JP7527011B2 - Method for measuring proteoglycan content - Google Patents
Method for measuring proteoglycan content Download PDFInfo
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- JP7527011B2 JP7527011B2 JP2021083758A JP2021083758A JP7527011B2 JP 7527011 B2 JP7527011 B2 JP 7527011B2 JP 2021083758 A JP2021083758 A JP 2021083758A JP 2021083758 A JP2021083758 A JP 2021083758A JP 7527011 B2 JP7527011 B2 JP 7527011B2
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- JP
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- Prior art keywords
- proteoglycan
- buffer
- ion exchange
- exchange resin
- solution
- Prior art date
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- 238000010494 dissociation reaction Methods 0.000 description 1
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- KXCLCNHUUKTANI-RBIYJLQWSA-N keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 description 1
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- CSSYQJWUGATIHM-IKGCZBKSSA-N l-phenylalanyl-l-lysyl-l-cysteinyl-l-arginyl-l-arginyl-l-tryptophyl-l-glutaminyl-l-tryptophyl-l-arginyl-l-methionyl-l-lysyl-l-lysyl-l-leucylglycyl-l-alanyl-l-prolyl-l-seryl-l-isoleucyl-l-threonyl-l-cysteinyl-l-valyl-l-arginyl-l-arginyl-l-alanyl-l-phenylal Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 CSSYQJWUGATIHM-IKGCZBKSSA-N 0.000 description 1
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- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
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Images
Landscapes
- Peptides Or Proteins (AREA)
Description
本発明は、サプリメントなどの飲食品等の組成物に含有されているプロテオグリカンを簡便に測定する方法（例えば、コラーゲンやゼラチンなどの高分子タンパク質と共に組成物に含まれるプロテオグリカン含有量の測定方法）、などに関する。 The present invention relates to a method for easily measuring the proteoglycan contained in a composition such as a food or beverage, such as a supplement (e.g., a method for measuring the proteoglycan content contained in a composition together with a high molecular weight protein such as collagen or gelatin), etc.
プロテオグリカンは、コラーゲンやヒアルロン酸と共に細胞外マトリックス中の基質を形成する主要な生体高分子である。ヒトや牛などの哺乳動物、鮭や鮫など魚類の軟骨に含まれていることが知られており、プロテオグリカンを含むサプリメントなどの食品、プロテオグリカンの製造方法等が数多く報告されている。また、プロテオグリカン自体の有用性も数多く報告されており、プロテオグリカンは軟骨分化促進作用やひざ関節改善作用、皮膚色素沈着抑制作用など報告されている（特許文献１参照）。 Proteoglycans are major biopolymers that form the substrate in the extracellular matrix together with collagen and hyaluronic acid. They are known to be contained in the cartilage of mammals such as humans and cows, and fish such as salmon and sharks, and many foods such as supplements containing proteoglycans and methods for producing proteoglycans have been reported. There have also been many reports on the usefulness of proteoglycans themselves, and proteoglycans have been reported to promote cartilage differentiation, improve knee joints, and inhibit skin pigmentation (see Patent Document 1).
上述のように、プロテオグリカンは複合体であり、サプリメントなどの飲食品等の組成物に含有されているプロテオグリカンの含有量を正確に測定することは難しい。そのため、組成物に含まれているプロテオグリカンの含有量について、未だその測定技術が確立されていないのが現実である。例えば、カルバゾール硫酸法は、プロテオグリカン糖鎖の主成分のひとつであるウロン酸の呈色反応を利用した定量法であり、サンプル水溶液にカルバゾール溶液と濃硫酸を添加すると、サンプル水溶液にウロン酸が含まれていれば赤紫色に呈色する。呈色度合はウロン酸含量に比例するが、カルバソール硫酸法による分析では、組成物中に含まれるコンドロイチン硫酸とプロテオグリカンの分別定量が難しい、と考えられている。 As mentioned above, proteoglycan is a complex, and it is difficult to accurately measure the amount of proteoglycan contained in a composition such as a food or beverage supplement. Therefore, the reality is that no technology has yet been established to measure the amount of proteoglycan contained in a composition. For example, the carbazole sulfate method is a quantitative method that utilizes the color reaction of uronic acid, one of the main components of proteoglycan sugar chains. When a carbazole solution and concentrated sulfuric acid are added to a sample aqueous solution, the sample aqueous solution turns reddish purple if it contains uronic acid. The degree of coloration is proportional to the uronic acid content, but it is considered difficult to quantitatively separate chondroitin sulfate and proteoglycan contained in a composition using the carbazole sulfate method.
プロテオグリカンは巨大な分子であるため凝集しやすく、抽出溶媒や抽出方法の違いにより立体構造の変化を起こしてその物理的性質が変化すると考えられる。例えば、ブタ軟骨組織に存在するタンパク質－コンドロイチン硫酸複合体は、カルシウムイオンに対する選択的な親和性を有し、当該タンパク質部分の分解によりカルシウムイオンを放出することが示唆されている（非特許文献１参照）。また、プロテオグリカンに結合するカルシウムイオンをナトリウムイオンに置換すると、プロテオグリカンの保水性が向上することも報告されている（特許文献２参照）。 Because proteoglycans are large molecules, they tend to aggregate, and it is believed that differences in extraction solvents and extraction methods cause changes in their three-dimensional structure, which in turn changes their physical properties. For example, it has been suggested that the protein-chondroitin sulfate complex present in porcine cartilage tissue has a selective affinity for calcium ions, and that decomposition of the protein portion releases calcium ions (see Non-Patent Document 1). It has also been reported that the water retention of proteoglycans is improved when calcium ions bound to proteoglycans are replaced with sodium ions (see Patent Document 2).
本発明により解決しようとする課題は、サプリメントなどの飲食品等の組成物に含有されているプロテオグリカンを簡便に測定する方法を見出すことなど、である。 The problem that the present invention aims to solve is to find a method for easily measuring proteoglycans contained in compositions such as food and beverage supplements.
上記課題を解決するために本発明者は鋭意検討を行った結果、プロテオグリカンを含む測定試料を調製する際に、プロテオグリカンの構造を適切な状態に維持すること、例えば、適切なイオンや化合物を含み中性領域の緩衝液を用いて測定試料を調製することで、イオン交換樹脂への吸着及び脱着により、他の夾雑タンパク質と分離することができ、測定試料中のプロテオグリカンを定量しうることを見出した。 As a result of intensive research conducted by the present inventors to solve the above problems, it was discovered that when preparing a measurement sample containing proteoglycan, the structure of the proteoglycan can be maintained in an appropriate state, for example, by preparing the measurement sample using a buffer solution in the neutral range containing appropriate ions or compounds, the proteoglycan can be separated from other contaminating proteins by adsorption to and desorption from an ion exchange resin, and the amount of proteoglycan in the measurement sample can be quantified.
すなわち、本発明は以下の実施形態を含む。
（１）組成物に含まれるプロテオグリカン含有量の測定方法であって、組成物を、プロテオグリカンの官能基の解離に適した緩衝液に溶解し、測定試料を調製する工程と、上記緩衝液を含む平衡化緩衝液でイオン交換樹脂を平衡化する工程と、測定試料をイオン交換樹脂に負荷してプロテオグリカンを吸着させる工程と、プロテオグリカンが吸着したイオン交換樹脂を、上記緩衝液を含む洗浄緩衝液で洗浄する工程と、イオン交換樹脂から溶出緩衝液でプロテオグリカンを溶出する工程と、溶出されたプロテオグリカンを定量する工程と、を含む測定方法。
（２）緩衝液が、ナトリウムイオン及び／又はカリウムイオンを含む（１）に記載の測定方法。
（３）緩衝液が、アミノスルホン酸誘導体を含む（１）又は（２）に記載の測定方法。
（４）緩衝液がｐＨ６．６～７．５を示す（１）～（３）のいずれかに記載の測定方法。
（５）緩衝液が、ＮａＯＨで中性化したＰＩＰＥＳ緩衝剤を含む（１）～（４）のいずれかに記載の測定方法。
（６）イオン交換樹脂が、弱塩基性陰イオン交換樹脂である（１）～（５）のいずれかに記載の測定方法。
（７）組成物が、コラーゲン又はゼラチンを含む（１）～（６）のいずれかに記載の測定方法。
（８）プロテオグリカンを定量する工程がＨＰＬＣ法により行う（１）～（７）のいずれかに記載の測定方法。
That is, the present invention includes the following embodiments.
(1) A method for measuring the proteoglycan content in a composition, comprising the steps of: dissolving the composition in a buffer suitable for dissociating the functional groups of proteoglycan to prepare a measurement sample; equilibrating an ion exchange resin with an equilibration buffer containing the above buffer; loading the measurement sample onto the ion exchange resin to adsorb the proteoglycan; washing the ion exchange resin with the adsorbed proteoglycan using a washing buffer containing the above buffer; eluting the proteoglycan from the ion exchange resin with an elution buffer; and quantifying the eluted proteoglycan.
(2) The measurement method according to (1), wherein the buffer solution contains sodium ions and/or potassium ions.
(3) The method according to (1) or (2), wherein the buffer solution contains an aminosulfonic acid derivative.
(4) The method according to any one of (1) to (3), wherein the buffer solution has a pH of 6.6 to 7.5.
(5) The method according to any one of (1) to (4), wherein the buffer solution contains a PIPES buffer neutralized with NaOH.
(6) The method according to any one of (1) to (5), wherein the ion exchange resin is a weakly basic anion exchange resin.
(7) The method according to any one of (1) to (6), wherein the composition contains collagen or gelatin.
(8) The method according to any one of (1) to (7), wherein the step of quantifying proteoglycan is carried out by HPLC.
本発明の方法によれば、サプリメントなどの飲食品等の組成物に含有されているプロテオグリカンを簡便に測定することができる。 The method of the present invention makes it possible to easily measure proteoglycans contained in compositions such as food and beverage supplements.
最初に、本明細書で用いる用語について簡単に説明する。
（組成物）
本発明の方法により測定対象となる組成物は、例えば、化粧品、及びサプリメントなどの飲食品、特に、ゼリーやゲル状の健康食品並びにそれらに含有される素材等が挙げられ、液体、固体又は半固体の形態を含む。また、この組成物に含まれるプロテオグリカンの含有量は、０μｇ／ｍｇ以上、好ましくは０．１μｇ／ｍｇ以上、より好ましくは１μｇ／ｍｇ以上である。組成物には、コラーゲン又はゼラチンを含んでもよく、このような組成物でも夾雑タンパク質の影響を受けずにプロテオグリカン含量を測定することができる点で、本発明の方法を用いることが好ましい。ゼラチンはコラーゲンの熱変性物質であり、通常の市販ゼラチンは、数万～数百万の分子量分布をもっている。
First, a brief explanation of the terms used in this specification will be provided.
(Composition)
Compositions to be measured by the method of the present invention include, for example, cosmetics, foods and beverages such as supplements, in particular health foods in the form of jellies or gels, and ingredients contained therein, and include liquid, solid, or semi-solid forms. The content of proteoglycan contained in this composition is 0 μg/mg or more, preferably 0.1 μg/mg or more, and more preferably 1 μg/mg or more. The composition may contain collagen or gelatin, and it is preferable to use the method of the present invention in such a composition because the proteoglycan content can be measured without being affected by contaminating proteins. Gelatin is a thermally denatured substance of collagen, and ordinary commercially available gelatin has a molecular weight distribution of tens of thousands to millions.
（プロテオグリカン）
プロテオグリカンはコアタンパク質にコンドロイチン硫酸、デルマタン硫酸等のグリコサミノグリカン（以下ＧＡＧと表す。）と呼ばれる糖鎖が共有結合した糖タンパク質である。プロテオグリカンは、細胞外マトリックスの主要構成成分の一つとして皮膚や軟骨など体内に広く分布している。ＧＡＧ鎖は分岐を持たない長い直鎖構造を持つ。多数の硫酸基とカルボキシル基を持つため負に荷電しており、ＧＡＧ鎖はその電気的反発力のために延びた形状をとる。また、プロテオグリカンは、糖の持つ水親和性により、多量の水を保持することができる。プロテオグリカンに含まれる多数のＧＡＧ鎖群はスポンジのように水を保持することで、弾性や衝撃への耐性といった軟骨特有の機能を担っている。
(Proteoglycan)
Proteoglycans are glycoproteins in which sugar chains called glycosaminoglycans (GAGs), such as chondroitin sulfate and dermatan sulfate, are covalently bonded to a core protein. Proteoglycans are one of the main components of the extracellular matrix and are widely distributed throughout the body, including in skin and cartilage. GAG chains have a long linear structure with no branches. They are negatively charged due to the presence of many sulfate and carboxyl groups, and the GAG chains take on an extended shape due to their electrical repulsive force. Proteoglycans can also retain a large amount of water due to the water affinity of sugars. The large number of GAG chains contained in proteoglycans retain water like a sponge, providing cartilage with functions unique to cartilage, such as elasticity and resistance to impact.
本発明の方法で測定しうるプロテオグリカンの種類としては、特に制限されるものではなく、サケ鼻軟骨などから抽出されるコンドロイチン硫酸プロテオグリカン、デルマタン硫酸プロテオグリカン、ヘパラン硫酸プロテオグリカン、又はケラタン硫酸プロテオグリカンのいずれに分類されるものであってもよい。具体的には、アグリカン、バーシカン、ニューロカン、ブレビカン、デコリン、ビグリカン、セルグリシン、パールカン、シンデカン、グリピカン、ルミカン、ケラトカン等が例示される。これらの中でも、本発明の方法には、好ましくはコンドロイチン硫酸プロテオグリカン、更に好ましくはアグリカン含量の測定に適している。 The type of proteoglycan that can be measured by the method of the present invention is not particularly limited, and may be classified as any of chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, or keratan sulfate proteoglycans extracted from salmon nasal cartilage, etc. Specific examples include aggrecan, versican, neurocan, brevican, decorin, biglycan, serglycin, perlecan, syndecan, glypican, lumican, and keratocan. Among these, the method of the present invention is preferably suitable for measuring the content of chondroitin sulfate proteoglycans, and more preferably aggrecan.
本発明の方法で測定される組成物において、組成物に含有されるプロテオグリカン含有量の下限は、０μｇ／ｍｇ以上、好ましくは０．１μｇ／ｍｇ、より好ましくは１μｇ／ｍｇである。 In the composition measured by the method of the present invention, the lower limit of the proteoglycan content contained in the composition is 0 μg/mg or more, preferably 0.1 μg/mg, and more preferably 1 μg/mg.
（タンパク質）
本発明の方法で測定される組成物において、組成物に含有されることがあるタンパク質は、例えば、乳タンパク質、植物由来のプロテイン（例えば、ソイプロテイン、エンドウ豆由来のプロテイン）、魚介類由来のプロテイン、がある。乳タンパク質は、例えば脱脂粉乳、全脂粉乳、トータルミルクプロテイン、ホエイプロテイン、カゼインプロテイン、カゼインナトリウム、乳清蛋白質、バターミルクパウダー、牛乳、全脂濃縮乳、脱脂濃縮乳、ナチュラルチーズ、である。例えば、Ｗｈｅｙｃｏ社の販売するＷ８０ Ｉｎｓｔａｎｔは、チーズホエイ由来のホエイタンパク質であり、主成分のβ－ラクトグロブリン（分子量３６６００の２量体）、α－ラクトアルブミン（分子量１４２００）に加え、ＢＳＡやラクトフェリンなどから構成されている。
(protein)
In the composition measured by the method of the present invention, the protein that may be contained in the composition is, for example, milk protein, vegetable-derived protein (e.g., soy protein, pea-derived protein), seafood-derived protein, etc. Milk protein is, for example, skim milk powder, whole milk powder, total milk protein, whey protein, casein protein, sodium caseinate, whey protein, buttermilk powder, cow's milk, whole fat concentrated milk, skim milk concentrated milk, natural cheese, etc. For example, W80 Instant sold by Wheyco is a whey protein derived from cheese whey, and is composed of β-lactoglobulin (a dimer with a molecular weight of 36,600) and α-lactalbumin (molecular weight of 14,200) as main components, as well as BSA, lactoferrin, etc.
（緩衝液）
緩衝液は、その酸－塩基結合成分の作用によって、ｐＨの変化に抵抗する溶液である。１つの実施形態において緩衝液は、（例えば、以下の実施例１～５のように）約６．０～約８．０の範囲のｐＨを有する。この範囲でｐＨを制御する緩衝液の例としては、ＭＥＳ、ＰＩＰＥＳ、ＭＯＰＳ、ＭＯＰＳＯ、リン酸、酢酸、クエン酸、コハク酸及びアンモニウム緩衝液、ならびにそれらの組み合わせが挙げられる。本発明の方法に使用される緩衝液は、塩（例えば、ＮａＣｌ、ＫＣｌ、またはＮａＯＡｃ）を同時に含んでもよい。
Buffer Solution
A buffer is a solution that resists changes in pH by the action of its acid-base binding components. In one embodiment, the buffer has a pH in the range of about 6.0 to about 8.0 (e.g., as in Examples 1-5 below). Examples of buffers that control pH in this range include MES, PIPES, MOPS, MOPSO, phosphate, acetate, citrate, succinate and ammonium buffers, and combinations thereof. Buffers used in the methods of the invention may also contain a salt (e.g., NaCl, KCl, or NaOAc).
「平衡化緩衝液」は、イオン交換樹脂を平衡化するために使用される緩衝液である。平衡化緩衝液はまた、測定対象のプロテオグリカン及び夾雑物を含む組成物をイオン交換樹脂に負荷するために使用される。平衡化緩衝液は、好ましくは、プロテオグリカンがイオン交換樹脂に結合するようなイオン強度及び／又はｐＨを有する。 An "equilibration buffer" is a buffer used to equilibrate an ion exchange resin. The equilibration buffer is also used to load a composition containing the proteoglycan to be measured and contaminants onto the ion exchange resin. The equilibration buffer preferably has an ionic strength and/or pH such that the proteoglycan binds to the ion exchange resin.
「洗浄緩衝液」は、測定試料の負荷後及び溶出前にイオン交換樹脂を通る緩衝液である。洗浄緩衝液は、１種類以上の夾雑物をイオン交換樹脂から溶出させるように働く。洗浄緩衝液のイオン強度及び／又はｐＨは、夾雑物はイオン交換樹脂から溶出されるが、プロテオグリカンは溶出されないような、イオン強度及び／又はｐＨである。洗浄緩衝液は、場合により所定の塩（例えば０．４Ｍ未満）を含むこともある。 A "wash buffer" is a buffer passed through the ion exchange resin after loading and before elution of the measurement sample. The wash buffer serves to elute one or more contaminants from the ion exchange resin. The ionic strength and/or pH of the wash buffer are such that the contaminants are eluted from the ion exchange resin but the proteoglycans are not. The wash buffer may optionally contain a certain salt (e.g., less than 0.4 M).
「溶出緩衝液」とはプロテオグリカンを固相から溶出するために使用される。溶出緩衝液のイオン強度及び／又はｐＨは、プロテオグリカンがイオン交換樹脂から溶出されるようなイオン強度及び／又はｐＨである。溶出緩衝液は、場合により所定の塩（例えば０．４Ｍ以上）を含むこともある。 An "elution buffer" is used to elute the proteoglycan from the solid phase. The ionic strength and/or pH of the elution buffer is such that the proteoglycan is eluted from the ion exchange resin. The elution buffer may optionally contain a certain salt (e.g., 0.4 M or more).
（イオン交換樹脂）
用語、イオン交換樹脂とは、負に荷電した固相（すなわち、陽イオン交換樹脂）、又は正に荷電した固相（すなわち、陰イオン交換樹脂）をいう。この荷電は、１つ以上の荷電したイオン基を（例えば、共有結合によって）固相に結合させることによって提供される。あるいは、この電荷は、全体として正電荷を有するキチンやキトサンのように固相の内在的性質であり得る。
(Ion exchange resin)
The term ion exchange resin refers to a negatively charged (i.e., a cation exchange resin) or positively charged (i.e., an anion exchange resin) solid phase. The charge is provided by attaching (e.g., by covalent bonding) one or more charged ionic groups to the solid phase. Alternatively, the charge can be an intrinsic property of the solid phase, such as chitin or chitosan, which have an overall positive charge.
固相とは、１つ以上の荷電したイオン基が接着し得る非水性マトリックスを意味する。この固相は、精製カラム、個々の粒子の非連続的な相、膜、またはフィルターなどであり得る。固相を形成するための材料の例としては、多糖類（例えば、アガロースおよびセルロース）、及び他の機械的に安定なマトリックス（例えば、シリカ（例えば、制御された孔を有するガラス）、ポリ（スチレンジビニル）ベンゼン、ポリアクリルアミド、セラミック粒子、および上記のいずれかの誘導体）が挙げられる。 By solid phase is meant a non-aqueous matrix to which one or more charged ionic groups can adhere. The solid phase can be a purification column, a discontinuous phase of discrete particles, a membrane, or a filter, etc. Examples of materials for forming the solid phase include polysaccharides (e.g., agarose and cellulose) and other mechanically stable matrices (e.g., silica (e.g., controlled pore glass), poly(styrenedivinyl)benzene, polyacrylamide, ceramic particles, and derivatives of any of the above).
陰イオン交換樹脂は、正に荷電した（例えば、固相に結合された４級アミノ基のような、１つ以上の正に荷電したイオン基を有する）固相を指す。市販の陰イオン交換樹脂としては、ＤＥＡＥセルロース、ＱＡＥ ＳＥＰＨＡＤＥＸＴＭ、及びＦＡＳＴ Ｑ ＳＥＰＨＡＲＯＳＥＴＭ（Ｐｈａｒｍａｃｉａ）が挙げられる。陰イオン交換樹脂は、そのイオン基の解離性により強塩基性又は弱塩基性に分けられる。 Anion exchange resin refers to a solid phase that is positively charged (e.g., has one or more positively charged ionic groups, such as quaternary amino groups, attached to the solid phase). Commercially available anion exchange resins include DEAE cellulose, QAE SEPHADEX ™ , and FAST Q SEPHAROSE ™ (Pharmacia). Anion exchange resins are classified as strong or weak basic depending on the dissociation of their ionic groups.
弱塩基性陰イオン交換樹脂とは、例えば、１～３級アミノ基を官能基として持つ樹脂のように、弱塩基性を示す陰イオン交換樹脂を意味する。弱塩基の官能基はアルカリ溶液中では解離せずイオン交換能を示さないため中性領域で使用することが好ましい。また、プロテオグリカン中のスルホ基のような比較的強い酸性基とは交換するが、夾雑タンパク質中のカルボキシ基のような弱酸性基とは交換しにくいと考えられる。 Weakly basic anion exchange resin means an anion exchange resin that exhibits weak basicity, such as a resin that has primary, secondary, or tertiary amino groups as functional groups. Since weakly basic functional groups do not dissociate in alkaline solutions and do not exhibit ion exchange capacity, it is preferable to use them in the neutral region. In addition, although they will exchange with relatively strong acidic groups such as sulfo groups in proteoglycans, they are thought to have difficulty exchanging with weakly acidic groups such as carboxy groups in impurity proteins.
以下に本発明を、図面を参照して説明する。図１は、一実施形態におけるプロテオグリカン含有量の測定方法の流れを示す工程図である。図１に示すように、本実施形態の測定方法は、測定対象となる組成物を緩衝液に加温溶解して測定試料を調製する測定試料の調製工程（Ｓ１）と、イオン交換樹脂にプロテオグリカンを吸着及び脱着させるイオン交換工程（Ｓ２）と、イオン交換樹脂から溶出されたプロテオグリカンを含む溶出物を脱塩するよう溶出物の脱塩工程（Ｓ３）と、ＨＰＬＣ分析により脱塩後のプロテオグリカンを定量する定量工程（Ｓ４）と、を含む。 The present invention will be described below with reference to the drawings. FIG. 1 is a process diagram showing the flow of a method for measuring proteoglycan content in one embodiment. As shown in FIG. 1, the measurement method of this embodiment includes a measurement sample preparation step (S1) in which a composition to be measured is dissolved by heating in a buffer solution to prepare a measurement sample, an ion exchange step (S2) in which proteoglycan is adsorbed and desorbed to an ion exchange resin, an eluate desalting step (S3) in which the eluate containing proteoglycan eluted from the ion exchange resin is desalted, and a quantification step (S4) in which the amount of desalted proteoglycan is quantified by HPLC analysis.
ここで、測定試料の調製工程Ｓ１は、本発明の方法における最も重要な工程である。上述したように、プロテオグリカンは極めて高分子量の糖タンパク質であるため溶液中での凝集を防ぐ必要がある。このため、測定対象の組成物を、プロテオグリカンの官能基の解離に適した緩衝液に溶解する。このような緩衝液としては、例えば、ナトリウムイオン及び／又はカリウムイオンを含む緩衝液が挙げられる。好ましくは、ナトリウムイオン及び／又はカリウムイオンを含み、及びカルシウムイオンを含まない緩衝液である。ナトリウムイオン及びカリウムイオンの濃度は特に限定されないが、５ｍＭ以上の濃度であることが好ましく、さらに好ましくは１０ｍＭ以上である。 Here, the measurement sample preparation step S1 is the most important step in the method of the present invention. As described above, proteoglycan is a glycoprotein with an extremely high molecular weight, and therefore it is necessary to prevent aggregation in the solution. For this reason, the composition to be measured is dissolved in a buffer suitable for dissociating the functional groups of proteoglycan. Examples of such buffers include buffers containing sodium ions and/or potassium ions. A buffer containing sodium ions and/or potassium ions and not containing calcium ions is preferable. The concentrations of sodium ions and potassium ions are not particularly limited, but are preferably 5 mM or more, and more preferably 10 mM or more.
本発明の他の好ましい実施形態では、緩衝液にはアミノスルホン酸誘導体を含む。アミノスルホン酸誘導体としては、Ｎ，Ｎ－ビス（２－ハイドロキシエチル）－２－アミノエタンスルホン酸（以下ＢＥＳと略す）、Ｎ－シクロヘキシル－３－アミノプロパンスルホン酸（以下ＣＡＰＳと略す）、Ｎ－シクロヘキシル－２－ハイドロキシ－３－アミノプロパンスルホン酸（以下ＣＡＰＳＯと略す）、Ｎ－シクロヘキシル－２－アミノエタンスルホン酸（以下ＣＨＥＳと略す）、３－｛Ｎ，Ｎ－ビス（２－ハイドロキシエチル）アミノ｝－２－ハイドロキシプロパンスルホン酸（以下ＤＩＰＳＯと略す）、Ｎ－トリス（ハイドロキシメチル）メチル－３－アミノプロパンスルホン酸（以下ＴＡＰＳと略す）、２－ハイドロキシ－Ｎ－トリス（ハイドロキシメチル）メチル－３－アミノプロパンスルホン酸（以下ＴＡＰＳＯと略す）、Ｎ－トリス（ハイドロキシメチル）メチル－２－アミノエタンスルホン酸（以下ＴＥＳと略す）等を挙げることができる。 In another preferred embodiment of the present invention, the buffer solution contains an aminosulfonic acid derivative. Examples of the aminosulfonic acid derivative include N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (hereinafter abbreviated as BES), N-cyclohexyl-3-aminopropanesulfonic acid (hereinafter abbreviated as CAPS), N-cyclohexyl-2-hydroxy-3-aminopropanesulfonic acid (hereinafter abbreviated as CAPSO), N-cyclohexyl-2-aminoethanesulfonic acid (hereinafter abbreviated as CHES), 3-{N,N-bis(2-hydroxyethyl) Examples of such compounds include N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (hereinafter abbreviated as DIPSO), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (hereinafter abbreviated as TAPS), 2-hydroxy-N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (hereinafter abbreviated as TAPSO), and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (hereinafter abbreviated as TES).
また、他の実施形態におけるアミノスルホン酸誘導体としては、ピペラジン環を有する化合物である、３－｛４－（２－ハイドロキシエチル）－１－ピペラジニル｝プロパンスルホン酸（以下ＥＰＰＳと略す）、２－｛４－（２－ハイドロキシエチル）－１－ピペラジニル｝エタンスルホン酸（以下ＨＥＰＥＳと略す）、２－ハイドロキシ－３－｛４－（２－ハイドロキシエチル）－１－ピペラジニル｝プロパンスルホン酸（以下ＨＥＰＰＳＯと略す）、ピペラジン－１，４－ビス（２－エタンスルホン酸）（以下ＰＩＰＥＳと略す）、ピペラジン－１，４－ビス（２－ハイドロキシ－３－プロパンスルホン酸）（以下ＰＯＰＳＯと略す）等を挙げることができる。 In addition, examples of aminosulfonic acid derivatives in other embodiments include compounds having a piperazine ring, such as 3-{4-(2-hydroxyethyl)-1-piperazinyl}propanesulfonic acid (hereinafter abbreviated as EPPS), 2-{4-(2-hydroxyethyl)-1-piperazinyl}ethanesulfonic acid (hereinafter abbreviated as HEPES), 2-hydroxy-3-{4-(2-hydroxyethyl)-1-piperazinyl}propanesulfonic acid (hereinafter abbreviated as HEPPSO), piperazine-1,4-bis(2-ethanesulfonic acid) (hereinafter abbreviated as PIPES), and piperazine-1,4-bis(2-hydroxy-3-propanesulfonic acid) (hereinafter abbreviated as POPSO).
また、他の実施形態におけるアミノスルホン酸誘導体としては、モルホリン環を含む化合物である、２－モルホリノエタンスルホン酸（以下ＭＥＳと略す）、３－モルホリノプロパンスルホン酸（以下ＭＯＰＳと略す）、２－ハイドロキシ－３－モルホリノプロパンスルホン酸（以下ＭＯＰＳＯと略す）等を挙げることができる。 In other embodiments, examples of aminosulfonic acid derivatives include compounds containing a morpholine ring, such as 2-morpholinoethanesulfonic acid (hereinafter abbreviated as MES), 3-morpholinopropanesulfonic acid (hereinafter abbreviated as MOPS), and 2-hydroxy-3-morpholinopropanesulfonic acid (hereinafter abbreviated as MOPSO).
これらの緩衝液の濃度は、溶液のｐＨ値を約６．０～約８．０、好ましくは、６．６～７．５の間に安定的に保持できる濃度であれば特に制限されないが、好ましくは、５ｍＭ～１００ｍＭ，さらに好ましくは、１０ｍＭ～５０ｍＭである。 The concentration of these buffer solutions is not particularly limited as long as it can stably maintain the pH value of the solution between about 6.0 and about 8.0, preferably between 6.6 and 7.5, but is preferably 5 mM to 100 mM, more preferably 10 mM to 50 mM.
図２は、上記イオン交換工程（Ｓ２）をさらに詳細に説明したものである。イオン交換工程（Ｓ２）は、最初に、平衡化緩衝液でイオン交換樹脂を平衡化する平衡化工程（Ｓ２１）と、測定試料をイオン交換樹脂に負荷してプロテオグリカンを吸着させる吸着工程（Ｓ２２）と、プロテオグリカンが吸着したイオン交換樹脂を、上記緩衝液を含む洗浄緩衝液で洗浄する洗浄工程（Ｓ２３）と、イオン交換樹脂から溶出緩衝液でプロテオグリカンを溶出する及び／又は脱着する工程（Ｓ２４、図２では「溶出」と標記）と、を含む。 Figure 2 is a more detailed explanation of the ion exchange step (S2). The ion exchange step (S2) includes an equilibration step (S21) in which the ion exchange resin is equilibrated with an equilibration buffer, an adsorption step (S22) in which a measurement sample is loaded onto the ion exchange resin to adsorb proteoglycan, a washing step (S23) in which the ion exchange resin with the adsorbed proteoglycan is washed with a washing buffer containing the buffer, and a step (S24, labeled "Elution" in Figure 2) in which the proteoglycan is eluted and/or desorbed from the ion exchange resin with an elution buffer.
平衡化工程（Ｓ２１）では、通常、カラム容積の１０倍以上の平衡化緩衝液を流すことにより平衡化を行う。あるいは、カラムから溶出される平衡化緩衝液のベースラインが安定すること、カラム圧変動が安定すること、同じ成分を繰り返し分析した際に保持時間の移動がないことなどが目安となる。本実施形態の方法では、平衡化緩衝液は、測定試料の調製に用いた緩衝液と同じ緩衝液を用いることが、プロテオグリカンのカラムへの吸着を促進するために好ましい。 In the equilibration step (S21), equilibration is usually performed by passing an equilibration buffer solution with a volume 10 times or more the column volume. Alternatively, the baseline of the equilibration buffer solution eluted from the column should be stable, the column pressure fluctuation should be stable, and there should be no shift in retention time when the same components are repeatedly analyzed. In the method of this embodiment, it is preferable to use the same equilibration buffer solution as the buffer solution used to prepare the measurement sample in order to promote adsorption of proteoglycan to the column.
吸着工程（Ｓ２２）において負荷される試料中のプロテオグリカンは、官能基の解離に適した緩衝液に溶解されているため効率よくイオン交換樹脂に吸着される。プロテオグリカンの吸着を促進するために、室温ないし低温（４℃～１０℃）で、低流速で測定試料をチャージすることが好ましい。 The proteoglycan in the sample loaded in the adsorption step (S22) is dissolved in a buffer suitable for dissociating functional groups, and is therefore efficiently adsorbed to the ion exchange resin. To promote the adsorption of proteoglycan, it is preferable to charge the measurement sample at room temperature or low temperature (4°C to 10°C) and at a low flow rate.
洗浄工程（Ｓ２３）で用いられる洗浄緩衝液は、測定試料の調製に用いた緩衝液及び／又は平衡化緩衝液と同じ緩衝液が好ましい。洗浄緩衝液には、夾雑物をカラムから除去するために溶液のイオン強度を上げるための塩を含んでもよい。例えば、ＮａＣｌを添加する場合の濃度は０～０．４Ｍ未満であり、好ましくはＮａＣｌを含まない緩衝液と約０．２ＭのＮａＣｌを含む緩衝液を逐次的に用いてもよい。 The washing buffer used in the washing step (S23) is preferably the same as the buffer used in preparing the measurement sample and/or the equilibration buffer. The washing buffer may contain a salt to increase the ionic strength of the solution in order to remove impurities from the column. For example, when NaCl is added, the concentration is 0 to less than 0.4 M, and preferably a buffer containing no NaCl and a buffer containing about 0.2 M NaCl may be used sequentially.
溶出工程（Ｓ２４）で用いられる溶出緩衝液は、測定試料の調製に用いた緩衝液を含んでも含まなくてもよいが、溶出されるプロテオグリカンの凝集を抑止するために、平衡化緩衝液と同じ緩衝液を用いることが好ましい。溶出液のイオン強度は、イオン交換樹脂に対するプロテオグリカンの吸着力を減弱させる程度のイオン強度が好ましく、例えば、ＮａＣｌを添加する場合の濃度は０．４Ｍ以上であり、好ましくは約０．５ＭのＮａＣｌを含む緩衝液を用いることができる。 The elution buffer used in the elution step (S24) may or may not contain the buffer used in preparing the measurement sample, but it is preferable to use the same buffer as the equilibration buffer in order to prevent aggregation of the eluted proteoglycan. The ionic strength of the elution solution is preferably such that it weakens the adsorption force of the proteoglycan to the ion exchange resin. For example, when NaCl is added, the concentration is 0.4 M or more, and a buffer containing about 0.5 M NaCl can be used.
図１に戻って、脱塩工程（Ｓ３）は、本発明の方法に必ずしも必須ではないが、後続するＨＰＬＣ工程での検出を容易にする上で、溶出液に含まれる塩濃度を低下させることが好ましい。 Returning to FIG. 1, the desalting step (S3) is not essential to the method of the present invention, but it is preferable to reduce the salt concentration in the eluate in order to facilitate detection in the subsequent HPLC step.
イオン交換樹脂からの溶出物中のプロテオグリカンを定量する定量工程（Ｓ４）は、特に限定されないが、本発明の一実施形態では、図１に示すＨＰＬＣ分析を行うことが好ましい。この工程におけるサイズ排除ゲルクロマトグラフィー用のカラムとしては、分析可能な分子量が１０万～２００万に対応しているカラムを用いればよいが、分離能がよくなることから粒子径１０μｍ以上の充填剤を含むカラムやタンパク分析用のカラムを使用することが好ましく、特に基材がメタクリレートポリマーのカラムを用いることが好ましい。 The quantitative determination step (S4) for quantifying the amount of proteoglycan in the eluate from the ion exchange resin is not particularly limited, but in one embodiment of the present invention, it is preferable to perform the HPLC analysis shown in Figure 1. As the column for size exclusion gel chromatography in this step, a column with an analyzable molecular weight of 100,000 to 2 million may be used, but it is preferable to use a column containing a packing material with a particle size of 10 μm or more or a column for protein analysis, as this improves separation ability, and it is particularly preferable to use a column whose base material is a methacrylate polymer.
ＨＰＬＣ法における検出は、溶媒と比較した被分析物の屈折率を利用する示唆屈折率検出器や、ＵＶ検出器を使用して２００～２８０ｎｍの吸光度を検出する方法等が使用でき、特に２００～２２０ｎｍのＵＶ波長におけるＵＶ検出器が好ましい。また、プロテオグリカンのピークは標準品であるプロテオグリカン、サケ鼻軟骨由来（富士フイルム和光純薬工業製）を用意し、分析対象の試料とクロマトグラムを重ねほぼ同じ溶出範囲にピークがあれば、そのピークがプロテオグリカンを検出したピークであることが分かる。また、予めプロテオグリカンの標準品で検量線を作成することで、試料中のプロテオグリカンの定量を行う。 Detection in the HPLC method can be performed using a refractive index detector that utilizes the refractive index of the analyte compared to the solvent, or a UV detector that detects absorbance at 200 to 280 nm, with a UV detector with a UV wavelength of 200 to 220 nm being particularly preferred. Proteoglycan peaks can be detected by preparing a standard proteoglycan derived from salmon nasal cartilage (manufactured by Fujifilm Wako Pure Chemical Industries), overlaying the chromatogram with the sample to be analyzed, and if there is a peak in roughly the same elution range, it can be determined that this peak is the peak where proteoglycan has been detected. Proteoglycan in a sample can be quantified by creating a calibration curve in advance using a proteoglycan standard.
以下に示す実施例は、単なる例示であって、上述した実施形態と共に本発明を詳細に説明することのみを意図しており、本発明を限定するものではない。当業者は、本発明の意義を逸脱することなく様々な態様に本発明を変更することができ、係る変更も本発明の範囲に含まれる。なお、以下の実施例において、プロテオグリカン含有率（「ＰＧ含有率」という場合もある。）を示す数値の単位％は、質量％を意味する。 The examples shown below are merely illustrative and are intended only to explain the present invention in detail together with the above-mentioned embodiments, and are not intended to limit the present invention. Those skilled in the art can modify the present invention in various ways without departing from the meaning of the present invention, and such modifications are also included in the scope of the present invention. In the following examples, the unit % of the numerical values indicating the proteoglycan content (sometimes called "PG content") means % by mass.
［実施例１］１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液によるプロテオグリカン含有量の測定
１．組成物の調製
９７．４５ｇの水を入れたビーカー内に、２．０ｇの市販ゼリー素（森永製菓株式会社製クックゼラチン）と、０．５５ｇの「プロテオグリカンＦ（プロテオグリカン含量２０．０％以上、一丸ファルコス株式会社）」とを添加し、合計１００ｇとした。これを約５０℃のウォータバスにて攪拌しながら完全に溶解した。この溶解液を型容器に入れ、冷蔵庫にて一晩冷やしてプロテオグリカン含有ゼリーを作製した。
[Example 1] Measurement of proteoglycan content using 10 mM PIPES-NaOH buffer 1. Preparation of composition 2.0 g of commercially available jelly base (Cook Gelatin, manufactured by Morinaga & Co., Ltd.) and 0.55 g of "Proteoglycan F (proteoglycan content 20.0% or more, Ichimaru Pharcos Co., Ltd.)" were added to a beaker containing 97.45 g of water, making a total of 100 g. This was completely dissolved while stirring in a water bath at about 50°C. The solution was placed in a mold container and cooled in a refrigerator overnight to prepare a proteoglycan-containing jelly.
２．測定試料の調製
３．０５ｇのＰＩＰＥＳ（ＤＯＪＩＮＤＯ）に、精製水約８００ｍＬを加えて、室温で攪拌し、５ＮのＮａＯＨにてｐＨ７．０に調整した。これに精製水を加えて全容を１０００ｍＬとし、１０ｍＭのＰＩＰＥＳ溶液を調製した。このようにして調製した１０ｍＭのＰＩＰＥＳ－ＮａＯＨ（ｐＨ７．０）緩衝液１０ｍＬに、上記で作成した約１８０ｍｇのゼリー組成物（ＰＧとして約２００μｇ含有）を正確に加え、約４０℃で加温しながら攪拌して完全に溶解した。
2. Preparation of measurement sample About 800 mL of purified water was added to 3.05 g of PIPES (DOJINDO), stirred at room temperature, and adjusted to pH 7.0 with 5N NaOH. Purified water was added to this to make the total volume 1000 mL, and a 10 mM PIPES solution was prepared. About 180 mg of the jelly composition (containing about 200 μg of PG) prepared above was accurately added to 10 mL of the 10 mM PIPES-NaOH (pH 7.0) buffer solution thus prepared, and the mixture was stirred while being heated at about 40° C. to completely dissolve it.
３．イオン交換処理
弱塩基性陰イオン交換用ゲルであるＴＯＹＯＰＡＫ ＤＥＡＥ Ｍ（ゲル量１．０ｍＬ、東ソー株式会社製）のカラムに、精製水１０ｍＬを流した後、上記で調製した１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液（ｐＨ７．０）１０ｍＬを流してイオン交換樹脂を平衡化した。このカラムに同緩衝液に溶解した上記測定試料１０ｍＬを負荷した。続いて、５ｍＬの同緩衝液でカラムを洗浄後、０．２ＭのＮａＣｌを含む同緩衝液１０ｍＬを流してさらに洗浄した。洗浄後のカラムに、０．５ＭのＮａＣｌを含む同緩衝液１０ｍＬを流してプロテオグリカンをカラムから脱着させた。
3. Ion exchange treatment After 10 mL of purified water was poured into a column of TOYOPAK DEAE M (gel volume 1.0 mL, manufactured by Tosoh Corporation), which is a weakly basic anion exchange gel, 10 mL of the 10 mM PIPES-NaOH buffer (pH 7.0) prepared above was poured to equilibrate the ion exchange resin. 10 mL of the measurement sample dissolved in the same buffer was loaded onto this column. Next, the column was washed with 5 mL of the same buffer, and then further washed with 10 mL of the same buffer containing 0.2 M NaCl. After washing, 10 mL of the same buffer containing 0.5 M NaCl was poured into the column to desorb the proteoglycan from the column.
４．脱塩処理
上記イオン交換処理で回収した脱着画分１０ｍＬを、アミコンウルトラ遠心式フィルターユニット（分画分子量５０Ｋ、メルク株式会社製）を用いて最初に脱着液で１回洗浄後、精製水で３回洗浄して脱塩した。遠心後のフィルターカップから濃縮液を回収し、リン酸緩衝液（ｐＨ６．８）を加えて５ｍＬに定容した。
4. Desalting Treatment 10 mL of the desorption fraction recovered in the above ion exchange treatment was desalted by first washing once with the desorption solution and then washing three times with purified water using an Amicon Ultra centrifugal filter unit (molecular weight cutoff 50K, manufactured by Merck Ltd.). The concentrated solution was recovered from the filter cup after centrifugation, and the volume was adjusted to 5 mL by adding phosphate buffer (pH 6.8).
５．ＨＰＬＣ分析
上記で回収した溶液を、以下の操作条件でＨＰＬＣを行い標準品の検量線からプロテオグリカン量を求めた。
カラム ：ＴＳＫｇｅｌ Ｇ５０００ＰＷＸＬ（東ソー株式会社製）
カラム温度：４０℃
試料注入量：１００μＬ
移動相 ：リン酸緩衝液（ｐＨ６．８）
流量 ：０．５ｍＬ／ｍｉｎ
検出器 ：示差屈折率検出器（ＲＩＤ－１０Ａ、株式会社島津製作所製）
5. HPLC Analysis The solution collected above was subjected to HPLC under the following operating conditions, and the amount of proteoglycan was determined from the calibration curve of a standard product.
Column: TSKgel G5000PWXL (manufactured by Tosoh Corporation)
Column temperature: 40°C
Sample injection volume: 100 μL
Mobile phase: Phosphate buffer (pH 6.8)
Flow rate: 0.5mL/min
Detector: Differential refractive index detector (RID-10A, manufactured by Shimadzu Corporation)
なお、標準品によるプロテオグリカンの検量線は、以下のように作成した。プロテオグリカンの標準品（富士フイルム和光純薬工業製）２ｍｇを精密に量り、リン酸緩衝液（ｐＨ６．８）を加えて１０ｍＬ容量に調製した（Ａ液）。Ａ液５ｍＬを取り、リン酸緩衝液（ｐＨ６．８）を加えて１０ｍＬ容量に調製したもの、Ａ液２ｍＬを取り、リン酸緩衝液（ｐＨ６．８）を加えて１０ｍＬ容量に調製したもの及びＡ液をそれぞれ検量線作成用標準液とする。検液及び標準液について０．４５μｍのメンブレンフィルターを通した後、次の操作条件で液体クロマトグラフィーを行い、得られた検量線からプロテオグリカン量を求めた。なお、標準品：プロテオグリカン，サケ鼻軟骨由来（富士フイルム和光純薬工業製）は、室温減圧デシケーター（シリカゲル）で３時間乾燥してから採取した。 The calibration curve for proteoglycan using the standard product was created as follows. 2 mg of the proteoglycan standard product (manufactured by Fujifilm Wako Pure Chemical Industries) was precisely weighed and phosphate buffer (pH 6.8) was added to make a volume of 10 mL (liquid A). 5 mL of liquid A was taken and phosphate buffer (pH 6.8) was added to make a volume of 10 mL, 2 mL of liquid A was taken and phosphate buffer (pH 6.8) was added to make a volume of 10 mL, and liquid A was used as the standard solution for creating the calibration curve. The test solution and the standard solution were passed through a 0.45 μm membrane filter, and then liquid chromatography was performed under the following operating conditions, and the amount of proteoglycan was calculated from the obtained calibration curve. The standard product: proteoglycan, derived from salmon nasal cartilage (manufactured by Fujifilm Wako Pure Chemical Industries) was dried for 3 hours in a vacuum desiccator (silica gel) at room temperature before collection.
ＨＰＬＣ分析の結果を図３に示す。図３（Ａ）は、上記イオン交換処理における洗浄画分を濃縮したものであり、夾雑タンパク質の大きなピークが認められた。一方、図３（Ｂ）は脱着画分を分析した結果であり、夾雑タンパク質と分離されたプロテオグリカンのピークが認められた。検量線のピーク面積から計算した組成物のＰＧ含有率は０．１２３％であった。 The results of the HPLC analysis are shown in Figure 3. Figure 3 (A) shows the concentrated washing fraction from the above ion exchange treatment, and a large peak of contaminating proteins was observed. Meanwhile, Figure 3 (B) shows the results of analyzing the desorption fraction, and a peak of proteoglycan separated from the contaminating proteins was observed. The PG content of the composition calculated from the peak area of the calibration curve was 0.123%.
［比較例１］
実施例１における１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液の代わりに、精製水を用いて実施例１と同様の方法にて測定試料を定量した。すなわち、精製水を用いてカラムの平衡化を行い、カラムの洗浄液及び脱着液は、精製水にＮａＣｌをそれぞれ０．２Ｍ及び０．５Ｍ添加したものを用いた。ＨＰＬＣ分析の結果を図４に示す。図４（Ａ）は、上記イオン交換処理における洗浄画分の濃縮物であり、夾雑タンパク質の大きなピークが認められた。一方、図４（Ｂ）は脱着画分を分析した結果であり、夾雑タンパク質と共にプロテオグリカンの小さなピークが認められた。検量線のピーク面積から計算した組成物のＰＧ含有率は、０．０７０％であった。
[Comparative Example 1]
Instead of the 10 mM PIPES-NaOH buffer solution in Example 1, purified water was used to quantify the measurement sample in the same manner as in Example 1. That is, the column was equilibrated with purified water, and the column washing solution and desorption solution were purified water to which NaCl was added at 0.2 M and 0.5 M, respectively. The results of the HPLC analysis are shown in FIG. 4. FIG. 4(A) shows the concentrate of the washing fraction in the above ion exchange treatment, and a large peak of impurity proteins was observed. On the other hand, FIG. 4(B) shows the results of the analysis of the desorption fraction, and a small peak of proteoglycan was observed together with the impurity proteins. The PG content of the composition calculated from the peak area of the calibration curve was 0.070%.
［比較例２］
実施例１における１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液の代わりに、４Ｍのグアニジン塩酸を用いて実施例１と同様の方法にて測定試料を定量した。すなわち、４Ｍのグアニジン塩酸水溶液を用いてカラムの平衡化を行い、カラムの洗浄液及び脱着液は、４Ｍのグアニジン塩酸水溶液にＮａＣｌをそれぞれ０．２Ｍ及び０．５Ｍ添加したものを用いた。ＨＰＬＣ分析の結果を図５に示す。図５（Ａ）は、上記イオン交換処理における洗浄画分の濃縮物であり、図５（Ｂ）は脱着画分を分析した結果である。何れの画分からもタンパク質は検出されず、４Ｍグアニジン塩酸を用いた方法ではプロテオグリカンの定量ができないことが分かった。
[Comparative Example 2]
Instead of the 10 mM PIPES-NaOH buffer solution in Example 1, 4 M guanidine hydrochloride was used to quantify the measurement sample in the same manner as in Example 1. That is, the column was equilibrated with 4 M guanidine hydrochloride aqueous solution, and the column washing solution and desorption solution were 4 M guanidine hydrochloride aqueous solution to which NaCl was added at 0.2 M and 0.5 M, respectively. The results of the HPLC analysis are shown in FIG. 5. FIG. 5(A) shows the concentrate of the washing fraction in the above ion exchange treatment, and FIG. 5(B) shows the results of the analysis of the desorption fraction. No protein was detected in any of the fractions, and it was found that the method using 4 M guanidine hydrochloride did not allow the quantification of proteoglycan.
［実施例２］
実施例１における１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液の代わりに、日局法に基づき、リン酸二水素カリウム３．４０ｇと、無水リン酸水素二ナトリウム３．５５ｇとを水に溶かし、１０００ｍＬとして調製したリン酸塩緩衝液（ｐＨ６．８）を用いて実施例１と同様の方法にて測定試料を定量した。すなわち、カラムの平衡化液、洗浄液及び脱着液に用いた緩衝液をリン酸塩緩衝液（ｐＨ６．８）に置換した。０．５Ｎ－ＮａＣｌを含むリン酸塩緩衝液（ｐＨ６．８）で脱着した画分のＨＰＬＣ分析の結果を図６に示す。図６に示したように、プロテオグリカンのピークが認められ、検量線のピーク面積から計算した組成物のＰＧ含有率は、０．１１０％であった。
[Example 2]
Instead of the 10 mM PIPES-NaOH buffer in Example 1, 3.40 g of potassium dihydrogen phosphate and 3.55 g of anhydrous disodium hydrogen phosphate were dissolved in water to prepare 1000 mL of phosphate buffer (pH 6.8) according to the Japanese Pharmacopoeia, and the measurement sample was quantified in the same manner as in Example 1. That is, the buffers used for the equilibration solution, washing solution and desorption solution of the column were replaced with phosphate buffer (pH 6.8). The results of HPLC analysis of the fraction desorbed with phosphate buffer (pH 6.8) containing 0.5 N-NaCl are shown in FIG. 6. As shown in FIG. 6, a proteoglycan peak was observed, and the PG content of the composition calculated from the peak area of the calibration curve was 0.110%.
［実施例３］
実施例１における１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液の代わりに、７Ｍ尿素／５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）を用いて実施例１と同様の方法にて測定試料を定量した。すなわち、カラムの平衡化液、洗浄液及び脱着液に用いた緩衝液をすべて７Ｍ尿素／５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）に置換した。０．５Ｎ－ＮａＣｌを含む７Ｍ尿素／５０ｍＭトリス塩酸緩衝液（ｐＨ７．４）で脱着した画分のＨＰＬＣ分析の結果を図７に示す。図７に示したように、プロテオグリカンのピークが認められ、検量線のピーク面積から計算した組成物のＰＧ含有率は、０．１０２％であった。
[Example 3]
The measurement sample was quantified in the same manner as in Example 1, except that 7M urea/50 mM Tris-HCl buffer (pH 7.4) was used instead of the 10 mM PIPES-NaOH buffer in Example 1. That is, all of the buffers used for the equilibration solution, washing solution and desorption solution of the column were replaced with 7M urea/50 mM Tris-HCl buffer (pH 7.4). The results of HPLC analysis of the fraction desorbed with 7M urea/50 mM Tris-HCl buffer (pH 7.4) containing 0.5N-NaCl are shown in FIG. 7. As shown in FIG. 7, a proteoglycan peak was observed, and the PG content of the composition calculated from the peak area of the calibration curve was 0.102%.
［実施例４］
実施例１における１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液の代わりに、酢酸ナトリウム三水和物０．３ｇに水８０ｍＬ加え溶解し、酢酸でｐＨ６．０に調製後、水を加え１００ｍＬとして調製した酢酸塩緩衝液（ｐＨ６．０）を用いて実施例１と同様の方法にて測定試料を定量した。すなわち、カラムの平衡化液、洗浄液及び脱着液に用いた緩衝液をすべて酢酸塩緩衝液（ｐＨ６．０）に置換した。０．５Ｎ－ＮａＣｌを含む酢酸塩緩衝液（ｐＨ６．０）で脱着した画分のＨＰＬＣ分析の結果を図８に示す。図８に示したように、プロテオグリカンのピークが認められ、検量線のピーク面積から計算した組成物のＰＧ含有率は、０．１０８％であった。
[Example 4]
Instead of the 10 mM PIPES-NaOH buffer in Example 1, 0.3 g of sodium acetate trihydrate was dissolved in 80 mL of water, adjusted to pH 6.0 with acetic acid, and then water was added to make 100 mL to prepare an acetate buffer (pH 6.0). The measurement sample was quantified in the same manner as in Example 1. That is, all of the buffers used for the equilibration solution, washing solution, and desorption solution of the column were replaced with acetate buffer (pH 6.0). The results of HPLC analysis of the fraction desorbed with acetate buffer (pH 6.0) containing 0.5 N-NaCl are shown in FIG. 8. As shown in FIG. 8, a proteoglycan peak was observed, and the PG content of the composition calculated from the peak area of the calibration curve was 0.108%.
［実施例５］
実施例１における１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液の代わりに、１０ｍＭのＰＩＰＥＳ－ＫＯＨ緩衝液（ｐＨ７．０）を用いて実施例１と同様の方法にて測定試料を定量した。すなわち、カラムの平衡化液、洗浄液及び脱着液に用いた緩衝液をすべて１０ｍＭのＰＩＰＥＳ－ＫＯＨ緩衝液（ｐＨ７．０）に置換した。０．５Ｎ－ＮａＣｌを含む１０ｍＭのＰＩＰＥＳ－ＫＯＨ緩衝液（ｐＨ７．０）で脱着した画分のＨＰＬＣ分析の結果を図９に示す。図８に示したように、プロテオグリカンのピークが認められ、検量線のピーク面積から計算した組成物のＰＧ含有率は、０．１１６％であった。
[Example 5]
The measurement sample was quantified in the same manner as in Example 1, except that 10 mM PIPES-KOH buffer (pH 7.0) was used instead of the 10 mM PIPES-NaOH buffer in Example 1. That is, all of the buffers used for the equilibration solution, washing solution and desorption solution of the column were replaced with 10 mM PIPES-KOH buffer (pH 7.0). The results of HPLC analysis of the fraction desorbed with 10 mM PIPES-KOH buffer (pH 7.0) containing 0.5 N-NaCl are shown in FIG. 9. As shown in FIG. 8, a proteoglycan peak was observed, and the PG content of the composition calculated from the peak area of the calibration curve was 0.116%.
実施例１～５並びに比較例１及び２の結果を以下の表１にまとめた。表１には、それぞれの組成物中のＰＧ含量（組成物のＰＧ含有率）とともに、実施例１で検出されたプロテオグリカン（ＰＧ）の回収率を１００％としたときの各実施例及び比較例の回収率相対値（％）も示した。これらの結果より、比較例１及び２と比べて、中性領域の緩衝液であって、ナトリウムイオン及び／又はカリウムイオンを含む実施例１～５の緩衝液を用いた場合に効率よくプロテオグリカンを回収できることが分かった、特に緩衝液としてＰＩＰＥＳを用いた場合の回収率が優れていることが分かる。 The results of Examples 1 to 5 and Comparative Examples 1 and 2 are summarized in Table 1 below. Table 1 shows the PG content in each composition (PG content in the composition), as well as the relative recovery rate (%) for each Example and Comparative Example when the recovery rate of proteoglycan (PG) detected in Example 1 is taken as 100%. These results show that proteoglycan can be recovered more efficiently when the buffer solutions of Examples 1 to 5, which are in the neutral range and contain sodium ions and/or potassium ions, are used compared to Comparative Examples 1 and 2, and that the recovery rate is particularly excellent when PIPES is used as the buffer solution.
［実施例６］組成物中のプロテオグリカンの測定
１．粉末の調製
２種類の粉末を作製した。
図１０（Ａ）で示す測定試料作製用に、プロテオグリカンを０．４４質量％含有、所定の乳タンパク質を所定量含有された試料粉末約４５４．５ｍｇを準備した。
図１０（Ｂ）で示す測定試料（ブランクの試料）作製用に、プロテオグリカンを含有せず、所定の乳タンパク質を所定量含有された試料粉末約４５４．５ｍｇを準備した。
[Example 6] Measurement of proteoglycan in the composition 1. Preparation of powder Two types of powder were prepared.
To prepare the measurement sample shown in FIG. 10(A), about 454.5 mg of sample powder containing 0.44 mass % proteoglycan and a given amount of a given milk protein was prepared.
To prepare the measurement sample (blank sample) shown in FIG. 10(B), about 454.5 mg of sample powder containing no proteoglycan but a given amount of a given milk protein was prepared.
ここで、当該粉末に含有された乳タンパク質は、具体的な素材としては脱脂粉乳、全脂粉乳、トータルミルクプロテイン、カゼインナトリウム、乳清タンパク質、バターミルクパウダー、牛乳、全脂濃縮乳、脱脂濃縮乳、ナチュラルチーズ等である。 Specific examples of the milk proteins contained in the powder include skim milk powder, whole milk powder, total milk protein, sodium caseinate, whey protein, buttermilk powder, milk, whole fat concentrated milk, skim milk concentrated milk, and natural cheese.
２．溶液の作製
当該粉末を精密に量り、１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液（ｐＨ７．０）１５ｍＬを加え、溶液を作製した。次にこの溶液を所定条件で超音波処理を行い、超音波処理後、遠心分離により上清液を回収した。当該上清液を濾紙でろ過し、５０ｍＬの溶液を調製した。
2. Preparation of solution The powder was precisely weighed and 15 mL of 10 mM PIPES-NaOH buffer (pH 7.0) was added to prepare a solution. This solution was then subjected to ultrasonic treatment under specified conditions, and after ultrasonic treatment, the supernatant was collected by centrifugation. The supernatant was filtered through filter paper to prepare 50 mL of solution.
３．イオン交換処理
弱塩基性陰イオン交換用ゲルであるＴＯＹＯＰＡＫ ＤＥＡＥ Ｍ（ゲル量２．０ｍＬ、東ソー株式会社製）のカラムに、精製水１０ｍＬを流した後、上記で調製した１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液（ｐＨ７．０）１０ｍＬを流してイオン交換樹脂を平衡化した。このカラムに同緩衝液に溶解した上記測定試料１０ｍＬを負荷した。続いて、５ｍＬの同緩衝液でカラムを洗浄後、０．３５ＭのＮａＣｌを含む同緩衝液１０ｍＬを流してさらに洗浄した。洗浄後のカラムに、０．５ＭのＮａＣｌを含む同緩衝液１０ｍＬを流してプロテオグリカンをカラムから脱着させた。
3. Ion exchange treatment After 10 mL of purified water was poured into a column of TOYOPAK DEAE M (gel volume 2.0 mL, manufactured by Tosoh Corporation), which is a weakly basic anion exchange gel, 10 mL of the 10 mM PIPES-NaOH buffer (pH 7.0) prepared above was poured to equilibrate the ion exchange resin. 10 mL of the measurement sample dissolved in the same buffer was loaded onto this column. Next, the column was washed with 5 mL of the same buffer, and then further washed with 10 mL of the same buffer containing 0.35 M NaCl. After washing, 10 mL of the same buffer containing 0.5 M NaCl was poured into the column to desorb the proteoglycan from the column.
４．脱塩処理
上記イオン交換処理で回収した脱着画分１０ｍＬを、アミコンウルトラ遠心式フィルターユニット（分画分子量１００Ｋ、メルク株式会社）を用いて最初に脱着液で１回洗浄後、精製水で洗浄して脱塩した。遠心後のフィルターカップから濃縮液を回収し、リン酸緩衝液（ｐＨ６．８）を加えて５ｍＬの溶液（測定試料）とした。
4. Desalting Treatment 10 mL of the desorption fraction recovered in the above ion exchange treatment was desalted by first washing once with the desorption solution and then with purified water using an Amicon Ultra centrifugal filter unit (molecular weight cutoff 100K, Merck Ltd.). The concentrated solution was recovered from the filter cup after centrifugation, and phosphate buffer (pH 6.8) was added to make 5 mL of solution (measurement sample).
別に、プロテオグリカンの標準品（富士フイルム和光純薬工業製）約１ｍｇを精密に量り、リン酸緩衝液（ｐＨ６．８）を加えて１０ｍＬ（標準液）とした。 Separately, approximately 1 mg of a proteoglycan standard (manufactured by Fujifilm Wako Pure Chemical Industries) was precisely weighed and phosphate buffer (pH 6.8) was added to make 10 mL (standard solution).
５．ＨＰＬＣ分析
上述で作製した測定試料及び標準液を用いて、以下の操作条件でＨＰＬＣ分析を行い、測定試料中のプロテオグリカンの含有の有無を測定した。
検出器：ＵＶ－ＶＩＳ検出器ＳＰＤ－２０Ａ（株式会社島津製作所製）
測定波長：２１５ｎｍ
カラム：ＴＳＫｇｅｌ Ｇ５０００－ＰＷＸＬ φ７．８ｍｍ×３００ｍｍ（東ソー株式会社製）
カラム温度：４０℃
移動相：リン酸緩衝液（ｐＨ６．８）
流速：０．５ｍＬ／分
注入量：１００μＬ
5. HPLC Analysis Using the measurement samples and standard solutions prepared above, HPLC analysis was carried out under the following operating conditions to determine whether the measurement samples contained proteoglycan.
Detector: UV-VIS detector SPD-20A (manufactured by Shimadzu Corporation)
Measurement wavelength: 215nm
Column: TSKgel G5000-PWXL φ7.8 mm × 300 mm (Tosoh Corporation)
Column temperature: 40°C
Mobile phase: phosphate buffer (pH 6.8)
Flow rate: 0.5mL/min Injection volume: 100μL
この測定の結果を図１０に示す。図１０（Ａ）の矢印は、プロテオグリカンの溶出位置（１２．７３３ｍｉｎ）を示す。なお、図示しないが当該標準液を用いてのＨＰＬＣ分析では、プロテオグリカンの溶出位置が１２．７ｍｉｎ付近であった。
一方、図１０（Ｂ）は、プロテオグリカンを含まないブランクの試料を分析した結果である。プロテオグリカンの溶出位置が確認できなかった。
The results of this measurement are shown in Figure 10. The arrow in Figure 10(A) indicates the elution position of proteoglycan (12.733 min). Although not shown, in HPLC analysis using the standard solution, the elution position of proteoglycan was around 12.7 min.
On the other hand, Fig. 10(B) shows the results of an analysis of a blank sample that does not contain proteoglycan. The elution position of proteoglycan could not be confirmed.
［実施例７］組成物中のプロテオグリカンの測定
１．試料カプセルの調製
２種類の試料溶液を作製した。
図１１（Ａ）で示す測定試料作製用に、プロテオグリカンを１．６６質量％含有、所定のヒアルロン酸Ｎａを所定量含有された試料粉末約１６０ｍｇを充填したハードカプセルを準備した。
図１１（Ｂ）で示す測定試料（ブランクの試料）作製用に、プロテオグリカンを含有せず、所定のヒアルロン酸Ｎａを所定量含有された試料粉末約１６０ｍｇを充填したハードカプセルを準備した。
[Example 7] Measurement of proteoglycan in a composition 1. Preparation of sample capsules
Two types of sample solutions were prepared.
To prepare the measurement sample shown in FIG. 11(A), a hard capsule was prepared, filled with approximately 160 mg of sample powder containing 1.66% by mass of proteoglycan and a specified amount of sodium hyaluronate.
To prepare the measurement sample (blank sample) shown in FIG. 11(B), a hard capsule was prepared, filled with approximately 160 mg of sample powder that did not contain proteoglycan but contained a specified amount of sodium hyaluronate.
２．標準溶液の調製
標準品のプロテオグリカンとして、サケ鼻軟骨由来（富士フイルム和光純薬株式会社製）を精密に量り、「食品添加物公定書、一般試験法、試薬・試液」に基づいて調製したリン酸緩衝液（ｐＨ６．８）に溶解し、０．０４、０．１及び０．２ｍｇ／ｍＬとなるように調製したものを標準溶液とした。なお、使用時には、室温減圧デシケーター（シリカゲル）で３時間乾燥したものを用いた。
2. Preparation of standard solutions Standard proteoglycan was precisely measured from salmon nasal cartilage (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), dissolved in phosphate buffer (pH 6.8) prepared according to the "Food Additives Standards, General Testing Methods, Reagents and Test Solutions" to prepare standard solutions at concentrations of 0.04, 0.1, and 0.2 mg/mL. The standard solutions were dried in a vacuum desiccator (silica gel) at room temperature for 3 hours before use.
３．試料溶液の調製と分析方法
試料カプセルを粉砕し、その粉末約１２．８ｍｇを精密に量り、酢酸緩衝液（ｐＨ６．０）（酢酸ナトリウム三水和物０．３ｇに水８０ｍＬを加え溶解し、酢酸にてｐＨ６．０に調製し、水を加えて１００ｍＬにしたもの。）１０ｍＬと、ヒアルロニダーゼ溶液（ヒアルロニダーゼ“アマノ”Ｓｔｒｅｐｔｏｍｙｃｅｓ ａｌｂｏｇｒｉｓｅｏｌｕｓ由来（富士フイルム和光純薬株式会社製）に酢酸塩緩衝液（ｐＨ６．０）を加えて、１０ｍＬにしたもの。）を０．１～０．２ＴＲＵ量の力価になるように加え、酵素処理した。酵素失活後、遠心分離し、上清液を回収した。残留物に対してリン酸緩衝液（ｐＨ６．８）を加えて抽出し、遠心分離後、上清液を回収した。
3. Preparation of sample solution and analysis method The sample capsule was crushed, and about 12.8 mg of the powder was precisely weighed, and 10 mL of acetate buffer (pH 6.0) (0.3 g of sodium acetate trihydrate was added to 80 mL of water to dissolve, adjusted to pH 6.0 with acetic acid, and water was added to make 100 mL.) and hyaluronidase solution (hyaluronidase "Amano" derived from Streptomyces albogriseolus (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added with acetate buffer (pH 6.0) to make 10 mL.) were added to a titer of 0.1 to 0.2 TRU, and enzyme treatment was performed. After enzyme inactivation, the mixture was centrifuged and the supernatant was collected. The residue was extracted with phosphate buffer (pH 6.8), and the supernatant was collected after centrifugation.
上清液は分子量分画膜（Ｍｏｌｅｃｕｌａｒ Ｗｅｉｇｈｔ Ｃｕｔ Ｏｆｆ（以下、ＭＷＣＯと記載）が、５～１０万）を用いて、遠心分離し、不透過側のプロテオグリカン画分を精製した。得られたプロテオグリカン濃縮液に塩化ナトリウム含有１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液（ｐＨ７．０）（ＰＩＰＥＳ３．０５ｇに水約８００ｍＬを加え、ＮａＯＨ溶液にてｐＨ７．０に調製し、水を加えて１０００ｍＬとしたもの。）１０ｍＬを加えて、溶解し、平衡化した陰イオン交換樹脂カラム（イオン交換容量：０．２ミリ当量以上）に供し、塩化ナトリウム濃度が０．２Ｍ～１Ｍの濃度勾配溶出でプロテオグリカンを脱着した。この脱着液を、分子量分画膜（ＭＷＣＯ５～１０万）を用いて遠心分離し、精製水で洗浄して脱塩した。得られたプロテオグリカン濃縮液にリン酸緩衝液（ｐＨ６．８）を加えて正確に５ｍＬの溶液（測定試料）とした。 The supernatant was centrifuged using a molecular weight cutoff membrane (Molecular Weight Cut Off (MWCO) 50,000-100,000) to purify the non-permeable proteoglycan fraction. The resulting proteoglycan concentrate was dissolved in 10 mM PIPES-NaOH buffer (pH 7.0) (3.05 g of PIPES was added to approximately 800 mL of water, adjusted to pH 7.0 with NaOH solution, and water was added to make 1000 mL). The solution was then applied to an equilibrated anion exchange resin column (ion exchange capacity: 0.2 milliequivalents or more), and the proteoglycan was desorbed by concentration gradient elution with a sodium chloride concentration of 0.2 M to 1 M. The desorbed liquid was centrifuged using a molecular weight cutoff membrane (MWCO 50,000-100,000) and desalted by washing with purified water. Phosphate buffer (pH 6.8) was added to the resulting proteoglycan concentrate to make exactly 5 mL of solution (measurement sample).
試料溶液及び標準溶液を次の操作条件で液体クロマトグラフィーにより試験を行い、検量線から試料カプセル中のプロテオグリカン量を求めた。
計算方法
プロテオグリカン含量（％）＝Ａ×Ｖ×Ｎ×１／Ｗ×１００
Ａ：試験溶液中のプロテオグリカン濃度（ｍｇ／ｍＬ）
Ｖ：定容量（ｍＬ）
Ｎ：希釈倍数
Ｗ：試験品採取量（ｍｇ）
The sample solution and the standard solution were tested by liquid chromatography under the following operating conditions, and the amount of proteoglycan in the sample capsule was determined from the calibration curve.
Calculation method: Proteoglycan content (%) = A x V x N x 1/W x 100
A: Proteoglycan concentration in the test solution (mg/mL)
V: constant volume (mL)
N: Dilution factor W: Amount of test sample collected (mg)
操作条件
検出器：示差屈折率検出器（ＲＩＤ－１０Ａ、株式会社島津製作所製）
カラム：ゲルろ過カラム（東ソー株式会社製、ＴＳＫｇｅｌ Ｇ５０００ＰＷＸＬ）
カラム管：内径約７．８ｍｍ、長さ３０ｃｍのステンレス管
カラム温度：４０℃
移動相：リン酸緩衝液（ｐＨ６．８）
流量：０．５ｍＬ／分
注入量：５０μＬまたは、１００μＬ
Operating conditions Detector: Differential refractive index detector (RID-10A, manufactured by Shimadzu Corporation)
Column: gel filtration column (TSKgel G5000PWXL, manufactured by Tosoh Corporation)
Column tube: Stainless steel tube with inner diameter of about 7.8 mm and length of 30 cm Column temperature: 40°C
Mobile phase: phosphate buffer (pH 6.8)
Flow rate: 0.5 mL/min Injection volume: 50 μL or 100 μL
４．結果
試料溶液及びブランク品のクロマトグラムを図１１に示す。図１１（Ａ）の矢印（１３．２９２ｍｉｎ）は、標準品を用いた分析で検出されたプロテオグリカンの溶出位置を示す。一方、図１１（Ｂ）は、プロテオグリカンを含まないブランク品を分析した結果である。これらの結果より、検量線のピーク面積から計算した組成物中に含まれるプロテオグリカンの含有率は、１．８５質量％であった。
4. Results Chromatograms of the sample solution and the blank are shown in FIG. 11. The arrow (13.292 min) in FIG. 11(A) indicates the elution position of the proteoglycan detected in the analysis using the standard. Meanwhile, FIG. 11(B) shows the results of the analysis of the blank that does not contain proteoglycan. From these results, the content of proteoglycan in the composition calculated from the peak area of the calibration curve was 1.85% by mass.
［実施例８］組成物中のプロテオグリカンの測定
１．試料粉末の調製
２種類の試料溶液を作製した。
図１２（Ａ）で示す測定試料作製用に、プロテオグリカンを０．１３９質量％含有、所定の青汁粉末を所定量含有された試料粉末約９０００ｍｇを準備した。
図１２（Ｂ）で示す測定試料（ブランクの試料）作製用に、プロテオグリカンを含有せず、所定の青汁粉末を所定量含有された試料粉末約９０００ｍｇを準備した。
ここで、当該粉末に含有された青汁粉末は、具体的な素材としては緑黄色野菜のしぼり汁を乾燥させた粉末であり、より具体的には大麦若葉末、ケール末、明日葉末、よもぎ末、抹茶末、小松菜末、桑葉末等である。
[Example 8] Measurement of proteoglycan in a composition 1. Preparation of sample powder
Two types of sample solutions were prepared.
To prepare the measurement sample shown in FIG. 12(A), about 9,000 mg of sample powder containing 0.139% by mass of proteoglycan and a predetermined amount of a predetermined green juice powder was prepared.
To prepare the measurement sample (blank sample) shown in FIG. 12(B), about 9000 mg of sample powder containing no proteoglycan but a predetermined amount of a predetermined green juice powder was prepared.
Here, the green juice powder contained in the powder is, specifically, a powder made by drying the squeezed juice of green and yellow vegetables, and more specifically, barley leaf powder, kale powder, angelica leaf powder, mugwort powder, matcha powder, komatsuna powder, mulberry leaf powder, etc.
２．標準溶液の調製
標準品のプロテオグリカンとして、サケ鼻軟骨由来（富士フイルム和光純薬株式会社製）を精密に量り、リン酸緩衝液（ｐＨ６．８）に溶解し、０．０４、０．１及び０．２ｍｇ／ｍＬとなるように調製したものを標準溶液とした。なお、使用時には、室温減圧デシケーター（シリカゲル）で３時間乾燥したものを用いた。
2. Preparation of standard solutions Standard proteoglycan was prepared by precisely weighing salmon nasal cartilage-derived proteoglycan (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) and dissolving it in phosphate buffer (pH 6.8) to prepare standard solutions with concentrations of 0.04, 0.1, and 0.2 mg/mL. The standard solutions were dried in a vacuum desiccator (silica gel) at room temperature for 3 hours before use.
３．試料溶液の調製と分析方法
試料約７１９．４ｍｇを精密に量り、含水エタノール溶液１５ｍＬを加え、よく撹拌し、遠心分離後、上清を除去した。残留物に対して精製水１５ｍＬを加えて、所定条件で超音波処理にて抽出した。この抽出液を遠心分離後、上清液を回収した。上清液をろ紙ろ過し、そのろ液を分子量分画膜（ＭＷＣＯ５～１０万）を用いて、遠心分離し、不透過側のプロテオグリカン画分を精製し、精製水で洗浄した。含水エタノールのエタノール濃度の下限は５０％であり、好ましくは５５％、より好ましくは６０％である。また、含水エタノールのエタノール濃度の上限は９９．９％％であり、好ましくは９０％、より好ましくは８０％である。
3. Preparation of sample solution and analysis method Approximately 719.4 mg of sample was precisely weighed, 15 mL of aqueous ethanol solution was added, and the mixture was thoroughly stirred and centrifuged, after which the supernatant was removed. 15 mL of purified water was added to the residue, and the mixture was extracted by ultrasonic treatment under specified conditions. After centrifuging this extract, the supernatant was collected. The supernatant was filtered through a filter paper, and the filtrate was centrifuged using a molecular weight cutoff membrane (MWCO 50,000 to 100,000), and the proteoglycan fraction on the non-permeable side was purified and washed with purified water. The lower limit of the ethanol concentration of aqueous ethanol is 50%, preferably 55%, and more preferably 60%. The upper limit of the ethanol concentration of aqueous ethanol is 99.9%, preferably 90%, and more preferably 80%.
得られたプロテオグリカン濃縮液に１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液（ｐＨ７．０）を加えて正確に１０ｍＬに調製した。この調製液２ｍＬを、１０ｍＭのＰＩＰＥＳ－ＮａＯＨ緩衝液（ｐＨ７．０）で平衡化した陰イオン交換樹脂カラム（イオン交換容量：０．２ミリ当量以上）に供し、塩化ナトリウム濃度が０．２Ｍ～１Ｍ濃度勾配溶出でプロテオグリカンを脱着した。この脱着液を、分子量分画膜（ＭＷＣＯ５～１０万）を用いて、遠心分離し、精製水で洗浄して脱塩した。得られたプロテオグリカン濃縮液にリン酸緩衝液（ｐＨ６．８）を加えて正確に５ｍＬの溶液（測定試料）とした。 10 mM PIPES-NaOH buffer (pH 7.0) was added to the resulting proteoglycan concentrate to make exactly 10 mL. 2 mL of this preparation was applied to an anion exchange resin column (ion exchange capacity: 0.2 milliequivalents or more) equilibrated with 10 mM PIPES-NaOH buffer (pH 7.0), and proteoglycan was desorbed by gradient elution with sodium chloride concentrations of 0.2 M to 1 M. This desorbed liquid was centrifuged using a molecular weight cutoff membrane (MWCO 50,000 to 100,000) and washed with purified water for desalting. Phosphate buffer (pH 6.8) was added to the resulting proteoglycan concentrate to make exactly 5 mL of solution (measurement sample).
試料溶液及び標準溶液を次の操作条件で液体クロマトグラフィーにより試験を行い、検量線から試験溶液中のプロテオグリカン濃度を算出し、次式により試料溶液中のプロテオグリカン含量を求めた。
計算方法
プロテオグリカン含量（％）＝Ａ×Ｖ×Ｎ×１／Ｗ×１００
Ａ：試験溶液中のプロテオグリカン濃度（ｍｇ／ｍＬ）
Ｖ：定容量（ｍＬ）
Ｎ：希釈倍数
Ｗ：試験品採取量（ｍｇ）
The sample solution and the standard solution were tested by liquid chromatography under the following operating conditions, the proteoglycan concentration in the test solution was calculated from the calibration curve, and the proteoglycan content in the sample solution was calculated by the following formula.
Calculation method: Proteoglycan content (%) = A x V x N x 1/W x 100
A: Proteoglycan concentration in the test solution (mg/mL)
V: constant volume (mL)
N: Dilution factor W: Amount of test sample collected (mg)
操作条件
検出器：示差屈折率検出器（ＲＩＤ－１０Ａ、株式会社島津製作所製）
カラム：ゲルろ過カラム（東ソー株式会社製、ＴＳＫｇｅｌ Ｇ５０００ＰＷＸＬ）
カラム管：内径約７．８ｍｍ、長さ３０ｃｍのステンレス管
カラム温度：４０℃
移動相：リン酸緩衝液（ｐＨ６．８）
流量：０．５ｍＬ／分
注入量：５０μＬまたは、１００μＬ
Operating conditions Detector: Differential refractive index detector (RID-10A, manufactured by Shimadzu Corporation)
Column: gel filtration column (TSKgel G5000PWXL, manufactured by Tosoh Corporation)
Column tube: Stainless steel tube with inner diameter of about 7.8 mm and length of 30 cm Column temperature: 40°C
Mobile phase: phosphate buffer (pH 6.8)
Flow rate: 0.5 mL/min Injection volume: 50 μL or 100 μL
４．結果
試料溶液及びプラセボ品のクロマトグラムを図１２に示す。図１２（Ａ）の矢印（１３．４１７ｍｉｎ）は、標準品を用いた分析で検出されたプロテオグリカンの保持時間を示す。一方、図１２（Ｂ）は、プロテオグリカンを含まないブランク品を分析した結果である。これらの結果より、検量線のピーク面積から計算した組成物中に含まれるプロテオグリカンの含有率は、０．１３５質量％であった。
4. Results Chromatograms of the sample solution and the placebo product are shown in Figure 12. The arrow (13.417 min) in Figure 12 (A) indicates the retention time of the proteoglycan detected in the analysis using the standard product. Meanwhile, Figure 12 (B) shows the results of the analysis of a blank product that does not contain proteoglycan. From these results, the content of proteoglycan contained in the composition calculated from the peak area of the calibration curve was 0.135% by mass.
本発明により、サプリメントなどの飲食品等の組成物に含有されているプロテオグリカンを簡便に測定する方法が提供でき、例えば、コラーゲンやゼラチン等のタンパク質を含む食品等の品質管理分析に役立つ可能性がある。
The present invention provides a method for easily measuring proteoglycans contained in compositions such as food and beverage supplements, which may be useful, for example, for quality control analysis of foods containing proteins such as collagen and gelatin.
Claims (7)
前記組成物を、プロテオグリカンの官能基の解離に適した緩衝液に溶解し、測定試料を調製する工程と、
前記緩衝液を含む平衡化緩衝液でイオン交換樹脂を平衡化する工程と、
前記測定試料を前記イオン交換樹脂に負荷して前記プロテオグリカンを吸着させる工程と、
前記プロテオグリカンが吸着したイオン交換樹脂を、前記緩衝液を含む洗浄緩衝液で洗浄する工程と、
前記イオン交換樹脂から溶出緩衝液でプロテオグリカンを溶出する工程と、
前記溶出されたプロテオグリカンを定量する工程と、
を含む、
前記緩衝液が、ｐＨが中性領域の緩衝液であって、ナトリウムイオン及び／又はカリウムイオンを含む緩衝液である、
測定方法。 A method for measuring a proteoglycan content in a composition, comprising the steps of:
dissolving the composition in a buffer suitable for dissociating functional groups of proteoglycans to prepare a measurement sample;
equilibrating the ion exchange resin with an equilibration buffer containing the buffer;
A step of loading the measurement sample onto the ion exchange resin to adsorb the proteoglycan;
washing the ion exchange resin to which the proteoglycan has been adsorbed with a washing buffer solution containing the buffer solution;
eluting the proteoglycan from the ion exchange resin with an elution buffer;
Quantifying the eluted proteoglycan;
including,
The buffer solution has a pH in the neutral range and contains sodium ions and/or potassium ions.
Measuring method.
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| JP2007204473A (en) | 1995-11-13 | 2007-08-16 | Seikagaku Kogyo Co Ltd | Novel chondroitin sulfate proteoglycan, core protein thereof, dna encoding the same, and antibody against the same |
| US20100151584A1 (en) | 2007-04-16 | 2010-06-17 | Momenta Pharmaceuticals, Inc. | Multi-dimensional chromatographic methods for separating n-glycans |
| JP2018151207A (en) | 2017-03-10 | 2018-09-27 | 一丸ファルコス株式会社 | Method for analyzing proteoglycan |
| JP2019162056A (en) | 2018-03-19 | 2019-09-26 | 地方独立行政法人青森県産業技術センター | Encapsulated proteoglycan, manufacturing method therefor, and proteoglycan added food product |
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| JP2007204473A (en) | 1995-11-13 | 2007-08-16 | Seikagaku Kogyo Co Ltd | Novel chondroitin sulfate proteoglycan, core protein thereof, dna encoding the same, and antibody against the same |
| US20100151584A1 (en) | 2007-04-16 | 2010-06-17 | Momenta Pharmaceuticals, Inc. | Multi-dimensional chromatographic methods for separating n-glycans |
| JP2018151207A (en) | 2017-03-10 | 2018-09-27 | 一丸ファルコス株式会社 | Method for analyzing proteoglycan |
| JP2019162056A (en) | 2018-03-19 | 2019-09-26 | 地方独立行政法人青森県産業技術センター | Encapsulated proteoglycan, manufacturing method therefor, and proteoglycan added food product |
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