JP3142415B2 - High acetylation rate hyaluronic acid and method for producing the same - Google Patents
High acetylation rate hyaluronic acid and method for producing the sameInfo
- Publication number
- JP3142415B2 JP3142415B2 JP05112226A JP11222693A JP3142415B2 JP 3142415 B2 JP3142415 B2 JP 3142415B2 JP 05112226 A JP05112226 A JP 05112226A JP 11222693 A JP11222693 A JP 11222693A JP 3142415 B2 JP3142415 B2 JP 3142415B2
- Authority
- JP
- Japan
- Prior art keywords
- hyaluronic acid
- acid
- acetylation
- producing
- acetylation rate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Polysaccharides And Polysaccharide Derivatives (AREA)
- Emulsifying, Dispersing, Foam-Producing Or Wetting Agents (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明はアセチル化ヒアルロン酸
及びその製造方法、特にヒアルロン酸のアルコール性水
酸基にアセチル基を高率で結合させた高アセチル化率ヒ
アルロン酸及びその製造方法の改良に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an acetylated hyaluronic acid and a method for producing the same, and more particularly to an improved acetylated hyaluronic acid in which an acetyl group is bonded to an alcoholic hydroxyl group of the hyaluronic acid at a high rate and an improved method for producing the same.
【０００２】[0002]
【従来の技術】ヒアルロン酸は生体由来の高分子物質で
あり、高い増粘性、粘張性、曳糸性等の特異的な物性を
有しており、しかも生体適合性が高いことから各種分野
での応用が期待されている。2. Description of the Related Art Hyaluronic acid is a high-molecular substance derived from living organisms, has specific physical properties such as high viscosity, stickiness, and spinnability, and has high biocompatibility. Application in is expected.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、ヒアル
ロン酸は強水溶性であり、このため有機溶媒系での増粘
剤、油性基剤中での各種乳化安定剤、リポソームの被覆
強化剤、生体への埋め込み基剤、カプセル基剤等への用
途が期待されていながら、充分な応用が出来ないもので
あった（特開平３−１４３５４０、特開昭５４−３６３
８８等参照）。すなわち、ヒアルロン酸をアセチル化す
る工程を想定すると、一般の低分子物質のアセチル化
は、無水酢酸とピリジンの系で行なわれることが多い
が、前述したようにヒアルロン酸は強水溶性であり、無
水酢酸−ピリジン系でのアセチル化は困難であり、しか
も反応工程中でのヒアルロン酸の分解を免れない。その
他、数十種類に及ぶ方法で検討したことが、結果は同様
であった。このため、高アセチル化率のヒアルロン酸誘
導体を製造することは極めて困難であり、しかもヒアル
ロン酸の本来有する特性自体を失うことが多かった。However, hyaluronic acid is strongly water-soluble, and therefore, it is used as a thickener in an organic solvent system, various emulsion stabilizers in an oily base, a coating enhancer for liposomes, Although it is expected to be used as an embedding base, a capsule base, etc., it cannot be sufficiently applied (JP-A-3-143540, JP-A-54-363).
88 etc.). That is, assuming a step of acetylating hyaluronic acid, acetylation of general low-molecular substances is often performed in a system of acetic anhydride and pyridine, but as described above, hyaluronic acid is strongly water-soluble, Acetylation in an acetic anhydride-pyridine system is difficult, and the degradation of hyaluronic acid in the reaction process is unavoidable. In addition, the results were the same as those examined in dozens of other methods. For this reason, it is extremely difficult to produce a hyaluronic acid derivative having a high acetylation rate, and the original properties of hyaluronic acid are often lost.
【０００４】一方、例えば特開平３−１４３５４０号公
報には、ヒアルロン酸の繰り返し単位にアセチル基等の
アシル基を導入した乳化安定剤が示されている。しかし
ながら、このヒアルロン酸誘導体は修飾率が極めて低
く、アシル基／Ｎ−アセチル基の比率が数分の１以下で
ある。すなわち、数個ないし数十個の繰り返し単位に一
つのアシル基が導入されているのみであり、このような
修飾率では、パルミトイル基等の高油性アシル基を導入
しなければ乳化剤としての機能を事実上奏することは出
来ず、しかも前記ピリジン系を用いるため、修飾率を高
くしようとすればヒアルロン酸の分解等を生じてしま
う。本発明は前記従来技術の課題に鑑みなされたもので
あり、その目的はヒアルロン酸の本来有する機能を保持
し、各種特異な物性を有する高アセチル化率ヒアルロン
酸及びその製造方法を提供することにある。On the other hand, for example, JP-A-3-143540 discloses an emulsion stabilizer in which an acyl group such as an acetyl group is introduced into a repeating unit of hyaluronic acid. However, the modification ratio of this hyaluronic acid derivative is extremely low, and the ratio of acyl group / N-acetyl group is a fraction or less. That is, only one acyl group is introduced into several to several tens of repeating units, and at such a modification rate, the function as an emulsifier is not provided unless a highly oily acyl group such as a palmitoyl group is introduced. In fact, it cannot be performed, and since the pyridine system is used, an attempt to increase the modification rate results in the decomposition of hyaluronic acid. The present invention has been made in view of the above-mentioned problems of the prior art, and an object of the present invention is to provide a hyaluronic acid having a high acetylation rate having various unique physical properties while retaining the inherent functions of hyaluronic acid, and a method for producing the same. is there.
【０００５】[0005]
【課題を解決するための手段】前記目的を達成するため
に本発明者が鋭意検討した結果、ヒアルロン酸を酢酸と
共存させ、しかも触媒として無水トリフルオロ酢酸、な
いしｐ−トルエンスルホン酸と無水酢酸を加えることに
より、温和な条件で高アセチル化率ヒアルロン酸が製造
出来ることを見出し、本発明を完成するに至った。即
ち、本出願の請求項１記載の高アセチル化率ヒアルロン
酸は、次の化２を構造を有することを特徴とする。Means for Solving the Problems As a result of intensive studies conducted by the present inventors to achieve the above object, hyaluronic acid is allowed to coexist with acetic acid, and trifluoroacetic anhydride or p-toluenesulfonic acid and acetic anhydride are used as catalysts. It has been found that by adding, a high acetylation rate hyaluronic acid can be produced under mild conditions, and the present invention has been completed. That is, the high acetylation rate hyaluronic acid according to claim 1 of the present application is characterized by having the following structure.
【０００６】[0006]
【化２】 尚、上記化２中、Ｒ1，Ｒ2，Ｒ3，Ｒ4は、水素またはエ
ステル結合されたアセチル基を示し、且つ平均すると各
繰り返し構造においてＲ1〜Ｒ4の少なくとも１以上がア
セチル基である。Ｒ5は水素又はアルカリ金属原子を示
す。なお、以下においては、Ｒ1〜Ｒ4のうちの１がアセ
チル基である場合をアセチル化度１と表現する。請求項
２記載の高アセチル化率ヒアルロン酸の製造方法は、粉
末状ヒアルロン酸を酢酸に懸濁し、触媒としてトリフル
オル酢酸を加え反応させることを特徴とする。請求項３
記載の高アセチル化率ヒアルロン酸の製造方法は、粉末
状ヒアルロン酸を酢酸に分散させ、ｐ−トルエンスルホ
ン酸を加えて、前記微粉末ヒアルロン酸を膨潤させ、更
に無水酢酸を加え反応させることを特徴とする。以下、
本発明の構成を更に詳細に説明する。本発明において、
ヒアルロン酸とは、ヒアルロン酸及びヒアルロン酸塩を
意味し、各種分子量のものを用いることができる。Embedded image In the above chemical formula 2 , R 1 , R 2 , R 3 , and R 4 represent hydrogen or an acetyl group having an ester bond, and on average, at least one of R 1 to R 4 in each repeating structure is an acetyl group. It is. R 5 represents hydrogen or an alkali metal atom. In the following, the case where one of R 1 to R 4 is an acetyl group is referred to as “acetylation degree 1”. A method for producing hyaluronic acid having a high acetylation ratio according to claim 2 is characterized in that hyaluronic acid in powder form is suspended in acetic acid, and trifluoroacetic acid is added as a catalyst to cause a reaction. Claim 3
The method for producing high acetylation rate hyaluronic acid according to the present invention is to disperse powdery hyaluronic acid in acetic acid, add p-toluenesulfonic acid, swell the fine powdered hyaluronic acid, and further react by adding acetic anhydride. Features. Less than,
The configuration of the present invention will be described in more detail. In the present invention,
Hyaluronic acid means hyaluronic acid and hyaluronic acid salt, and those having various molecular weights can be used.
【０００７】又、本発明にかかる高アセチル化率ヒアル
ロン酸の製造方法において、ヒアルロニダーゼ等の酵素
処理により、オリゴヒアルロン酸から分子量１０，００
０ｋｄ以上におよぶ広範囲の高アセチル化率ヒアルロン
酸を得ることができ、又エステル化反応時間を変えるこ
とにより修飾化率を大幅に変更することができる。ま
た、本出願の請求項２記載の方法において、トリフルオ
ロ酢酸は触媒として機能し、ヒアルロン酸と酢酸の反応
は、例えば室温にて数時間行なえば済み、温和な条件で
ヒアルロン酸自体の構造に変化を与えることなく、高ア
セチル化率ヒアルロン酸を製造することができる。又、
本出願の請求項３記載の方法において、ｐ−トルエンス
ルホン酸は触媒として機能し、反応は例えば５０℃程度
にて行えばすみ、前記請求項２記載の方法と同様に温和
な条件でヒアルロン酸自体の構造に変化を与えることな
く、しかも高アセチル化率のヒアルロン酸を製造するこ
とができる。In the method for producing hyaluronic acid having a high acetylation rate according to the present invention, the molecular weight of oligohyaluronic acid is reduced to 10,000 by enzymatic treatment such as hyaluronidase.
A wide range of hyaluronic acid having a high acetylation ratio of 0 kd or more can be obtained, and the modification ratio can be drastically changed by changing the esterification reaction time. Further, in the method according to claim 2 of the present application, trifluoroacetic acid functions as a catalyst, and the reaction between hyaluronic acid and acetic acid may be performed at room temperature for several hours, for example. Highly acetylated hyaluronic acid can be produced without any change. or,
In the method according to claim 3 of the present application, p-toluenesulfonic acid functions as a catalyst, and the reaction may be carried out, for example, at about 50 ° C. It is possible to produce hyaluronic acid having a high acetylation rate without changing its structure.
【０００８】[0008]
【実施例】以下、本発明を実施例に基づき説明する。
尚、本発明は以下の実施例に限定されるものではない。実施例１ アセチル化ヒアルロン酸 ５００mlのガラス製ビーカーに市販の特級氷酢酸を入
れ、２ｇのバイオヒアロ１２（分子量約１２００kdのヒ
アルロン酸 資生堂（株）製）の微細粉末を攪拌しなが
ら少しずつ加える。続いて、２０mlの無水トリフルオロ
酢酸をゆっくり加え、室温で１時間攪拌してアセチル化
反応を行なわせる。次に２００mlのピリジンを加え中和
後、２００mlの水を加え攪拌し、沈殿物を完全に溶解さ
せる。４００mlのアセトンを攪拌しながら徐々に加え生
成物を沈殿させる。沈殿は、３，０００rpm１０分間遠
心分離することにより分取する。沈殿は再度２００mlの
１％酢酸ナトリウムに溶解後、前記同様にアセトン沈殿
させる。この溶剤沈殿操作を２度繰り返すことにより、
純水なアセチル化ヒアルロン酸を得る。続いて、アセト
ン洗浄により完全に脱水させた後、減圧乾燥し、アセチ
ル化ヒアルロン酸の白色粉末を得る。DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on embodiments.
The present invention is not limited to the following embodiments. Example 1 A commercially available special grade glacial acetic acid is placed in a glass beaker of acetylated hyaluronic acid ( 500 ml), and 2 g of biohyalo 12 (hyaluronic acid manufactured by Shiseido Co., Ltd. having a molecular weight of about 1200 kd) is added little by little with stirring. Subsequently, 20 ml of trifluoroacetic anhydride is slowly added, and the mixture is stirred at room temperature for 1 hour to cause an acetylation reaction. Next, 200 ml of pyridine was added and neutralized, and then 200 ml of water was added and stirred to completely dissolve the precipitate. 400 ml of acetone is slowly added with stirring to precipitate the product. The precipitate is collected by centrifugation at 3,000 rpm for 10 minutes. The precipitate is dissolved again in 200 ml of 1% sodium acetate, and then precipitated with acetone as described above. By repeating this solvent precipitation operation twice,
Obtain pure water acetylated hyaluronic acid. Subsequently, after completely dehydrated by washing with acetone, drying is performed under reduced pressure to obtain a white powder of acetylated hyaluronic acid.
【０００９】本実施例品は、高分子量のアセチル化ヒア
ルロン酸となる。本実施例にかかるアセチル化ヒアルロ
ン酸を、０．１〜１％（w/v）の濃度となるように水に
溶解させ、１／２〜３倍量のエタノール等の有機溶剤を
加え、攪拌すると増粘しゲル状となる。又、本実施例の
１％水溶液は、図１に示すように紫外部に明瞭な吸収ピ
ークを示さず、原料としたヒアルロン酸水溶液の紫外部
吸収スペクトルに酷似する。実施例２ アセチル化ヒアルロン酸 ５００mlのガラス製ビーカーに４００mlの市販の特級氷
酢酸を入れ、５ｇのバイオヒアルロン酸（分子量約２，
２００kdのヒアルロン酸 資生堂（株）製）の微細粉末
を攪拌しながら少しずつ加える。これに１００mlの無水
トリフルオロ酢酸をゆっくり加え、室温で一晩攪拌して
反応させる。反応液は粘張な無色透明な液体となる。２
ｌのガラス製ビーカーに、予め７００mlの精製水を氷冷
しておく。攪拌しながら上記反応液をゆっくり加える。
生じたアセチル化ヒアルロン酸の沈殿を濾別して分取す
る。沈殿は精製水で洗浄液が中性となるまで繰り返し洗
浄する。そして、真空乾燥機で脱水することにより、ア
セチル化ヒアルロン酸の白色粉末を得る事が出来る。The product of this example is a high molecular weight acetylated hyaluronic acid. The acetylated hyaluronic acid according to this example is dissolved in water to a concentration of 0.1 to 1% (w / v), and a 1/2 to 3 times the amount of an organic solvent such as ethanol is added thereto, followed by stirring. Then, it thickens and becomes gel-like. Further, the 1% aqueous solution of this example does not show a clear absorption peak in the ultraviolet as shown in FIG. 1, and resembles very closely the ultraviolet absorption spectrum of the hyaluronic acid aqueous solution used as the raw material. Example 2 400 ml of commercial grade glacial acetic acid was placed in a 500 ml acetylated hyaluronic acid glass beaker, and 5 g of biohyaluronic acid (molecular weight of about 2,
200 kd of hyaluronic acid (Shiseido Co., Ltd.) fine powder is added little by little with stirring. 100 ml of trifluoroacetic anhydride is slowly added thereto, and the mixture is stirred at room temperature overnight and reacted. The reaction liquid becomes a viscous colorless and transparent liquid. 2
1 ml of purified water is previously ice-cooled in a 1 glass beaker. The above reaction solution is slowly added with stirring.
The resulting precipitate of acetylated hyaluronic acid is separated by filtration. The precipitate is washed repeatedly with purified water until the washing solution becomes neutral. Then, a white powder of acetylated hyaluronic acid can be obtained by dehydrating with a vacuum dryer.
【００１０】本実施例品は高分子量で且つ高アセチル化
度のアセチル化ヒアルロン酸となる。本実施例品はジメ
チルスルホキサイド、ジメチルホルムアミド等の有機溶
媒に可溶である。又、少量の水を含むエタノール、アセ
トン等にも可溶である。又、本実施例品の希薄な有機溶
媒溶液を薄く平らにのばし、溶媒を蒸発させることによ
り、アセチル化ヒアルロン酸のフィルムを得ることが出
来る。更に、本実施例品は有機溶剤可溶性であるので、
カルボジイミド、クロロ蟻酸イソブチル等を触媒とし
て、アミノ基、水酸基等を有する多くの生理活性物質を
共有結合により導入することができる。本実施例品の赤
外吸収スペクトルを図２に示す。同図より明らかな様
に、Ｏ−アセチル基のカルボニルの伸縮振動に基づく吸
収を１，７３８cm-1に、同様にエーテル結合に基づく吸
収を１，２４８cm-1に認める。尚、１，６５２cm-1の吸
収は、Ｎ−アセチル基のカルボニルによる伸縮振動に由
来する。The product of this example is acetylated hyaluronic acid having a high molecular weight and a high acetylation degree. The product of this example is soluble in organic solvents such as dimethylsulfoxide and dimethylformamide. It is also soluble in ethanol, acetone and the like containing a small amount of water. Further, a thin organic solvent solution of the product of this example is spread thinly and flatly, and the solvent is evaporated to obtain a film of acetylated hyaluronic acid. Furthermore, since the product of this example is soluble in an organic solvent,
Many physiologically active substances having an amino group, a hydroxyl group or the like can be introduced by covalent bond using carbodiimide, isobutyl chloroformate or the like as a catalyst. FIG. 2 shows the infrared absorption spectrum of the product of this example. As is apparent from the figure, absorption based on the stretching vibration of the carbonyl of the O-acetyl group is observed at 1,738 cm −1 , and absorption based on the ether bond is similarly observed at 1,248 cm −1 . The absorption at 1,652 cm -1 is derived from stretching vibration of N-acetyl group due to carbonyl.
【００１１】同図より、本実施例品が極めてアセチル化
度の高いものであることが確認された。実施例３ アセチル化ヒアルロン酸 前記バイオヒアロ１２ ２ｇを、１０mg/mlの濃度に１
００mM酢酸ナトリウム緩衝液（ｐＨ５．０）に溶解させ
る。攪拌しながら３７℃に加温し、１０Ｕ/mlの牛睾丸
由来のヒアルロニダーゼを加え、この温度で４時間反応
させる。１００℃５分間加熱し、酵素を失活させる。４
００mlのアセトンを攪拌させながら徐々に加え、低分子
化したヒアルロン酸を沈殿させる。続いてアセトン洗浄
により脱水させる。粉末粉砕機により微粉にした後、実
施例１ないし実施例２と同様な方法により高アセチル化
率ヒアルロン酸を得る。本実施例においては、分子量１
００kd程度の高アセチル化率ヒアルロン酸を得ることが
できる。From the figure, it was confirmed that the product of this example had an extremely high acetylation degree. Example 3 Acetylated Hyaluronic Acid 2 g of the above biohyalo 12 was added to a concentration of 10 mg / ml.
Dissolve in 00 mM sodium acetate buffer (pH 5.0). The mixture is heated to 37 ° C. with stirring, 10 U / ml of bovine testis-derived hyaluronidase is added, and the mixture is reacted at this temperature for 4 hours. Heat at 100 ° C. for 5 minutes to inactivate the enzyme. 4
00 ml of acetone is gradually added with stirring to precipitate low molecular weight hyaluronic acid. Subsequently, dehydration is performed by washing with acetone. After pulverizing with a powder grinder, hyaluronic acid with a high acetylation rate is obtained in the same manner as in Examples 1 and 2. In this embodiment, the molecular weight is 1
Hyaluronic acid having a high acetylation rate of about 00 kd can be obtained.
【００１２】本実施例品の１％溶液の粘度は、高分子量
のものと比べてかなり低い。又、ゲルを形成させた場
合、高分子量のものと比べて緩やかであるので、乳液な
どにも容易に配合され得る。実施例４ アセチル化ヒアルロン酸 前記バイオヒアロ１２ ２ｇを１０mg/mlの濃度となる
ように、１００mM酢酸ナトリウム緩衝液（ｐＨ５．０）
に加熱溶解（１００℃）させ、加熱を３０分間維持す
る。３７℃に冷却したのち、攪拌させながら１０Ｕ/ml
の牛睾丸由来のヒアルロニダーゼを、滅菌された０．２
２μのメンブランフィルタを介して加える。２時間毎に
この操作を４回繰り返し（ヒアルロニダーゼの終濃度５
０Ｕ/ml）、更に一晩反応させる。１００℃で５分間加
熱し酵素を失活させる。そして、６００mlのアセトンを
攪拌させながら徐々に加え、低分子化したヒアルロン酸
を沈殿させ、アセトン洗浄により脱水させる。この状態
で、既にヒアルロン酸は微粉であるので、前記実施例１
ないし実施例２と同様な方法によりアセチル化ヒアルロ
ン酸を得る。The viscosity of a 1% solution of the product of the present invention is considerably lower than that of a high molecular weight product. Further, when a gel is formed, the gel is looser than a high molecular weight one, so that it can be easily blended in an emulsion or the like. Example 4 Acetylated Hyaluronic Acid A 100 mM sodium acetate buffer (pH 5.0) was used so that 2 g of the above-mentioned biohyaluronan 12 had a concentration of 10 mg / ml.
And heat (100 ° C.) and maintain the heating for 30 minutes. After cooling to 37 ° C, 10 U / ml with stirring
Hyaluronidase from bovine testes was sterilized 0.2%
Add through a 2μ membrane filter. This operation was repeated 4 times every 2 hours (final concentration of hyaluronidase 5
0 U / ml) and further react overnight. Heat at 100 ° C. for 5 minutes to inactivate the enzyme. Then, 600 ml of acetone is gradually added with stirring to precipitate low molecular weight hyaluronic acid, and dehydrated by washing with acetone. In this state, since hyaluronic acid is already a fine powder,
Or acetylated hyaluronic acid is obtained in the same manner as in Example 2.
【００１３】本実施例においては、分子量１０kd程度又
はそれ以下の低分子量アセチル化ヒアルロン酸を得るこ
とが出来る。実施例５ 三角フラスコ内に酢酸約２００ｍｌを取り、これに分子
量２００万の微粉末ヒアルロン酸２ｇを分散させ、更に
ｐ−トルエンスルホン酸を４ｇを加え５０℃で４時間攪
拌しながら放置した。膨潤したところで、無水酢酸１０
０ｍｌを３０分かけて滴下したのち、５０℃で保ったま
ま２８時間攪拌しながら反応させた。反応液を水中に投
入し、繊維上に沈殿してきた反応精製物を１００倍量の
純粋で３回水洗後、真空乾燥してアセチル化ヒアルロン
酸を得た。図３に以上のようにして得たアセチル化ヒア
ルロン酸の1Ｈ−ＮＭＲ分析結果が示されている。同図
において、（Ａ）〜（Ｆ）に移行するに従い、アセチル
化反応時間を短くしている。同図より明らかなように、
アセチル化反応時間が短い場合にはヒアルロン酸自体に
由来するＮ−アセチル基を示すピークが大きいが、反応
時間を長くするに従って他の部位に結合したアセチル基
を示すピークが顕著に増大し、該Ｎ−アセチル基の存在
量以上に他のアルコール性水酸基のアセチル化が進んで
いることが理解される。このように、本発明においては
アセチル基／Ｎ−アセチル基の比率が１以上となってお
り、従来事実上不可能であったアセチル化度１以上の高
アセチル化率ヒアルロン酸が得られている。In this embodiment, low molecular weight acetylated hyaluronic acid having a molecular weight of about 10 kd or less can be obtained. Example 5 About 200 ml of acetic acid was placed in an Erlenmeyer flask, 2 g of fine powdered hyaluronic acid having a molecular weight of 2,000,000 was dispersed therein, and 4 g of p-toluenesulfonic acid was further added. When swollen, acetic anhydride 10
After dropwise adding 0 ml over 30 minutes, the reaction was carried out with stirring at 50 ° C. for 28 hours. The reaction solution was poured into water, and the purified reaction product precipitated on the fiber was washed three times with 100 volumes of pure water, and then dried under vacuum to obtain acetylated hyaluronic acid. FIG. 3 shows the results of 1 H-NMR analysis of the acetylated hyaluronic acid obtained as described above. In the figure, the acetylation reaction time is shortened as the process proceeds to (A) to (F). As is clear from the figure,
When the acetylation reaction time is short, a peak indicating an N-acetyl group derived from hyaluronic acid itself is large, but as the reaction time is increased, a peak indicating an acetyl group bonded to another site is significantly increased. It is understood that the acetylation of other alcoholic hydroxyl groups has progressed beyond the amount of N-acetyl groups. Thus, in the present invention, the ratio of acetyl group / N-acetyl group is 1 or more, and a high acetylation rate hyaluronic acid having an acetylation degree of 1 or more, which has been practically impossible in the past, is obtained. .
【００１４】尚、次の表１には前記図３に示した各高ア
セチル化率ヒアルロン酸の反応時間と概略アセチル化率
の関係が示されている。The following Table 1 shows the relationship between the reaction time of each high acetylation rate hyaluronic acid shown in FIG. 3 and the approximate acetylation rate.
【表１】 ──────────────────────────────────── アセチル化ＨＡ 反応時間 アセチル化度 水溶性 アルコール溶解性 ──────────────────────────────────── （Ａ） 168時間 3.1 × ○ （Ｂ） 74 2.9 × ○ （Ｃ） 32 2.5 △ △ （Ｄ） 20 2.0 △ △ （Ｅ） 8 1.5 △ × （Ｆ） 5 1.2 ○ × ──────────────────────────────────── 以上のようにして得られた高アセチル化率ヒアルロン酸
は、分子量あるいは修飾化率等によって著しく物性が異
なり、高分子量で中程度の修飾度のものは、少量の有機
溶剤の添加のより安定なゲルをつくる等、その化粧品基
剤、あるいはドラッグデリバリーシステムの基剤として
その応用が期待される。又、高分子量で高度アセチル化
等のものや、低分子量のものは、かなりの濃度の有機溶
剤に可溶であり、乳液などにも容易に配合され得る。そ
して、乳液等に配合された場合、使用時の滑らかさを増
すなど、種々の効果を発揮することが出来る。又、アセ
チル化等により脂溶性を増加させれば、表面が脂質膜で
ある角質層との親和性が増し、生体適合性を向上させる
ことが出来る。[Table 1] Acetylation HA Reaction time Acetylation degree Water solubility Alcohol solubility ──────────────────────────────────── (A) 168 hours 3.1 × ○ (B) 74 2.9 × ○ (C) 32 2.5 △ △ (D) 20 2.0 △ △ (E) 8 1.5 △ × (F) 5 1.2 ○ × ──────────────────高 The high acetylation rate hyaluronic acid obtained as described above has remarkably different physical properties depending on the molecular weight, modification rate, etc. Those having a small degree of modification are expected to be applied as cosmetic bases or bases for drug delivery systems, such as forming more stable gels with the addition of small amounts of organic solvents. Those having a high molecular weight and high acetylation and those having a low molecular weight are soluble in a considerable concentration of an organic solvent and can be easily blended in an emulsion or the like. And when it is blended in an emulsion or the like, various effects can be exhibited, such as increasing smoothness during use. In addition, if lipophilicity is increased by acetylation or the like, affinity with the stratum corneum having a lipid membrane on the surface is increased, and biocompatibility can be improved.
【００１５】また、本発明にかかる高アセチル化ヒアル
ロン酸は、化粧品などに配合された場合にヒアルロン酸
の欠点でもあった曳糸性が著しく低下するという利点を
有する。尚、次の表２に高アセチル化率ヒアルロン酸
（アセチル化度約１．５）の各種溶媒に対する溶解性試
験の結果を示す。Further, the highly acetylated hyaluronic acid according to the present invention has an advantage that when blended in cosmetics or the like, the spinnability, which is a disadvantage of hyaluronic acid, is significantly reduced. Table 2 below shows the results of the solubility test of the high acetylation ratio hyaluronic acid (acetylation degree: about 1.5) in various solvents.
【表２】 [Table 2]
【００１６】[0016]
【発明の効果】以上説明したように本発明に係る高アセ
チル化率ヒアルロン酸は、温和な条件で各種分子量ヒア
ルロン酸にアセチル化率を各繰り返し構造において１以
上の割合で結合させることが出来る。As described above, the hyaluronic acid having a high acetylation rate according to the present invention can be bonded to hyaluronic acid of various molecular weights under a mild condition at a acetylation rate of 1 or more in each repeating structure.
【図１】 実施例１にかかるアセチル化ヒアルロン酸の
紫外部吸収スペクトル説明図である。FIG. 1 is an explanatory diagram of an ultraviolet absorption spectrum of an acetylated hyaluronic acid according to Example 1.
【図２】 実施例２にかかるアセチル化ヒアルロン酸の
赤外吸収スペクトル図である。FIG. 2 is an infrared absorption spectrum of the acetylated hyaluronic acid according to Example 2.
【図３】 実施例５にかかるアセチル化ヒアルロン酸の
反応時間とアセチル化率の時間を示す説明図である。FIG. 3 is an explanatory diagram showing the reaction time of acetylated hyaluronic acid and the time of acetylation rate according to Example 5.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 山口 道広 神奈川県横浜市港北区新羽町1050番地 株式会社 資生堂 第一リサーチセンタ ー内 (72)発明者 秋間 和雄 神奈川県横浜市港北区新羽町1050番地 株式会社 資生堂 第一リサーチセンタ ー内 (58)調査した分野(Int.Cl.7，ＤＢ名) C08B 37/08 ＣＡ（ＳＴＮ)──────────────────────────────────────────────────続 き Continuing on the front page (72) Michihiro Yamaguchi 1050 Nippa-cho, Kohoku-ku, Yokohama, Kanagawa Prefecture Inside the Shiseido First Research Center (72) Kazuo Akima 1050 Nippa-cho, Kohoku-ku, Yokohama, Kanagawa Shiseido Co., Ltd. First Research Center (58) Field surveyed (Int. Cl. 7 , DB name) C08B 37/08 CA (STN)
Claims (3)
ル化率ヒアルロン酸。 【化１】 なお、上記化１において、Ｒ1，Ｒ2，Ｒ3，Ｒ4は水素ま
たはエステル結合されたアセチル基を意味し、且つ平均
すると各繰り返し構造においてＲ1〜Ｒ4の少なくとも１
はアセチル基である。Ｒ5は水素またはアルカリ金属原
子を示す。1. A highly acetylated hyaluronic acid having a structure represented by the following general formula 1. Embedded image In the above formula 1 , R 1 , R 2 , R 3 , and R 4 represent hydrogen or an acetyl group bonded by an ester bond, and on average, at least one of R 1 to R 4 in each repeating structure.
Is an acetyl group. R 5 represents hydrogen or an alkali metal atom.
媒として無水トリフルオロ酢酸を加えて反応させること
を特徴とする高アセチル化率ヒアルロン酸の製造方法。2. A process for producing hyaluronic acid having a high acetylation rate, comprising suspending powdery hyaluronic acid in acetic acid and adding trifluoroacetic anhydride as a catalyst to cause a reaction.
ｐ−トルエンスルホン酸を加え、前記微粉末ヒアルロン
酸を膨潤させ、更に無水酢酸を加えて反応させることを
特徴とする高アセチル化率ヒアルロン酸の製造方法。3. Dispersing powdered hyaluronic acid in acetic acid,
A method for producing hyaluronic acid having a high acetylation rate, comprising adding p-toluenesulfonic acid, swelling the fine powdered hyaluronic acid, and further reacting by adding acetic anhydride.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP05112226A JP3142415B2 (en) | 1992-04-21 | 1993-04-15 | High acetylation rate hyaluronic acid and method for producing the same |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4-128199 | 1992-04-21 | ||
| JP12819992 | 1992-04-21 | ||
| JP05112226A JP3142415B2 (en) | 1992-04-21 | 1993-04-15 | High acetylation rate hyaluronic acid and method for producing the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH069707A JPH069707A (en) | 1994-01-18 |
| JP3142415B2 true JP3142415B2 (en) | 2001-03-07 |
Family
ID=26451449
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP05112226A Expired - Fee Related JP3142415B2 (en) | 1992-04-21 | 1993-04-15 | High acetylation rate hyaluronic acid and method for producing the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3142415B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013146376A1 (en) | 2012-03-27 | 2013-10-03 | テルモ株式会社 | Coating composition and medical device |
Families Citing this family (20)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3563440B2 (en) * | 1994-05-16 | 2004-09-08 | 生化学工業株式会社 | Method for producing acylated hyaluronic acid |
| JP3556975B2 (en) * | 1994-08-11 | 2004-08-25 | 株式会社資生堂 | Production method and purification method of acetylated hyaluronic acid |
| DE69524197T2 (en) * | 1994-08-11 | 2002-08-14 | Shiseido Co. Ltd., Tokio/Tokyo | LOW MOLECULAR WEIGHT ACETYLATED HYALURONIC ACID; SOFTENERS AND METHOD FOR PRODUCING AND PURIFYING THIS ACID |
| JP3634113B2 (en) * | 1997-03-31 | 2005-03-30 | 株式会社資生堂 | Topical skin preparation |
| JPH10279418A (en) * | 1997-03-31 | 1998-10-20 | Shiseido Co Ltd | Preparation for external use for skin |
| JP2004262777A (en) * | 2003-02-27 | 2004-09-24 | Shiseido Co Ltd | Acetylated hyaluronic acid-containing ocular medicinal preparation |
| JP5160040B2 (en) * | 2005-02-28 | 2013-03-13 | 千葉製粉株式会社 | Method for producing sugar fatty acid ester |
| JP5022618B2 (en) * | 2005-04-05 | 2012-09-12 | 株式会社 資生堂 | Hydroxyalkylated hyaluronic acid |
| AU2011270180A1 (en) | 2010-06-23 | 2013-01-24 | Shiseido Company, Ltd. | Oxidative hair dye composition |
| JP5955646B2 (en) * | 2011-09-01 | 2016-07-20 | 株式会社 資生堂 | Hair color pretreatment composition |
| JP6071123B2 (en) | 2012-02-10 | 2017-02-01 | 株式会社 資生堂 | Oil-in-water emulsified skin cosmetic |
| CN104550279A (en) * | 2014-11-26 | 2015-04-29 | 南京钢铁股份有限公司 | De-scaling method for steel plate at TMCP delivery state |
| JP2016163696A (en) * | 2015-02-27 | 2016-09-08 | 大日精化工業株式会社 | Medical material and adhesion prevention material |
| JP6474360B2 (en) * | 2015-02-27 | 2019-02-27 | 大日精化工業株式会社 | Manufacturing method of medical material, medical material, and anti-adhesion material |
| GB201703850D0 (en) * | 2017-03-10 | 2017-04-26 | Givaudan Sa | Improvements in or relating to organic compounds |
| KR102371689B1 (en) * | 2017-04-26 | 2022-03-07 | 주식회사 엘지생활건강 | A manufacturing method of acetylation hyaluronic acid comprising activation process |
| CN113045686B (en) * | 2021-03-03 | 2022-09-23 | 杭州华玮生物科技有限公司 | Preparation method of acetylated hyaluronic acid |
| CN113150184B (en) * | 2021-04-14 | 2022-12-06 | 浙江理工大学 | Method for preparing acetylated sodium hyaluronate |
| CN113121721B (en) * | 2021-04-21 | 2022-02-15 | 山东焦点福瑞达生物股份有限公司 | Preparation method of acetylated sodium hyaluronate |
| CN115975070B (en) * | 2022-12-18 | 2023-11-14 | 南京乐韬生物科技有限公司 | Preparation method of acetylated hyaluronate |
-
1993
- 1993-04-15 JP JP05112226A patent/JP3142415B2/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013146376A1 (en) | 2012-03-27 | 2013-10-03 | テルモ株式会社 | Coating composition and medical device |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH069707A (en) | 1994-01-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3142415B2 (en) | High acetylation rate hyaluronic acid and method for producing the same | |
| RU2230073C2 (en) | Method for cross-linking carboxylated polysaccharides | |
| KR100674177B1 (en) | Cross-linked hyaluronic acids and medical uses thereof | |
| US5652347A (en) | Method for making functionalized derivatives of hyaluronic acid | |
| Luo et al. | Cross-linked hyaluronic acid hydrogel films: new biomaterials for drug delivery | |
| AU773628B2 (en) | Process for cross-linking hyaluronic acid to polymers | |
| DE69232790T2 (en) | WATER-INSOLUBLE DERIVATIVES OF POLYANIONIC POLYSACCHARIDES | |
| CA2621899C (en) | Aryl/alkyl succinic anhydride hyaluronan derivatives | |
| JP4460617B2 (en) | Swellable crosslinked hyaluronic acid powder and method for producing the same | |
| EP1493754B1 (en) | Polysaccharide containing phosphorylcholine group and process for producing the same | |
| AU2007296939A1 (en) | Hyaluronic acid derivatives obtained via "click chemistry" crosslinking | |
| JP2012510535A (en) | Method for producing hyaluronic acid functionalized derivatives and formation of hydrogels thereof | |
| HUE031496T2 (en) | Hyaluronic acid derivative, method of preparation thereof, method of modification thereof and use thereof | |
| WO1996005233A1 (en) | Low-molecular-weight acetylated hyaluronic acid, emollient, and processes for producing and purifying the acid | |
| KR101571385B1 (en) | Mixed butyric-formic esters of acid polysaccharides, and their preparation and use as skin cosmetics | |
| ES2261681T3 (en) | NEW DERIVATIVES OF THE HIALURONAN. | |
| JP3282729B2 (en) | Self-crosslinked gellan gum | |
| Mocanu et al. | Associative pullulan gels and their interaction with biological active substances | |
| JP3556975B2 (en) | Production method and purification method of acetylated hyaluronic acid | |
| JP2003522255A (en) | Gel of hyaluronic acid cross-linked with bifunctional L-amino acid or L-amino ester or a mixture thereof | |
| JPH0971602A (en) | Low molecular weight acetylated hyaluronic acid and emollient | |
| EP0603264A1 (en) | Derivatives of carboxyl group containing polysaccharides | |
| Jabeen et al. | Crosslinking of alginic acid/chitosan matrices using bis phenol-F-diglycidyl ether: mechanical, thermal and water absorption investigation | |
| JP4958368B2 (en) | Cross-linked hyaluronic acid | |
| JPH05301904A (en) | Polysaccharide and water-absorbing, hygroscopic, moisture-retaining and thickening agent composed mainly of the polysaccharide |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20001205 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20071222 Year of fee payment: 7 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20081222 Year of fee payment: 8 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20081222 Year of fee payment: 8 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20091222 Year of fee payment: 9 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20101222 Year of fee payment: 10 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111222 Year of fee payment: 11 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20111222 Year of fee payment: 11 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20121222 Year of fee payment: 12 |
|
| LAPS | Cancellation because of no payment of annual fees |