JP2009155486A - Depolymerized hyaluronic acid and production method thereof - Google Patents
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本発明は、低分子化ヒアルロン酸とその塩及びその製造方法に関する。 The present invention relates to a low molecular weight hyaluronic acid, a salt thereof, and a production method thereof.
ヒアルロン酸は、生体に存在するムコ多糖であり、化粧料、医薬品及び飲食品などの分野において広く利用されている。医薬品においては関節痛緩和効果が、化粧料においては肌の保湿効果が、飲食品においては肌の改善効果が期待されている。 Hyaluronic acid is a mucopolysaccharide present in the living body and is widely used in the fields of cosmetics, pharmaceuticals, foods and drinks, and the like. Expected to have a joint pain relieving effect in pharmaceuticals, a skin moisturizing effect in cosmetics, and a skin improving effect in foods and beverages.
従来、ヒアルロン酸は、鶏の鶏冠からの抽出、又は微生物を培養して培養液より採取する方法により工業的に製造されている。しかし、これらのヒアルロン酸は、高分子量であるために、溶液中に高濃度に溶解させることができない。そのため化粧料として肌に使用する際には肌がつっぱる、医薬品や飲食品において使用する際には吸収性が悪いなどの問題がある。しかしながら、分子量を１０，０００以下にまで低分子化することにより、腸管膜吸収性が飛躍的に向上することが示されている（例えば、非特許文献１参照）。 Conventionally, hyaluronic acid has been industrially produced by extraction from a chicken's crown or by culturing microorganisms and collecting them from a culture solution. However, since these hyaluronic acids have a high molecular weight, they cannot be dissolved in a high concentration in the solution. Therefore, there is a problem that the skin becomes tight when used on the skin as a cosmetic, and the absorbability is poor when used in medicines and foods and drinks. However, it has been shown that by reducing the molecular weight to 10,000 or less, the intestinal membrane absorbability is dramatically improved (see, for example, Non-Patent Document 1).
ヒアルロン酸を低分子化する方法として、水溶液中で酸分解する方法や、酵素分解する方法などが報告されている（例えば、特許文献１〜３参照）。しかし、分子量１０，０００以下まで低分子化する場合、溶液中での酸分解ではヒアルロン酸が褐変してしまう、あるいは酵素分解ではコストが高くなるという問題がある。また、報告された上記の方法においては、ヒアルロン酸の分子量や色にのみ注目し、その分子構造の変性に注意が払われていない（例えば、特許文献１〜３参照）。
As a method for reducing the molecular weight of hyaluronic acid, a method of acid decomposition in an aqueous solution, a method of enzymatic decomposition, and the like have been reported (for example, see
本発明は、ヒアルロン酸の褐変を生じずに、工業的な生産に好適である低分子化ヒアルロン酸又はその塩を製造する方法を提供する。 The present invention provides a method for producing low molecular weight hyaluronic acid or a salt thereof suitable for industrial production without causing browning of hyaluronic acid.
本発明者は、分子量や色だけでなく、その分子構造についても考慮しながら鋭意検討を行い、ある特定の酸濃度と温度において、ヒアルロン酸を変性させることなく、低分子化ヒアルロン酸を製造できることを見出し、本発明を完成した。
すなわち本発明は、以下に関する。
（１）０．１〜０．６Ｎの酸濃度の条件下、５５℃以上９５℃以下の温度条件でヒアルロン酸又はその塩を低分子化することにより得られる、２〜６量体のヒアルロン酸の含有率が１０％以上である低分子化ヒアルロン酸。
（２）平均分子量が１０，０００以下である上記（１）に記載の低分子化ヒアルロン酸。
（３）ヒアルロン酸又はその塩に、０．１〜０．６Ｎの酸を添加し、５５℃以上９５℃以下の条件で、ヒアルロン酸又はその塩を低分子化することを特徴とする、低分子化ヒアルロン酸の製造方法。
The present inventor has conducted intensive studies while considering not only the molecular weight and color but also the molecular structure thereof, and can produce low molecular weight hyaluronic acid at a specific acid concentration and temperature without denaturing hyaluronic acid. The present invention has been completed.
That is, the present invention relates to the following.
(1) 2 to 6-mer hyaluronic acid obtained by reducing the molecular weight of hyaluronic acid or a salt thereof under a temperature condition of 55 ° C. or higher and 95 ° C. or lower under an acid concentration of 0.1 to 0.6N Low molecular weight hyaluronic acid having a content of 10% or more.
(2) The low molecular weight hyaluronic acid according to (1), wherein the average molecular weight is 10,000 or less.
(3) Add 0.1 to 0.6N acid to hyaluronic acid or a salt thereof, and reduce the molecular weight of hyaluronic acid or a salt thereof under the condition of 55 ° C or higher and 95 ° C or lower. A method for producing molecular hyaluronic acid.
本発明によれば、高分子のヒアルロン酸を低分子化処理しても、変色や分子構造の変性を生じることなく、安定かつ安価に効率よく低分子化ヒアルロン酸又はその塩を製造することができる。 According to the present invention, low molecular weight hyaluronic acid or a salt thereof can be produced stably and inexpensively and efficiently without causing discoloration or modification of molecular structure even when polymer hyaluronic acid is subjected to low molecular weight treatment. it can.
本発明の低分子化ヒアルロン酸は、０．１〜０．６Ｎの酸濃度の条件下、５５℃以上９５℃以下の温度条件でヒアルロン酸又はその塩を低分子化することを特徴とする。
本発明における温度としては、５５℃以上９５℃以下、好ましくは６０℃以上８０℃以下であることが好ましい。５５℃未満では、低分子化の反応が効率よく進まず、９５℃を超える条件下では、褐変が進んでしまうためである。
本発明に用いる酸の種類としては、例えば、塩酸、硫酸若しくはリン酸から選択される１種又は２種以上の酸を使用することができる。
本発明の低分子化に要する時間は、任意であるが、例えば、目的の温度に達してから１〜４８時間であることが好ましい。
The low molecular weight hyaluronic acid of the present invention is characterized by reducing the molecular weight of hyaluronic acid or a salt thereof under a temperature condition of 55 ° C. or higher and 95 ° C. or lower under an acid concentration of 0.1 to 0.6N.
As temperature in this invention, it is 55 to 95 degreeC, Preferably it is 60 to 80 degreeC. When the temperature is lower than 55 ° C., the reaction for reducing the molecular weight does not proceed efficiently, and when the temperature exceeds 95 ° C., the browning proceeds.
As a kind of acid used for this invention, the 1 type (s) or 2 or more types of acid selected from hydrochloric acid, a sulfuric acid, or phosphoric acid can be used, for example.
The time required for the molecular weight reduction of the present invention is arbitrary, but for example, it is preferably 1 to 48 hours after reaching the target temperature.
本発明において、「ヒアルロン酸」とは、Ｎ−アセチルグルコサミンとグルクロン酸がつながった糖を基本単位とし、その基本単位又は基本単位が繰り返し重合したものをいう。また、本発明において、「ヒアルロン酸塩」とは、薬学上許容し得る全てのヒアルロン酸塩を意味し、例えば、ナトリウム塩、カリウム塩、マグネシウム塩、カルシウム塩、アンモニウム塩などが挙げられる。
本発明の「濃度」と表記した部分について、特に記載がない限りは、溶液の容量に対する重量とする。
In the present invention, “hyaluronic acid” refers to a sugar in which N-acetylglucosamine and glucuronic acid are connected as a basic unit, and the basic unit or the basic unit is repeatedly polymerized. In the present invention, “hyaluronic acid salt” means all pharmaceutically acceptable hyaluronic acid salts such as sodium salt, potassium salt, magnesium salt, calcium salt, ammonium salt and the like.
Unless otherwise specified, the portion indicated as “concentration” in the present invention is the weight relative to the volume of the solution.
本発明の低分子ヒアルロン酸及びその塩の原料であるヒアルロン酸又はその塩の製法としては、特に限定されるものではないが、例えば、鶏冠、皮膚、関節、眼球などの動物組織、又はストレプトコッカス属の微生物若しくはその培養液などを原料として抽出又は精製して得られる。 The method for producing hyaluronic acid or a salt thereof, which is a raw material of the low molecular weight hyaluronic acid and the salt thereof of the present invention, is not particularly limited. For example, animal tissue such as chicken crown, skin, joint, eyeball, or Streptococcus genus It is obtained by extracting or purifying the microorganism or its culture solution as a raw material.
本発明における低分子化ヒアルロン酸の分子量は、平均分子量１０，０００以下、好ましくは平均分子量３，０００以下であり、その平均分子量は、Ｍｏｒｇａｎ−Ｅｌｓｏｎ法とＣａｒｂａｚｏｌ硫酸法の組み合わせなどにより求めることができる。
Ｍｏｒｇａｎ−Ｅｌｓｏｎ法とは、ＤＭＡＢ（ｐ−Ｄｉｍｅｔｈｙｌａｍｉｎｏｂｅｎｚａｌｄｅｈｙｄｅ）標識による発色により還元末端のＮ−アセチルグルコサミン（以下、「末端ＮＡＧ」と表記する）を定量するものである（Ｂｉｏｃｈｉｍｉｃａ Ｅｔ Ｂｉｏｐｈｙｓｉｃａ Ａｃｔａ ４２（１９６０） ｐ４７６−４８５参照）。
Ｃａｒｂａｚｏｌ硫酸法は、Ｃａｒｂａｚｏｌ標識による発色により、ヒアルロン酸に含まれる全グルクロン酸量（以下、「全ＧｌｃＵＡ」と表記する）を定量するものである。
The molecular weight of the low molecular weight hyaluronic acid in the present invention is 10,000 or less, preferably 3,000 or less, and the average molecular weight can be determined by a combination of the Morgan-Elson method and the Carbazol sulfuric acid method. it can.
The Morgan-Elson method is a method for quantifying reducing N-acetylglucosamine (hereinafter referred to as “terminal NAG”) by color development using DMAB (p-dimethylaminobenzaldehyde) labeling (Biochimica Et Biophysica Acta 42 (1960). p476-485).
The Carbazol sulfuric acid method quantifies the total amount of glucuronic acid contained in hyaluronic acid (hereinafter referred to as “total GlcUA”) by color development using Carbazol labeling.
Ｍｏｒｇａｎ−Ｅｌｓｏｎ法により求められた「末端ＮＡＧ」のモル濃度は、ヒアルロン酸糖鎖のモル濃度に相当する。また、Ｃａｒｂａｚｏｌ硫酸法により求められた「全ＧｌｃＵＡ」のモル濃度は、ＮＡＧとＧｌｃＵＡがβグルコシド結合した、ヒアルロン酸の基本ユニットのモル濃度に相当する。下記の式の通り、ヒアルロン酸の平均分子量（ナトリウム塩として）はこれらの値を使って求めることができる。
平均分子量 ＝ 平均重合度／２ × ４０１．３
＝ ヒアルロン酸ユニット数／糖鎖数 × ４０１．３
＝ 全ＧｌｃＵＡ濃度（Ｍ）／末端ＮＡＧ濃度（Ｍ） × ４０１．３
The molar concentration of “terminal NAG” determined by the Morgan-Elson method corresponds to the molar concentration of the hyaluronic acid sugar chain. The molar concentration of “total GlcUA” determined by the Carbazol sulfuric acid method corresponds to the molar concentration of the basic unit of hyaluronic acid in which NAG and GlcUA are β-glucoside-bonded. The average molecular weight (as sodium salt) of hyaluronic acid can be determined using these values as shown in the following formula.
Average molecular weight = average degree of polymerization / 2 x 401.3
= Number of hyaluronic acid units / number of sugar chains x 401.3
= Total GlcUA concentration (M) / terminal NAG concentration (M) x 401.3
本発明における低分子化ヒアルロン酸の２量体、４量体及び６量体のヒアルロン酸の含有率は、１０％以上、好ましくは２０％以上であることが好ましい。
また、２量体、４量体及び６量体のヒアルロン酸の含有率は、ゲルろ過カラムを用いたＨＰＬＣ法（カラム：ＧＬ−Ｗ５２０−Ｓ（日立化成社製）、溶離液等張リン酸緩衝液（ｐＨ７．１）、流速：０．４ｍｌ／ｍｉｎ検出；２０６ｎｍ、カラム温度：４０℃）により得られるチャートにおいて、２量体、４量体及び６量体におけるピークの面積を、全てのピークの面積で除することにより求めることができる。
また、２量体、４量体及び６量体のピークの同定は、各ピークを分取し、質量分析計で分析することにより行うことができる。
In the present invention, the dimer, tetramer and hexamer hyaluronic acid content of the low molecular weight hyaluronic acid is 10% or more, preferably 20% or more.
The dimer, tetramer and hexamer hyaluronic acid contents are determined by HPLC using a gel filtration column (column: GL-W520-S (manufactured by Hitachi Chemical Co., Ltd.), eluent isotonic phosphoric acid. In the chart obtained by the buffer solution (pH 7.1), flow rate: 0.4 ml / min detection; 206 nm, column temperature: 40 ° C.), the peak areas in the dimer, tetramer and hexamer are all It can be obtained by dividing by the area of the peak.
In addition, identification of dimer, tetramer and hexamer peaks can be carried out by fractionating each peak and analyzing it with a mass spectrometer.
なお、本発明により生成した低分子化ヒアルロン酸又はその塩は、スプレードライ、凍結乾燥などにより粉末化することができる。また、得られた低分子化ヒアルロン酸又はその塩は、有機溶媒による晶析などにより精製することができるが、その精製方法は、任意に選ぶことができ、以下の実施例に制限されるものではない。 The low molecular weight hyaluronic acid or salt thereof produced by the present invention can be pulverized by spray drying, freeze drying or the like. The obtained low molecular weight hyaluronic acid or a salt thereof can be purified by crystallization using an organic solvent, but the purification method can be arbitrarily selected and is limited to the following examples. is not.
（塩酸を用いた酸加熱処理１）
（１）酸加熱処理による分子量の変化
粉末状のヒアルロン酸（以下、「ＦＣＨ−Ａ」という。紀文フードケミファ社製、分子量２０，８１６）を、６．２５％（ｗ／ｗ）の濃度で溶解させ、さらに種々の濃度の塩酸を１／１０量加え、塩酸の終濃度が０．１、０．２５、１．０Ｎのいずれかになるようにした。この溶液を６０℃、７０℃、８０℃のいずれかで、１８時間処理した。処理後、水酸化ナトリウムを加えて中和し、凍結乾燥により粉末化した。低分子化ヒアルロン酸水溶液の平均分子量を糖鎖数定量法により測定した。結果を図１に示す。
(Acid heat treatment with hydrochloric acid 1)
(1) Change in molecular weight by acid heat treatment Powdered hyaluronic acid (hereinafter referred to as “FCH-A”, manufactured by Kibun Food Chemifa Corporation, molecular weight 20,816) at a concentration of 6.25% (w / w) Further, 1/10 amount of hydrochloric acid having various concentrations was added to make the final concentration of hydrochloric acid 0.1, 0.25, or 1.0N. This solution was treated at 60 ° C., 70 ° C., or 80 ° C. for 18 hours. After the treatment, sodium hydroxide was added to neutralize, and pulverized by freeze drying. The average molecular weight of the low molecular weight hyaluronic acid aqueous solution was measured by the sugar chain number determination method. The results are shown in FIG.
０．１Ｎ塩酸、８０℃、又は０．２５Ｎ塩酸、７０℃で処理したサンプルでは、平均分子量３，０００以下にまで低分子化していた。サンプルの分子量分布を、ゲルろ過ＨＰＬＣ（カラム：Ｇ６０００ＰＷＸＬ（７．８×３００ｍｍ）＋Ｇ４０００ＰＷＸＬ（７．８×３００ｍｍ）＋Ｇ２５００ＰＷＸＬ（７．８×３００ｍｍ）（東ソー社製）、流速：１ｍｌ／ｍｉｎ、溶離液：０．２Ｍ ＮａＣｌ、検出：２１０ｎｍ、カラム温度：３０℃）で測定した。 In samples treated with 0.1N hydrochloric acid, 80 ° C., or 0.25N hydrochloric acid, 70 ° C., the molecular weight was reduced to an average molecular weight of 3,000 or less. The molecular weight distribution of the sample was determined by gel filtration HPLC (column: G6000PWXL (7.8 × 300 mm) + G4000PWXL (7.8 × 300 mm) + G2500PWXL (7.8 × 300 mm) (manufactured by Tosoh Corporation), flow rate: 1 ml / min, eluent : 0.2 M NaCl, detection: 210 nm, column temperature: 30 ° C.).
図２に示すように、ピークが一つである分子量分布であり、エンド型の分解が起こっていることがわかった。それに対して、１Ｎ塩酸で処理したサンプルでは、複数のピークがみられる分子量分布であり、ランダムなエンド型分解ではない分解が起こっていることが示唆された。また、これらのサンプルでは酢酸様の匂いがあり、化粧料、医薬品及び飲食品として利用するには不適なものであった。 As shown in FIG. 2, it was a molecular weight distribution with one peak, and it was found that end-type decomposition occurred. On the other hand, the sample treated with 1N hydrochloric acid has a molecular weight distribution in which a plurality of peaks are observed, suggesting that decomposition is not random end-type decomposition. In addition, these samples had an acetic acid-like odor and were unsuitable for use as cosmetics, pharmaceuticals, and foods and drinks.
（２）白色度の検討
上記（１）の酸加熱処理により得られた低分子化ヒアルロン酸粉末の白色度を、測色色差計（日本電色社製、Ｚ−１００１ＤＰ）を用いて測定した。
０．２５Ｎ塩酸、７０℃で処理したサンプルの白色度（Ｌ値）は、９５．０６であった。
(2) Examination of whiteness The whiteness of the low molecular weight hyaluronic acid powder obtained by the acid heat treatment of (1) above was measured using a colorimetric color difference meter (Nippon Denshoku Co., Ltd., Z-1001DP). .
The whiteness (L value) of the sample treated with 0.25N hydrochloric acid at 70 ° C. was 95.06.
（３）分子構造の変性について
低分子化ヒアルロン酸の分析を、陰イオン交換カラムを用いたＨＰＬＣ（カラム；ＴＳＫ−ｇｅｌ Ｓｕｐｅｒ−Ｑ（東ソー社製）、溶離液；０−２００ｍＭ（０−８０分）ＮａＣｌ、カラム温度；４０℃、検出；２１０ｎｍ）により実施した。０．２５Ｎ、７０℃での酸加熱処理により得られた低分子化ヒアルロン酸について測定したところ、図３のようなチャートが得られた。
(3) Modification of molecular structure Analysis of low molecular weight hyaluronic acid was performed by HPLC using an anion exchange column (column; TSK-gel Super-Q (manufactured by Tosoh Corporation), eluent: 0-200 mM (0-80). Min) NaCl, column temperature; 40 ° C., detection; 210 nm). When the low molecular weight hyaluronic acid obtained by acid heat treatment at 0.25 N and 70 ° C. was measured, a chart as shown in FIG. 3 was obtained.
さらにピーク画分を分取し、脱塩した後に、ＥＳＩ−ＴＯＦ−ＭＳ（ＡｐｐｌｉｅｄＢｉｏｓｙｓｔｅｍｓ社製・ＱＳＴＡＲ Ｅｌｉｔｅ）により質量分析を行った結果、グルクロン酸とＮ−アセチルグルコサミンから構成される２糖単位のヒアルロン酸に相当する値が得られた。 Further, after fractionating and desalting the peak fraction, mass spectrometry was performed by ESI-TOF-MS (Applied Biosystems QSTAR Elite). As a result, a disaccharide unit composed of glucuronic acid and N-acetylglucosamine was obtained. A value corresponding to hyaluronic acid was obtained.
上記のＨＰＬＣにおいて、Ｎ−アセチル基の脱離等、分子構造の変性を示すピークは極めて少なく、１％以下であった。このことから、本発明で得られる低分子化ヒアルロン酸は、ヒアルロン酸の基本単位である２糖の加水分解により生じており、Ｎ−アセチル基の脱離等、変性したヒアルロン酸は極めて少ないことが確認された。 In the above-mentioned HPLC, the peak indicating the modification of the molecular structure such as elimination of the N-acetyl group was extremely small and was 1% or less. From this, the low molecular weight hyaluronic acid obtained in the present invention is produced by hydrolysis of disaccharide, which is a basic unit of hyaluronic acid, and there is very little modified hyaluronic acid such as elimination of N-acetyl group. Was confirmed.
（４）２量体、４量体及び６量体の含有量
酸加熱処理を施したヒアルロン酸中の、２量体、４量体及び６量体のヒアルロン酸の含有量を、ゲルろ過クロマトグラフィー（カラム：ＧＬ−Ｗ５２０−Ｓ（日立化成社製）、溶離液等張リン酸緩衝液（ｐＨ７．１）、流速：０．４ｍｌ／ｍｉｎ検出；２０６ｎｍ、カラム温度：４０℃）で分析した。その結果、０．２５Ｎ、７０℃で１８時間処理したサンプルでは、２量体、４量体及び６量体の含有量は、２８．９％であった（図４）。
(4) Content of dimer, tetramer and hexamer The content of dimer, tetramer and hexamer hyaluronic acid in hyaluronic acid subjected to acid heat treatment was analyzed by gel filtration chromatography. Analysis by chromatography (column: GL-W520-S (manufactured by Hitachi Chemical Co., Ltd.), eluent isotonic phosphate buffer (pH 7.1), flow rate: 0.4 ml / min detection; 206 nm, column temperature: 40 ° C.) . As a result, in the sample treated at 0.25 N and 70 ° C. for 18 hours, the content of the dimer, tetramer and hexamer was 28.9% (FIG. 4).
（塩酸を用いた酸加熱処理２）
粉末状のＦＣＨ−Ａを、６．２５％（ｗ／ｗ）の濃度で溶解させ、さらに種々の濃度の塩酸を１／１０量加え、塩酸の終濃度が０．３、０．４、又は０．６Ｎのいずれかになるようにした。
上記溶液を７０℃、７５℃、８０℃のいずれかの温度条件で、４時間処理した。処理後、水酸化ナトリウムを加えて中和し、凍結乾燥により粉末化した。実施例１と同様の分析で、これらの条件下でも低分子化が起こることを確認した（図５）。
(Acid heat treatment 2 using hydrochloric acid)
Powdered FCH-A is dissolved at a concentration of 6.25% (w / w), and 1/10 amount of hydrochloric acid of various concentrations is further added, so that the final concentration of hydrochloric acid is 0.3, 0.4, or It was set to either 0.6N.
The solution was treated for 4 hours under any temperature condition of 70 ° C, 75 ° C, or 80 ° C. After the treatment, sodium hydroxide was added to neutralize, and pulverized by freeze drying. In the same analysis as in Example 1, it was confirmed that a decrease in molecular weight occurred even under these conditions (FIG. 5).
（硫酸を用いた酸加熱処理の検討）
粉末状のＦＣＨ−Ａを、６．２５％（ｗ／ｗ）の濃度で溶解させ、さらに終濃度が０．１Ｎになるように硫酸を１／１０量加え、７０℃で１８時間処理した。得られた処理物の分子量は、１，５６１であり、ゲルろ過分析での分子量分布は単一のピークを示した（図６）。
硫酸処理後、水酸化ナトリウムを加えて中和し、凍結乾燥により粉末化した。
また、実施例１と同様の陰イオン交換カラムを用いたＨＰＬＣにより、塩酸を用いた酸加熱処理と同様の、２糖単位の加水分解が起こっていることを確認した（図６）。
（比較例１）
(Examination of acid heat treatment using sulfuric acid)
Powdered FCH-A was dissolved at a concentration of 6.25% (w / w), and 1/10 amount of sulfuric acid was further added to a final concentration of 0.1 N, followed by treatment at 70 ° C. for 18 hours. The molecular weight of the obtained processed product was 1,561, and the molecular weight distribution in gel filtration analysis showed a single peak (FIG. 6).
After the sulfuric acid treatment, the mixture was neutralized by adding sodium hydroxide and powdered by freeze drying.
Moreover, it was confirmed by HPLC using the same anion exchange column as in Example 1 that hydrolysis of a disaccharide unit occurred as in the acid heat treatment using hydrochloric acid (FIG. 6).
(Comparative Example 1)
（水酸化ナトリウムを用いたヒアルロン酸の分解）
ＦＣＨ−Ａを、６．２５％で溶解させ、さらに終濃度が０．１Ｎとなるように水酸化ナトリウム溶液を１／１０量加え、７０℃又は４３℃の温度条件で１８時間処理した。７０℃の温度条件で処理したサンプルは、溶液が褐色に変化していた。４３℃の温度条件で処理したサンプルは、褐変していなかったものの、ゲルろ過ＨＰＬＣでの分析で、複数のピークがみられ、非特異的なヒアルロン酸の分解が起こっていることが推察された（図７）。
（比較例２）
(Decomposition of hyaluronic acid using sodium hydroxide)
FCH-A was dissolved at 6.25%, and a 1/10 amount of sodium hydroxide solution was added to a final concentration of 0.1N, followed by treatment at 70 ° C or 43 ° C for 18 hours. In the sample treated under the temperature condition of 70 ° C., the solution turned brown. Although the sample treated under the temperature condition of 43 ° C. was not browned, the analysis by gel filtration HPLC showed a plurality of peaks, which suggests that non-specific decomposition of hyaluronic acid occurred. (FIG. 7).
(Comparative Example 2)
（過酸化水素を用いた分解）
ＦＣＨ−Ａを、６．２５％で溶解させ、さらに終濃度が０．１Ｍとなるように過酸化水素液を１／１０量加え、７０℃で１８時間処理した。分子量は、１，５０４であり、ゲルろ過ＨＰＬＣでの分析で、単一のピークがみられたが、陰イオンクロマトグラフィー分析で、２糖単位ではなくランダムなヒアルロン酸の分解が起こっていることが示唆される結果が得られた（図８）。
(Decomposition using hydrogen peroxide)
FCH-A was dissolved at 6.25%, and a hydrogen peroxide solution was added in an amount of 1/10 so that the final concentration was 0.1M, followed by treatment at 70 ° C. for 18 hours. The molecular weight was 1,504, and a single peak was observed in the gel filtration HPLC analysis. However, in the anion chromatography analysis, random hyaluronic acid was decomposed instead of a disaccharide unit. The result which suggested was obtained (FIG. 8).
Claims (3)
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6279790A (en) * | 1985-09-30 | 1987-04-13 | Toyo Jozo Co Ltd | Production of modified hyaluronic acid |
| JPS62292710A (en) * | 1986-06-12 | 1987-12-19 | Lion Corp | Skin cosmetic |
| JPS6357602A (en) * | 1986-08-29 | 1988-03-12 | Q P Corp | Production of low-molecular hyaluronic acid |
| US6018035A (en) * | 1997-07-21 | 2000-01-25 | Baxter International Inc. | Reagents for isotropic size enhancement of a peptide, protein, nucleotide or other substrate |
| JP2001081103A (en) * | 1999-09-13 | 2001-03-27 | Denki Kagaku Kogyo Kk | Hyaluronic acid-bound medicinal agent |
| JP2004043645A (en) * | 2002-07-11 | 2004-02-12 | Seikagaku Kogyo Co Ltd | Method for producing oligomerized saccharide |
| JP2006265287A (en) * | 2005-03-22 | 2006-10-05 | Q P Corp | Low molecular weight hyaluronic acid and/or its salt, its manufacturing process and cosmetic and food composition containing it |
| JP2009051877A (en) * | 2007-08-23 | 2009-03-12 | Q P Corp | New hyaluronic acid and/or salt thereof, food composition and oral medicinal composition using the same, and oral skin ameliorating agent or oral skin water content-increasing agent comprising the hyaluronic acid and/or the salt thereof as active ingredient |
-
2007
- 2007-12-27 JP JP2007335626A patent/JP2009155486A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6279790A (en) * | 1985-09-30 | 1987-04-13 | Toyo Jozo Co Ltd | Production of modified hyaluronic acid |
| JPS62292710A (en) * | 1986-06-12 | 1987-12-19 | Lion Corp | Skin cosmetic |
| JPS6357602A (en) * | 1986-08-29 | 1988-03-12 | Q P Corp | Production of low-molecular hyaluronic acid |
| US6018035A (en) * | 1997-07-21 | 2000-01-25 | Baxter International Inc. | Reagents for isotropic size enhancement of a peptide, protein, nucleotide or other substrate |
| JP2001081103A (en) * | 1999-09-13 | 2001-03-27 | Denki Kagaku Kogyo Kk | Hyaluronic acid-bound medicinal agent |
| JP2004043645A (en) * | 2002-07-11 | 2004-02-12 | Seikagaku Kogyo Co Ltd | Method for producing oligomerized saccharide |
| JP2006265287A (en) * | 2005-03-22 | 2006-10-05 | Q P Corp | Low molecular weight hyaluronic acid and/or its salt, its manufacturing process and cosmetic and food composition containing it |
| JP2009051877A (en) * | 2007-08-23 | 2009-03-12 | Q P Corp | New hyaluronic acid and/or salt thereof, food composition and oral medicinal composition using the same, and oral skin ameliorating agent or oral skin water content-increasing agent comprising the hyaluronic acid and/or the salt thereof as active ingredient |
Cited By (13)
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|---|---|---|---|---|
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| CN102648214A (en) * | 2009-12-08 | 2012-08-22 | 丘比株式会社 | Method for manufacturing purified hyaluronic acids |
| US8829180B2 (en) | 2009-12-08 | 2014-09-09 | Kewpie Corporation | Method of purifying a low molecular weight hyaluronic acid or cationized hyaluronic acid via precipitation from aqueous solution by addition of alcohol or acetone followed by ph adjustment |
| JP2015507070A (en) * | 2012-02-17 | 2015-03-05 | キムフレクサー エセ．エレ． | Partially depolymerized glycosaminoglycan silver and gold salts |
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| JP5824599B1 (en) * | 2014-01-14 | 2015-11-25 | キユーピー株式会社 | Method for producing hyaluronic acid and / or salt thereof |
| WO2015108029A1 (en) | 2014-01-14 | 2015-07-23 | キユーピー株式会社 | Hyaluronic acid and/or salt thereof, method for producing same, and food, cosmetic, and pharmaceutical containing said hyaluronic acid and/or salt thereof |
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