JP7197891B2 - Method for producing proteoglycan and/or glycosaminoglycan - Google Patents

Description

The present invention relates to methods for producing proteoglycans and/or glycosaminoglycans, and the like.

Proteoglycans are a type of macromolecules that constitute the extracellular matrix of animals. Proteoglycans are known to have high water retention and various physiological functions such as wound healing, anti-inflammatory, and cell growth promoting effects. can be expected to be used in a wide range of areas. Therefore, the development of a method for producing proteoglycans that can be used for these in a simple and efficient manner is desired.

Conventionally, as a method for producing proteoglycan, a method of extracting cartilage of fish such as salmon with a guanidine hydrochloride solution has been reported (Patent Document 1). However, this method requires a degreasing process using chloroform, methanol, or the like to remove the unpleasant odors unique to fish (beast odor, fishy odor), and the degreasing process completely removes the unpleasant odor. was impossible. In addition, guanidine hydrochloride, chloroform, methanol and the like used in this method are considered to be harmful to the human body, so the use of proteoglycans obtained by this method in cosmetics, food and drink, etc. has been restricted.

As a method for solving such problems, a method using acetic acid as an extraction solution has been reported (Patent Document 2). However, the proteoglycan obtained by this method has a problem of emitting an unpleasant odor, which is considered to be caused by acetic acid (Patent Document 3).

Japanese Patent Application Laid-Open No. 2001-172296 Japanese Patent Application Laid-Open No. 2002-069097 JP 2009-173702 A

An object of the present invention is to provide a method for producing proteoglycans and/or glycosaminoglycans that are highly safe for living organisms. Preferably, the present invention also aims to provide a method for producing proteoglycan and/or glycosaminoglycan with reduced unpleasant odor, and a more convenient and efficient method for producing proteoglycan and/or glycosaminoglycan. can be

As a result of intensive research by the present inventors, (a) proteoglycans and/or glycosaminoglycans, including extracting proteoglycans and/or glycosaminoglycans from proteoglycan- and/or glycosaminoglycan-containing biological raw materials, with a solution containing magnesium sulfate; or a method for producing a glycosaminoglycan. As a result of further research based on this knowledge, the present invention was completed.

That is, the present invention includes the following aspects.

Section 1. (a) A method for producing proteoglycan and/or glycosaminoglycan, which comprises extracting proteoglycan and/or glycosaminoglycan from a proteoglycan- and/or glycosaminoglycan-containing biological material with a magnesium sulfate-containing solution.

Section 2. Item 2. The production method according to Item 1, wherein the magnesium sulfate-containing solution has a magnesium sulfate concentration of 0.5 to 5M.

Item 3. Item 3. The production method according to Item 1 or 2, wherein the magnesium sulfate-containing solution has a magnesium sulfate concentration of 2.5 to 5M.

Section 4. Items 1 to 3, wherein the biological raw material is at least one selected from the group consisting of cartilage, connective tissue, healthy tissue, cornea, atrium, basement membrane, brain, skin, and processed products thereof. manufacturing method.

Item 5. Item 5. The production method according to any one of Items 1 to 4, wherein the organism derived from the biological raw material is at least one species selected from the group consisting of fish, mammals, mollusks, and echinoderms.

Item 6. Item 6. The production method according to any one of Items 1 to 5, wherein the weight ratio of the biological raw material to the magnesium sulfate-containing solution (biological raw material:magnesium sulfate-containing solution) is 1:0.5 to 1:30.

Item 7. Item 7. The production method according to any one of Items 1 to 6, wherein the weight ratio of the biological raw material to the magnesium sulfate-containing solution (biological raw material:magnesium sulfate-containing solution) is 1:1.5 to 1:6.

Item 8. Item 8. The production method according to any one of Items 1 to 7, wherein the extraction temperature is 10 to 40°C.

Item 9. Item 9. The production method according to any one of Items 1 to 8, further comprising (b) removing insoluble matter from the extract obtained in step a.

Item 10. Item 10. The production method according to Item 9, further comprising (c) purifying proteoglycans and/or glycosaminoglycans after step b.

Item 11. A proteoglycan and/or a glycosaminoglycan obtained by the production method according to any one of Items 1 to 10.

Item 12. A proteoglycan obtained by the production method according to any one of Items 1 to 10.

Item 13. Item 13. The proteoglycan according to Item 12, which has an average molecular weight of 30 kDa or more.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for producing proteoglycans and/or glycosaminoglycans that are highly safe for living organisms. In addition, according to the present invention, it is possible to provide a method for producing proteoglycan and/or glycosaminoglycan with less unpleasant odor, and a simpler and more efficient method for producing proteoglycan and/or glycosaminoglycan. is.

HPLC results of proteoglycan obtained in Example 1 are shown (Test Example 1). The results of cellulose acetate membrane electrophoresis of the proteoglycan obtained in Example 1 are shown (Test Example 2).

As used herein, the expressions "contain" and "include" include the concepts "contain", "include", "consist essentially of" and "consist only of".

As one aspect of the present invention, (a) a proteoglycan and/or glycosaminoglycan extracting proteoglycan and/or glycosaminoglycan from a proteoglycan- and/or glycosaminoglycan-containing biological raw material with a magnesium sulfate-containing solution. The present invention relates to a method for producing saminoglycan (also referred to herein as the “production method of the present invention”). This will be explained below.

Biological materials containing proteoglycans and/or glycosaminoglycans (hereinafter sometimes simply referred to as "biological materials") are biological tissues and their processed materials containing proteoglycans and/or glycosaminoglycans. is not particularly limited as long as

The organisms from which the biological raw material is derived are not particularly limited, and include, for example, fish, mammals, molluscs such as Aplysia, and echinoderms such as sea cucumbers. Among these, fish are preferred.

Fishes are not particularly limited, and include a wide range of bony fishes, cartilaginous fishes, and the like. Examples of bony fish include cod, tuna, salmon, trout, bonito, flounder, and yellowtail, and examples of cartilaginous fish include sharks and rays.

Examples of biological tissue include, but are not limited to, cartilage, connective tissue, healthy tissue, cornea, atrium, basement membrane, brain, and skin. Among these, cartilage is preferred.

As the fish biological tissue, fish cartilage is preferable. Although the cartilage is not particularly limited, head cartilage, especially nasal cartilage, is preferable. In addition, since the heads of fish are usually discarded when they are processed into food products and the like, there is also the advantage that the acquisition cost of head cartilage is low and a large amount can be stably supplied.

The processed biological tissue is not particularly limited, and examples thereof include dried biological tissue, crushed biological tissue, and dried crushed biological tissue. Processing methods such as drying, crushing, and cutting into small pieces are not particularly limited, and known methods can be employed.

From the viewpoint of extraction efficiency, etc., the biological raw material is preferably a processed biological tissue, more preferably a processed material processed to increase the solvent contact area, and even more preferably a crushed material, a dried crushed material, or the like.

Biological raw materials may be used singly or in combination of two or more.

The magnesium sulfate-containing solution is not particularly limited as long as it contains magnesium sulfate (MgSO ₄ ) and partially or wholly dissolved therein.

The solvent for the magnesium sulfate-containing solution is typically water. As the solvent, alcohols such as ethanol may be included in addition to water. The content of solvents other than water is, for example, 20% by mass or less, preferably 10% by mass or less, more preferably 3% by mass or less, still more preferably 1% by mass or less, and even more preferably 100% by mass or less of the solvent. 0.3% by mass or less, particularly preferably 0.1% by mass or less. The lower limits of the content are, for example, 0% by mass, 0.1% by mass, 0.3% by mass, 1% by mass, and 3% by mass. The solvent may be used singly or in combination of two or more.

The magnesium sulfate concentration of the magnesium sulfate-containing solution is not particularly limited. The concentration is for example 0.5-5M. From the viewpoint of extraction efficiency, suppression of protein contamination, etc., the concentration is preferably 1 M or more, more preferably 1.5 M or more, still more preferably 2 M or more, still more preferably 2.5 M or more, and particularly preferably is greater than or equal to 2.8M. From the viewpoint of extraction cost, efficiency of solution preparation, etc., the concentration is preferably 4.5M or less, more preferably 4M or less, even more preferably 3.5M or less, and even more preferably 3.2M or less. From the above viewpoint, the concentration range is preferably 1.5 to 5 M, more preferably 2.5 to 5 M, still more preferably 2.5 to 3.5 M, still more preferably 2.8 to 3.2 M. is.

In addition to magnesium sulfate, the magnesium sulfate-containing solution may contain other components as long as they do not significantly deteriorate the extraction efficiency and degree of purification of proteoglycans and/or glycosaminoglycans. Other components include, for example, additives that can be added to the extraction solvent (eg, acids, bases, preservatives such as toluene, protease inhibitors, etc.). Other components may be used singly or in combination of two or more. The content of other components is, for example, 20% by mass or less, 10% by mass or less, 5% by mass or less, or 1% by mass or less with respect to 100% by mass of the magnesium sulfate-containing solution. The lower limits of the content are, for example, 0% by mass, 0.1% by mass, 0.3% by mass, 1% by mass, and 3% by mass.

Extraction of proteoglycan and/or glycosaminoglycan can be performed according to a known extraction method. For example, it can be carried out by mixing the biological raw material and the magnesium sulfate-containing solution, and then allowing the mixture to stand (preferably while stirring).

The weight ratio between the biological material and the magnesium sulfate-containing solution is not particularly limited, but the weight ratio is preferably such that the biological material is immersed in the magnesium sulfate-containing solution. Specifically, the weight ratio (biological raw material:magnesium sulfate-containing solution) is, from the viewpoint of extraction efficiency, for example, 1:0.5 to 1:50, preferably 1:0.5 to 1:30, more preferably. is 1:1 to 1:20, more preferably 1:1 to 1:10, even more preferably 1:1 to 1:6, even more preferably 1:1.5 to 1:6, particularly preferably 1 :2 to 1:5.

The extraction time is not particularly limited as long as the proteoglycan can be extracted. The extraction time is, for example, 3-72 hours, preferably 8-48 hours, more preferably 12-36 hours, still more preferably 18-30 hours. If the extraction time is the above time, the proportion of proteoglycan in the extracted components can be further increased.

The extraction temperature is not particularly limited as long as the proteoglycan can be extracted. The extraction temperature can be, for example, 10-40°C, preferably 15-30°C.

A proteoglycan- and/or glycosaminoglycan-containing extract is obtained by the above step a. This proteoglycan and/or glycosaminoglycan-containing extract can be used as it is as "proteoglycan and/or glycosaminoglycan", or the product obtained through the following steps can be used as "proteoglycan and/or glycosaminoglycan It can also be used as a glycan.

In addition to step a, it is preferable to further perform (b) removing insoluble matter (extraction residue) from the extract obtained in step a (step b). The method for removing insoluble matter is not particularly limited, and known methods such as filtration and centrifugation can be employed.

In order to further increase the purity of the resulting proteoglycan, it is preferable to perform (c) after step b, purifying the proteoglycan and/or glycosaminoglycan (step c) after step a or b. Purification methods include, for example, alcohol precipitation, dialysis, column (preferably anion exchange column) chromatography, gel filtration chromatography, affinity column chromatography, ultrafiltration, electrodialysis and the like. These purification methods may be used singly or in combination of two or more.

In one aspect of the present invention, ultrafiltration can be employed as the purification method in step c from the viewpoints of convenience, cost, and the like. Ultrafiltration can remove most of the solvents, salts, etc., and purify substances with high molecular weights (proteoglycans and/or glycosaminoglycans).

Ultrafiltration can be performed according to known methods. After ultrafiltration, drying (for example, freeze-drying) is preferred.

In one aspect of the present invention, alcohol precipitation can be employed as the purification method of step c. When performing alcohol precipitation, depending on the concentration of magnesium sulfate, its precipitation may become a problem. In that case, before alcohol precipitation, the salt concentration is lowered or the salt is removed by other purification methods such as dialysis and recrystallization. It is desirable to

Alcohol precipitation can be carried out according to known methods. Typically, the proteoglycan- and/or glycosaminoglycan-containing extract obtained through step b is mixed with 1 to 5 times (preferably 2 to 4 times) alcohol, and then allowed to stand for a certain period of time. to form a precipitate, followed by centrifugation and recovery of the obtained pellet.

Alcohol is not particularly limited as long as it can precipitate proteoglycans and/or glycosaminoglycans. Examples of alcohols include ethanol, isopropanol, etc. Among these, ethanol is preferred from the viewpoint of lower toxicity. One type of alcohol may be used alone, or two or more types may be used in combination.

The alcohol preferably contains a salt. The salt is not particularly limited and includes common salts used for alcohol precipitation, such as sodium chloride, sodium acetate, ammonium acetate, lithium chloride, magnesium chloride and the like. Salts may be used singly or in combination of two or more. The concentration of the salt in the alcohol is not particularly limited, and may be a normal salt concentration employed in alcohol precipitation, such as the saturation concentration for alcohol in the case of sodium chloride.

The temperature when left to stand to form a precipitate is not particularly limited as long as the proteoglycan can be precipitated. The temperature can be, for example, about -80°C to room temperature, preferably about 0 to 10°C.

The time for standing to form a precipitate is not particularly limited as long as the proteoglycan can be precipitated, and is appropriately set according to the temperature. The time can be, for example, 4-24 hours, preferably 8-16 hours, provided that the temperature is between 0 and 10°C.

Centrifugal force is not particularly limited as long as it can form pellets. The centrifugal force can be, for example, 700-2500 g.

The production method of the present invention may include steps other than the steps described above as long as the effects of the present invention are not impaired.

The type of proteoglycan obtained by the production method of the present invention depends on the raw material and is not particularly limited. The proteoglycan is not particularly limited as long as it is a compound in which a glycosaminoglycan is bound to a core protein. Proteoglycans include, for example, aggrecan, biglycan, versican, neurocan, decorin, biglycan, fibromodulin, lumican, perlecan, syndecan, serglycine, brevican, keratocan, mimecan, vermacan, agrin, or degradation products thereof. mentioned.

The production method of the present invention can suppress the decomposition of proteoglycans and obtain proteoglycans with a higher molecular weight. From this viewpoint, the proteoglycan obtained by the production method of the present invention preferably has an average molecular weight of 250 kDa or more, more preferably 300 kDa or more, still more preferably 350 kDa or more, and even more preferably 370 kDa or more. is. The upper limit of the average molecular weight is not particularly limited, and is, for example, 5000 kDa, 2000 kDa, 1000 kDa , 700 kDa , 500 kDa . The average molecular weight of proteoglycan can be measured by fractionation using chromatography, as in Test Example 2, and comparison with the retention time of a molecular weight standard such as a pullulan standard.

The glycosaminoglycan that proteoglycan has as a partial structure and the glycosaminoglycan obtained by the production method of the present invention are not particularly limited. Examples of the glycosaminoglycan include chondroitin sulfate, chondroitin, heparin, heparan sulfate, keratan sulfate, dermatan sulfate and the like. In addition, these glycosaminoglycans may have chains containing sugars such as fucose as branched chains. In this case, the number of sugar residues constituting the branched chain may be one, or two or more. In addition, the sugar residues constituting the glycosaminoglycan chain (including sugar residues such as fucose residues in branched chains) may be sulfated.

Conventional methods for extracting proteoglycans and glycosaminoglycans often use toxic reagents such as guanidine hydrochloride as raw materials for extraction, and require complicated procedures to remove the reagents after extraction, resulting in cost problems. . In addition, since reagents used for extraction are harmful to the human body, use of the extracted proteoglycans and glycosaminoglycans in cosmetics or foods may be restricted.

As an alternative means, there is an extraction method with acetic acid, but in this case, the extracted proteoglycans and glycosaminoglycans have the drawback that the odor of acetic acid used during the extraction process adheres to them, giving off an unpleasant odor during handling.

On the other hand, the magnesium sulfate used in the production method of the present invention is widely used in foods such as "bitter" and is also used as a bathing agent, and is familiar to our daily lives. Moreover, since magnesium sulfate is odorless, it is possible to suppress adhesion of unpleasant odors such as acetic acid odors to the proteoglycans and glycosaminoglycans obtained by the production method of the present invention.

Applications of proteoglycans and/or glycosaminoglycans obtained by the production method of the present invention are not particularly limited. For example, it can be preferably used as a raw material for foods, cosmetics, pharmaceuticals, etc., and as a reagent for biochemical and medical research. In addition, medical applications are possible in anticipation of the physiological activities of proteoglycans and glycosaminoglycans (wound healing action, anti-inflammatory action, human epithelial cell proliferation activity). Furthermore, it can be used as a cosmetic raw material utilizing the water retention capacity of proteoglycans and glycosaminoglycans. It can also be used as a sugar chain supplement, the market of which is expanding significantly in recent years.

Proteoglycans and/or glycosaminoglycans obtained by the production method of the present invention are not particularly limited in usage. Since proteoglycans and/or glycosaminoglycans have effects such as those described above, they are preferably used as external or oral compositions. That is, it is preferably used as an external composition or an oral composition containing the proteoglycan and/or glycosaminoglycan obtained by the production method of the present invention. In addition, the external composition or oral composition can be used as a pharmaceutical composition, a quasi-drug composition, a cosmetic composition, a food composition, and the like. These can be produced by conventional methods using proteoglycans and/or glycosaminoglycans obtained by the production method of the present invention. In particular, these compositions can be preferably used in the fields of cosmetics and foods and drinks.

A cosmetic composition containing proteoglycan and/or glycosaminoglycan obtained by the production method of the present invention (hereinafter sometimes referred to as "cosmetic composition according to the present invention") used in the field of cosmetics is the proteoglycan It may be the proteoglycan itself, or it may be produced according to a conventional method by appropriately blending the proteoglycan with a cosmetically acceptable medium, base, carrier, additive, and other cosmetically acceptable components and materials. can be anything. Specifically, emulsions, lotions, creams, beauty essences, foundations, packs, sunscreens, etc., produced containing the proteoglycan can be mentioned. Such a cosmetic composition according to the present invention can be preferably used for ameliorating inflammation or anti-aging. For example, it can be preferably used for prevention or improvement of dry skin, rough skin, wrinkles, sagging skin, etc.).

Food and drink compositions containing proteoglycans and/or glycosaminoglycans obtained by the production method of the present invention, which are used in the field of food and drink (beverages and foods) (hereinafter referred to as "food and drink compositions according to the present invention") ) may be the proteoglycan and / or glycosaminoglycan itself, or the proteoglycan and / or glycosaminoglycan, food hygiene acceptable bases, carriers, additives, In addition, ingredients, materials, etc. that can be used as foods may be appropriately blended. For example, processed foods, beverages, health foods (nutrition functional foods, foods for specified health uses, etc.), supplements, beauty foods, foods for sick people, and the like. Furthermore, moisturizing agents and anti-skin aging agents comprising such food and drink compositions according to the present invention are also included in the present invention. The moisturizing agents and anti-skin aging agents are used for cosmetics and anti-skin aging (for example, for preventing or improving dry skin, rough skin, wrinkles, sagging skin, etc.) Drinks, tablets, tablets, capsules, granules , jellies, lozenges, and the like.

The pharmaceutical composition, quasi-drug composition, cosmetic composition, or food and drink composition according to the present invention is not particularly limited, but the proteoglycan and/or glycosamine contained in these compositions The amount of noglycan is, for example, generally 0.001 to 100% by mass, preferably 0.01 to 95% by mass, based on the total composition.

EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited by these examples.

Example 1. Extraction of proteoglycans from cartilage tissue
Fish nasal cartilage (10 g) sliced into 3 cm squares was immersed in 3 M magnesium sulfate solution (30 mL), gently stirred at room temperature, and allowed to stand for 24 hours. The resulting mixture was filtered through filter paper to remove insoluble components. The resulting filtrate was transferred to a dialysis cellulose tube (dialysis molecular weight MWCO 12000-16000), and dialysis was performed using tap water as the dialysate. Next, ethanol "salt-saturated ethanol" (90 mL, 3 times the volume of the dialysis internal solution) in which a saturated amount of salt was dissolved was added to the dialysis internal solution, and the mixture was allowed to stand at 4°C for 12 hours. After centrifuging the resulting white precipitate (8000 rpm×10 min), the pellet (white) was collected. The pellet was washed with ethanol (20 mL) and air-dried to obtain a white powder of proteoglycan (126.2 mg).

Comparative Example 1: Extraction of proteoglycan using acetic acid Proteoglycan was obtained in the same manner as in Example 1, except that a 4% aqueous acetic acid solution was used instead of the magnesium sulfate solution.

Test example 1. Molecular Weight Determination of Proteoglycan The proteoglycan obtained in Example 1 was dissolved in water to prepare a 0.1% proteoglycan aqueous solution, of which (50 μL) was subjected to high performance liquid chromatography analysis. Analysis conditions are as shown below.
Detector: Differential refractometer (RI) 5450 RI Detector (manufactured by Hitachi High-Technologies Corporation)
Column: TOSOH TSK-gel G5000PWXL
Eluent: 0.2M-NaCl
Flow rate: 1 mL/min
Column temp.: 40°C.

The results are shown in FIG. As shown in Figure 1, a peak was observed at a retention time of 7.113 minutes, and its average molecular weight was calculated to be 400,000. The average molecular weight was calculated using a pullulan standard (manufactured by Shodex, P-82).

Test example 2. Cellulose acetate membrane electrophoresis of proteoglycan The proteoglycan obtained in Example 1 was dissolved in water to prepare a 0.1% proteoglycan aqueous solution. (0.1 M formic acid-pyridine buffer (pH = 3.0)) was spotted onto a cellulose acetate membrane pre-soaked. Then, the cellulose acetate membrane was placed on the migration layer and electrophoresis was performed at 1 mA/cm 2 for 15 minutes. After electrophoresis, the cellulose acetate membrane was subjected to alcian blue staining, destained with 70% ethanol, and the stained band was visually observed.

The results are shown in FIG. As shown in FIG. 2, the proteoglycan of Example 1 exhibited a mobility equivalent to that of the standard proteoglycan, confirming that the target proteoglycan was certainly extracted by this extraction method.

In Example 1, since the raw material for extraction was fish cartilage containing a large amount of proteoglycan, proteoglycan was mainly obtained. Obtainable.

Test example 3. Odor Test of Proteoglycan The odor of the proteoglycan obtained in Example 1 and the proteoglycan obtained in Comparative Example 1 was evaluated by 7 evaluators in three grades (no odor, slight unpleasant odor, strong unpleasant odor). ) was evaluated. Table 1 shows the results.

As shown in Table 1, the proteoglycan obtained in Comparative Example 1 was evaluated as having an unpleasant odor by more than half (4/7) of the evaluators, whereas the proteoglycan obtained in Example 1 was evaluated as having an unpleasant odor. All of the panelists (7/7) rated it as odorless.

Claims (10)

(a) proteoglycans and/or glycosaminoglycans, comprising extracting proteoglycans from a proteoglycan-containing biological raw material with a magnesium sulfate-containing solution, and wherein the magnesium sulfate-containing solution has a magnesium sulfate concentration of 0.5 to 5M; Production method. The production method according to claim 1, wherein the magnesium sulfate-containing solution has a magnesium sulfate concentration of 1.5 to 5M. The production method according to claim 1 or 2, wherein the magnesium sulfate-containing solution has a magnesium sulfate concentration of 2.5 to 5M. Any one of claims 1 to 3, wherein the biological raw material is at least one selected from the group consisting of cartilage, connective tissue, healthy tissue, cornea, atrium, basement membrane, brain, skin, and processed products thereof. Method of manufacture as described. The production method according to any one of claims 1 to 4, wherein the organism derived from the biological raw material is at least one species selected from the group consisting of fish, mammals, mollusks, and echinoderms. The production method according to any one of claims 1 to 5, wherein the weight ratio of the biological raw material to the magnesium sulfate-containing solution (biological raw material:magnesium sulfate-containing solution) is 1:0.5 to 1:30. The production method according to any one of claims 1 to 6, wherein the weight ratio of the biological raw material to the magnesium sulfate-containing solution (biological raw material:magnesium sulfate-containing solution) is 1:1.5 to 1:6. The production method according to any one of claims 1 to 7, wherein the extraction temperature is 10 to 40°C. The production method according to any one of claims 1 to 8, further comprising (b) removing insoluble matter from the extract obtained in step a. 10. The production method according to claim 9, further comprising (c) purifying proteoglycans and/or glycosaminoglycans after step b.

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