JP2007039424A - Dipeptidyl peptidase iv inhibitor - Google Patents
Dipeptidyl peptidase iv inhibitor Download PDFInfo
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- JP2007039424A JP2007039424A JP2005261209A JP2005261209A JP2007039424A JP 2007039424 A JP2007039424 A JP 2007039424A JP 2005261209 A JP2005261209 A JP 2005261209A JP 2005261209 A JP2005261209 A JP 2005261209A JP 2007039424 A JP2007039424 A JP 2007039424A
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Abstract
Description
本発明は、インスリン非依存性糖尿病（ＮＩＤＤＭ）と称される２型糖尿病やこの病気に関連する疾患（例えば、肥満及び脂質障害）の治療において治療用化合物として使用される、新規なジペプチジルペプチダーゼIV（ＤＰＰIV）阻害剤に関する。また、本発明は、チーズの水溶性画分に存在するＤＰＰIV阻害活性を有するペプチドに関する。本発明のＤＰＰIV阻害剤は、チーズの水溶性画分に存在するペプチドまたはチーズの水溶性画分を有効成分とし、ＤＰＰIVに拮抗的に作用することで血糖値を低下させることを特徴とする。
The present invention relates to novel dipeptidyl peptidases used as therapeutic compounds in the treatment of
糖尿病は、複数の原因から誘発され、空腹状態時または経口ブドウ糖負荷試験中にグルコース投与後の血漿グルコースレベルが高いこと、すなわち高血糖により特徴づけられる病気である。高血糖が持続し、血糖値が正常にコントロールされないと、他の代謝及び血行動態的疾患の罹患率及び死亡率が高くなる。グルコースホメオスタシスが異常であると、脂質、リポタンパク質及びアポリポタンパク質代謝が変化し、他の代謝及び血行動態的疾患が発症する。よって、糖尿病患者は、冠状心疾患、卒中、末梢血管疾患、高血圧、腎症、神経障害及び網膜症を含めた大血管及び微小血管合併症を併発する危険性が特に高い。従って、糖尿病を臨床的に管理及び治療する際にはグルコースホメオスタシス、脂質代謝及び高血圧をコントロールすることが重要である。 Diabetes is a disease that is induced by multiple causes and is characterized by high plasma glucose levels following glucose administration during fasting or during an oral glucose tolerance test, ie, hyperglycemia. If hyperglycemia persists and blood glucose levels are not normally controlled, the morbidity and mortality of other metabolic and hemodynamic diseases increases. Abnormal glucose homeostasis alters lipid, lipoprotein and apolipoprotein metabolism and develops other metabolic and hemodynamic diseases. Thus, diabetic patients are particularly at risk of developing macrovascular and microvascular complications including coronary heart disease, stroke, peripheral vascular disease, hypertension, nephropathy, neuropathy and retinopathy. Therefore, it is important to control glucose homeostasis, lipid metabolism and hypertension when clinically managing and treating diabetes.
糖尿病に２つの型があることが認められている。１型糖尿病、すなわちインスリン依存性糖尿病（ＩＤＤＭ）では、患者はグルコース利用を調節するホルモンであるインスリンをほとんど乃至全く生成しない。２型糖尿病、すなわちインスリン非依存性糖尿病（ＮＩＤＤＭ）では、患者の血漿インスリンレベルは糖尿病でない人と同等かまたは高いことが多い。しかしながら、これらの患者は主なインスリン感受性組織である筋肉、肝臓や脂肪組織におけるグルコース及び脂質代謝に対するインスリン刺激作用に対して抵抗性を有しており、血漿インスリンレベルは高いが顕著なインスリン抵抗性を解決するには十分でない。
It is recognized that there are two types of diabetes. In
インスリン抵抗性は、主にインスリン受容体の数が減少したことではなく、ポストインスリン受容体結合欠陥に起因していると言われている。このインスリン応答性に対して抵抗性が生ずると、筋肉におけるグルコース摂取、酸化及び貯蔵のインスリン活性化は不十分となり、脂肪組織における脂肪分解及び肝臓におけるグルコース産生及び分泌に対するインスリン抑制は不十分となる。
現在、２型糖尿病に対する治療は何年にもわたり実質的に変わっていないが、限界があることは認められている。運動及び食事のカロリー摂取制限により糖尿病状態は改善されるが、この治療のコンプライアンスは非常に低い。なぜならば、非活動的なライフスタイルが確立されており、食品、特に飽和脂肪を多く含む食品が過剰に消費されているからである。より多くのインスリンを分泌するように膵臓β細胞を刺激するスルホニル尿素（例えば、トルブタミド及びグリピジド）またはメグリチニドを投与することにより、インスリン濃度は高インスリン抵抗性組織を刺激するのに十分なレベルの高さとなる。スルホニル尿素またはメグリチニドが有効でなかったときには、インスリンを注射することによりインスリンの血漿レベルを上昇させる。しかしながら、インスリンまたはインスリン分泌促進薬（スルホニル尿素及びメグリチニド）の投与により血漿グルコースレベルが非常に低くなる恐れがあり、血漿インスリンレベルが高いためにインスリン抵抗性が増す恐れがある。ビグアニドはインスリン感受性を増加させて高脂血症を若干改善する。しかしながら、２種のビグアニドのフェンホルミン及びメトホルミンはそれぞれ乳酸アシドーシス及び悪心、下痢を誘発する恐れがある。
Insulin resistance is said to be mainly due to a post-insulin receptor binding defect, not a decrease in the number of insulin receptors. When resistance to this insulin responsiveness occurs, insulin activation of glucose uptake, oxidation and storage in muscle is insufficient, and insulin suppression of lipolysis in adipose tissue and glucose production and secretion in the liver is insufficient. .
Currently, treatment for
膵臓β細胞から分泌されるインスリンは、血糖を降下させる唯一のホルモンとして生体の糖代謝の制御にかかわり、グルコースホメオスタシスに重要な役割を果たしている。インスリンの分泌には、インクレチンが関与していると考えられる。インクレチンとは、消化管ホルモンの総称であり、食物を摂取したという情報がインクレチンの分泌を介して速やかに膵臓β細胞へと伝達され、インスリン分泌を刺激し、食後の血糖値上昇を抑制し、血糖を一定に保つ役割を有している。これまでにインクレチンとして胃抑制性ポリペプチド（ＧＩＰ）およびグルカゴン様ペプチド１（ＧＬＰ−1）の2つのホルモンが知られている。よって、インクレチンの作用により、血糖が上昇し始める食後早期にインスリン分泌を効率的に上昇させ食後の高血糖を抑えることができる。２型糖尿病患者では、食後の高血糖が一つの特徴であり、この一因としてインクレチンの作用障害が報告されている。
Insulin secreted from pancreatic β cells plays an important role in glucose homeostasis, as it is the only hormone that lowers blood sugar and is involved in the regulation of glucose metabolism in the body. Incretin is thought to be involved in insulin secretion. Incretin is a general term for gastrointestinal hormones, and information that food has been consumed is quickly transmitted to the pancreatic β-cells via incretin secretion, stimulating insulin secretion and suppressing postprandial blood glucose levels. And has a role of keeping blood sugar constant. So far, two hormones of gastric inhibitory polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are known as incretins. Therefore, by the action of incretin, insulin secretion can be efficiently increased early after meals when blood sugar begins to rise, and postprandial hyperglycemia can be suppressed. In patients with
血中に存在するＤＰＰIVは、特異的にＮ末端から2番目のプロリンまたはアラニンを持つ生理活性ペプチドに対して作用し、Ｎ末端からジペプチドを遊離させる酵素であり、各種のホルモンやケモカインの分解に関与していることが知られている。ＧＩＰやＧＬＰ−1も血中においてＤＰＰIVにより速やかに分解され、活性を失う。このことは、ＤＰＰIV欠損マウスを用いた試験で、経口ブドウ糖負荷試験（ＯＧＴＴ）において耐糖能の悪化が見られ、ＤＰＰIVがＧＬＰ−1やＧＩＰなどの分解を介して、生体内の糖代謝に影響を及ぼしていることが報告されている（例えば、非特許文献１参照。）。
よって、血中のＤＰＰIVによるインクレチン分解活性を阻害することで血糖値を低下させることが可能となる。
DPPIV present in the blood is an enzyme that specifically acts on a physiologically active peptide having the second proline or alanine from the N-terminus and releases the dipeptide from the N-terminus, and is used for the degradation of various hormones and chemokines. It is known to be involved. GIP and GLP-1 are also rapidly degraded by DPPIV in the blood and lose their activity. This is a test using DPPIV-deficient mice, in which glucose tolerance was deteriorated in the oral glucose tolerance test (OGTT), and DPPIV affected in vivo glucose metabolism through degradation of GLP-1 and GIP. (For example, refer nonpatent literature 1).
Therefore, the blood glucose level can be lowered by inhibiting the incretin degradation activity by DPPIV in the blood.
ＤＰＰIV阻害剤としては、化学合成物が多数報告されおり、たとえばフルオロピリジン、ピラジン、スルフォスチンなどがありすでに経口剤として実用化されているものもある（例えば、特許文献１、２、３、４参照。）。しかし、これらの化学合成品は、常に安全性の問題に注意を払わなければならない。
天然物由来では納豆抽出液がＤＰＰIV阻害活性を持つと報告されているのみである（例えば、非特許文献２参照。）。
As DPPIV inhibitors, many chemical compounds have been reported. For example, there are fluoropyridines, pyrazines, sulfostins, and the like, and some of them have already been put into practical use as oral agents (for example, see
Only natural natto extract has been reported to have DPPIV inhibitory activity (see, for example, Non-Patent Document 2).
チーズの水溶性画分についてはカルシウム吸収促進作用、細胞増殖作用、抗菌作用等の生理作用が明らかにされている。しかしながら、チーズの水溶性画分がＤＰＰIV阻害活性を持つという報告は見当たらない。また、チーズの生理機能に関しては、抗腫瘍作用、抗変異原作用等が報告されているものの、ＤＰＰIV阻害活性を持つことについては知られていない。このように、チーズの水溶性画分がＤＰＰIV阻害活性を持つという報告は見当たらない。
本発明は、天然物から得られ、毒性が低く安全性の高い、顕著なＤＰＰIV阻害活性を有するＤＰＰIV阻害剤およびその製造方法を提供することを課題とする。また、医薬品、特定保健用食品、健康食品等に利用できるＤＰＰIV阻害剤を提供することを課題とする。 An object of the present invention is to provide a DPPIV inhibitor obtained from a natural product, having low toxicity and high safety, having a remarkable DPPIV inhibitory activity, and a method for producing the same. It is another object of the present invention to provide a DPPIV inhibitor that can be used for pharmaceuticals, foods for specified health use, health foods, and the like.
本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、チーズを水性溶媒に懸濁した後、不溶性物質の除去を行って得られた水溶性画分に、顕著なＤＰＰIV阻害活性を有する物質が存在することを見出した。また、上記水溶性画分をＯＡＳＩＳカラムで処理したＯＡＳＩＳ溶出画分にＤＰＰIV阻害活性があり、チーズの熟成期間が増加すると共に阻害活性が増加する傾向があった。さらに、活性の高かったゴーダタイプ12ヶ月熟成チーズのＯＡＳＩＳ溶出画分をＨＰＬＣで分画し、フラクションの活性を測定した結果、全体的に活性はあるが、特に活性の強い画分が数個あり、活性成分が分画された。この活性の高い画分をＭＳで解析し、カゼイン(α,β)由来ペプチドの存在が確認された。確認されたいくつかのペプチドを合成し、そのＩＣ50を求めた結果、市販の阻害剤であるDiprotin Aの1／8〜1／500倍の活性があることを見出した。
すなわち、本発明は、チーズの水溶性画分を有効成分とするＤＰＰIV阻害剤に関する。
また、本発明は、熟成ナチュラルチーズの水溶性画分を有効成分とするＤＰＰIV阻害剤に関する。
また、本発明は、ＤＰＰIV阻害活性を有する以下のいずれかの配列からなるペプチドに関する。
(1) Ile-Pro-Asn
(2) Gly-Pro-Ile-Pro-Asn
(3) Val-Pro-Ile-Thr-Pro-Thr
(4) Val-Pro-Gly-Glu-Ile-Val-Glu
(5) Leu-Pro-Gln-Asn-Ile-Pro-Pro
(6) Thr-Pro-Val-Val-Val-Pro-Pro
(7) Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro
(8) Val-Ala-Pro-Phe-Pro-Glu
(9) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(10) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser
(11) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn
(12) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val
(13) Met-Pro-Phe-Pro-Lys-Tyr
(14) Phe-Pro-Gly-Pro-Ile-Pro-Asn
(15) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(16) Val-Pro-Ile-Thr-Pro-Thr-Leu
(17) Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn
(18) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
(19) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu
(20) Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
(21) Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys
(22) Thr-Pro-Val-Val-Val-Pro-Pro-Phe
(23) Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp
(24) Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
また、本発明は、チーズの水溶性画分に存在するＤＰＰIV阻害活性を有する前記のいずれかの配列からなるペプチドに関する。
また、本発明は、ＤＰＰIV阻害活性を有する前記のいずれか1以上のペプチドを有効成分とするＤＰＰIV阻害剤に関する。
As a result of intensive studies to solve the above problems, the present inventors have found that the water-soluble fraction obtained by suspending cheese in an aqueous solvent and then removing insoluble substances has significant DPPIV inhibitory activity. It was found that a substance having Further, the OASIS elution fraction obtained by treating the water-soluble fraction with an OASIS column has DPPIV inhibitory activity, and the inhibitory activity tends to increase as the ripening period of cheese increases. Furthermore, OASIS elution fraction of Gouda type 12-month-aged cheese with high activity was fractionated by HPLC and the activity of the fraction was measured. As a result, there were several fractions that were particularly active. The active ingredient was fractionated. This highly active fraction was analyzed by MS, and the presence of casein (α, β) -derived peptide was confirmed. As a result of synthesizing several confirmed peptides and determining their IC50s, they found that they were 1/8 to 1/500 times more active than Diprotin A, a commercially available inhibitor.
That is, this invention relates to the DPPIV inhibitor which uses the water-soluble fraction of cheese as an active ingredient.
Moreover, this invention relates to the DPPIV inhibitor which uses the water-soluble fraction of ripening natural cheese as an active ingredient.
The present invention also relates to a peptide having any of the following sequences having DPPIV inhibitory activity.
(1) Ile-Pro-Asn
(2) Gly-Pro-Ile-Pro-Asn
(3) Val-Pro-Ile-Thr-Pro-Thr
(4) Val-Pro-Gly-Glu-Ile-Val-Glu
(5) Leu-Pro-Gln-Asn-Ile-Pro-Pro
(6) Thr-Pro-Val-Val-Val-Pro-Pro
(7) Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro
(8) Val-Ala-Pro-Phe-Pro-Glu
(9) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(10) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser
(11) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn
(12) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val
(13) Met-Pro-Phe-Pro-Lys-Tyr
(14) Phe-Pro-Gly-Pro-Ile-Pro-Asn
(15) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(16) Val-Pro-Ile-Thr-Pro-Thr-Leu
(17) Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn
(18) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
(19) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu
(20) Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
(21) Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys
(22) Thr-Pro-Val-Val-Val-Pro-Pro-Phe
(23) Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp
(24) Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
Moreover, this invention relates to the peptide which consists of one of the said sequences which has DPPIV inhibitory activity which exists in the water-soluble fraction of cheese.
The present invention also relates to a DPPIV inhibitor comprising as an active ingredient any one or more of the aforementioned peptides having DPPIV inhibitory activity.
本発明のチーズの水溶性画分またはチーズの水溶性画分に存在するＤＰＰIV阻害活性を有するペプチドは、ＤＰＰIV阻害活性を有する高血糖防止剤等として利用可能な、安全性が高く、副作用のない画分であり、数々の医薬品、特定用保健食品、機能性食品等への利用が期待できる。また、本発明のＤＰＰIV阻害剤は、チーズを水性溶媒に懸濁した後、不溶性物質の除去を行って得られるチーズの水溶性画分をそのまま利用するという簡便な製造方法で得ることができ、さらに原料となるチーズも容易に、しかも安価に入手することができるという利点があり、実用上極めて利用価値が高い。 The water-soluble fraction of the cheese of the present invention or the peptide having DPPIV inhibitory activity present in the water-soluble fraction of cheese can be used as a hyperglycemia inhibitor having DPPIV inhibitory activity, etc., and has high safety and no side effects It is a fraction and can be used for various medicines, health foods for specific use, functional foods, etc. The DPPIV inhibitor of the present invention can be obtained by a simple production method in which the water-soluble fraction of cheese obtained by suspending cheese in an aqueous solvent and then removing insoluble substances is used as it is, Furthermore, cheese as a raw material has an advantage that it can be easily obtained at low cost, and is extremely useful in practical use.
本発明のＤＰＰIV阻害剤はチーズの水溶性画分またはチーズの水溶性画分に存在するＤＰＰIV阻害活性を有するペプチドを有効成分とする。チーズの水溶性画分が熟成ナチュラルチーズの水溶性画分であればより好ましい。
本発明のＤＰＰIV阻害活性を有するチーズの水溶性画分は、例えばチーズを水性溶媒に懸濁した後、不溶性物質の除去を行って得ることができる。本発明のチーズの水溶性画分においてチーズを水性溶媒に懸濁するということは、チーズに水性溶媒を加えてホモジナイザーなどで均質化したり、または水性溶媒中で破砕したりして、水溶性画分を得やすい大きさにすることをいう。チーズを水性溶媒に懸濁した後、遠心分離して得た上清、あるいは、その上清をさらにろ過して不溶性物質を除去したものが本発明のチーズの水溶性画分である。この水溶性画分を限外ろ過に通して得られる透過液であっても良い。また、上記水溶性画分を透析膜やイオン交換樹脂等によって脱塩処理を行っても良いし、ＯＡＳＩＳカラム等に通して吸着した画分を溶出した画分であっても良い。また、ＤＰＰIV阻害活性を有するチーズの水溶性画分は、C18カラムを用いて、式（１）〜（24）で表されるアミノ酸配列のいずれかの配列からなるペプチドを多く含む画分に分画することも可能である。
また、本発明に用いることができる配列式（1）〜（24）で表されるアミノ酸配列のいずれかの配列からなるＤＰＰIV阻害活性を有するペプチドは、例えば上記水溶性画分をC18カラムを用いて分画することにより得ることができる。さらに、合成して得られたペプチドについても利用可能である。水性溶媒としては、水、リン酸緩衝液等の溶媒を用いることができる。この水溶性画分を凍結乾燥や噴霧乾燥等によって乾燥させることにより粉末化しても良い。
The DPPIV inhibitor of the present invention contains, as an active ingredient, a water-soluble fraction of cheese or a peptide having DPPIV-inhibiting activity present in the water-soluble fraction of cheese. More preferably, the water-soluble fraction of cheese is a water-soluble fraction of ripened natural cheese.
The water-soluble fraction of cheese having DPPIV inhibitory activity of the present invention can be obtained, for example, by suspending cheese in an aqueous solvent and then removing insoluble substances. Suspending cheese in an aqueous solvent in the water-soluble fraction of the cheese of the present invention means adding an aqueous solvent to the cheese and homogenizing it with a homogenizer or crushing it in an aqueous solvent, To make it easy to get the minutes. The supernatant obtained by suspending cheese in an aqueous solvent and then centrifuging, or the supernatant further filtered to remove insoluble substances is the water-soluble fraction of the cheese of the present invention. A permeate obtained by passing this water-soluble fraction through ultrafiltration may be used. Further, the water-soluble fraction may be desalted with a dialysis membrane, an ion exchange resin, or the like, or may be a fraction obtained by eluting the fraction adsorbed through an OASIS column or the like. In addition, the water-soluble fraction of cheese having DPPIV inhibitory activity is separated into a fraction containing a large amount of peptide consisting of any one of the amino acid sequences represented by formulas (1) to (24) using a C18 column. It is also possible to draw.
A peptide having DPPIV inhibitory activity consisting of any one of the amino acid sequences represented by the sequence formulas (1) to (24) that can be used in the present invention uses, for example, the above water-soluble fraction using a C18 column. Can be obtained by fractionation. Furthermore, the peptide obtained by synthesis can also be used. As an aqueous solvent, solvents such as water and phosphate buffer can be used. The water-soluble fraction may be pulverized by drying by freeze drying or spray drying.
本発明のＤＰＰIV阻害活性を有するチーズの水溶性画分または配列式（1）〜（24）で表されるアミノ酸配列のいずれかの配列からなるチーズの水溶性画分に存在するＤＰＰIV阻害活性を有するペプチドを得るためのチーズ原料としては、ナチュラルチーズ、及びナチュラルチーズを原料としたプロセスチーズ等を用いることができるが、パルメザンチーズ、グリュイエールチーズ、マリボーチーズ、ゴーダチーズ、チェダーチーズ、エメンタールチーズ、エダムチーズ、カマンベールチーズ、ブリーチーズ、マンステールチーズ、ポン・レヴェックチーズ、スチルトンチーズ、ダナブルーチーズ、ブルーチーズ等の熟成ナチュラルチーズ、及びこれらの熟成ナチュラルチーズを原料としたプロセスチーズ等を用いることが望ましく、より熟成期間の長い熟成ナチュラルチーズを用いることがより望ましい。
このように、本発明のＤＰＰIV阻害剤は、チーズを原料とするので、原料の入手が極めて容易であり、安価であるという利点を有する。しかも、チーズを原料とし、水性溶媒を利用するだけであり、安全性、毒性の面でも極めて有利である。
The DPPIV inhibitory activity present in the water-soluble fraction of cheese having DPPIV inhibitory activity of the present invention or the water-soluble fraction of cheese comprising any one of the amino acid sequences represented by the sequence formulas (1) to (24) Natural cheese and processed cheese made from natural cheese can be used as the cheese raw material for obtaining the peptide having, but Parmesan cheese, Gruyère cheese, Maribo cheese, Gouda cheese, Cheddar cheese, Emmental cheese, Edam cheese, Camembert It is desirable to use aged cheeses such as cheese, brie cheese, manstail cheese, pon levec cheese, stilton cheese, dana blue cheese, blue cheese, and processed cheese made from these aged natural cheeses. Ripening period It is more desirable to use a long ripened natural cheese.
Thus, since the DPPIV inhibitor of this invention uses cheese as a raw material, it has the advantage that acquisition of a raw material is very easy and it is cheap. In addition, cheese is used as a raw material and only an aqueous solvent is used, which is extremely advantageous in terms of safety and toxicity.
本発明のＤＰＰIV阻害剤は、チーズを水性溶媒に懸濁した後、脱脂、遠心分離、ろ過等によって不溶性物質の除去を行って得られるチーズの水溶性画分をそのまま利用することができるし、透析膜やイオン交換樹脂等によって脱塩を行ったもの、さらに、凍結乾燥や噴霧乾燥等によって乾燥を行い粉末化したものも利用することができる。
また、本発明のＤＰＰIV阻害活性を有する画分は、飲食品に配合してＤＰＰIV阻害用飲食品として使用することができる。飲食品に配合する場合は、チーズの水溶性画分をそのまま配合することができるし、透析膜やイオン交換樹脂等によって脱塩を行ったもの、さらに、凍結乾燥や噴霧乾燥等によって乾燥を行い粉末化したものも配合することができる。
このように、本発明のＤＰＰIV阻害剤は、チーズを水性溶媒に懸濁した後、脱脂、遠心分離、ろ過等によって不溶性物質の除去を行って得られるチーズの水溶性画分をそのまま利用するという簡便な製造方法で得ることもできるので、実用上極めて利用価値が高い。さらに原料となるチーズも容易に入手することができるという利点がある。
The DPPIV inhibitor of the present invention can utilize the water-soluble fraction of cheese obtained by suspending cheese in an aqueous solvent and then removing insoluble substances by degreasing, centrifugation, filtration, etc. Those obtained by desalting with a dialysis membrane or an ion exchange resin or the like, and further powdered by drying by freeze drying or spray drying can be used.
Moreover, the fraction which has DPPIV inhibitory activity of this invention can be mix | blended with food-drinks, and can be used as food-drinks for DPPIV inhibition. When blended in food or drink, the water-soluble fraction of cheese can be blended as it is, desalted with a dialysis membrane, ion exchange resin, etc., and further dried by freeze drying, spray drying, etc. Powdered products can also be blended.
Thus, the DPPIV inhibitor of the present invention uses the water-soluble fraction of cheese obtained by suspending cheese in an aqueous solvent and then removing insoluble substances by degreasing, centrifugation, filtration, etc. Since it can be obtained by a simple production method, it is extremely useful in practical use. Furthermore, there is an advantage that cheese as a raw material can be easily obtained.
本発明のＤＰＰIV阻害剤は、経口的に投与して、生体においてＤＰＰIVを阻害することにより血糖値を低下させることが可能となる。経口的に投与する場合、本発明のＤＰＰIV阻害剤の剤形としては、錠剤、カプセル剤、細粒剤、散剤、丸剤、トローチ、舌下剤または液剤等の経口投与用の製剤を例示することができる。 The DPPIV inhibitor of the present invention can be orally administered to inhibit blood glucose level by inhibiting DPPIV in a living body. When administered orally, examples of the dosage form of the DPPIV inhibitor of the present invention include tablets, capsules, fine granules, powders, pills, troches, sublingual or liquid preparations. Can do.
本発明のＤＰＰIV阻害剤の経口による投与量は、治療や予防の目的、症状、体重、年齢や性別等を考慮して適宜決定すればよいが、通常、成人1日あたりチーズの水溶性画分の固形分として50mg〜100g投与すれば、ＤＰＰIVを阻害する治療または予防効果が得られる。 The oral dose of the DPPIV inhibitor of the present invention may be appropriately determined in consideration of the purpose of treatment and prevention, symptoms, body weight, age, sex, etc., but usually the water soluble fraction of cheese per adult day When administered as a solid content of 50 mg to 100 g, a therapeutic or prophylactic effect inhibiting DPPIV can be obtained.
本発明のＤＰＰIV阻害剤、あるいはそれらを配合した飲食品を経口摂取することによって生体内で血糖値を低下させる作用を発揮する。本発明のＤＰＰIV阻害剤を配合した飲食品としては、チーズ、バター、乳飲料、ジュース、ヨーグルト、ゼリー、パン、アイスクリーム、麺、ソーセージ、育児用調製乳や離乳食等を挙げることができる。 By ingesting the DPPIV inhibitor of the present invention, or a food or drink containing them, the effect of lowering blood glucose level in vivo is exhibited. Examples of the food and drink containing the DPPIV inhibitor of the present invention include cheese, butter, milk drink, juice, yogurt, jelly, bread, ice cream, noodles, sausage, infant formula and baby food.
ＤＰＰIVの活性測定は、例えば‐ＧｌｏTM プロテアーゼ法を用いて行うことができる。基質としてＤＰＰIV‐ＧｌｏTM Reagent（プロメガ社）を用いる。この方法は、ＤＰＰIVに被験サンプルを加え、基質溶液を添加し、混和して、一定時間後の発光量を測定することにより、ＤＰＰIVの活性を測定する。このように、添加‐混和‐測定の操作により、ＤＰＰIV‐ＧｌｏTM Reagentを加えると、ＤＰＰIVによる基質の切断に引き続き、ルシフェラーゼ反応による発光シグナルが生じる。この酵素反応のカップリングによるホモジニアスタイプアッセイで発生するシグナルは、ＤＰＰIV活性量に比例するので、これによりＤＰＰIVの活性を測定することができる。 The activity of DPPIV can be measured using, for example, the -Glo ™ protease method. DPPIV-Glo ™ Reagent (Promega) is used as the substrate. In this method, the activity of DPPIV is measured by adding a test sample to DPPIV, adding a substrate solution, mixing, and measuring the amount of luminescence after a certain time. Thus, when DPPIV-Glo ™ Reagent is added by the operation of addition-mixing-measurement, a luminescence signal is generated by luciferase reaction following the cleavage of the substrate by DPPIV. Since the signal generated in the homogeneous type assay due to the coupling of the enzyme reaction is proportional to the amount of DPPIV activity, it is possible to measure the activity of DPPIV.
以下に実施例及び試験例を示し、本発明をより詳細に説明するが、これらは単に例示するのみであり、本発明はこれらによって何ら限定されるものではない。 EXAMPLES The present invention will be described in more detail below with reference to examples and test examples, but these are merely illustrative and the present invention is not limited by these.
（チーズの水溶性画分の調製）
ラクトコッカス・ラクチス・サブスピーシズ・ラクチス（Lactococcus lactis subsp. lactis）、ラクトコッカス・ラクチス・サブスピーシズ・クレモリス（Lactococcus lactis subsp. cremoris）、ロイコノストック・メセンテロイデス（Leuconostoc mesenteroides）を乳酸菌スターターとして常法により製造したゴーダタイプチーズを原料として用いた。このチーズを細かく切断し、10倍量の水を加え、ホモジナイザー（AM-3: 株式会社日本精機製作所）で粉砕（10,000rpm、10分間）して懸濁した後、遠心分離（6,000rpm、10分間、4℃）で沈殿を除いた上清をさらにろ過（No．2ろ紙:ADVANTEC）して不溶性物質を除去後、限外ろ過（Centriplus YM-30 :Millipore）をして透過液を得、これをチーズの水溶性画分とした。このようにして得られたチーズの水溶性画分は、そのままＤＰＰIV阻害剤として利用可能である。
(Preparation of water-soluble fraction of cheese)
Lacticococcus lactis subsp. Gouda type cheese was used as a raw material. This cheese is cut into fine pieces, 10 times the amount of water is added, pulverized (10,000 rpm, 10 minutes) with a homogenizer (AM-3: Nippon Seiki Seisakusho), suspended, and then centrifuged (6,000 rpm, 10 The supernatant from which the precipitate was removed at 4 ° C for 4 minutes was further filtered (No. 2 filter paper: ADVANTEC) to remove insoluble substances, followed by ultrafiltration (Centriplus YM-30: Millipore) to obtain a permeate, This was made into the water-soluble fraction of cheese. The water-soluble fraction of cheese thus obtained can be used as it is as a DPPIV inhibitor.
（チーズの水溶性画分の調製）
乳酸菌スターターとしてストレプトコッカス・サーモフィルス(Streptococcus thermophilus)及びラクトバチラス・ヘルベティカス（Lactobacillus helveticus）を用いて常法により製造したイタリアンタイプチーズを原料として用いた以外は実施例１と同様にして、チーズの水溶性画分を調製した。このようにして得られたチーズの水溶性画分は、そのままＤＰＰIV阻害剤として利用可能である。
(Preparation of water-soluble fraction of cheese)
A water-soluble image of cheese in the same manner as in Example 1 except that Italian type cheese produced by a conventional method using Streptococcus thermophilus and Lactobacillus helveticus as a lactic acid bacteria starter was used as a raw material. Minutes were prepared. The water-soluble fraction of cheese thus obtained can be used as it is as a DPPIV inhibitor.
[試験例１]
（ＤＰＰIVの活性測定）
実施例１で得られたゴーダタイプチーズと実施例２で得られたイタリアンタイプチーズのそれぞれの水溶性画分について、ＤＰＰIV‐ＧｌｏTM プロテアーゼ法により、ＤＰＰIV阻害活性の測定を行った。ゴーダタイプチーズは、製造直後、1、2、6、及び12ヶ月熟成させたもののそれぞれの水溶性画分を実施例１に記載のとおり調製し、試験に供した。基質としてＤＰＰIV‐ＧｌｏTM Reagent（G8350；プロメガ社）を用い、酵素はＤＰＰIV （317630、ヒト胎盤由来；Calbiochem社）、対照阻害剤としてディプロチンＡ (DiprotinＡ、 4132−v ；Peptide社）を使用した。すなわち、100μl 、96穴ブラックプレートに、1μlの ＤＰＰIV（10μU/μl）及び前記のゴーダタイプチーズ水溶性画分 6μlを添加して、超純水で50μlにメスアップし、さらに基質溶液50μlを添加し、混和した。30分後に発光量を測定した。サンプル無添加のものの活性（発光量）を100％とし、サンプルを添加した際の相対的な活性を算出した。
また、イタリアンタイプチーズは、製造直後、1、2、6、及び12ヶ月熟成させたもののそれぞれの水溶性画分を実施例２に記載のとおり調製し、ゴーダタイプチーズの水溶性画分と同様の試験に供した。
結果を図１に示す。
[Test Example 1]
(Measurement of DPPIV activity)
About each water-soluble fraction of the Gouda type cheese obtained in Example 1, and the Italian type cheese obtained in Example 2, the DPPIV inhibitory activity was measured by the DPPIV-Glo ™ protease method. Gouda-type cheese was prepared as described in Example 1 for each of the water-soluble fractions that had been ripened immediately after production, for 1, 2, 6, and 12 months, and used for the test. DPPIV-Glo ™ Reagent (G8350; Promega) was used as the substrate, DPPIV (317630, derived from human placenta; Calbiochem) was used as the enzyme, and diprotin A (Diprotin A, 4132-v; Peptide) was used as the control inhibitor. That is, add 1 μl of DPPIV (10 μU / μl) and 6 μl of the Gouda-type cheese water-soluble fraction to 100 μl, 96-well black plate, make up to 50 μl with ultrapure water, and add 50 μl of substrate solution. And mixed. The amount of luminescence was measured after 30 minutes. The relative activity when the sample was added was calculated by setting the activity (luminescence amount) of the sample not added to 100%.
In addition, Italian type cheese is prepared as described in Example 2 for each of water-soluble fractions that have been ripened immediately after production, for 1, 2, 6, and 12 months, and is similar to the water-soluble fraction of Gouda-type cheese. The test was conducted.
The results are shown in FIG.
ゴーダタイプチーズの水溶性画分、イタリアンタイプチーズの水溶性画分のいずれにもＤＰＰIV活性を阻害する作用があることが確認された。
またそれぞれのチーズの熟成が進むにつれて阻害する作用は強くなっており、特に熟成期間が6カ月以上のチーズの水溶性画分は顕著なＤＰＰIV活性を阻害する作用を示した。これは熟成によって生じるペプチドが有効成分であると考えられる。このようにチーズの水溶性画分は、そのままＤＰＰIV阻害剤として利用可能である。
It was confirmed that both the water-soluble fraction of Gouda-type cheese and the water-soluble fraction of Italian-type cheese have an effect of inhibiting DPPIV activity.
Moreover, the inhibitory effect became stronger as the ripening of each cheese progressed, and especially the water-soluble fraction of cheese with a ripening period of 6 months or more showed a remarkable inhibitory effect on DPPIV activity. This is considered to be an active ingredient is a peptide produced by aging. Thus, the water-soluble fraction of cheese can be used as a DPPIV inhibitor as it is.
（チーズの水溶性画分の調製）
実施例１のゴーダタイプチーズ、実施例２のイタリアンタイプチーズと同じチーズを原料として用いた。これらのチーズを細かく切断し、10倍量の水を加え、ホモジナイザー（AM-3: 株式会社日本精機製作所）で粉砕（10,000rpm、10分間）して懸濁した後、スターラーで攪拌しながら40℃の恒温槽で1時間加温処理した。そして、遠心分離（6,000rpm、10分間、4℃）で沈殿を除いた上清をさらにろ過（No．2ろ紙:ADVANTEC）して不溶性物質を除去後、限外ろ過（Centriplus YM-30 :Millipore）をして透過液を得た。
この透過液を、蒸留水で平衡化したＯＡＳＩＳカラム（ＨＬＢ 6cc：ウォーターズ社）に供し、蒸留水で洗浄後、吸着画分を100％メタノールにて溶出した。回収した溶出液を遠心エバポレーター（CVE-200D：EYELA）で乾固した後、ＯＡＳＩＳカラムに供した量の1／10量の蒸留水に溶解した。
このようにして得られたＯＡＳＩＳ溶出画分は、チーズの水溶性画分として、そのまま本発明のＤＰＰIV阻害剤として利用可能である。
(Preparation of water-soluble fraction of cheese)
The same cheese as the gouda type cheese of Example 1 and the Italian type cheese of Example 2 was used as a raw material. After cutting these cheeses finely, adding 10 times the amount of water, pulverizing (10,000 rpm, 10 minutes) with a homogenizer (AM-3: Nippon Seiki Seisakusho Co., Ltd.) and suspending, then stirring with a
This permeate was applied to an OASIS column (HLB 6cc: Waters) equilibrated with distilled water, washed with distilled water, and the adsorbed fraction was eluted with 100% methanol. The recovered eluate was dried with a centrifugal evaporator (CVE-200D: EYELA), and then dissolved in 1/10 amount of distilled water supplied to the OASIS column.
The OASIS-eluted fraction thus obtained can be used as the water-soluble fraction of cheese as it is as the DPPIV inhibitor of the present invention.
[試験例２]
（ＤＰＰIVの活性測定）
実施例３で得られたゴーダタイプチーズ及びイタリアンタイプチーズの水溶性画分について、ＤＰＰIV‐ＧｌｏTMプロテアーゼキット（G8350、プロメガ社）により、ＤＰＰIV阻害活性の測定を行った。ゴーダタイプチーズ及びイタリアンタイプチーズは、製造直後、1、2、4、6、及び12ヶ月熟成させたもののそれぞれの水溶性画分を実施例３に記載のとおり調製し、試験に供した。基質としてＤＰＰIV‐ＧｌｏTM Reagent（プロメガ社）を用い、酵素はＤＰＰIV （317630、ヒト胎盤由来；Calbiochem社）を使用した。すなわち、96穴ブラックプレートに前記のゴーダタイプチーズ又はイタリアンタイプチーズ水溶性画分 10μlを添加し、基質溶液50μlを添加して混和した。さらに超純水39μlと1μlの ＤＰＰIV（10μU/μl）を添加して反応を開始し、30分後に発光量を測定した。サンプル無添加のものの発光量を100％とし、サンプルを添加した際の相対的な活性を算出した。ＤＰＰIV阻害活性の測定結果を図2に示す。
[Test Example 2]
(Measurement of DPPIV activity)
With respect to the water-soluble fraction of Gouda type cheese and Italian type cheese obtained in Example 3, DPPIV inhibitory activity was measured using a DPPIV-Glo ™ protease kit (G8350, Promega). Gouda-type cheese and Italian-type cheese were prepared as described in Example 3 for each water-soluble fraction after ripening for 1, 2, 4, 6 and 12 months immediately after production and subjected to the test. DPPIV-Glo ™ Reagent (Promega) was used as a substrate, and DPPIV (317630, derived from human placenta; Calbiochem) was used as an enzyme. That is, 10 μl of the Gouda type cheese or Italian type cheese water-soluble fraction was added to a 96-well black plate, and 50 μl of the substrate solution was added and mixed. Furthermore, 39 μl of ultrapure water and 1 μl of DPPIV (10 μU / μl) were added to start the reaction, and the amount of luminescence was measured after 30 minutes. The relative activity when the sample was added was calculated with the amount of luminescence of the sample not added as 100%. The measurement results of DPPIV inhibitory activity are shown in FIG.
（熟成期間の増加による活性の変化）
ゴーダタイプチーズ、イタリアンタイプチーズのいずれの水溶性画分にもＤＰＰIVの活性を阻害する作用があることが確認された。またそれぞれのチーズの熟成が進むにつれて阻害する作用が強くなっており、特にゴーダタイプ12ヶ月熟成チーズでは非常に顕著なＤＰＰIV活性を阻害する作用を示した。これは熟成によって生じるペプチドが有効成分であると考えられる。
(Change in activity due to increased aging period)
It was confirmed that any water-soluble fraction of Gouda-type cheese or Italian-type cheese has an effect of inhibiting DPPIV activity. Moreover, the inhibitory effect became stronger as the ripening of each cheese progressed, and in particular, the gouda-type 12-month-aged cheese showed a very remarkable effect of inhibiting DPPIV activity. This is considered to be an active ingredient is a peptide produced by aging.
（逆相ＨＰＬＣによる分画とＤＰＰIV阻害活性の測定）
阻害活性成分を同定するため、まずは阻害活性の高かったゴーダタイプ12ヶ月熟成チーズのＯＡＳＩＳ溶出画分を逆相ＨＰＬＣで分画した。ゴーダタイプ12ヶ月熟成チーズの水溶性画分を実施例３に記載のとおり調製し、試験に供した。分画にはHPLC system（HP 1050；ヒューレット・パッカード社）を用い、カラムはPROTEIN & PEPTIDE C18 カラム(4.6 mm ID×250mm: VYDAC) を30℃で使用した。溶出は5→65%アセトニトリル(0.1% TFA) /60分のグラジエント、1ml/分の流速で行い、UV220nmで検出した。220nmでの吸光度の変化を図3に示す。
ＯＡＳＩＳ溶出画分100μlをインジェクション後、3〜43分の間1分間隔で溶出液を分取した。それぞれ遠心エバポレーター（CVE-200D：EYELA）で乾固した後、10μlの蒸留水または1μlのDMSO（048-21985：和光純薬工業）に溶解し、1ウエル当たりその全量を添加して各々のＤＰＰIV阻害活性を求めた。HPLC画分のＤＰＰIV阻害活性の変化を図4に示す。
その結果、広く阻害活性が見られたが、いくつかのフラクションで特に活性が強かった。このようにして活性成分を分画することも可能である。
(Fractionation by reverse phase HPLC and measurement of DPPIV inhibitory activity)
In order to identify the inhibitory active ingredient, the OASIS elution fraction of Gouda type 12-month-aged cheese having a high inhibitory activity was first fractionated by reverse phase HPLC. A water soluble fraction of Gouda type 12 month ripened cheese was prepared as described in Example 3 and subjected to testing. For the fractionation, an HPLC system (HP 1050; Hewlett Packard) was used, and a PROTEIN & PEPTIDE C18 column (4.6 mm ID × 250 mm: VYDAC) was used at 30 ° C. Elution was performed at a gradient of 5 → 65% acetonitrile (0.1% TFA) / 60 minutes, a flow rate of 1 ml / minute, and detected at UV 220 nm. The change in absorbance at 220 nm is shown in FIG.
After injecting 100 μl of OASIS elution fraction, the eluate was collected at intervals of 1 minute for 3 to 43 minutes. Each was dried with a centrifugal evaporator (CVE-200D: EYELA), then dissolved in 10 μl of distilled water or 1 μl of DMSO (048-21985: Wako Pure Chemical Industries), and the total amount was added per well to add each DPPIV Inhibitory activity was determined. The change in DPPIV inhibitory activity of the HPLC fraction is shown in FIG.
As a result, a broad inhibitory activity was observed, but the activity was particularly strong in some fractions. It is also possible to fractionate the active ingredient in this way.
[試験例３]
（活性画分のＭＳによる解析）
活性が高かったフラクションについてマススペクトル（ＭＳ）による解析を行った。ＨＰＬＣで分画した各フラクションをバッファーで適宜希釈したのち、LCQ Advantage ＭＳsystemによりＭＳ分析を行った。ＭＳ/ＭＳ解析はXcalibur 1.3 & Bioworks 3.1により行った。データベースには nr.fasta (NCBI) よりBos taurus由来のタンパク配列のみを抽出して用いた。
ゴーダタイプ12ヶ月熟成チーズの水溶性画分をＨＰＬＣで分画したフラクションをＭＳ解析した結果、同定されたカゼイン由来ペプチドを表１に示す。
その結果、各フラクションにカゼイン由来のペプチドが多数存在することが示され、これらの同定されたペプチドがＤＰＰIV阻害活性に関与していると考えられる。
[Test Example 3]
(Analysis of active fraction by MS)
Fractions with high activity were analyzed by mass spectrum (MS). Each fraction fractionated by HPLC was appropriately diluted with a buffer, and then subjected to MS analysis by LCQ Advantage MSsystem. MS / MS analysis was performed with Xcalibur 1.3 & Bioworks 3.1. For the database, only protein sequences derived from Bos taurus were extracted from nr.fasta (NCBI) and used.
Table 1 shows the identified casein-derived peptides as a result of MS analysis of the fraction obtained by HPLC fractionation of the water-soluble fraction of Gouda type 12-month ripened cheese.
As a result, it was shown that there were many casein-derived peptides in each fraction, and these identified peptides are considered to be involved in DPPIV inhibitory activity.
[表１]
───────────────────────────────────
フラクシ アミノ酸配列 カゼイン 位置
ョン（分）
───────────────────────────────────
6 Ile-Pro-Asn β 66-68
───────────────────────────────────
10 Gly-Pro-Ile-Pro-Asn β 64-68
───────────────────────────────────
12 Val-Pro-Ile-Thr-Pro-Thr αS2 117-122
───────────────────────────────────
15 Val-Pro-Gly-Glu-Ile-Val-Glu β 8-14
Leu-Pro-Gln-Asn-Ile-Pro-Pro β 70-86
───────────────────────────────────
17 Thr-Pro-Val-Val-Val-Pro-Pro β 80-86
Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro β 178-186
───────────────────────────────────
18 Val-Ala-Pro-Phe-Pro-Glu αS1 25-30
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro β 74-81
───────────────────────────────────
21 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser β 60-69
Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn β 60-68
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val β 74-82
Met-Pro-Phe-Pro-Lys-Tyr β 109-114
───────────────────────────────────
22 Phe-Pro-Gly-Pro-Ile-Pro-Asn β 62-68
Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-P β 70-84
ro
Val-Pro-Ile-Thr-Pro-Thr-Leu αS2 117-123
───────────────────────────────────
23 Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn αS1 106-114
───────────────────────────────────
4 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn β 60-68
Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu β 70-78
Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu β 84-91
───────────────────────────────────
25 Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys αS1 26-34
───────────────────────────────────
27 Thr-Pro-Val-Val-Val-Pro-Pro-Phe β 80-87
───────────────────────────────────
29 Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp αS2 102-109
───────────────────────────────────
30 Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-G β 80-91
lu
───────────────────────────────────
[Table 1]
───────────────────────────────────
Flax amino acid sequence casein position (min)
───────────────────────────────────
6 Ile-Pro-Asn β 66-68
───────────────────────────────────
10 Gly-Pro-Ile-Pro-Asn β 64-68
───────────────────────────────────
12 Val-Pro-Ile-Thr-Pro-Thr αS2 117-122
───────────────────────────────────
15 Val-Pro-Gly-Glu-Ile-Val-Glu β 8-14
Leu-Pro-Gln-Asn-Ile-Pro-Pro β 70-86
───────────────────────────────────
17 Thr-Pro-Val-Val-Val-Pro-Pro β 80-86
Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro β 178-186
───────────────────────────────────
18 Val-Ala-Pro-Phe-Pro-Glu αS1 25-30
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro β 74-81
───────────────────────────────────
21 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser β 60-69
Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn β 60-68
Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val β 74-82
Met-Pro-Phe-Pro-Lys-Tyr β 109-114
───────────────────────────────────
22 Phe-Pro-Gly-Pro-Ile-Pro-Asn β 62-68
Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-P β 70-84
ro
Val-Pro-Ile-Thr-Pro-Thr-Leu αS2 117-123
───────────────────────────────────
23 Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn αS1 106-114
───────────────────────────────────
4 Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn β 60-68
Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu β 70-78
Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu β 84-91
───────────────────────────────────
25 Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys αS1 26-34
───────────────────────────────────
27 Thr-Pro-Val-Val-Val-Pro-Pro-Phe β 80-87
───────────────────────────────────
29 Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp αS2 102-109
───────────────────────────────────
30 Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-G β 80-91
lu
───────────────────────────────────
（ペプチドの合成と活性測定）
ＭＳで推定されたペプチドのうちのいくつかを合成し、蒸留水またはＤＭＳＯ（048-21985：和光純薬工業）に溶解してそのＤＰＰIV阻害活性を求めた。まず、各ペプチド溶液を段階希釈して各々の阻害活性を求め、阻害活性(％)とサンプル濃度の対数(Log10)の関係式から逆算して、各サンプルの50％阻害濃度(IC50)を求めた。対照阻害剤としてDiprotinＡ(4132−v ；Peptide社）の阻害活性も測定した。
ＭＳ解析の結果同定されたペプチドを合成したもののＤＰＰIV阻害活性を比較した。結果を表２に示す。
その結果、ペプチドにより阻害活性に違いが見られたが、特にLeu-Pro-Gln-Asn-Ile-Pro-Pro-Leuの配列を有するペプチド（#9）ではIC50=45.6μMと高い阻害活性を示した。これらの成分を含むことで各フラクションが阻害活性を示していると考えられる。
(Peptide synthesis and activity measurement)
Some of the peptides estimated by MS were synthesized and dissolved in distilled water or DMSO (048-21985: Wako Pure Chemical Industries) to determine their DPPIV inhibitory activity. First, serially dilute each peptide solution to determine the inhibitory activity of each peptide, and calculate the 50% inhibitory concentration (IC50) of each sample by calculating back from the relationship between the inhibitory activity (%) and the logarithm of the sample concentration (Log10). It was. The inhibitory activity of Diprotin A (4132-v; Peptide) was also measured as a control inhibitor.
DPPIV inhibitory activities of the peptides synthesized as a result of MS analysis were compared. The results are shown in Table 2.
As a result, there was a difference in the inhibitory activity depending on the peptide. In particular, the peptide with the sequence of Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu (# 9) has a high inhibitory activity of IC50 = 45.6μM. Indicated. It is thought that each fraction shows inhibitory activity by containing these components.
[試験例４]
（熟成期間による活性ペプチドの蓄積）
ＤＰＰIV阻害活性を示したペプチドのいくつかについて、チーズ熟成中における量変化を調べるため、各熟成期間のチーズ水溶性画分を適当に希釈後、LCQ Advantage ＭＳsystemによりＭＳ分析を行った。ＭＳ/ＭＳ解析はXcalibur 1.3 & Bioworks 3.1により行った。データベースには nr.fasta (NCBI) よりBos taurus由来のタンパク配列のみを抽出して用いた。得られたデータから、各ペプチドに相当するシグナルをそれぞれ抽出し、そのシグナルの強度を熟成期間毎に比較した。
その結果、これらのペプチドが熟成に伴い増加していることが示され、以上のことから熟成に伴って阻害活性を持ついくつかのペプチドが生成・増加し、そのトータルな活性によりチーズ上清中の阻害活性が増加していることが示された。
[Test Example 4]
(Accumulation of active peptide by aging period)
For some of the peptides that showed DPPIV inhibitory activity, in order to examine the amount change during cheese ripening, the cheese water-soluble fraction during each ripening period was appropriately diluted, and then subjected to MS analysis by LCQ Advantage MSsystem. MS / MS analysis was performed with Xcalibur 1.3 & Bioworks 3.1. For the database, only protein sequences derived from Bos taurus were extracted from nr.fasta (NCBI) and used. A signal corresponding to each peptide was extracted from the obtained data, and the intensity of the signal was compared for each aging period.
As a result, it was shown that these peptides increased with aging. From the above, some peptides with inhibitory activity were generated and increased with aging, and the total activity in the cheese supernatant It was shown that the inhibitory activity of was increased.
Claims (5)
(1) Ile-Pro-Asn
(2) Gly-Pro-Ile-Pro-Asn
(3) Val-Pro-Ile-Thr-Pro-Thr
(4) Val-Pro-Gly-Glu-Ile-Val-Glu
(5) Leu-Pro-Gln-Asn-Ile-Pro-Pro
(6) Thr-Pro-Val-Val-Val-Pro-Pro
(7) Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro
(8) Val-Ala-Pro-Phe-Pro-Glu
(9) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(10) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser
(11) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn
(12) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val
(13) Met-Pro-Phe-Pro-Lys-Tyr
(14) Phe-Pro-Gly-Pro-Ile-Pro-Asn
(15) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(16) Val-Pro-Ile-Thr-Pro-Thr-Leu
(17) Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn
(18) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
(19) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu
(20) Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
(21) Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys
(22) Thr-Pro-Val-Val-Val-Pro-Pro-Phe
(23) Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp
(24) Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu A peptide comprising any one of the following sequences having dipeptidyl peptidase IV inhibitory activity.
(1) Ile-Pro-Asn
(2) Gly-Pro-Ile-Pro-Asn
(3) Val-Pro-Ile-Thr-Pro-Thr
(4) Val-Pro-Gly-Glu-Ile-Val-Glu
(5) Leu-Pro-Gln-Asn-Ile-Pro-Pro
(6) Thr-Pro-Val-Val-Val-Pro-Pro
(7) Val-Pro-Tyr-Pro-Gln-Arg-Asp-Met-Pro
(8) Val-Ala-Pro-Phe-Pro-Glu
(9) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(10) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn-Ser
(11) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-His-Asn
(12) Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro-Val
(13) Met-Pro-Phe-Pro-Lys-Tyr
(14) Phe-Pro-Gly-Pro-Ile-Pro-Asn
(15) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu-Thr-Gln-Thr-Pro
(16) Val-Pro-Ile-Thr-Pro-Thr-Leu
(17) Val-Pro-Gln-Leu-Glu-Ile-Val-Pro-Asn
(18) Tyr-Pro-Phe-Pro-Gly-Pro-Ile-Pro-Asn
(19) Leu-Pro-Gln-Asn-Ile-Pro-Pro-Leu
(20) Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
(21) Ala-Pro-Phe-Pro-Glu-Val-Phe-Gly-Lys
(22) Thr-Pro-Val-Val-Val-Pro-Pro-Phe
(23) Gly-Pro-Ile-Val-Leu-Asn-Pro-Trp
(24) Thr-Pro-Val-Val-Val-Pro-Pro-Phe-Ile-Gln-Pro-Glu
A dipeptidyl peptidase IV inhibitor comprising any one or more peptides according to claim 3 as an active ingredient.
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| WO2013133032A1 (en) * | 2012-03-09 | 2013-09-12 | 森永乳業株式会社 | Dipeptidyl peptidase-iv inhibitor |
| US9617300B2 (en) | 2012-03-09 | 2017-04-11 | Morinaga Milk Industry Co., Ltd. | Dipeptidyl peptidase-IV inhibitor |
| WO2014199780A1 (en) * | 2013-06-12 | 2014-12-18 | マルハニチロ株式会社 | Peptide compound inhibiting dipeptidyl peptidase-iv (dppiv), composition containing same, and method for producing same |
| JP5952967B2 (en) * | 2013-06-12 | 2016-07-13 | マルハニチロ株式会社 | Dipeptidyl peptidase IV (DPPIV) inhibiting peptide compound, composition containing the same, and method for producing the same |
| US9868764B2 (en) | 2013-06-12 | 2018-01-16 | Maruha Nichiro Corporation | Dipeptidyl peptidase-IV (DPPIV), inhibitory peptide compound, composition containing the same, and production method for the same |
| US10513538B2 (en) | 2013-06-12 | 2019-12-24 | Maruha Nichiro Corporation | Dipeptidyl peptidase-IV (DPPIV), inhibitory peptide compound, composition containing the same, and production method for the same |
| JP2018537462A (en) * | 2015-12-16 | 2018-12-20 | ディーエスエム アイピー アセッツ ビー．ブイ．Ｄｓｍ Ｉｐ Ａｓｓｅｔｓ Ｂ．Ｖ． | New use |
| JP7115310B2 (en) | 2015-12-16 | 2022-08-09 | ディーエスエム アイピー アセッツ ビー．ブイ． | new use |
| CN114349818A (en) * | 2021-12-31 | 2022-04-15 | 华南理工大学 | Tripeptides with double-target glucose-reducing function and application thereof |
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