waters Co. Ltd.), pump (waters 515 HPLC pump, Waters Co. Ltd.), and autosampler (waters 717 plus Autosampler, Waters Co. Ltd.) in pH 7 buffer solution at 35 °C with flow rate of 1 ml/min using polyethyleneglycol (PEG) as standard sample (see figure 1).
- gel-permeation chromatography Waters Co. Ltd.
- RI detector waters 410 Differential Refractometer, Waters Co. Ltd.
- pump waters 515 HPLC pump, Waters Co. Ltd.
- autosampler waters 717 plus Autosampler, Waters Co. Ltd.
- fucoidan molecule was treated with 2 N pyruvic acid, it represented 15,000 Da.
- fucoidan molecule was treated with ION pyruvic acid it showed homogeneous molecular weight of 3,500 Da.
- fucoidan having different weight average molecular weight can be prepared by selecting the type or concentration of treated organic acid.
- Figure 2 represents the results of Fourier transformed FT-IR Spectrophotometer (FT-IR, Spectrum GX, PerkinElmer, USA) analysis for fucoidan, which has been prepared by treating 1% high molecular weight fucoidan solution with 10-fold concentrated solution of brewed vinegar having 6% acidity by the ratio of 50/100 (v/v). As showed in figure 2, it has been proved that the method of this invention did not induce structural changes in fucoidan molecule since the prepared fucoidan showed FT-IR spectrum patterns similar to those of untreated fucoidan molecule.
- FT-IR Fourier transformed FT-IR Spectrophotometer
- the content of sulfate group in fucoidan which has been prepared by treating 1% high molecular weight fucoidan solution with 10 fold concentrated solution of brewed vinegar having 6% acidity by the ratio of 50/100 (v/v), was determined.
- the content of sulfate group was analyzed as followings: The bioavailable fucoidan molecule was hydrolyzed with 0.5-2N hydrochlroric acid solution at 110 ° C for 4 hours, and then, 0.2ml of hydrolysis products was taken, added with 3.8 ml of 4% TCA solution and 1 ml of BaCI 2 -gelatin reagent (Difco, USA). Afterwards, it was incubated for 20 minutes at RT (room temperature), and the optical density
- the content of sulfate group which plays a pivotal role in physiological activities, was greater by 4.6% in bioavailable fucoidan molecule than un-hydrolyzed fucoidan molecule.
- fucoidan molecule was treated with ION pyruvic acid, its content of sulfate group was greater by 9.8% than those of untreated fucoidan.
- EXAMPLE 6 The solubility and viscosity of fucoidan molecule prepared by organic acid hydrolysis
- the fucoidan powders which have been prepared by treating 1% high molecular weight fucoidan solution with 10 fold concentrated solution of brewed vinegar having 6% acidity by the ratio of 50/100 (v/v), were dissolved in distilled water to produce fucoidan solutions ranging 1-10% (w/v) respectively.
- the viscosities of them were determined by viscometer (Viscometer, LVT, Brookfield, USA).
- the transparent solution of fucoidan molecule of this invention verified that the solubility of fucoidan molecule has been increased.
- the turbidity of high molecular weight fucoidan was remarkably high.
- the viscosity of fucoidan molecule of this invention is strikingly lower than those of high molecular weight fucoidan, and this demonstrates that the solubility of low molecular weight fucoidan of instant invention has been greatly increased.
- EXAMPLE 7 MTT assay for demonstrating anti proliferative activity in cancer cell
- bioavailable fucoidan molecule of this invention inhibited the proliferation of tumor cell in a concentration dependent manner. Furthermore, the low molecular weight fucoidan molecule exhibited higher antiproliferative activity by about 2-fold compared to that of high molecular weight fucoidan. In addition, anti-proliferative activitiy has almost not been shown where fucoidan molecule was treated with sole brewed vinegar.
- the method of present invention it is possible to prepare bioavailable fucoidan with improved efficiency, convenience and economical efficacy compared to the conventional enzymatic method.
- the method of this invention does not affect the basic structure of fucoidan molecule so that it does not make any damage to native physiological activities.
- differential activities of fucoidan owing to its various molecular weights are expected.
- the low molecular weighted fucoidan of the present invention has higher content of sulfate groups, low weight average molecular weight of less than 30,000 Da, low polydispersity and viscosity, and high water-solubility, its absorption rate in the body is greatly increased resulting in high improvement in its bioavailability. Accordingly, it would be appreciated that the present low molecular weight fucoidan sufficiently exerts its unique physiological activities in the body (inhibition of cancer cell proliferation, anti-oxidative activities, suppression of blood glucose level, anticoagulant effects, suppression of triglyceride and cholesterol level, and improvement in the therapeutic effect to gastric ulcer). In addition, the improved physical properties of bioavailable fucoidans of this invention ensure to overcome problems owing to non-homogeneous equality in conventional fucoidan products.
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Abstract
The present invention relates to a bioavailable fucoidan characterized in that the bioavailable fucoidan has (a) 20-40% of the content of sulfate group; (b) 1,000-30,000 Da of the weight average molecular weight; (c) 1-6 of polydispersity; and (d) 0-200 centi-poise of viscosity. Since the low molecular weighted fucoidan of the present invention has higher content of sulfate groups, low weight average molecular weight of less than 30,000 Da, low polydispersity and viscosity, and high water-solubility, its absorption rate in the body is greatly increased resulting in high improvement in its bioavailability. Accordingly, it would be appreciated that the present low molecular weight fucoidan sufficiently exerts its unique physiological activities in the body (inhibition of cancer cell proliferation, anti-oxidative activities, suppression of blood glucose level, anticoagulant effects, suppression of triglyceride and cholesterol level, and improvement in the therapeutic effect to gastric ulcer). In addition, the improved physical properties of bioavailable fucoidans of this invention ensure to overcome problems owing to non- homogeneous quality in conventional fucoidan products.
Description
BIOAVAILABLE FUCOIDAN AND METHODS FOR PREPARING THE SAME
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a bioavailable fucoidan and methods for preparing it.
DESCRIPTION OF THE RELATED ART
Functional polysaccharides which are widely used in the food industry are contained in algae. The representative of the functional polysaccharides includes agar, alginic acid, carrageenan, and fucoidan whose physiological activities are proved. Fucoidan is a complex sulfated polysaccharide which includes esterifed sulfate group containing L-fucose as a primary constituent sugar and small amount of galactose, xylose and glucuronic acid.
Fucoidan is a mucous high molecular weight polysaccharide having 40,000- 2,000,000 dalton of weight average MW (molecular weight). It is mainly contained in the mucous of Mollusca such as cuttlefish and abalone or in the brown algae such as undaria seaweeds and sea tangles. The weight average MW of fucoidan extracted from sporophyll of undaria seaweeds is 170,000 dalton. The acidic polysaccharide fucoidan derived from brown algae is functional food materials which have been developed in Japan and are in early stage of development in South Korea. Most of fucoidan on the market in South Korea is high molecular weight compound extracted from brown algae with hot water and has high viscosity. The functionalities of fucoidan derived from brown algae have been extensively studied in Japan from 1990s and the studies have revealed that fucoidan has the physiological activities such as enhancement of immunity, anti-tumor activity, (cancer cell apoptosis), suppression of blood glucose level increase, anticoagulant
effects, suppression of triglyceride and cholesterol level increase, anti-oxidative activity, and anti-ulcer effects (Scand. J. Urol. Nephrol., 40(1), 2006; Am. J. Hematol., 78(1), 2005; Journal of the Korean Chemical Society, 46(2); The Monthly Food Industry, November Special Issue, 2005).
However, in spite of these broad spectrums of functionalities of fucoidan, the problems such as high molecular weight, high viscosity, low absorption rate owing to low solubility, limitation of commercialization resulting from the inequality of product quality according to variability of starting materials and preparing process are left to be solved.
Accordingly, the process of low-molecularization of high molecular weight fucoidan to more fluent fucoidan is key technology for commercialization, which can expand the usefulness and maximize the value added of fucoidan because this process can ensure the uniformity and improve the demerits of physical properties.
For low-molecularization of fucoidan, the hydrolysis method employing enzyme is extensively studied. Takara Shuzo co. Ltd in Japan has obtained U.S. patent No. 6,054,577 on April 25, 2000 concerning enzymatic treatment product of fucoidan and U.S. patent 6,277,616 on August 21, 2001 claiming endo-fucoidan- lyase. After that, intensive researches to secure novel enzyme capable of hydrolysis of high MW fucoidan have been made in Japan and Russia. However, these technologies are in early stage of development.
Recently, since the particular physiological activities of low MW fucoidan, which are not present in High MW fucoidan, have been reported (The Journal of Pharmacology and -Experimental Therapeutics, 305(1), 2003; Carbohydrate Research, 289, 1996), in domestic and oversea industry, much research and commercial activities have converged on manufacturing low MW fucoidan having higher practical
value than high MW fucoidan.
Although preparation of fuco-oligosaccharide using enzymatic method has a merit of hydrolysis of specific bond, there are commercialization limitations such as difficulty of securing enzyme, problem of liquefaction of high MW polysaccharide, and high manufacturing cost because of using enzyme in industrialization process.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entities are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.
DETAILED DESCRIPTION OF THIS INVETNION
The present inventors have made intensive researches to prepare a low molecular weight bioavailable fucoidan which have improved physical properties and physiological activities, and to provide novel method for preparing the low molecular weight bioavailable fucoidan which overcomes the demerits and limitations of conventional enzymatic method. As a result, the present inventor has discovered that where the fucoidan polysaccharide is treated with proper organic acids such as pyruvic acid to effectively produce a low molecular weight fucoidan, its molecular weight can be controlled by regulating the concentration and type of organic acids, the hydrolyzed products of fucoidan polysaccharide have structural similarity to high molecular weight fucoidan molecules, and its physiological activities (for example, activity to inhibit the proliferation of cancer cells) has been remarkably improved.
Accordingly, it is an object of this invention to provide bioavailable fucoidan having excellent physical properties and physiological activities.
It is another object of this invention to provide methods for preparing the bioavailable fucoidan.
Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.
In one aspect of this invention, there is provided a bioavailable fucoidan characterized in that the bioavailable fucoidan has (a) 20-40% of the content of sulfate group; (b) 1,000-30,000 Da of the weight average molecular weight; (c) 1- 6 of polydispersity; and (d) 0-200 centi-poise of viscosity.
The present inventors have made intensive researches to prepare a low molecular weight bioavailable fucoidan which have improved physical properties and physiological activities, and to provide novel method for preparing the low molecular weight bioavailable fucoidan which overcomes the demerits and limitations of conventional enzymatic method. As a result, the present inventor has discovered that where the fucoidan polysaccharide is treated with proper organic acids such as pyruvic acid to effectively produce a low molecular weight fucoidan, its molecular weight can be controlled by regulating the concentration and type of organic acids, the hydrolyzed products of fucoidan polysaccharide have structural similarity to high molecular weight fucoidan molecules, and its physiological activities (for example, activity to inhibit the proliferation of cancer cells) has been remarkably improved.
The present invention relates to a bioavailable fucoidan which shows improved physical properties and physiological activities. The term "bioavailability" used herein with reference to fucoidan has been used to express low molecular weight fucoidan which has enhanced absorption rate and water solubility in the body, and shows higher physiological activities than those of naturally occurring fucoidan.
The bioavailable fucoidan of this invention has very low molecular weight with compared to the conventional high molecular weight fucoidan (more than 170,000 Da) derived from brown algae {e.g., Undaria pinnatifida).
According to a preferred embodiment of the present invention, the weight average molecular weight of hydrolyzed product of the fucoidan is 1,000-20,000 Da, more preferably, 3,000-10,000 Da, most preferably 1,500-3,000 Da.
The term "weight average molecular weight" used herein means that weight average molecular weight of fucoidan molecules which have various molecular weights and are present in the bioavailable fucoidan of this invention. For example, where the bioavailable fucoidan of this invention is obtained by treating the high molecular weight fucoidan with organic acids, the treated resulting products contain variable fucoidan molecules having diverse molecular weights. The "weight average molecular weight" used herein is the weight average molecular weight of these variable fucoidan molecules included in the treated resulting product. For instance, where the weight average molecular weight of the bioavailable fucoidan of the present invention ranges from 1,500 to 3,000 Da, it may be preferably a mixture of fucoidans having the weight average molecular weights of 3000-5000 Da:1250-1500 Da:600-750 Da:140-210 Da = 30-60:12-35:5-20:15-25.
According to a preferred embodiment of this invention, the content of sulfate group of the low molecular weight of the instance invention is 20-35%, most preferably, 20-30%. The sulfate group is a very pivotal moiety for fucoidan to exert its physiological activities (Biochem Pharmacol, 65, 2003). Accordingly, the physiological activities of the low molecular weight fucoidan of this invention can be greatly improved because of its high content of sulfate group. The bioavailable fucoidan of this invention has polydiversity of 1-6, preferably, 1-5, more preferably 1-4, most preferably 1-2. Such a low degree of polydiversity addresses that the size distribution of bioavailable fucoidan of this invention is significantly narrow. Accordingly, the bioavailable fucoidan of this
invention is the low molecular weight fucoidan having homogeneous physical and physiological properties. Where commercial products are produced using the bioavailable fucoidan of this invention, the equal quality of fucoidan products may become reliable. The bioavailable fucoidan of the instant invention (more specifically, aqueous solution of the bioavailable fucoidan) has considerably low viscosity, preferably, 0-100 centi-poise, most preferably 0-50 centi-poise. The very low viscosity of the bioavailable fucoidan demonstrates that its solubility to water is almost 100. According to a preferred embodiment of this invention, the ratio of the constituent elements of the bioavailable fucoidan is carbon:hydrogen:oxygen:nitrogen:sulfur = 20-30:3-5:45-60:0.05-1.0:5.7-20, more preferably 20-25:4-4.7:50-60:0.05-0.5:5.7-20, most preferably carbon:hydrogen:oxygen:nitrogen:sulfur = 23-25:4.2-4.7:52-58:0.07-0.2:7.0-10.
According to a preferred embodiment of this invention, the bioavailable fucoidan of the present invention is obtained by treating high molecular weight fucoidan with pyruvic acid, citric acid, acetic acid or mixtures thereof. The bioavailable fucoidan having desirable physical properties and physiological activities can be obtained by controlling the concentration and type of organic acid. The preparing method for the bioavailable fucoidan will be described in more detail hereinunder:
In another aspect of this invention, there is provided a method for preparing the bioavailable fucoidan having the afore-mentioned characteristics, which comprises the steps of: (a) hydrolyzing a high molecular weight fucoidan by treating the high molecular weight fucoidan with pyruvic acid, citric acid, acetic acid or mixtures thereof; and (b) recovering the hydrolyzed product of high
molecular weight fucoidan.
The term "high molecular weight fucoidan" used herein means fucoidan of at least 170,000 Da, preferably at least 150,000 Da, more preferably at least 100,000 Da, most preferably at least 40,000 Da. In another embodiment of this invention, the high molecular weight fucoidan is fucoidan molecules which are extracted from seaweeds such as sea mustard (Undaria pinnatifida), sea tangle {Laminaria), sea weed fusiforme (Hizikia fust ! forme) and sea oak (Eisenia bicyclis), and various ocean organisms such as abalone, cuttlefish and sea star.
The present invention is the process to prepare bioavailable fuco- oligosaccharides of interest by effectively hydrolyzing the high molecular weight fucoidan.
In the present invention, the organic acid used to hydrolyze fucoidan is pyruvic acid, citric acid, acetic acid or mixtures thereof. Preferably, the step (a) is carried out by using the mixture of pyruvic acid and acetic acid. More preferably, the volumetric ratio of pyruvic acid to acetic acid in the mixture of pyruvic acid and acetic acid is 1:2-1:20. The present inventors have made experimentation for fucoidan hydrolysis with various organic acids and have discovered that edible organic acids such as pyruvic acid, citric acid, acetic acid or mixtures thereof are remarkably effective to hydrolyze fucoidan polysaccharide. The inventors have verified that where pyruvic acid and citric acid are used to hydrolyze fucoidan, they have significant advantages in preparing foods by use of products of fucoidan hydrolysis. Furthermore, it has been discovered that pyruvic acid and citric acid make it possible to provide fucoidan molecules having desirable and controlled molecular weights because of their effective hydrolysis activities on high molecular weight fucoidan.
Acetic acid for hydrolysis can be used in the form of eatable vinegars such as brewed vinegar, synthetic vinegar or alcoholic vinegar. The vinegars are edible those containing acetic acid as a major component. The alcoholic vinegar comprises citric acid, lactic acid and succinic acid as well as acetic acid. The other
vinegars are mainly composed of acetic acid.
For hydrolyzing high molecular weight fucoidan with solely pyruvic acid, citric acid or acetic acid, the acid may be used in concentrations of typically 0.001- 10 N, preferably 0.01-10 N, more preferably 0.1-10 N, still more preferably 1-8 N, most preferably 2-7 N. In addition, in case that brewed vinegar, synthetic vinegar, alcoholic vinegar or other vinegars is solely used, a solution of high molecular weight fucoidan in concentrations of typically 0.1-2%, preferably 0.4-1.5%, more preferably 0.8-1.2%, most preferably about 1% is used and then the solution is treated with vinegar having 6% acidity in at least 20% (v/v), preferably at least 60% (v/v), more preferably at least 80% (v/v), still more preferably at least 100% (v/v), most preferably 50% (v/v) of 10-fold concentrated solution of 6% acidity vinegar.
If a mixture of (i) vinegar and (ii) pyruvic acid or citric acid is used to treat fucoidan, the volumetric ratio of organic acid solution and vinegar solution both of which are in the same concentration, is preferably 1:1-1:20, more preferably 1:1- 1:10, most preferably 1:1-1:5.
High molecular weight fucoidan as starting materials in the hydrolysis step of this invention is used in preferably 0.1-20% (w/v), more preferably 0.1-10 % (w/v), most preferably 0.1-6% (w/v). In the hydrolysis step, the reaction temperature is preferably 40-120 °C and the reaction time is preferably 1-10 hours.
In the method of the present invention, the step of recovering fucoidan hydrolysis product may be performed according to various protocols. For example, membrane filters having a constant molecular weight cutoff value {e.g., 1,000- 50,000 Da of cutoff value) are used to filter the hydrolysis product. Afterwards, the concentration under reduced pressure may be performed to evaporate water and organic acids and then to concentrate, providing final products in the form of liquid, or further processing such as ethanol washing, decolorizing and drying may be carried out to provide final fucoidan products in the form of powder.
According to a preferred embodiment of this invention, the weight average molecular weight of fucoidan hydrolysis product is 1,000-30,000 Da, more preferably 1,000-20,000 Da, still more preferably 3,000-10,000 Da, most preferably 1,500-3,000 Da. The molecular weight of bioavailable fucoidan prepared by the present method can be controlled by selecting the type or concentration of organic acids used in this invention, which is one of the prominent features of the instant invention. In other words, the striking feature of this invention is not to provide prepare fucoidan molecules with low molecular weight but to provide fucoidan molecules having molecular weight which exhibit higher bioavailability by treatment with suitable organic acids.
In the present invention, the fucoidan hydrolysis product has preferably sulfate group content of 20-40%, more preferably 20-35%, most preferably 20- 30%. According to the present method, the finally produced bioavailable fucoidan has higher content of sulfate groups compared to high molecular weight fucoidan molecules used as starting materials. Preferably, the sulfate group content in the fucoidan hydrolysis product is increased by 1-20%, more preferably 2-15%, most preferably 5-10% compared to high molecular weight fucoidan. The sulfate group is a very important moiety for fucoidan to exert physiological activity (Biochem Pharmacol, 65, 2003). Accordingly, the present bioavailable fucoidan molecules showing high sulfate group content have enhanced physiological activities {e.g., anti-tumor activity).
According to a preferred embodiment of this invention, the fucoidan hydrolysis product has polydispersity of preferably 1-6, more preferably 1-4, most preferably 1-2. This low degree of polydiversity shows that size distribution of the present bioavailable fucoidan is very narrow. Therefore, according to this method,
the bioavailable fucoidan having uniform size (molecular weight) can be obtained, and as a result, the low molecular weight fucoidan having homogeneous physical and physiological properties can be prepared. Where products are produced using the low molecular weight bioavailable fucoidan of this invention, the equal quality of products can be accomplished.
The bioavailable fucoidan of the instant invention (more specifically, aqueous solution of the bioavailable fucoidan) has very low viscosity. In other words, the present method can highly reduce the viscosity of high molecular weight fucoidan used as starting material. The bioavailable fucoidan shows viscosity preferably of 0-300 centi-poise, more preferably of 0-200 centi-poise, more and more preferably of 0-100 centi-poise, most preferably of 0-50 centi- poise. The very low viscosity of the bioavailable fucoidan means that its solubility to water is almost 100. From this view, the method of this invention is also expressed as 'method for regulating the solubility of fucoidan to water'.
In the bioavailable fucoidan prepared by the method of the present invention, its molecular weight is not just reduced but the ratio of its constituent element changed as a whole. According to a preferred embodiment of this invention, the ratio of the constituent elements of the present bioavailable fucoidan is carbon:hydrogen:oxygen -.nitrogen -.sulfur = 20-30:3-5:45-60:0.05-1.0:5.7-20, more preferably, 20-25:4-4.7:50-60:0.05-0.5:5.7-20, most preferably, carbon: hydrogen: oxygen: nitrogen: sulfur =23-25:4.2-4.7:52-58:0.07-0.2:7.0-10.
According to the method of this invention, physiological activity of the prepared bioavailable fucoidan has been improved compared to that of high molecular weight fucoidan used as starting material (see the example 7).
According to the method of the present invention, it is possible not only to
prepare bioavailable fucoidan with improved efficiency, convenience and economical efficacy compared to the conventional enzymatic method but also to regulate the molecular weight of bioavailable fucoidan. In addition, the method of this invention does not affect the basic construction of fucoidan (see the example 3) so that it does not make any damage to native physiological activities. Furthermore, differential activities of fucoidan owing to the variable molecular weights are expected.
High molecular weight fucoidan has various physiological activities. However, in spite of these broad functionalities, high molecular weight fucoidan has problems of high molecular weight and viscosity, and low absorption rate in the body because of the low solubility, limitation of commercialization owing to the inequality of product quality according to variability of starting materials and preparing process.
The present invention overcomes successfully shortcomings associated with high molecular weighted fucoidan molecules. Since the low molecular weighted fucoidan of the present invention has higher content of sulfate groups, low weight average molecular weight of less than 30,000 Da, low polydispersity and viscosity, and high water-solubility, its absorption rate in the body is greatly increased resulting in high improvement in its bioavailability. Accordingly, it would be appreciated that the present low molecular weight fucoidan sufficiently exerts its unique physiological activities in the body (inhibition of cancer cell proliferation, anti-oxidative activities, suppression of blood glucose level, anticoagulant effects, suppression of triglyceride and cholesterol level, and improvement in the therapeutic effect to gastric ulcer). In addition, the improved physical properties of bioavailable fucoidans of this invention ensure to overcome problems owing to non-homogeneous quality in conventional fucoidan products.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows results of gel-chromatography analysis of molecular weight of bioavailable fucoidan molecule prepared according to the method of this invention.
Rg. 2 shows results of Fourier transformed FT-IR Spectrophotometer analysis for verifying the structural properties of bioavailable fucoidan molecule of the present invention.
Fig. 3 shows results of analysis of the content of sulfate group in bioavailable fucoidan molecule of the instant invention.
Fig 4a and 4b show results of water-solubility and viscosity analysis of bioavailable fucoidan molecule of this invention.
Fig 5 shows the anti-proliferation activities of the present bioavailable fucoidan molecule in Squamous Cell Carcinoma compared to those of high molecular weight fucoidan molecule.
The present invention will now be described in further detail by examples. It would be obvious to those skilled in the art that these examples are intended to be more concretely illustrative and the scope of the present invention as set forth in the appended claims is not limited to or by the examples.
EXAMPLES
EXAMPLE 1: Preparation of low molecular weight fucoidan molecule by hydrolyzing high molecular weight fucoidan with organic acid
1% (w/v) of high molecular weight fucoidan of weight average molecular
weight of about 170,000 Da was added to (i) edible organic acids of 2N of pyruvic acid, citric acid, and acetic acid; (ii) 100% (v/v) brewed vinegar, synthetic vinegar, and alcoholic vinegar having 6% acidity; (iii) mixture (50/50, v/v) of 2N pyruvic acid and 100% vinegar. Afterwards, it was hydrolyzed at 100 °C for 4 hours.
The resulting mixture solution was filtrated using membrane filter having MW cut-off value of 30,000 Da. The resulting filtrated solution was concentrated under reduced pressure for 6 hours, and then powder form was obtained by drying it with heated air. The results of hydrolysis process are represented in Table 1. TABLE 1
As represented in Table 1, where brewed vinegar among organic acids was employed, the highest amount of 0.75g of fucoidan powder was obtained and its yield is 75%. However, where other organic acids were used, the yields of pyruvic acid, citric acid, acetic acid, synthetic vinegar, alcoholic vinegar, and other vinegars were 60%, 52%, 53%, 65%, 42% and 34% respectively. In addition, where 1:1 (v/v) mixtures of brewd vinegar and pyruvic acid, and of synthetic vinegar and pyruvic acid were used, their yields were 34% and 46% respectively, and these value of yield are lower compared to the case treated with single organic acid.
EXAMPLE 2: Weight average molecular weight of low molecular weight fucoidan molecule prepared by organic acid hydrolysis
The molecular weight of hydrolyzed products with organic acid of example 1 was analyzed by employing gel-permeation chromatography (Waters Co. Ltd.) equipped with column (PL aquagel,OH30*2, Waters Co. Ltd.), RI detector (waters 410 Differential Refractometer, Waters Co. Ltd.), pump (waters 515 HPLC pump, Waters Co. Ltd.), and autosampler (waters 717 plus Autosampler, Waters Co. Ltd.) in pH 7 buffer solution at 35 °C with flow rate of 1 ml/min using polyethyleneglycol (PEG) as standard sample (see figure 1). As demonstrated by results displayed in figure 1, the molecular weight of fucoidan, which has not been hydrolyzed, was about 170,000 Da, however, where fucoidan molecule has been hydrolyzed with brewed vinegar having 6% acidity, the products exhibited a mixed molecular weight constituting with the ratio of 3000- 5000 Da:1250-1500 Da:600-750 Da:140-210 Da = 30-60: 12-35:5-20:15-25 and the weight average molecular weight of about 1,800-2,300 Da. Where fucoidan molecule was treated with 2 N pyruvic acid, it represented 15,000 Da. Furthermore, where fucoidan molecule was treated with ION pyruvic acid, it showed homogeneous molecular weight of 3,500 Da.
Accordingly, it has been verified that the molecular weight of fucoidan is strikingly reduced when it is hydrolyzed by organic acid, and fucoidan having different weight average molecular weight can be prepared by selecting the type or concentration of treated organic acid.
EXAMPLE 3: FT-IR spectrum of fucoidan molecule prepared by organic acid hydrolysis
Figure 2 represents the results of Fourier transformed FT-IR Spectrophotometer (FT-IR, Spectrum GX, PerkinElmer, USA) analysis for fucoidan, which has been prepared by treating 1% high molecular weight fucoidan solution
with 10-fold concentrated solution of brewed vinegar having 6% acidity by the ratio of 50/100 (v/v). As showed in figure 2, it has been proved that the method of this invention did not induce structural changes in fucoidan molecule since the prepared fucoidan showed FT-IR spectrum patterns similar to those of untreated fucoidan molecule.
EXAMPLE 4: Sulfate group content of fucoidan molecule prepared by organic acid hydrolysis
The content of sulfate group in fucoidan, which has been prepared by treating 1% high molecular weight fucoidan solution with 10 fold concentrated solution of brewed vinegar having 6% acidity by the ratio of 50/100 (v/v), was determined.
The content of sulfate group was analyzed as followings: The bioavailable fucoidan molecule was hydrolyzed with 0.5-2N hydrochlroric acid solution at 110°C for 4 hours, and then, 0.2ml of hydrolysis products was taken, added with 3.8 ml of 4% TCA solution and 1 ml of BaCI2-gelatin reagent (Difco, USA). Afterwards, it was incubated for 20 minutes at RT (room temperature), and the optical density
(absorbance) was detected at 360nm. K2SO4 was used as standard sample.
As represented in figure 3, the content of sulfate group, which plays a pivotal role in physiological activities, was greater by 4.6% in bioavailable fucoidan molecule than un-hydrolyzed fucoidan molecule. In addition, where fucoidan molecule was treated with ION pyruvic acid, its content of sulfate group was greater by 9.8% than those of untreated fucoidan.
EXAMPLE 5: Analysis of elements mainly constituting fucoidan molecule prepared by organic acid hydrolysis
The contents of elements of carbon, hydrogen, oxygen, nitrogen and sulfur mainly constituting the fucoidan molecule, which has been prepared by treating 1% high molecular weight fucoidan solution with 10 fold concentrated solution of brewed
vinegar having 6% acidity by the ratio of 50/100 (v/v), were determined by Element Analyzer(Elemental Analyzer EA1108, Fisons instrument, Italy). The results are summarized in Table 2. Table 2
As represented in Table 2, the contents of major elements constituting the hydrolyzed low molecular weight fucoidan molecule have been changed compared to those of fucoidan molecule before hydrolyzed. Particularly, the contents of oxygen and sulfur have been greatly increased. These experimental data correspond to the results of increase in sulfate group content of low molecular weight fucoidan in the Example 4.
EXAMPLE 6: The solubility and viscosity of fucoidan molecule prepared by organic acid hydrolysis The fucoidan powders, which have been prepared by treating 1% high molecular weight fucoidan solution with 10 fold concentrated solution of brewed vinegar having 6% acidity by the ratio of 50/100 (v/v), were dissolved in distilled water to produce fucoidan solutions ranging 1-10% (w/v) respectively. The viscosities of them were determined by viscometer (Viscometer, LVT, Brookfield, USA).
As represented in figure 4a, the transparent solution of fucoidan molecule of this invention verified that the solubility of fucoidan molecule has been increased. However, the turbidity of high molecular weight fucoidan was remarkably high.
In addition, as proved with the result in figure 4b, the viscosity of fucoidan molecule of this invention is strikingly lower than those of high molecular weight fucoidan, and this demonstrates that the solubility of low molecular weight fucoidan of instant invention has been greatly increased.
EXAMPLE 7: MTT assay for demonstrating anti proliferative activity in cancer cell
In order to demonstrate anti-proliferative activity in malignant tumor cell of low molecular weight fucoidan prepared by treating 1% high molecular weight fucoidan solution with 10 fold concentrated solution of brewed vinegar having 6% acidity by the ratio of 50/100 (v/v), 20 ul of bioavailable fucoidan (ranging 0.5-50 mg/L) prepared according to the process of example 1 was added to the suspension of squamous cell carcinoma, and then, incubated for two days. After that, 100 ul of MTT (3-(4, 5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, Sigma, USA) was added into wells. After incubation for 4 hours, MTT diluted solution was removed carefully, 100 ul of DMSO (dimethylsulfoxide) was added into wells and the wells were agitated for 15-20 minutes. Anti-proliferative activity was analyzed by measuring optical density (absorbance) at 540 nm.
As represented in figure 5, bioavailable fucoidan molecule of this invention inhibited the proliferation of tumor cell in a concentration dependent manner. Furthermore, the low molecular weight fucoidan molecule exhibited higher antiproliferative activity by about 2-fold compared to that of high molecular weight fucoidan. In addition, anti-proliferative activitiy has almost not been shown where fucoidan molecule was treated with sole brewed vinegar.
According to the method of present invention, it is possible to prepare bioavailable fucoidan with improved efficiency, convenience and economical efficacy compared to the conventional enzymatic method. In addition, the method
of this invention does not affect the basic structure of fucoidan molecule so that it does not make any damage to native physiological activities. Furthermore, differential activities of fucoidan owing to its various molecular weights are expected.
Since the low molecular weighted fucoidan of the present invention has higher content of sulfate groups, low weight average molecular weight of less than 30,000 Da, low polydispersity and viscosity, and high water-solubility, its absorption rate in the body is greatly increased resulting in high improvement in its bioavailability. Accordingly, it would be appreciated that the present low molecular weight fucoidan sufficiently exerts its unique physiological activities in the body (inhibition of cancer cell proliferation, anti-oxidative activities, suppression of blood glucose level, anticoagulant effects, suppression of triglyceride and cholesterol level, and improvement in the therapeutic effect to gastric ulcer). In addition, the improved physical properties of bioavailable fucoidans of this invention ensure to overcome problems owing to non-homogeneous equality in conventional fucoidan products.
Having described a preferred embodiment of the present invention, it is to be understood that variants and modifications thereof falling within the spirit of the invention may become apparent to those skilled in the art, and the scope of this invention is to be determined by appended claims and their equivalents.
Claims
1. A bioavailable fucoidan characterized in that the bioavailable fucoidan has (a)
20-40% of the content of sulfate group; (b) 1,000-30,000 Da of the weight average molecular weight; (c) 1-6 of polydispersity; and (d) 0-200 centi-poise of viscosity.
2. The bioavailable fucoidan according to claim 1, wherein the weight average molecular weight of said bioavailable fucoidan is 3,000-10,000 Da.
3. The bioavailable fucoidan according to claim 2, wherein the weight average molecular weight of said bioavailable fucoidan is 1,500-3,000 Da.
4. The bioavailable fucoidan according to claim 3, wherein said bioavailable fucoidan is the mixture of fucoidans having the weight average molecular weights of 3000-5000 Da : 1250-1500 Da : 600-750 Da : 140-210 Da = 30-60 : 12-35 : 5- 20 : 15-25.
5. The bioavailable fucoidan according to claim 1, wherein the content of sulfate group of said bioavailable fucoidan is 20-30%.
6. The bioavailable fucoidan according to claim 1, wherein the polydispersity of said bioavailable fucoidan is 1-4.
7. The bioavailable fucoidan according to claim 1, wherein the viscosity of said bioavailable fucoidan is 0-50 centi-poise.
8. The bioavailable fucoidan according to claim 1, wherein the ratio of the constituent elements in said bioavailable fucoidan is carbon:hydrogen:oxygen:nitrogen:sulfur = 20-30:3-5:45-60:0.05-1.0:5.7-20.
9. The bioavailable fucoidan according to claim 6, wherein the ratio of the constituent elements in said bioavailable fucoidan is carbon:hydrogen:oxygen:nitrogen:sulfur = 23-25:4.2-4.7:52-58:0.07-0.2:7.0-10.
10. The bioavailable fucoidan according to claim 1, wherein said bioavailable fucoidan is obtained by treating high molecular weight fucoidan with pyruvic acid, citric acid, acetic acid or mixtures thereof.
11. A method for preparing the bioavailable fucoidan of any one of claims 1-10, which comprises the steps of:
(a) hydrolyzing a high molecular weight fucoidan by treating said high MW fucoidan with pyruvic acid, citric acid, acetic acid or mixtures thereof; and
(b) recovering the hydrolyzed product of high molecular weight fucoidan.
12. The method according to claim 11, wherein the step (a) is carried out by using a mixture of pyruvic acid and acetic acid.
13. The method according to claim 11, wherein the step (a) is carried out by using 0.1-10 N pyruvic acid, citric acid, acetic acid or mixtures thereof.
14. The method according to claim 13, wherein the volume ratio of pyruvic acid to acetic acid in the mixture of pyruvic acid and acetic acid is 1:2-1:20.
15. The method according to claim 11, wherein the acetic acid is provided in the form of brewed vinegar, synthetic vinegar or alcoholic vinegar.
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KR101156226B1 (en) * | 2009-05-06 | 2012-06-18 | (주)바이온 | Composition For Treating Obesity Including Bioavailable Fucoidan |
KR102642121B1 (en) * | 2021-03-31 | 2024-02-29 | 엠에스글로벌바이오주식회사 | Manufacturing method for cosmetic composition of fucoidan seaweed and functional and anti-aging cosmetic composition thereof |
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