WO2007069621A1

WO2007069621A1 - Novel composition and method for production thereof

Info

Publication number: WO2007069621A1
Application number: PCT/JP2006/324800
Authority: WO
Inventors: Kazuaki Kakehi; Yuki Matsuno; Yuji Matsuzaki; Junichi Kumada
Original assignee: Tokyo Cemical Industry Co., Ltd.
Priority date: 2005-12-14
Filing date: 2006-12-13
Publication date: 2007-06-21
Also published as: JPWO2007069621A1

Abstract

Disclosed are: a hyaluronic acid composition which is purified by, in a purification process, subjecting a biological tissue to a treatment with a protease and a molecular weight fractionation and concentrating or lyophilizing the resulting product without performing a treatment with a quaternary ammonium salt or an organic solvent (e.g., an alcohol); a hyaluronic acid composition having a resistance against the activity of a mammalian hyaluronidase; an inhibitor of the decomposition of a glucosaminoglycan comprising the composition as an active ingredient; and a pharmaceutical, food or cosmetic comprising the composition as an active ingredient.

Description

Specification

NOVEL COMPOSITION AND METHOD FOR PRODUCING THE SAME

Technical field

[0001] The present invention relates to a method for producing a hyaluronic acid composition having high resistance to hyaluronidase (enzymatic degradation) in hyaluronic acid applied to pharmaceuticals and cosmetics.

Background art

[0002] Hyaluronic acid is a kind of mucopolysaccharide in which a disaccharide in which D-glucuronic acid β-1,3 bonds are linked to N-acetylyl D-darcosamine in succession by ι8-1,4 bonds. Hyaluronic acid is present as a component in connective tissues such as joints, vitreous, umbilical cord, cartilage, skin, trachea, and bird's chicken crown, and functions important for tissue flexibility, structure maintenance, and cell metabolism regulation. Plays. In addition, hyaluronic acid is a very large polymer substance, and its solution has strong viscoelasticity and water retention, so it has a wide range of uses such as cosmetics, wound treatments, eye drops, and arthritis treatments.

[0003] Hyaluronic acid is known to have excellent biocompatibility even when transplanted or injected into a living body without having a polydisperse species or organ specificity due to its molecular weight. ing. However, since hyaluronic acid is susceptible to enzymatic degradation, there is a problem that it disappears at an early stage even if it is placed in the living body, and it is prepared by methods such as chemical modification and chemical cross-linking in order to solve the problem. Techniques for imparting enzyme resistance as modified hyaluronic acid have been studied (Patent Documents 1 to 10).

[0004] However, modified hyaluronic acid with improved biostability in vivo uses chemically reactive substances, so it inherently holds risks such as toxicity and biocompatibility due to new modifications. There was still a problem left to be solved.

[0005] On the other hand, as a general purification method in the production of hyaluronic acid, the following steps are exemplified for both animal tissue extraction and fermentation methods.

(1) Add an organic solvent such as ethanol to the crude product containing connective tissue or medium, and collect the precipitate using an organic solvent and thoroughly wash it. (2) Redissolve sodium hyaluronate precipitate in sodium chloride aqueous solution and add activated carbon.

(3) The resulting suspension is filtered using diatomaceous earth filter aid to remove activated carbon, microorganisms and other insoluble materials.

(4) Treat the clear filtrate with cetylpyridinum chloride to form insoluble cetylpyridinium salt of hyaluronic acid.

(5) Collect cetyl pyridinium salt, dissolve it in an aqueous sodium chloride solution containing ethanol, and recover sodium sodium hyaluronate by adding an organic solvent such as ethanol.

(5) Redissolve sodium hyaluronate again in an aqueous sodium chloride solution.

(6) Add cetyl pyridinium chloride to form cetyl pyridinium salt of hyaluronic acid again.

(7) Sodium hyaluronate is recovered by dissolving hyaluronate in an aqueous sodium chloride solution containing ethanol and adding an organic solvent.

(8) Dissolve the precipitate in an aqueous solution of sodium chloride and contact the resulting solution with a magnesium silicate adsorbent such as Florisil to remove impurities and residual cetylpyridinium ions.

(9) The solution is sterilized by filtration, and sodium hyaluronate is precipitated and obtained by adding a sterile organic solvent such as sterile ethanol.

[0006] However, such a purification method using a quaternary ammonium salt and an organic solvent is complicated, and the quaternary ammonium salt requires an operation for preventing the organic solvent from remaining. It had been.

[0007] Patent Document 1: Japanese Patent Laid-Open No. 3-105003

Patent Document 2: European publication 0341745

Patent Document 3: US Patent No. 4,582,865

Patent Document 4: Japanese Patent Publication No. 6-37575

Patent Document 5: JP-A-7-97401

Patent Document 6: JP-A-60-130601 Patent Document 7: JP-A-6-73103

Patent Document 8: JP-A-5-58881

Patent Document 9: European publication 0216453

Patent Document 10: International Publication W095Z25751 Pamphlet

Disclosure of the invention

Problems to be solved by the invention

[0008] When hyaluronic acid is used as a pharmaceutical or cosmetic product, the chemical cross-linking modification improves the stability of mammalian-derived hyaluronidase and other degrading enzymes. I couldn't ignore it. Accordingly, a hyaluronic acid composition having high stability against hyaluronic acid-degrading enzymes and the ability to perform such structural modification has been desired.

Means for solving the problem

[0009] The inventor has intensively studied the enzyme stability of begu hyaluronic acid itself, which achieves the above-mentioned problems. As a result, we have obtained knowledge that hyaluronic acid compositions with different biostability (resistance to enzymes) can be obtained due to differences in the production process of hyaluronic acid.

That is, the inventors have found a method for producing a hyaluronic acid composition exhibiting resistance to hydrolysis by mammalian-derived hyaluronan-dase, and have arrived at the present invention.

[0010] The present invention uses (1) a precipitation step in which an organic solvent such as ethanol is added, without using (1) a quaternary ammonium salt such as salt cetyl pyridinium in the production process. It has the following features:

[0011] The gist of the present invention is as follows.

(1) A hyaluronic acid composition obtained by the following steps <1> to <3>.

1) Process of degrading proteins by treating biological tissue with proteolytic enzymes; 2) Obtaining fractions by decomposing the degradation products obtained by 2X1> by molecular weight; 3) Fracturing power obtained by 3X2> Recovering the acid composition;

(2) A hyaluronic acid composition having the following properties.

( _a ) Under standard conditions, coexist with Hushi testis or Hedgei testis-derived hyaluronidase, boil for 5 minutes, and centrifuge the supernatant to be subjected to disaccharide analysis by capillary single electrophoresis Then no decomposition is allowed;

(b) When analyzed by the Raleigh method, the protein content is less than 0.1%;

(c) Heavy metal content of 20ppm or less as determined by the heavy metal test method described in the Japanese Pharmacopoeia;

(d) The arsenic content is 2ppm or less as measured by the arsenic test method described in the Japanese Pharmacopoeia.

(3) A method for producing a hyaluronic acid composition comprising the following steps <1> to <3>.

(4) A glycosaminodarican decomposition inhibitor comprising a hyaluronic acid composition as an active ingredient.

(5) Use of a hyaluronic acid composition as a glycosaminodarican decomposition inhibitor.

(6) A medicament comprising the hyaluronic acid composition according to (1) or (2).

(7) A food containing the hyaluronic acid composition according to (1) or (2).

(8) A cosmetic comprising the hyaluronic acid composition according to (1) or (2).

The invention's effect

[0012] According to the present invention, a hyaluronic acid composition having a hyaluronan-dase degradation resistance derived from a mammal can be obtained.

Brief Description of Drawings

[0013] FIG. 1 is a chart in which the capillary electrophoresis force after treatment with a guinea pig testis-derived hyaluronan-dase is also obtained. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

FIG. 2 is a chart obtained from capillary Pillar electrophoretic treatment after treatment with a hygiene testis-derived hyal-mouth-dase of the composition of the present invention. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

FIG. 3 is a chart in which capillary electrophoresis force was also obtained after treatment of hyaluronic acid, a hyaluronic acid derived from hygiene testis, which has been promoted as a pharmaceutical product. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

[Fig. 4] Treatment of microorganism-derived hyaluronan-dase (Streptomyces hyalurolyticus) of the composition of the present invention It is the chart from which the capillary electrophoresis force after treatment was also obtained. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

FIG. 5 is a chart showing the capillary electrophoresis force after treatment of the composition of the present invention with a microorganism-derived hyaluronan-dase (Streptococcus dysgalactiae). The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

FIG. 6 is a chart obtained by capillary electrophoresis after subjecting the composition of the present invention to EDTA (edetic acid) treatment and then to testicular-derived hyaluronidase treatment. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

FIG. 7 is a chart showing the capillary electrophoresis force obtained after treating the composition of the present invention with urea or guanidine hydrochloride and then treating with a ashi or a hygiene testis-derived hyal-mouth-dase.

. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

FIG. 8 is a chart obtained by capillary electrophoresis after treatment of the composition of the present invention and the control substance 1 with a honey or a testicle-derived hyaluronidase, respectively. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

[FIG. 9] A chart obtained from a single capillary electrophoresis after treating a mixture of conventional hyaluronic acid and the composition of the present invention with a ushi or hydrange testis-derived hyaluronidase. The broken line shows the chart before the enzyme reaction, and the solid line shows the chart after the enzyme reaction.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the hyaluronic acid composition of the present invention will be described in detail.

[0015] <1> Composition of the present invention

The composition of the present invention comprises hyaluronic acid which is known to have a polydisperse physical property, and has the characteristics described in the following (a) to (d).

[0016] (a) Under the standard conditions, when coexisting with the hyaluronidase derived from the urchin testis or the Hedgei testis, the digestion analysis of the treated composition by the capillary single electrophoresis method showed degradation. Absent.

(b) Protein content less than 0.1%

(c) Heavy metal content of 20 ppm or less

(d) Arsenic content is 2ppm or less Hereinafter, each characteristic of the above (a) force (d) will be described.

[0017] (a) The composition of the present invention is co-existing under standard conditions with a whip testis-derived or Hedgei testis-derived hyal-mouth-dase, followed by boiling for 5 minutes, centrifugation, and the supernatant. When disaccharide analysis is carried out by capillary electrophoresis, it has the property that no degradation is observed.

[0018] In the present invention, "under standard conditions" means 0.1 mol / l acetate buffer (pH 4.0, 0.1 mol / l NaCl) with respect to ultrapure water containing the composition of the present invention at 100 g / 100 1 10 / z 1, with 10% ultrapure water containing Hushi testis-derived hyaluronan-dase or Hedge testis-derived hyaluronan-dase (l, 000unit / ml) at 37 ° C for 10 hours This refers to the conditions for keeping warm.

[0019] The "centrifugation" in this property refers to centrifugation for 5 minutes under the condition of 12,000 Xg and 24 degrees.

In addition, “disaccharide analysis by capillary electrophoresis” in this property refers to the analysis method described in Measurement Example 9 described later.

[0020] (b) The composition of the present invention has a protein content of less than 0.1%.

The protein content in this property refers specifically to the content measured by the Raleigh method. The protein content can be measured by the Raleigh method by the method described in Measurement Example 5.

[0021] (c) The composition of the present invention has a heavy metal content of 20 ppm or less.

The heavy metal content in this property has a feature that it is 20 ppm or less as a result of measurement according to the second method of heavy metal test method of the Japanese Pharmacopoeia 14. General test method. The specific method of measurement is described in Measurement Example 6.

[0022] (d) The composition of the present invention has an arsenic content of 2 ppm or less.

The arsenic content in this property has a feature that it is 2 ppm or less as a result of measurement according to the third arsenic test method of the Japanese Pharmacopoeia 14. General Test Method. The specific method of measurement is described in Measurement Example 7.

The composition of the present invention preferably further has any of the following properties.

[0023] (e) Properties are white cotton powder

(β drying loss (60 degrees, reduced pressure 5 hours, phosphorus pentoxide) is 5% or more and less than 9%

(g) pH 5.8-6.5 (h) Intrinsic viscosity is 12.9 or more and less than 13.9

(0 weight average molecular weight 600,000 to 1.2 million

(j) Glucuronic acid content is 30% or more and less than 50%

[0024] (e) The composition of the present invention is a white cotton-like powder.

The dried product of the composition of the present invention has the properties of a white cotton-like powder.

(1) Loss on drying (60 ° C., reduced pressure 5 hours, phosphorus pentoxide) of the composition of the present invention is 5% or more and less than 9% The weight loss on drying of the composition of the present invention is 5% or more and less than 9%. Show. Such loss on drying can be measured by the method described in Measurement Example 1 according to Japanese Pharmacopoeia14.

[0026] (g) The composition of the present invention has a pH of 5.8 to 6.5.

The pH of the composition of the present invention is 5.8 to 6.5. The powerful pH can be determined by the method described in Measurement Example 2 according to the pH test method of Japanese Pharmacopoeia14.

[0027] (h) The intrinsic viscosity of the composition of the present invention is 12.9 dl / g or more and less than 13.9 dl / g.

The intrinsic viscosity of the composition of the present invention is 12.9 dl / g or more and less than 13.9 dl / g. Such intrinsic viscosity can be measured using an Ubbelohde viscometer according to the first method of viscosity measurement in the Japanese Pharmacopoeia 14. General Test Method.

(0) The weight average molecular weight of the composition of the present invention is 600,000 or more and 1.2 million or less.

The weight average molecular weight of the composition of the present invention is from 600,000 to 1,200,000. Such weight average molecular weight can be calculated according to the following formula from the value of intrinsic viscosity according to Biochem. Biophys. Acta. 42, 476 (1960).

[0029] (Formula 1)

[τ?] = 3.6 Χ 10 " ⁴ Μ ° · ⁷⁸ indicates intrinsic viscosity, Μ indicates weight average molecular weight) [0030] (j) The glucuronic acid content of the composition of the present invention is 30% or more and less than 50%. is there.

The composition of the present invention has a glucuronic acid content of 30% or more and less than 50% as measured by the force rubazole sulfate method. The glucuronic acid content can be measured by the following method using the force rubazole sulfate method.

[0031] That is, the sample is dried, about 50 mg thereof is accurately weighed, and purified water is added to dissolve to make a total volume of 1000 ml. Take 1 ml of this solution in a test tube and cool it in ice water. Mix 5 ml of sulfuric acid reagent and heat in a water bath for 10 minutes. Immediately cool in ice water, add 0.2 ml of strong rubazole reagent, mix, heat in water bath for 15 minutes, allow to cool, and measure absorbance at 530 nm. A standard solution of D-Dalcronolaton is prepared, and the amount of glucuronic acid can be calculated from the calibration curve force (D-Dalcronolaton is multiplied by 1.10 2 to The amount of acid). The ratio of the obtained glucuronic acid amount in the sample is calculated.

[0032] <2> Preparation of the composition of the present invention

The composition of the present invention can be obtained, for example, by the following steps (1) to (3).

[0033] (1) A process of degrading protein by treating a biological tissue with a proteolytic enzyme;

(2) a step of fractionating degradation products by molecular weight;

(3) A step of recovering the hyaluronic acid composition.

[0034] (1) Proteolytic process of biological tissue with proteolytic enzymes

The composition of the present invention can be prepared from an extract obtained by degrading protein such as vital tissue strength.

Examples of biological tissues include chicken crown, umbilical cord, skin, trachea and the like, and any of them can be used as mince. I also like the ease of handling and handling of chicken crowns.

[0035] Examples of proteolytic enzymes that degrade vital tissue include pronase, protease, proteinase, papain, and trypsin, and any of them can be used. However, the ability of pronase to exhibit the desired degradability is particularly preferable.

[0036] The reaction conditions and reaction time for the proteolytic enzyme can be appropriately adjusted by those skilled in the art according to the protein degrading enzyme used. For example, when pronase is used as a protein degrading enzyme, a temperature condition of about 35 to 60 ° C., preferably about pH 7 to 8 is preferred. The amount of proteolytic enzyme used can be adjusted by those skilled in the art depending on the type, and it is preferable to use 200,000 units or more per kg of living tissue. By using a large amount of proteolytic enzyme, the proteolytic enzyme treatment time can be reduced to 5 hours or less.

[0037] (2) Step of fractionating degradation products by molecular weight

The degradation product obtained in (1) is subjected to molecular weight fractionation. The molecular weight cutoff is 6,000 to 50,000. 8,000 to 30,000 is more preferred, and 10,000 to 20,000 is the most preferred range. The fractional molecular weight can be appropriately adjusted by those skilled in the art according to the average molecular weight of the hyaluronic acid composition to be obtained.

[0038] As a method of exerting molecular weight fractionation, known methods such as ultrafiltration and gel filtration can be appropriately used. Ultrafiltration is preferred because a large amount of sample can be processed relatively easily.

[0039] Prior to the fractionation step based on the strong molecular weight, for example, by using a technique such as filtration, the undegraded tissue is removed or contacted with celite, activated carbon, etc., and the molecular weight is 1,000 or less. By carrying out the treatment for removing loose impurities, a hyaluronic acid composition with fewer impurities can be obtained. Those skilled in the art can appropriately select and apply such processing according to the purpose.

[0040] (3) Step of recovering the hyaluronic acid composition

The hyaluronic acid composition can be recovered from the fraction subjected to molecular weight fractionation by a known method. Among known methods, the hyaluronic acid composition obtained by freeze-drying can be prevented from being modified, and the operation is simple.

[0041] The powerful method for preparing the composition of the present invention is characterized in that the quaternary ammonium salt and the organic solvent (alcohols) do not come into contact with the composition of the present invention before and after purification. is there

[0042] For example, the difference between the general purification method in the production of chicken crown strength hyaluronic acid and the production method of the composition in the present invention is summarized in Table 1 below.

[0043] [Table 1]

[0044] <3> Inhibitor of the present invention The inhibitor of the present invention comprises the composition of the present invention as an active ingredient.

[0045] The composition of the present invention has resistance to the degradation activity of the guinea pig testicular-derived hyaluronan-dase and the hygiene testis-derived hyaluronan-idase. Therefore, the composition of the present invention can be used as a glycosaminodarlican degradation inhibitor derived from such glycosaminodarican degrading enzyme activity.

[0046] The inhibitor of the present invention includes hyaluronic acid, chondroitin sulfate (chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, chondroitin sulfate D, chondroitin sulfate E), keratan sulfate, heparin, heparan sulfate, dermatan sulfate. It is useful as a decomposition inhibitor of hyaluronic acid, chondroitin sulfate, and dermatan sulfate, which are the targets of mammalian type endo hyaluro-dase (EC 3.2.1.35). is there.

[0047] When the inhibitor of the present invention is mixed with other glycosaminodarlicans to suppress degradation by the enzyme, the amount of the inhibitor of the present invention used is a weight ratio with respect to the other glycosaminodaricans. Forces exemplified by 0.01% to 80%, preferably 0.1% to 30%, more preferably 0.5% to 20%, depending on the coexisting enzyme concentration (concentration of glycosaminodarlican-degrading enzyme whose activity should be suppressed) It can be used after adjusting appropriately.

[0048] The inhibitor of the present invention can be added to pharmaceuticals, foods, and cosmetics to utilize the glycosaminodarican degradation inhibitory action of the present invention composition. The present invention composition is considered to be formed by hyaluronic acid. Hyaluronic acid is already listed in the pharmacopoeia as a sodium salt, and is also used as a medicine. Therefore, the safety | security of this invention inhibitor and this invention composition is also the same.

Example

[0049] Hereinafter, the present invention will be described more specifically by way of examples. Needless to say, the technical scope of the present invention is not limited to the scope of this embodiment.

[0050] <Measurement Example 1> Method for measuring loss on drying

Drying loss is determined according to the Japanese Pharmacopoeia 14 by accurately measuring sample O.lg and using phosphorus pentoxide as the desiccant, and the amount after drying at 95 ° C for 3 hours under reduced pressure.

[0051] The sample is pre-stretched to a constant weight and placed in a force bottle, and the surface of the sample is flattened and the mass is reduced to 0. Weigh to the order of lmg. Place the sample bottle into the vacuum dryer prepared at 95 ° C. Add an appropriate amount of phosphorus pentoxide, reduce the pressure in the vacuum dryer to O.lkPa, and dry for 3 hours. After drying, gradually reduce the pressure in the vacuum dryer to atmospheric pressure, quickly transfer the force bottle and lid to a desiccator and allow to cool to room temperature. After cooling, cover the force bottle and remove it from the desiccator, and weigh it to the order of 0.1 mg. The weight loss is calculated as a percentage by mass from the amount of sample before and after drying.

[0052] <Measurement example 2> pH measurement method

The pH is determined by the pH electrode method according to the pH test method of Japanese Pharmacopoeia 14 by adding 50 ml of freshly boiled and cooled distilled water to sample O.lg.

[0053] pH is measured using a roughness force Ji glass electrode and the _P H meter soaked several hours or more in order water.

Calibrate the pH meter using phosphate pH standard solution and phthalate pH standard solution, and add sample O.lg to a solution in which 50 ml of distilled water is boiled and cooled and then dissolved. Measure with a pH meter.

<Measurement Example 3> Method for Measuring Intrinsic Viscosity

First, the sample was dissolved in 0.05 mol / l Tris buffer (pH 8.0, 0.1 mol / l containing sodium chloride), diluted to 0.02 g / dl to 0.1 g / dl, and the concentration was changed to 10 Prepare several samples. Next, prepare an Ubbe mouth viscometer, and measure the intrinsic viscosity of the sample solution using an Ubbelohde viscometer by a conventional method.

<Measurement Example 4> Calculation Method of Weight Average Molecular Weight

The weight average molecular weight was calculated from the intrinsic viscosity [r?] Based on the description of C. Laurent, A. Pietuszklewicz, M. Ryan, Biochim. Biophys. Act a. 42,476 (1960).

[0056] (Formula 2)

[7?] = 3.6 X 10 " ⁴ Μ ° · ⁷⁸ (Μ indicates weight average molecular weight)

[0057] <Measurement example 5> Protein measurement by the Raleigh method

Protein is measured by the Raleigh method. Precisely weigh about 0.08 g of the sample, and add exactly 2 ml of dilute sodium hydroxide reagent to dissolve it. Accurately measure 1 ml of sample solution, accurately place 5 ml of alkaline copper reagent, leave it at 25 ° C for 10 minutes, leave it at 25 ° C for 0.5 minute, leave it at 25 ° C for 60 minutes, and measure the absorbance at a wavelength of 750 nm. . Bullying cow blood Prepare a standard solution of clean albumin and calculate the amount of protein in the sample solution using this calibration curve.

[0058] <Measurement example 6> Method for measuring heavy metal content

For heavy metals, a sample of 0.5 was taken and measured according to the second method of the heavy metal test method of the Japanese Pharmacopoeia 14 · General test method. A lead standard solution was used as a comparison solution.

[0059] 0.5 g of the sample is weighed in a magnetic crucible, and gently heated with a lid covered and weakly carbonized. After cooling, add 2 ml of nitric acid and 5 drops of sulfuric acid, heat carefully until no white smoke is formed, then ignite at 500-600 ° C to incinerate. After cooling, add 2 ml of hydrochloric acid, evaporate to dryness in a water bath, moisten the residue with 3 drops of hydrochloric acid, add 10 ml of hot water and warm for 2 minutes. Next, add 1 drop of phenolphthalein test solution, add ammonia test solution until the solution turns slightly red, add 2 ml of dilute hydrochloric acid, add this solution to a Nessler tube, and add water to make 50 ml. A sample solution was obtained.

[0060] Add a drop of sodium sulfate test solution to the sample and lead standard solution (containing 20 ppm of lead), mix, and let stand for 5 minutes. Compare the liquid colors. When the color of the liquid is observed and it is darker than the standard liquid, it means that heavy metal is contained at a higher concentration.

[0061] <Measurement example 7> Method for measuring arsenic content

Arsenic weighs 0.5g of sample and puts it in a magnetic crucible. In contrast, a solution (10 ml) obtained by diluting an ethanol (95%) solution of magnesium nitrate hexahydrate 50-fold with pure water was added, and the ethanol was ignited and burned, and then gradually heated. After that, it is ignited by igniting. After cooling, add 3 ml of hydrochloric acid to the residue and warm to dissolve in a water bath to make the sample solution. The sample solution is reacted using an arsenic test apparatus, and the content is measured by observing the color of the solution. The color observed in the standard solution prepared in advance (a solution containing arsenic at 2 ppm) is darker, and if a color is observed, it indicates that it contains arsenic at a higher concentration.

<Measurement Example 8> Method for measuring glucuronic acid content using the force rubazole sulfate method

The glucuronic acid content is measured using the force rubazole sulfate method. Dried the sample and about 5

Omg was precisely weighed and purified water was added to dissolve, making the total volume 1000 ml. Take 1 ml of this solution in a test tube, add 5 ml of sodium borate and sulfuric acid reagent while cooling in ice water, mix and heat for 10 minutes in a water bath. Immediately cool in ice water and add 0.2 ml of force rubazole reagent. Mix, heat in water bath for 15 minutes, allow to cool, and measure absorbance at 530 nm. A standard solution of D-Dalcronolatone was prepared in advance, and the calibration curve force was also used to calculate the amount of dalc carboxylic acid. (D-Dalcronolaton is multiplied by 1.102 to give the amount of glucuronic acid) Calculate the proportion of the glucuronic acid obtained in the sample.

[0063] Measurement Example 9> Disaccharide analysis by capillary electrophoresis

Use a capillary electrophoresis device (P / ACE 5010 model from Beckman) and a single capillary tube (40 cm x 50 m). The electrophoresis buffer is 50 nmol / l borate buffer (pH 8.5), lOOmmol / 1 SDS (sodium dodecyl sulfate), and analyzed at a temperature of 25 ° C in a single tube.

[0064] The capillary was washed with ultrapure water for 2 minutes by the pressurization method, and then washed with the electrophoresis buffer for 2 minutes by the pressurization method, and the inside of the capillary was filled with the electrophoresis buffer. An analytical sample was introduced into the capillary for 10 seconds by the pressurization method, and then a voltage was applied. The applied voltage was 20 kV and the analysis time was 10 minutes. Detect UV absorption at 200nm wavelength and examine enzymatic degradation.

<Example 1> Preparation of the composition of the present invention

-A 24 kg chicken crown was cut out from a chicken and minced and suspended in 160 L of deionized water. Salt (2.5 kg) was added and stirred in an extraction tank with heating at 105 ° C for 30 minutes. After cooling to room temperature, the pH was adjusted to 7.8, 50 g of calcium chloride and 6.5 million units of pronase (manufactured by Kaken Pharmaceutical) were added, and the mixture was stirred at 53 ° C. for 4 hours. Celite to this mixture (gemtuzumab write super one 3H: Hakusan Ltd.) 2 kg of activated carbon (Shirasagi - Yu Gold: Takeda Seiyaku) 2 kg was added, using a spark color filter (manufactured by Nissen), _No .5277 (Azumi filter paper manufactured by ) And Z-800 (Azumi filter paper

) And filtered through filter paper to obtain 170 L of extract from chicken crown. Adjust the pH of this extract to 4.8 with hydrochloric acid, add 2 kg of activated carbon (Shirakaba-Yu Gold: manufactured by Takeda Pharmaceutical), use a sparkler filter (manufactured by Nissen), No.5277 (manufactured by Azumi filter paper) and Z -800 (Azumi filter paper) was combined, filtered, and the pH was adjusted to 8.0. The obtained solution was subjected to ultrafiltration at 20 ° C. all day and night (ultrafiltration membrane ACV-3050, manufactured by Asahi Kasei Chemicals: molecular weight cut off 13000). The obtained ultrafiltrate was freeze-dried to obtain 120 g of the composition of the present invention. [0066] Each characteristic measured by the methods described in Measurement Examples 1 to 8 was as shown in Table 2 below.

[0067] [Table 2]

[0068] <Preparation example> Preparation of target substance 1

2 g of the composition of the present invention obtained in Example 1 was dissolved in 200 ml of a 2 mol / l aqueous sodium chloride solution, and then 600 ml of 10% cetylpyridydium chloride (containing sodium chloride at 2 mol / l of w / w) was added thereto. About 340 ml of 0.05% salt cetyl pyridinium solution was added to a concentration of 4 mol / l. The resulting precipitate was centrifuged at 5,000 × g for 30 minutes to obtain 1,000 ml of supernatant. To obtain a salt concentration of 1.2 mol / l in the supernatant, 160 ml of 0.05% salt cetyl pyridinium (manufactured by Tokyo Chemical Industry Co., Ltd.) is added, the resulting precipitate is centrifuged, and 1,100 ml of the supernatant is obtained. Got. To this supernatant, 1,540 ml of 0.05% cetylpyridyl chloride solution was added so that the sodium chloride concentration was 0.5 mol / l, followed by centrifugation to obtain 2,510 ml of supernatant. To this supernatant, 10,040 ml of 0.05% cetylpyridyl chloride solution was added and centrifuged so that the sodium chloride concentration was 0.1 mol / l. The obtained supernatant was dissolved again in 200 ml of 2 mol / l saline solution containing 15% ethanol (w / w) by heating at 40 ° C. Three times the amount of ethanol was precipitated again. The resulting precipitate was separated by centrifugation, the precipitate was dissolved in 100 ml of ultrapure water, and dialyzed overnight using a dialysis membrane (permeated molecular weight 14,000: made of regenerated cellulose). The obtained lysate was freeze-dried to obtain 95 Omg of control substance 1 (hyaluronic acid).

To 100 1 of ultrapure water containing 100 g of the composition of the present invention, 0.1 mol / l acetate buffer solution (containing pH 4.0, 0.1 mol / 1 sodium chloride) 10 1 Add 10 1 of dase (Sigma: lmg / ml), react at 37 ° C for 10 hours, boil the reaction solution for 5 minutes, centrifuge (12,000 X g, 24 ° C, 5 minutes), and remove the supernatant. Was analyzed by the method described in Measurement Example 9 (FIG. 1).

[0070] Hyaluronic acid shown in Table 3 below was used as a control substance. [0071] [Table 3]

[0072] As a result, it was revealed that only the composition of the present invention is resistant to urchin testis-derived hyaluronan-dase.

The experiment similar to that of Example 2 was performed in place of the Hushi testis-derived hyaluronan-dase in Example 2 instead of the Hygi testis-derived hyaluronidase (manufactured by Roche) (FIG. 2).

As a result, it was revealed that only the composition of the present invention exhibits resistance to hygiene testis-derived hyaluronan-dase.

[0075] In addition, the results of experiments using commercially available hyaluronic acid preparations (Table 4 below) according to the above procedure were also similarly degraded by Hedge testis-derived hyaluronan-dase (Fig. 3).

[0076] [Table 4]

2 μ 1 of microbial-derived hyaluro-dase (Streptomyces hyalurolyticus) (lmg / ml) dissolved in 0.1 mol / l acetate buffer (pH 6.0) to 20 1 of ultrapure water containing 20 g of hyaluronic acid composition The mixture was further reacted at 60 ° C for 10 hours. The reaction solution was boiled for 5 minutes, then centrifuged (12,000 X g, 24 ° C, 5 minutes), and the supernatant was analyzed by the method described in Measurement Example 9 (Fig. 4). ).

[0078] The same substance as in Example 2 was used as a control.

As a result, it was shown that the composition of the present invention was also decomposed similarly to the control. [0079] <Example 5>

100 μl of ultrapure water containing 100 g of hyaluronic acid composition 0.1 μl / l acetate buffer solution (pH 6.0) 10 μ1 10 / zl, react at 37 ° C for 10 hours, boil the reaction solution for 5 minutes, centrifuge (12,000 X g, 24 ° C, 5 minutes), and analyze the supernatant by capillary electrophoresis ( (Figure 5).

The same substance as in Example 2 was used as a control.

As a result, it was shown that the composition of the present invention was also decomposed in the same manner as the control.

Hyaluronan-dase is known to inhibit the enzymatic activity of metal ions. Thus, it was confirmed that the composition of the present invention is resistant to enzymatic degradation by hyaluro-dase derived from testes of ushi and hedges, regardless of the influence of metal ions. That is, 10 μl of lOOmmol / 1 edetic acid (chelating agent) was added to 100 μl of ultrapure water containing 100 μg of the composition of the present invention and treated at 25 ° C. for 10 hours. The sample was decomposed and analyzed according to Measurement Example 9 (Fig. 6). As a control, hyaluronic acid derived from Streptcoccus zooepidemi cus manufactured by Nacalai Testa Co. As a result, no degradation products were observed.

That is, it was found that the hyaluronan-dase resistance of the composition of the present invention was not due to contamination with metal ions.

The composition of the present invention has been isolated without any treatment such as treatment with a quaternary ammonium salt or fractionation with an organic solvent. Therefore, it is highly possible that the existing form in the chicken crown is maintained, and it is manufactured without damaging the three-dimensional higher order structure caused by hydrogen bonding. Therefore, it was known in protein studies and the like that it was known to cleave hydrogen bonds, and the effect of hydrogen bonds on hyaluronan-dase resistance was confirmed by treatment with urea or guanidine hydrochloride.

[0083] That is, after 100 μg of the composition of the present invention was treated with 6 mol / l guanidine solution and 8 mol / l urea solution for 100 hours at 25 ° C for 10 hours, 10 mg of sodium acetate and lml of each were treated. Ethanol was added. This solution is kept at −27 ° C. for 2 hours, and centrifuged at 12,000 × g for 10 minutes. The resulting precipitate is dissolved in 0.1 mol / l acetate buffer (pH 5.3) 100 1 and according to Example 2. Enzymatic degradation was performed and analyzed by the method described in Measurement Example 9 (FIG. 7).

As a result, when the composition of the present invention was treated with guanidine hydrochloride, degradation products were observed.

[0084] This indicates that the hyaluronan-dase resistance of the composition of the present invention is attributed to hydrogen bonds in the hyaluronic acid molecule. That is, the composition of the present invention is considered to differ from conventional hyaluronic acid in terms of the three-dimensional structure due to hydrogen bonding.

The composition of the present invention and the control substance 1 obtained in Preparation Example 2 were treated with hyaluronan-dase according to Examples 2 and 3, decomposed by the method described in Measurement Example 9, and their degradability was compared. (Figure 8).

[0086] As a result, it became clear that the control substance 1 was decomposed by both the testicular-derived hyaluronan-dase and the hygiene testis-derived hyaluronan-dase.

[0087] That is, treating the composition of the present invention with a quaternary ammonium salt and an organic solvent eliminates the resistance to hyaluro-dase, and there is a difference in the characteristics of the product depending on the intensive manufacturing process. It was found that this occurred.

90 mg of cultured hyaluronic acid (manufactured by Kibun Food Chemifa Corporation, molecular weight 1.84 million) and lOmg of the composition of the present invention were dissolved in 40 ml of ultrapure water, and then lyophilized.

[0089] Using this mixture, hyaluro-dase treatment was carried out using hyaluro-dase derived from testes of ushi and hidge according to Examples 2 and 3, respectively, and by the method described in Measurement Example 9 Analyzed (Figure 9).

[0090] As a result, it became apparent that the mixture was suppressed from being decomposed by hyaluronan-dase, even though the composition of the present invention was 10% and contained no force.

[0091] Similarly, the same result was obtained when 5% of the composition of the present invention was added.

Industrial applicability

[0092] The hyaluronic acid composition having resistance to mammalian hyaluronan-dase obtained according to the present invention avoids adverse effects on biocompatibility resulting from alteration of the molecular structure of hyaluronic acid, and is a practical degradation. It has superiority in enzyme stability and can be applied in the fields of pharmaceuticals, quasi drugs, cosmetics, and foods.

Claims

Claims [1] A hyaluronic acid composition obtained by the following steps (1) to (3). (1) A process of degrading a protein by treating a biological tissue with a proteolytic enzyme; (2) A process of fractionating the degradation product obtained in (1) by molecular weight to obtain a fraction; (3) By (2) Step of recovering the obtained fractional force hyaluronic acid composition. [2] A hyaluronic acid composition having the following properties. (a) Under standard conditions, when coexisting with the hyaluronidase derived from the urchin testis or the Hedgei testis, boiled for 5 minutes, and the supernatant after centrifugation was analyzed by disaccharide analysis using a capillary single electrophoresis. (B) Protein content of less than 0.1% when analyzed by the Raleigh method; (c) Heavy metal content of 20 ppm or less as determined by the heavy metal test method described in the Japanese Pharmacopoeia; (d) Arsenic as described in the Japanese Pharmacopoeia Arsenic content is 2ppm or less as measured by the test method. [3] A method for producing a hyaluronic acid composition comprising the following steps (1) to (3):

(1) a process of degrading protein by treating biological tissue with a proteolytic enzyme;

(2) A step of fractionating the degradation product obtained in (1) by molecular weight to obtain a fraction;

(3) The step of recovering the fractional force hyaluronic acid composition obtained in (2).

[4] A glycosaminodarican decomposition inhibitor comprising a hyaluronic acid composition as an active ingredient.

[5] Use of a hyaluronic acid composition as a glycosaminodarican decomposition inhibitor.

[6] A medicament comprising the hyaluronic acid composition according to claim 1 or 2.

[7] A food comprising the hyaluronic acid composition according to claim 1 or 2.

[8] A cosmetic comprising the hyaluronic acid composition according to claim 1 or 2.