KR20120137091A - The method of extracting carotinoid pigments of micro algae and composition comprising the extracted fucoxanthin for preventing or treating obesity or diabetes - Google Patents

Abstract

PURPOSE: A method for extracting pigment from microalgae and a composition containing fucoxanthin for preventing or treating obesity or diabetes are provided to enhance stability. CONSTITUTION: A method for extracting pigment from microalgae comprises a step of performing accelerated solvent extraction using 10-100% ethanol at 0-1,500 psi and 40-160 Deg.C. for 1-30 minutes. The microalgae include Chlorophyte, Phaeophyceae, Rhodophyte, Cyanophyta, Bacillariophycea, Dinophyta, or Haptophyta. The pigment is fucoxanthin. A pharmaceutical composition for preventing or treating obesity or diabetes contains fucoxanthin.

Description

Method of extracting carotinoid pigments of micro algae and composition comprising the extracted fucoxanthin for preventing or treating obesity or diabetes}

The present invention relates to a method for extracting a pigment from microalgae using an accelerated solvent treatment method and a composition for the prevention or treatment of obesity or diabetes comprising fucoxanthin extracted by the above method.

Microalgae are aquatic organisms in freshwater and seawater, including blue algae, diatoms, coarse algae, green algae, red algae, yellow brown algae, and silver algae, and there are about 35,000 species reported worldwide. It is estimated that abnormality exists. Microalgae, like other plants, are photosynthetic and biosynthesize sugars and proteins in the interior, and are therefore responsible for basic production in freshwater and the ocean, and are important producers of food chains.

Most microalgae contain unique bioactive substances inside the cell walls of each species and can produce large quantities of specific substances depending on the culture environment, so carotenoid pigments, antioxidants, fatty acids, enzymes, functional peptides, biotoxins Industrially produced for the purpose of using sterols and the like. In addition, it is used for a variety of uses, such as the production of fish and bio-energy of juvenile and sexual organs.

Among the carotenoid pigments, fucoxanthin is a bioactive pigment with strong antioxidant and anticancer activity.

Most of the production of fucoxanthin has been extracted from the land plants and marine macroalgae (Kashima, etc.), or the intestines of the organisms that consume it, but the production cost is very high because the content is very small. Recent studies have confirmed that microalgae contain 10 to 200 times more than conventional raw materials, and the production of these pigments is expanding from land plants and macro algae to microalgae.

In order to extract useful components of such microalgae, extraction has been preceded by an organic solvent extraction method or the like. The organic solvent extraction method takes a long time and has a disadvantage of showing low yield due to the toxicity by the solvent and the loss in the post- extraction process.

On the other hand, there is an increase in the incidence of adult diseases due to overnutrition and lack of exercise due to improvement of national income and living standard, which poses a threat to national health. The main causes of death of Koreans in 2002 were cancer of the first place, cerebrovascular disease of the second place, heart disease of the third place, diabetes of the fourth place, and diabetes in the fifth place.

The incidence of adult disease is increasing in children as well as adults in advanced countries with low intake of vegetables and high intake of meat, that is, overnutrition and obesity due to high fat diet.

α-glucosidase is an enzyme that catalyzes the reaction of hydrolysis of glucosides to form sugars and aglycones. It is widely distributed in microorganisms and higher animals and plants. It is secreted by the gut membrane and is involved in the digestion and absorption of disaccharides. In general, carbohydrates ingested are absorbed by saliva and small intestine by α-amylase and α-glucosidase, which are absorbed by glucose.In the case of diabetics, when glucose is absorbed in large amounts at once, blood glucose cannot be used properly, resulting in high blood sugar. The condition worsens. Therefore, by inhibiting the α-amylase and α-glucosidase of the small intestine, it is possible to delay the absorption of glucose through the small intestine wall and prevent postprandial hyperglycemia of diabetic patients.

In addition, the remaining monosaccharides used as energy are converted into aliphatic compounds called glycogen between muscle and skin, which is one of the causes of obesity. Inhibition of α-glucosidase and α-amylase delays the digestion of carbohydrates in the small intestine. Because it is possible to prevent the conversion to fat, it is possible to prevent obesity.

β-Glucuronidase is a generic term for enzymes that hydrolyze glucuronic bonds by acting on glycosides in which non-saccharides are bonded to β-form 1 carbon of D-gluconic acid, and are widely distributed in higher plants, microorganisms, and animals. In animals, it may be distributed in all tissues. Especially, it is always detected in body fluids such as plasma, and high activity is observed in the spleen, liver, and kidney. Hepatic injury, hyperthyroidism, diabetes, Cushing's disease, essential hypertension, urinary tract infections, cervical cancer, etc., are known to increase the activity of these enzymes. Toxic substances such as benzo (a) pyrene in the liver are known as glucuronic acid. When detoxified as a conjugate, it breaks this bond and provides a carcinogen, which is known to cause colon cancer. Its inhibition is known to prevent colon cancer caused by β-glucosidase.

Several inhibitors such as acarbose and saccharic acid 1,4-lactone have been developed and used as medicines for the prevention, improvement and treatment of these diseases. However, most of them are synthetic drugs. Although strong, the continuous dose is reported to be accompanied by side effects such as diarrhea and abdominal pain or drug resistance, there is a need for prevention, improvement or treatment derived from natural substances that can replace it.

In order to solve the above problems, the present inventors used a method of extracting pigments from natural materials, in particular microalgae, using an accelerated solvent extraction method and prevention of obesity or diabetes, including fucoxanthin extracted by the method. Or to develop a composition for treatment / improvement.

The present invention provides a method for extracting a pigment from microalgae using an accelerated solvent extraction method and a composition for preventing or treating or improving obesity or diabetes, including fucoxanthin extracted by the above method.

The present invention relates to a method for extracting the pigment of microalgae using an accelerated solvent extraction method.

The present invention also relates to a pharmaceutical composition for the prevention or treatment of obesity or diabetes, comprising fucoxanthin extracted by the above method.

In addition, the present invention relates to a cosmetic composition and a food composition for preventing or improving obesity or diabetes, including fucoxanthin extracted by the above method.

The composition comprising the microalgae-derived fucoxanthin according to the present invention has a very high safety feature because it uses natural materials as compared to the low safety of conventional synthetic diabetes treatment and obesity improving agents.

1 Phaeodactylum Correlation between time and temperature for accelerated solvent extraction of trocornutum is shown.
Figure 2 shows the structure of the microalgae-derived fucoxanthin.
Figure 3 shows the α-glucosidase inhibitory activity of the microalgae-derived fucoxanthine complex extract.
Figure 4 shows the β-glucosidase inhibitory activity of the microalgae-derived fucoxanthine complex extract.
Figure 5 shows the glycemic control effect of the microalgae-derived fucoxanthine complex extract.
Figure 6 shows the neutral lipid control effect of the microalgae-derived fucoxanthine complex extract.

The present invention relates to a method for extracting the pigment of microalgae using an accelerated solvent extraction method.

The accelerated solvent extraction method is a method of extracting a material at a higher speed than a general solvent extraction method by accelerating the speed of the fluid by high pressure and high temperature, and is generally very effective to extract a material that is stable to heat.

The accelerated solvent treatment may be treated with a pressure of 0-1,500 psi, a temperature of 40-160 ℃, 1-30 minutes using an accelerated solvent extractor using 10-100% ethanol.

The microalgae may be, but are not limited to, green algae (Chlorophyte), brown algae (Phaeophyceae), red algae (Rhodophyte), cyanophyta, diatoms (Bacillariophycea), diaphragm algae (Dinophyta), or flax algae (Haptophyta). The diatoms may be Phaeodactylum tricornutum .

The pigment is not limited thereto, but may be a carotenoid. Carotinoids are yellow, orange, and pink pigments, which are widely distributed in the flora and fauna, especially in red and orange vegetables and fruits. These ingredients are known to have antioxidant activity that inhibits cancer and inhibits the production of lipid peroxide, which is the body's rust.

In addition, Among the pigments of the carotenoid series Fucoxanthin. Foucault xanthine (fucoxanthin) (galjoso) is brown algae strip, causative agent of brown as the portion of the dye of the chloroplast, it is contained only galjo plants and yellow plants, of xanthophylls of the carotenoid dyes of the formula C ₄₂ H ₅₈ O ₆ It is a kind of reddish brown crystals and is a bioactive pigment with strong antioxidant and anticancer activity.

In one embodiment of the present invention, when the fucoxanthin extracted by the accelerated solvent extraction method, it was found that the efficacy in extraction yield and extraction time is superior to the general solvent extraction and ultrasonic extraction method (Table 6).

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity or diabetes, including fucoxanthin extracted by the above method.

The pharmaceutical composition of the present invention may include the microalgae-derived fucoxanthin alone as a pharmacological component exhibiting a pharmacological effect on obesity or diabetes, and additional ingredients, ie, pharmaceuticals, depending on the formulation, method of use, and purpose of use. It may further comprise a carrier, excipient, diluent or accessory ingredients that are acceptable or nutritionally acceptable.

More specifically, the pharmaceutical composition is a microalgae-derived fucoxanthin, in addition to nutrients, vitamins, electrolytes, flavors, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like may further be contained. In addition, the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acycia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline In the group consisting of cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline It may be one or more selected, but is not limited to any conventional carrier, excipient or diluent can be used. The components can be added independently or in combination.

The microalgae-derived fucoxanthin may be present in an amount of 0.001% to 99.9%, 0.1% to 99%, and 1% to 10% by weight based on the total weight of the pharmaceutical composition of the present invention. In addition, the content of the additional component is preferably added in the range of 0.1 to 20000 parts by weight per 100 parts by weight of the microalgae-derived fucoxanthin.

In addition, the pharmaceutical composition of the present invention may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives, and can be used orally or parenterally. have.

Specifically, the solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, in the composition for improving, treating or preventing colon cancer. Calcium carbonate, sucrose or sucrose, lactose, gelatin and the like are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients may include, for example, wetting agents, sweeteners, fragrances, and preservatives. have.

In addition, the formulation of the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, in particular by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated. Examples of specific formulations include warnings, granules, lotions, linings, limonades, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, liquid extracts, emulsions, Suspensions, premises, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules.

Furthermore, the pharmaceutical compositions of the present invention are preferably formulated using appropriate methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. Can be converted.

The dosage of the pharmaceutical composition of the present invention depends on the condition and weight, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. The microalgae-derived fucoxanthin derived from the composition of the present invention is preferably administered at 0.0001 to 12 g / kg and 0.01 to 10 g / kg per day. Administration may be administered once a day or may be divided several times. The dose is not intended to limit the scope of the invention in any way.

The present invention also provides a cosmetic composition for the prevention or improvement of obesity or diabetes comprising fucoxanthin (fucoxanthin) extracted by the above method.

The cosmetic composition may be a fatty substance, an organic solvent, a dissolving agent, a thickening agent, a gelling agent, a softening agent, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance, a surfactant, water, an ionic or nonionic emulsifier, Cosmetics or skin, such as fillers, metal ion sequestrants, chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic active agents, lipid vesicles or any other ingredients commonly used in cosmetics It may contain adjuvants commonly used in the scientific field. Such adjuvants are introduced in amounts commonly used in the cosmetics or dermatological fields.

In addition, the appearance of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example, emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing the oil phase in solutions, gels, solids, pasty anhydrous products, aqueous phases, and It may be provided in the form of a nonionic vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. These compositions may be prepared according to conventional methods in the art. The composition according to the invention may also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

In addition, the formulation is not particularly limited, for example, supple cosmetics, astringent cosmetics, nourishing cosmetics, nourishing cream, massage cream, essence, eye cream, eye essence, cleansing cream, cleansing foam, cleansing water, pack, It can be formulated into cosmetics such as powders, body lotions, body creams, body oils and body essences.

The cosmetic composition as described above may be applied in a form applied to the skin, or may be applied in a form that is absorbed into the skin using a microneedle or the like.

The present invention also provides a food composition for preventing or improving obesity or diabetes, including fucoxanthin extracted by the above method.

In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages.

Examples of the food to which fucoxanthin derived from microalgae of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and functional foods. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce, sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional production method.

In the present invention, the functional food refers to a food group which is imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques and the like, the regulation of the biological defense rhythm of the food composition, Means a food which is processed and designed so that the body control function regarding the body is sufficiently expressed to the living body. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

In the present invention, beverage is a generic term for drinking or enjoying a taste, and is intended to include functional beverages. The beverage is not particularly limited to other ingredients other than including the active ingredient as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates, and the like as additional ingredients, as in general drinks. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally 0.01 to 15 g, 0.05 to 10 g per 100 ml of the composition of the present invention, in addition to the composition of the present invention is added to the flesh for the production of natural fruit juices, fruit juice drinks, vegetable drinks It may contain.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and aggravating agents (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 20,000 parts by weight per 100 parts by weight of the microalgae derived fucoxanthin.

Functional beverage in the present invention is a biological defense rhythm control, disease prevention and the like having a beverage group or a beverage composition that has added value to the beverage by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the beverage to a specific purpose It means a beverage that is designed and processed to fully express the function of the gymnastics for recovery.

The functional beverage is not particularly limited to other ingredients except the microalgae-derived fucoxanthin of the present invention as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates are added like ordinary drinks. It may contain as a component. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, 5-12 g per 100 ml of the composition of the present invention.

In the present invention, natural seasoning means a generic term for improving the flavor of food using natural products. The natural seasonings include the microalgae-derived fucoxanthin as essential ingredients in the indicated ratios, and there are no special limitations on the other ingredients, and various natural complexes, flavoring agents, or natural carbohydrates, such as conventional seasonings, may be used. It may contain as an additional component. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 0.01-15 g, 0.05-10 g per 100 ml of the composition of the present invention.

In addition, in the food composition, the amount of the microalgae-derived fucoxanthin may include 0.01 to 15% by weight of the total food weight, the beverage composition is 0.02 to 5 g, 0.3 to 100 ml based on 100 ml It may be included in a ratio of 1g, the natural seasoning composition may be included in a ratio of 0.01 to 5g, 0.1 to 2g based on 100g.

Example  1> Origin of microalgae Fucoxanthine  extraction

Example  1-1. Preparation of Microalgal Powders

Phaeodactylum tricornutum , a microalgae with very high content of fucoxanthine used in the experiment, was used directly in the marine biology research education center (Gangneung, Gangwon-do). The cultured microalgae were centrifuged to remove the culture solution. More specifically, the microalgae purchased and cultured were centrifuged at 7,000 rpm for 15 minutes to separate the supernatant and solids, the supernatant was removed, and only the solids were lyophilized (-80 ° C., 72 h), followed by low temperature freezing (- 40 ° C.) was used for the experiment.

Example  1-2. Origin of microalgae Fucoxanthine  extraction

The obtained algae powder was accelerated solvent extraction, more specifically, 0.5 g of the microalgae powder in a stainless cell of 11 mL capacity, using a pressure of 1,500 psi, temperature of 40-160 ℃ using 90% ethanol, Extracted for 1-30 minutes, the results are shown in Table 1 below.

Accelerated Solvent Extraction of Fucoxanthin from Microalgae by Response Surface Methodology Expt. Actual variables Carotenoids
(mg / g fresh microalgae) Fucoxanthin
(mg / g fresh microalgae) Temperature
(° C) Time (min) Actual
value Predicted value Actual
value Predicted value One 140 25 0.775 ± 0.018 0.849 1.373 ± 0.363 1.350 2 100 15 1.008 ± 0.083 0.992 0.922 ± 0.178 0.897 3 100 30 0.762 ± 0.018 0.740 1.274 ± 0.313 1.285 4 60 5 0.746 ± 0.030 0.683 0.766 ± 0.097 0.785 5 100 One 0.711 ± 0.094 0.731 1.149 ± 0.152 1.141 6 140 5 0.884 ± 0.018 0.923 1.188 ± 0.229 1.179 7 60 25 0.840 ± 0.013 0.812 0.749 ± 0.138 0.753 8 100 15 0.980 ± 0.096 0.992 0.922 ± 0.178 0.897 9 100 15 0.980 ± 0.096 0.992 0.922 ± 0.178 0.897 10 40 15 0.704 ± 0.012 0.767 0.475 ± 0.266 0.459 11 100 15 0.988 ± 0.116 0.992 0.858 ± 0.069 0.897 12 100 15 1.008 ± 0.083 0.992 0.858 ± 0.069 0.897 13 160 15 1.061 ± 0.004 0.974 1.182 ± 0.185 1.202

The result of extraction by setting the temperature of 40-160 ℃ and the time of 1-30 min showed similar results with the theoretical value and the extraction value.The reaction surface analysis to set the optimum condition for fucoxanthine and total carotenoid content The results are shown in Figure 1, the optimum conditions are shown in Table 2.

Optimum Conditions for Accelerated Solvent Extraction of Fucoxanthin from Microalgae Optimum conditions Predicted values
(mg / g fresh
microalgae) Experimentalal
values
(mg / g fresh
microalgae) Temperature
(X1, ºC) Time
(X2, min) Carotenoids 125 15 1.0002 0.910 ± 0.1845 Fucoxanthin 140 25 1.3499 1.420 ± 0.0120

The optimal extraction conditions for fucoxanthin were 25 minutes at 140 ° C and total carotenoids were 15 minutes at 125 ° C. The theoretical and actual extraction values were similar.

Example  1-3. Origin of microalgae Fucoxanthine  Structural analysis

In order to confirm the safety of the microalgae-derived fucoxanthin was purified and structural analysis by nuclear magnetic resonance analysis (NMR). More specifically, the fucoxanthine complex prepared in 1-2 was dissolved in a nucleic acid: acetone (2: 1, v / v) solvent and purified using silica gel. This was dissolved in CDCl ₃ and the results of structural analysis by NMR are shown in Table 3 and FIG. 1. Table 3 below shows the structural analysis of the microalgae-derived fucoxanthin.

As a result of structural analysis of fucoxanthin, it was determined to be all trans-fucoxanthin, and its structure is the same as that of commercial plant-derived fucoxanthin.

< Example  2> derived from microalgae Fucoxanthin of  Extraction optimization

Example  2-1. By accelerated solvent extraction Fucoxanthin  Extraction optimization

Phaeodactylum, a microalgal species with very high content of fucoxanthin Accelerated solvent extraction of tricornutum , the results are shown in Table 4.

Accelerated Solvent Extraction of Fucoxanthin from Microalgae by Response Surface Methodology Expt. Actual variables Carotenoids
(mg / g fresh microalgae) Fucoxanthin
(mg / g fresh microalgae) Temperature
(° C) Time (min) Actual
value Predicted value Actual
value Predicted value One 140 25 0.775 ± 0.018 0.849 1.373 ± 0.363 1.350 2 100 15 1.008 ± 0.083 0.992 0.922 ± 0.178 0.897 3 100 30 0.762 ± 0.018 0.740 1.274 ± 0.313 1.285 4 60 5 0.746 ± 0.030 0.683 0.766 ± 0.097 0.785 5 100 One 0.711 ± 0.094 0.731 1.149 ± 0.152 1.141 6 140 5 0.884 ± 0.018 0.923 1.188 ± 0.229 1.179 7 60 25 0.840 ± 0.013 0.812 0.749 ± 0.138 0.753 8 100 15 0.980 ± 0.096 0.992 0.922 ± 0.178 0.897 9 100 15 0.980 ± 0.096 0.992 0.922 ± 0.178 0.897 10 40 15 0.704 ± 0.012 0.767 0.475 ± 0.266 0.459 11 100 15 0.988 ± 0.116 0.992 0.858 ± 0.069 0.897 12 100 15 1.008 ± 0.083 0.992 0.858 ± 0.069 0.897 13 160 15 1.061 ± 0.004 0.974 1.182 ± 0.185 1.202

As a result of setting the temperature of 40-160 ℃ and the time of 1-30 min, the theoretical value and the actual extraction value were similar, and the response surface analysis to set the optimal condition for the fucoxanthine and total carotenoid content was performed. The results are shown in Figure 3, the optimum conditions are shown in Table 5.

Optimum Conditions for Accelerated Solvent Extraction of Fucoxanthin from Microalgae Optimum conditions Predicted values
(mg / g fresh
microalgae) Experimentalal
values
(mg / g fresh
microalgae) Temperature
(X1, ºC) Time
(X2, min) Carotenoids 125 15 1.0002 0.910 ± 0.1845 Fucoxanthin 140 25 1.3499 1.420 ± 0.0120

The optimal extraction conditions for fucoxanthin were 25 minutes at 140 ° C and total carotenoids were 15 minutes at 125 ° C. The theoretical and actual extraction values were similar.

Example  2-2. For various extraction methods Fucoxanthin of Extraction yield  compare

Comparison of the general organic solvent extraction, ultrasonic and accelerated solvent extraction efficiency is shown in Table 6.

Extraction Efficiency of Fucoxanthin Derived from Microalgae Using Various Extraction Methods Extraction method Time Fucoxanthin (mg / g fresh microalgae) Conventional solvent extraction (CSE) 4 h 0.75 ± 0.055b Ultrasound assisted extraction (UAE) 2 h 1.53 ± 0.097a Pressurized liquid extraction (PLE) 25 min 1.42 ± 0.012a

As a result of analyzing the extraction efficiency by applying various extraction methods, the efficiency of general solvent extraction was possible to extract 0.75 mg / g in 4 hours, and ultrasonic extraction was possible to extract 1.53 mg / g in 2 hours.

On the contrary, the accelerated solvent extraction was able to extract 1.42 mg / g for 25 minutes, so the extraction time was very short and the extraction yield was similar to the ultrasonic extraction. Accordingly, it is determined that the accelerated solvent extraction enables efficient extraction of fucoxanthin as compared to other extraction methods.

< Example  3> Origin of microalgae Fucoxanthin of  Physiological activity

Example  3-1. Origin of microalgae Fucoxanthin of  α-glucosidase inhibitory activity

Α-glucosidase inhibitory activity of the fucoxanthine complex extract prepared in Example 1-2 was analyzed.

Determination of α-glucosidase enzyme inhibitory activity of fucoxanthin was performed by yeast ( Saccharomyces) cerevisiae ) and α-glucosidase from adult rats (Sigma, USA).

More specifically, 0.1 mL of 3 mM p-nitrophenyl α-D-glucoside (pNPG, Sigma, USA) dissolved in the same buffer as 0.1 mL of fucoxanthine complex extract adjusted in concentration in 2.2 ml of 0.01 M phosphate buffer (pH 7.0) and 0.1 mL of α-glucosidase was added, followed by reaction at 37 ° C. for 60 minutes. After the reaction was stopped by adding 1.5 mL of 0.1 M Na ₂ CO ₃ , inhibition was measured at 405 nm. The α-glucosidase inhibitory activity of the fucoxanthine complex extract was shown in FIG. 2.

The inhibitory activity of α-glucosidase from microorganisms and mammalian intestines of the fucoxanthine complex extract increased concentration-dependently, showing 97.88 and 91.81% of inhibitory activity at 0.5 mg / mL, respectively. It is considered that the microalgae-derived fucoxanthin complex extract can be developed as a natural-derived anti-obesity and anti-diabetic material.

Example  3-2. Origin of microalgae Fucoxanthin of  α-glucuronidase Inhibitory Activity

Β-glucuronidase inhibitory activity of the fucoxanthine complex extract prepared in Example 1-2 was analyzed.

The β-glucuronidase enzyme inhibitory activity of the fucoxanthine complex extract was measured using β-glucuronidase (Sigma, USA) derived from Escherichia coli .

More specifically, 2.2 mL of 10 mM phosphate buffer (pH 7.0) and 20 U / mL E. coli β-glucuronidase in 0.1 mL of fucoxanthine complex extract adjusted to 10 mM phosphate buffer (pH 7.0). mL was added and pre-reacted at 37 degreeC for 5 minutes. Thereafter, 0.1 mL of 40 mM p-nitrophenyl-β-D-glucuronide was added and reacted at 37 ° C. for 30 minutes. The reaction was stopped by adding 1.5 mL of 0.25 M Na ₂ CO _3, and then the inhibition was measured at 405 nm. As a positive control, saccharic acid (saccharic acid 1,4-lactone) was analyzed by the same method, and β-glucuronidase inhibitory activity of the fucoxanthine complex extract is shown in Table 7 and FIG. 4.

Β-glucuronidase inhibitory activity of the microalgae-derived fucoxanthine complex extract β-Glucuronidase inhibitory activity (%) 0.1 mg / mL 1.0 mg / mL Crude extract 41.39 ± 0.38 50.65 ± 1.31 Purified extract 42.59 ± 0.75 69.93 ± 2.36 D-saccharic acid 1,4-lactone 48.58 ± 0.75 61.76 ± 0.65

Β-glucuronidase inhibitory activity of the fucoxanthine complex extract increased concentration-dependently, and showed similar activity to saccharic acid, a positive control at a concentration of 1.0 mg / mL. The fucoxanthin purified product had higher β-glucuronidase inhibitory activity than the positive control sugar acid at the concentration of 1.0 mg / mL, and the IC ₅₀ value that inhibited 50% was 2.32 mM, which was 4.29 of the sugar acid used as the positive control. Higher than mM, the microalgae-derived fucoxanthine complex extract can be developed into a natural-derived anti-colon cancer material.

< Example  4> Origin of microalgae Fucoxanthin of  Obesity Improvement Effect in vivo )

In order to confirm the obesity improvement effect of the microalgae-derived fucoxanthine complex extract, the obesity of the animal was induced by diet, and the microalgae-derived fucoxanthine complex extract was added to a certain amount, and then the obesity improvement effect was confirmed.

More specifically, four-week-old male C57BL / 6J mice (n = 21) were purchased and after one week of adaptation, animals were divided into three groups according to the egg mass method. The ND group provided AIN-76 diets with soybean oil as a source of fat (5% of the diet), while the high fat group (HFD group) had high fat diets with 3% soybean oil and 17% Uji as a source of fat. For this, the Microalgae group (HFD + MA group) added dietary supplements of fucoxanthin-containing Microalgae extract to 0.7% of the diet for 8 weeks, ad libitum. The composition was supplied as shown in Table 5 below.

Experimental Dietary Composition Table Ingredient ND (%) HFD (%) HFD + MA (%) Corn starch 15.0 12.1 11.4 Casein 20.0 20.0 20.0 Sucrose 50.0 37.0 37.0 Cellulose 5.0 5.0 5.0 AIN-76 mineral mixture 3.5 4.2 4.2 AIN-76 vitamin mixture 1.0 1.2 1.2 DL-Methionine 0.3 0.3 0.3 Choline bitartrate 0.2 0.2 0.2 Tert-butylhydroquinone 0.001 0.004 0.004 Corn oil 5.0 3.0 3.0 Lard - 17.0 17.0 Microalgae extrct - - 0.7

Eight weeks after the start of the diet, the animals were fasted for 12 hours and the animals were sacrificed by cardiac sampling. Epididymal adipose tissue was collected and weighed, and blood was centrifuged at 3,000 × g for 15 minutes and then plasma was collected and stored at -70 ° C. Blood glucose, plasma triglyceride and cholesterol concentrations were measured by enzyme method.

㉮ Weight and epididymal fat weight

The results of measuring the weight, dietary intake and epididymal fat weight are shown in Table 9 below.

Body weight and epidydimal fat pad weight Group Body weight
(g) Food intake
(g) Epidydimal fat pad
weight (g) Relative Epidydimal fat pad (%) * ND 25.4 ±± 0.4 ^a 3.5 ±± 0.4 ^{ns **} 0.51 ±± 0.08 ^a 2.00 ±± 0.30 ^a HFD 29.7 ±± 2.1 ^b 3.3 ±± 0.3 0.78 ±± 0.15 ^b 2.62 ±± 0.37 ^b HFD + MA 27.3 ±± 1.1 ^a 3.2 ±± 0.3 0.59 ±± 0.10 ^a 2.15 ±± 0.31 ^a

* (Epidydimal fat pad weight / body weight) × 100

** not significant

The weights of normal, high fat and MA groups were 25.4 ± 0.4, 29.7 ± 2.1 and 27.3 ± 1.1 g, respectively. The body weight of the high fat group was significantly increased compared to the normal group (p <0.01), and the weight loss of the MA group was significantly lower than that of the high fat group (p <0.05), and there was no significant difference from the normal group. . There was no significant difference in dietary intake between normal, high fat and MA groups.

The epididymal fat weight of the high fat group was 0.78 ± 0.15 g, which was significantly increased compared to the normal group (0.51 ± 0.08 g) (p <0.01). The epididymal fat weight of the MA group was 0.59 ± 0.10 g, which was significantly decreased compared to the high fat group (p <0.05). The relative epididymal fat weights were 2.00 ± 0.30, 2.62 ± 0.37, and 2.15 ± 0.31% in the normal, high fat, and MA groups, respectively, and significantly increased in the high fat group (p <0.01). The MA group was significantly decreased compared to the high fat group (p <0.05), and there was no significant difference from the normal group.

조절 Blood glucose control effect

The blood glucose control effect of the microalgae-derived fucoxanthine complex is shown in FIG. 4.

Blood glucose levels in the normal, high fat and MA groups were 102.5 ± 10.5, 135.2 ± 15.1 and 113.1 ± 11.6 mg / dL, respectively (Fig. 12). The blood sugar level of the high fat group was significantly higher than that of the normal group (p <0.01). The blood sugar level of the MA group was significantly lower than that of the high fat group (p <0.05), and there was no significant difference from the normal group. .

개선 Improvement of lipid metabolism

Lipid metabolism improving effect of the microalgae-derived fucoxanthine complex is shown in FIG. 5.

The plasma triglyceride concentrations of the normal, high fat and MA groups were 77.4 ± 12.7, 99.7 ± 14.9 and 82.4 ± 9.7 mg / dL, respectively. The triglyceride concentration of the high fat group was significantly higher than that of the normal group (p <0.05). The triglyceride concentration of the MA group was significantly lower than that of the high fat group (p <0.05). There was no difference.

Ingestion of microalgae extract containing microalgae-derived fucoxanthin complex in high fat diet-induced obesity showed anti-obesity effect to reduce body weight and epididymal fat, and decreased blood glucose and plasma triglyceride concentration Indicated.

Claims (9)

A method of extracting pigments of microalgae using accelerated solvent extraction. The method of claim 1, wherein the accelerated solvent treatment using a 10-100% ethanol of the microalgae, the pressure of 0-1,500 psi, the temperature of 40-160 ℃, 1-30 minutes using an accelerated solvent extractor How to extract the pigment. The method of claim 1, wherein the microalgae are green algae (Chlorophyte), brown algae (Phaeophyceae), red algae (Rhodophyte), cyanophyta, diatoms (Bacillariophycea), algae (Dinophyta), or harpophyte algae (Haptophyta). Method for extracting the pigment of the microalgae characterized in that. The method of claim 3, wherein the diatoms are Phaeodactylum tricornutum . The method according to claim 1, wherein the dye is a carotenoid. 6. The method of claim 5, wherein the carotenoid is fucoxanthin. A pharmaceutical composition for preventing or treating obesity or diabetes, comprising fucoxanthin extracted by the method according to claim 6. Cosmetic composition for the prevention or improvement of obesity or diabetes comprising fucoxanthin extracted by the method according to claim 6. Food composition for the prevention or improvement of obesity or diabetes comprising fucoxanthin extracted by the method according to claim 6

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