JP2022101904A - Agent for preventing and improving gingivitis - Google Patents
Agent for preventing and improving gingivitis Download PDFInfo
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- JP2022101904A JP2022101904A JP2020216279A JP2020216279A JP2022101904A JP 2022101904 A JP2022101904 A JP 2022101904A JP 2020216279 A JP2020216279 A JP 2020216279A JP 2020216279 A JP2020216279 A JP 2020216279A JP 2022101904 A JP2022101904 A JP 2022101904A
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- collagen
- gingival
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- agent
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Abstract
Description
本発明は、コラーゲン加水分解物を含む、歯肉炎の発症予防剤および／または症状改善剤に関する。 The present invention relates to an agent for preventing the onset of gingival inflammation and / or an agent for improving symptoms, including a collagen hydrolyzate.
歯周組織とは歯を支える周辺組織の総称であり、歯肉、歯槽骨、歯根膜、セメント質で構成される。歯肉はその中で唯一外部から観察することが可能な組織であり、健常であるとピンク色や淡い赤色を呈する。歯周病の初期症状である歯肉炎は、歯の表面に沈着した歯垢中の歯周病原細菌により引き起こされる。歯肉は、歯や歯槽骨にしっかりと付着し非可動性の付着歯肉、歯の接触点下の下部歯間鼓形空隙を埋める乳頭歯肉、歯肉縁から遊離歯肉溝までを構成し、歯と付着しない辺縁歯肉（遊離歯肉）から成る。歯肉の約６割がコラーゲンから成り、歯茎の弾力や結びつきを担う。またコラーゲンの生合成は線維芽細胞が担う。 Periodontal tissue is a general term for peripheral tissues that support teeth, and is composed of gingiva, alveolar bone, periodontal ligament, and cementum. The gingiva is the only tissue that can be observed from the outside, and when it is healthy, it exhibits a pink color or a pale red color. Periodontitis, an early symptom of periodontal disease, is caused by periodontopathic bacteria in plaque deposited on the surface of teeth. The gingiva constitutes the non-movable adherent gingiva that firmly adheres to the tooth and alveolar bone, the papillary gingiva that fills the lower intergingival drum-shaped void below the contact point of the tooth, and the gingival margin to the free gingival groove, which adheres to the tooth. Consists of non-marginal gingiva (free gingiva). Approximately 60% of the gingiva is made of collagen, which is responsible for the elasticity and binding of the gums. Fibroblasts are responsible for collagen biosynthesis.
歯肉は血管が密集した組織でもある。歯肉の血管には、細動脈から細静脈にかけて中心毛細血管、真性毛細血管、細動静脈吻合の３つの経路がある。そしてこれらの血管は、分岐、吻合を繰り返しながら歯肉縁に向かって走り、血管網を形成する。そしてそこから外縁上皮に向けてヘアピン形の血管の係蹄が突出し、歯肉乳頭内へと入り込む。歯肉の微小循環系は、このような血管網が適切に作動することにより保たれている。歯肉炎の代表的な所見は歯肉の発赤や腫脹、出血であるが、これは毛細血管形態そのものに変化（毛細血管係蹄の拡張、屈曲、ねじれ）が生じることによる。 The gingiva is also a tissue with dense blood vessels. There are three routes of gingival blood vessels from arterioles to venules: central capillaries, true capillaries, and venule anastomosis. Then, these blood vessels run toward the gingival margin while repeating branching and anastomosis to form a vascular network. Then, the hooves of hairpin-shaped blood vessels protrude toward the outer peripheral epithelium and enter the gingival papilla. The gingival microcirculatory system is maintained by the proper operation of such a vascular network. Typical findings of gingival inflammation are redness, swelling, and bleeding of the gingiva due to changes in the capillary morphology itself (dilation, flexion, and twisting of the capillary hoof).
また炎症部では、炎症と同時にコラーゲン繊維の破壊・減少が起こる。まず炎症により線維芽細胞からのコラーゲンの分泌は抑制される。そして、線維芽細胞自身とマクロファージが分泌するｃｏｌｌａｇｅｎａｓｅがコラーゲン繊維を切断し、この断片を種々の分解酵素がさらに小さな断片やアミノ酸に分解する。さらに線維芽細胞とマクロファージはコラーゲン原繊維を貪食し、細胞内でも消化する（非特許文献１）。これらの作用によりコラーゲン繊維の緻密で規則的な配列が破壊されると、歯肉の機能は低下し細菌からの防御力が低下する。炎症が歯周組織まで波及し歯周炎に至ると、病態はより深刻となる。歯肉炎では炎症により歯肉溝（歯周ポケット）に２～３ｍｍの隙間ができるが、一般的にその原因菌は好気性菌であり、炎症も酸素がある表面付近に留まる。それに対し歯周炎の原因菌は嫌気性菌であり、歯周ポケットの奥深くで繁殖する。そして防御力の落ちた歯肉内へ深く侵入し、炎症を歯槽骨まで広げる。歯周ポケットの深さは４ｍｍ以上となり、最終的には歯槽骨も破壊され歯が抜け落ちてしまう。また歯茎の色も赤紫色に変色し、強い口臭が発生する場合もある。歯周炎に至り歯槽骨が吸収されると正常な状態への回復が困難になる（非特許文献２）ことから、歯肉炎の段階で早期に発見し、治療することが望ましい（非特許文献３）。 In the inflamed area, collagen fibers are destroyed or reduced at the same time as inflammation. First, inflammation suppresses the secretion of collagen from fibroblasts. Then, collagenase secreted by fibroblasts themselves and macrophages cleaves collagen fibers, and various degrading enzymes decompose these fragments into smaller fragments and amino acids. Furthermore, fibroblasts and macrophages phagocytose collagen fibrils and digest them intracellularly (Non-Patent Document 1). When these actions disrupt the dense and regular arrangement of collagen fibers, the function of the gingiva is reduced and the defense against bacteria is reduced. When the inflammation spreads to the periodontal tissue and leads to periodontitis, the condition becomes more serious. In gingival inflammation, inflammation creates a gap of 2 to 3 mm in the gingival sulcus (periodontal pocket), but the causative bacterium is generally aerobic, and inflammation also remains near the oxygen-containing surface. On the other hand, the causative bacteria of periodontitis are anaerobic bacteria, which propagate deep in the periodontal pocket. Then, it penetrates deeply into the weakened gingiva and spreads inflammation to the alveolar bone. The depth of the periodontal pocket becomes 4 mm or more, and eventually the bundle bone is also destroyed and the tooth falls out. In addition, the color of the gums may change to purplish red, and strong bad breath may occur. When periodontitis occurs and the alveolar bone is absorbed, it becomes difficult to recover to the normal state (Non-Patent Document 2). Therefore, it is desirable to detect and treat the periodontitis at an early stage (Non-Patent Document 2). 3).
歯肉炎の予防法・治療法としては、歯垢を取り除くことが最も有効である。すなわち定期的な歯科医院の受診、適切なブラッシングといった日々の歯科保健行動が不可欠である（非特許文献４）。これら歯科保健行動等によって炎症の原因が取り除かれると、歯肉の発赤や腫脹が消失し、ブラッシングの際の歯肉からの出血も軽快する。歯肉内では毛細血管の形態が改善し、コラーゲン繊維の量も回復する。しかし、日々の歯科保健行動の適切な実行が難しい場合もある。 Removing plaque is the most effective way to prevent and treat periodontitis. That is, daily dental health activities such as regular visits to the dental clinic and appropriate brushing are indispensable (Non-Patent Document 4). When the cause of inflammation is removed by these dental health behaviors, the redness and swelling of the gingiva disappear, and the bleeding from the gingiva during brushing is alleviated. In the gingiva, the morphology of capillaries improves and the amount of collagen fibers also recovers. However, proper implementation of daily dental health practices can be difficult.
なおコラーゲン繊維の消失は老化によっても進行しやすく、口腔用組成物及びコラーゲン合成促進剤を開示する特許文献１、正常ヒト歯肉線維芽細胞におけるＩ型コラーゲン産生促進剤及び口腔用剤を開示する特許文献２ではそのコラーゲン産生を高める手段が提供されている。しかし、上述の通り歯周病には感染源、炎症、血管の形態異常などコラーゲン繊維の消失以外にも多くの要素が関わることから、コラーゲン産生を促進させることだけで健常歯肉を回復することはできないと考えられる。しかもコラーゲン産生の過剰な促進によるコラーゲンの過形成には注意が必要である。過形成により生じた組織は従来のコラーゲン線維の配列と異なった方向に形成され、その後瘢痕化するので、コラーゲン原繊維配列にはモザイク構造が生じる。瘢痕は血管も少なく、血流が悪い上、一見して健常であってもモザイク構造の隙間からの再感染が起こりやすく、歯肉炎が再発しやすい。 It should be noted that the disappearance of collagen fibers easily progresses even with aging, and Patent Document 1 which discloses an oral composition and a collagen synthesis promoter, and a patent which discloses a type I collagen production promoter and an oral agent in normal human gingival fibroblasts. Document 2 provides a means for enhancing the collagen production. However, as mentioned above, periodontal disease involves many factors other than the disappearance of collagen fibers such as infection sources, inflammation, and abnormal morphology of blood vessels. Therefore, it is not possible to recover healthy gingiva simply by promoting collagen production. It is thought that it cannot be done. Moreover, caution is required for collagen hyperplasia due to excessive promotion of collagen production. The tissue produced by hyperformation is formed in a direction different from that of the conventional collagen fiber arrangement, and then scarred, so that a mosaic structure is formed in the collagen fibrillar arrangement. Scars have few blood vessels, poor blood flow, and even if they are seemingly healthy, reinfection from the gaps in the mosaic structure is likely to occur, and gingival inflammation is likely to recur.
厚生労働省「平成２９年患者調査の概況」によると、歯肉炎および歯周疾患の総患者数は３９８万人と、高血圧疾患に次いで２番目に罹患者数が多い疾患である上、調査のたびに増加しており、歯科保健行動の啓蒙が功を奏していないことを示唆する。その最大の理由として、歯科保健行動が技術的な側面と自己管理力の２面から構成されるため（非特許文献５）、個人の生活環境、自覚性、習得力などの生活要因を受けやすく、個人差が生じやすいという点が挙げられる。さらに、叢生、ドライマウス、喫煙、老化、ストレスによる免疫力の低下、糖尿病をはじめとする疾病などは口腔内環境の悪化を招きやすく、歯肉炎を発症しやすくなる。そのため、口腔内環境の悪化による歯肉炎を予防し、症状を改善する手段が必要とされている。
よって、本発明の課題は、歯肉炎の発症予防剤および／または症状改善剤を提供することである。
According to the Ministry of Health, Labor and Welfare "Overview of the 2017 Patient Survey", the total number of patients with gingival inflammation and periodontal disease is 3.98 million, which is the second most affected disease after hypertension disease, and every time the survey is conducted. It suggests that the enlightenment of dental health behavior has not been successful. The main reason for this is that dental health behavior is composed of two aspects, technical aspects and self-management ability (Non-Patent Document 5), so it is susceptible to living factors such as individual living environment, awareness, and learning ability. , The point that individual differences are likely to occur. Furthermore, crowding, dry mouth, smoking, aging, weakened immunity due to stress, diseases such as diabetes are likely to cause deterioration of the oral environment, and gingival inflammation is likely to occur. Therefore, there is a need for a means for preventing gingival inflammation due to deterioration of the oral environment and improving the symptoms.
Therefore, an object of the present invention is to provide an agent for preventing the onset of gingival inflammation and / or an agent for improving symptoms.
本発明者らは、上記課題を解決するために鋭意検討を重ねた結果、平均分子量３００～９００を有するコラーゲン加水分解物を２ヶ月間毎日経口投与したイヌの歯肉炎モデルでは、非投与群と比較して、歯肉の毛細血管係蹄の形態が正常に回復し、さらにコラーゲン繊維の面積が大きくなり、瘢痕化することなく正常に回復することが明らかとなった。このような知見に基づいて本発明は完成された。 As a result of diligent studies to solve the above problems, the present inventors in the canine gingival inflammation model in which collagen hydrolysate having an average molecular weight of 300 to 900 was orally administered daily for 2 months, was compared with the non-administered group. In comparison, it was clarified that the morphology of the capillaries of the gingiva was restored to normal, the area of collagen fibers was further increased, and the gingival was restored to normal without scarring. The present invention has been completed based on such findings.
本発明によれば、以下の発明が提供される。
［１］ コラーゲン加水分解物を含む、歯肉炎の発症予防剤および／または症状改善剤であって、前記コラーゲン加水分解物の平均分子量が３００～９００である、剤。
［２］ 前記コラーゲン加水分解物が分子量４００以下の低分子画分を２０質量％以上含む、［１］に記載の剤。
［３］ 歯肉中毛細血管係蹄の拡張、屈曲および／またはねじれを予防および／または改善することにより、歯肉炎の発症が予防されおよび／または症状が改善される、［１］または［２］に記載の剤。
［４］ 歯肉組織中コラーゲンの産生を亢進し、且つコラーゲン繊維の過形成または瘢痕化を抑制することにより、歯肉炎の発症が予防されおよび／または症状が改善される、［１］から［３］のいずれか一に記載の剤。
［５］ 歯肉炎に伴う歯肉からの出血を予防および／または改善する、［１］から［４］のいずれか一に記載の剤。
［６］ 歯肉炎に伴う歯肉組織中コラーゲンの消失を抑制する、［１］から［５］のいずれか一に記載の剤。
［７］歯肉炎の再発を予防する、［１］から［６］のいずれか一に記載の剤。
According to the present invention, the following inventions are provided.
[1] An agent containing a collagen hydrolyzate, which is an agent for preventing the onset of gingival inflammation and / or an agent for improving symptoms, wherein the average molecular weight of the collagen hydrolyzate is 300 to 900.
[2] The agent according to [1], wherein the collagen hydrolyzate contains 20% by mass or more of a small molecule fraction having a molecular weight of 400 or less.
[3] Preventing and / or ameliorating dilation, flexion and / or twisting of the gingival capillaries prevents the onset of gingival inflammation and / or improves symptoms, [1] or [2]. The agent described in.
[4] By enhancing the production of collagen in the gingival tissue and suppressing the hyperplasia or scarring of collagen fibers, the onset of gingival inflammation is prevented and / or the symptoms are improved, [1] to [3]. ] The agent according to any one of.
[5] The agent according to any one of [1] to [4], which prevents and / or improves gingival bleeding associated with gingival inflammation.
[6] The agent according to any one of [1] to [5], which suppresses the disappearance of collagen in the gingival tissue associated with gingival inflammation.
[7] The agent according to any one of [1] to [6], which prevents the recurrence of gingival inflammation.
本発明の歯肉炎の発症予防剤および／または症状改善剤は、歯肉への炎症細胞の浸潤を抑制し、歯肉の毛細血管係蹄の形態を回復し、および／または歯肉のコラーゲン繊維の産生を正常に促進することにより、歯肉炎の発症を予防し、および／または症状を改善することができる。 The prophylactic and / or symptom-improving agents for the onset of periodontitis of the present invention suppress the infiltration of inflammatory cells into the gingiva, restore the morphology of the gingival capillaries, and / or produce collagen fibers in the gingiva. By promoting normally, the onset of gingival inflammation can be prevented and / or the symptoms can be ameliorated.
以下に記載する本発明の説明は、代表的な実施形態や具体例に基づいてなされることがあるが、本発明はそのような実施形態に限定されるものではない。なお、本明細書において「～」を用いて表される数値範囲は「～」の前後に記載される数値を下限値および上限値として含む範囲を意味する。本明細書中、コラーゲン加水分解のペプチド含有率（％）は、ＬＣ－ＭＳ法を用いて絶対検量線法にて求めた。 The description of the present invention described below may be based on typical embodiments and specific examples, but the present invention is not limited to such embodiments. In the present specification, the numerical range represented by using "-" means a range including the numerical values before and after "-" as the lower limit value and the upper limit value. In the present specification, the peptide content (%) of collagen hydrolysis was determined by the absolute calibration curve method using the LC-MS method.
本発明は、コラーゲン加水分解物を含む、歯肉炎の発症予防剤および／または症状改善剤であって、前記コラーゲン加水分解物の平均分子量が３００～９００である、剤を提供する。歯肉炎の発症を予防するとは、歯肉炎が顕在化する可能性を低減すること、および／または歯肉炎の発症を遅延させることを意味する。歯肉炎の症状を改善するとは、歯肉炎の症状を低減または除去することを意味する。 The present invention provides an agent containing a collagen hydrolyzate, which is an agent for preventing the onset of gingival inflammation and / or an agent for improving symptoms, wherein the average molecular weight of the collagen hydrolyzate is 300 to 900. Preventing the onset of periodontitis means reducing the likelihood that the onset of periodontitis will manifest and / or delaying the onset of periodontitis. Improving the symptoms of periodontitis means reducing or eliminating the symptoms of periodontitis.
本明細書中「歯肉炎」は、歯肉に炎症がある状態を指し、歯と歯ぐきの境目が赤く腫れたり、触れると出血することもある。歯肉炎の歯ぐき（歯肉）が腫れて盛り上がると、歯肉溝が深くなる（仮性ポケット）。歯肉炎は、通常、歯の表面に沈着した歯垢中の歯周病原細菌により引き起こされる。さらに、歯肉炎がさらに進行すると歯周炎となる。歯肉炎は、歯ぐきの腫れや出血だけでなく、歯と歯ぐきの境目の部分が壊れて隙間が深くなり、歯周ポケット（真性ポケット）を形成する。プラークがさらにこの隙間に沿って侵入すると、根の先の方へとさらに破壊が進行する。 As used herein, "gingival inflammation" refers to a condition in which the gums are inflamed, and the boundary between the teeth and gums may be red and swollen, or bleeding may occur when touched. When the gums (gingiva) of periodontitis swell and swell, the gingival sulcus becomes deeper (pseudo-pocket). Gingival inflammation is usually caused by periodontopathic bacteria in plaque deposited on the surface of the tooth. Furthermore, when gingival inflammation progresses further, it becomes periodontitis. In gingival inflammation, not only swelling and bleeding of the gums, but also the boundary between the teeth and the gums is broken and the gap becomes deep, forming a periodontal pocket (intrinsic pocket). As plaque invades further along this gap, further destruction progresses towards the tip of the root.
本発明の歯肉炎の発症予防剤および／または症状改善剤に含有されるコラーゲン加水分解物は、特に製造方法が限定されるものではなく、例えば、タンパク質加水分解法、化学合成法、酵素法、発酵法などによって製造することができる。具体的には、ゼラチンまたはコラーゲンの酵素消化精製物であってもよいし、ゼラチンまたはコラーゲンの酵素消化精製物に化学合成したペプチドを添加したものであってよい。コラーゲン加水分解物は、例えば特許第３１４６２５１号公報に記載の方法で調製でき、ある程度精製したコラーゲンあるいは変性コラーゲン（ゼラチン）などを原材料として、プロテアーゼ、ペプチダーゼ等の酵素で加水分解することによって得られる。 The collagen hydrolyzate contained in the gingival inflammation onset preventive agent and / or symptom ameliorating agent of the present invention is not particularly limited in the production method, and for example, a protein hydrolysis method, a chemical synthesis method, an enzyme method, etc. It can be produced by a fermentation method or the like. Specifically, it may be an enzymatically digested and purified product of gelatin or collagen, or it may be a product obtained by adding a chemically synthesized peptide to an enzymatically digested and purified product of gelatin or collagen. The collagen hydrolyzate can be prepared, for example, by the method described in Japanese Patent No. 3146251, and is obtained by hydrolyzing collagen or denatured collagen (gelatin) purified to some extent with an enzyme such as protease or peptidase.
本発明のコラーゲン加水分解物は、平均分子量３００～９００を有することができる。平均分子量３００～９００のコラーゲン加水分解物は、トリペプチド類及びジペプチド類を多く含み、これらが直接吸収、あるいは簡単な消化分解で吸収されるので、体内に取り入れられやすいという特徴を有する。コラーゲン加水分解物の平均分子量は、加水分解物処理に用いる酵素の種類、添加量、および／または反応時間を変えることにより、調整することができる。 The collagen hydrolyzate of the present invention can have an average molecular weight of 300-900. Collagen hydrolysates having an average molecular weight of 300 to 900 contain a large amount of tripeptides and dipeptides, and since these are directly absorbed or absorbed by simple digestive decomposition, they have a characteristic that they are easily taken into the body. The average molecular weight of the collagen hydrolyzate can be adjusted by varying the type, amount and / or reaction time of the enzyme used in the hydrolyzate treatment.
本発明のコラーゲン加水分解物の平均分子量は、８５０以下、８００以下、７５０以下、７００以下、６５０以下、６００以下、５５０以下、５００以下または４５０以下であってもよい。さらに平均分子量の下限値は、３００以上、３５０以上、４００以上、４５０以上、５００以上または５５０以上であってもよい。
コラーゲン加水分解物の平均分子量（重量平均分子量値）は、標準品にＧｌｙ－Ｐｒｏ－Ｈｙｐを加えた上で写真用ゼラチン試験法（ＰＡＧＩ法）第１０版に記載されている方法に沿って決定することが出来る。
The average molecular weight of the collagen hydrolyzate of the present invention may be 850 or less, 800 or less, 750 or less, 700 or less, 650 or less, 600 or less, 550 or less, 500 or less or 450 or less. Further, the lower limit of the average molecular weight may be 300 or more, 350 or more, 400 or more, 450 or more, 500 or more, or 550 or more.
The average molecular weight (weight average molecular weight value) of the collagen hydrolyzate is determined according to the method described in the 10th edition of the photographic gelatin test method (PAGI method) after adding Gly-Pro-Hyp to the standard product. Can be done.
本発明の実施形態の一つにおいて、本発明のコラーゲン加水分解物は分子量４００以下の低分子画分を２０質量％以上含むことができる。分子量４００以下の低分子画分は、主にトリペプチド類及びジペプチド類である。本発明のコラーゲン加水分解物は、分子量４００以下の低分子画分を２０質量％以上を含み、より好ましくは２５質量％以上、３０質量％以上、３５質量％以上、４０質量％以上、４５質量％以上、５０質量％以上含むことができる。コラーゲン加水分解物における分子量４００以下の低分子画分の含有率は、例えば２０～８０質量％、２０～７５質量％、２０～７０質量％、２０～６５質量％、２０～６０質量％、２０～５５質量％または２０～５０質量％の範囲であってもよい。本発明のコラーゲン加水分解物は、分子量１５０～４００の低分子画分を２０質量％以上を含み、より好ましくは２５質量％以上、３０質量％以上、３５質量％以上、４０質量％以上、４５質量％以上、５０質量％以上含むことができる。コラーゲン加水分解物における分子量１５０～４００の低分子画分の含有率は、例えば２０～８０質量％、２０～７５質量％、２０～７０質量％、２０～６５質量％、２０～６０質量％、２０～５５質量％または２０～５０質量％の範囲であってもよい。 In one of the embodiments of the present invention, the collagen hydrolyzate of the present invention can contain 20% by mass or more of a small molecule fraction having a molecular weight of 400 or less. Small molecule fractions with a molecular weight of 400 or less are mainly tripeptides and dipeptides. The collagen hydrolyzate of the present invention contains 20% by mass or more of a low molecular weight fraction having a molecular weight of 400 or less, more preferably 25% by mass or more, 30% by mass or more, 35% by mass or more, 40% by mass or more, and 45% by mass. % Or more and 50% by mass or more can be contained. The content of the low molecular weight fraction having a molecular weight of 400 or less in the collagen hydrolyzate is, for example, 20 to 80% by mass, 20 to 75% by mass, 20 to 70% by mass, 20 to 65% by mass, 20 to 60% by mass, 20. It may be in the range of to 55% by mass or 20 to 50% by mass. The collagen hydrolyzate of the present invention contains 20% by mass or more of a low molecular weight fraction having a molecular weight of 150 to 400, more preferably 25% by mass or more, 30% by mass or more, 35% by mass or more, 40% by mass or more, 45. It can contain mass% or more and 50% by mass or more. The content of the low molecular weight fraction having a molecular weight of 150 to 400 in the collagen hydrolyzate is, for example, 20 to 80% by mass, 20 to 75% by mass, 20 to 70% by mass, 20 to 65% by mass, 20 to 60% by mass. It may be in the range of 20 to 55% by mass or 20 to 50% by mass.
コラーゲン加水分解物に含まれる各種ペプチドの含有率は、例えば、液体クロマトグラフィー質量分析（ＬＣ－ＭＳ）法により分析することができる。ＬＣ－ＭＳ法は、以下に記載の条件で実施することができる。装置としてＡＣＱＵＩＴＹ ＵＹＰＬＣ Ｈ－Ｃｌａｓｓ Ｓｙｓｔｅｍ ｗｉｔｈ ＳＱ Ｄｅｔｅｃｔｏｒ２（ウォーターズ社）、カラムとしてＡＣＣＱ－ＴＡＧ ＵＬＴＡ Ｃ１８（ウォーターズ社、直径２．１×１００ｍｍ、粒子径１．７μｍ）を用いることができる。溶媒Ａに０．１％ギ酸水溶液を、溶媒Ｂとして０．１％ギ酸を含む５０％アセトニトリル水溶液を用い、カラム温度４５℃の条件で０．６ｍＬ／ｍｉｎの速度で溶媒を流すことができる。グラジエント条件として、０～５分まで溶媒Ａを１００％、５～１０分まで溶媒Ｂを徐々に２５％まで上昇させることができる。これに０．１あるいは０．０２％で溶媒Ａに溶解したコラーゲン加水分解物水溶液を注入し、各トリペプチドの質量を検出し、それらの含有率を計算することで分析することができる。 The content of various peptides contained in the collagen hydrolyzate can be analyzed by, for example, a liquid chromatography-mass spectrometry (LC-MS) method. The LC-MS method can be carried out under the conditions described below. ACQUITY UYPLC H-Class System with SQ Detector2 (Waters) can be used as an apparatus, and ACCQ-TAG ULTA C18 (Waters, diameter 2.1 × 100 mm, particle diameter 1.7 μm) can be used as a column. A 0.1% formic acid aqueous solution is used as the solvent A, and a 50% acetonitrile aqueous solution containing 0.1% formic acid is used as the solvent B, and the solvent can be flowed at a rate of 0.6 mL / min under the condition of a column temperature of 45 ° C. As a gradient condition, the solvent A can be gradually increased to 100% from 0 to 5 minutes, and the solvent B can be gradually increased to 25% from 5 to 10 minutes. It can be analyzed by injecting an aqueous collagen hydrolyzate solution dissolved in solvent A at 0.1 or 0.02%, detecting the mass of each tripeptide, and calculating their content.
コラーゲン加水分解物は、Ｉ型コラーゲンのペプチド結合をグリシンのアミノ末端で切断する細菌コラゲナーゼを用いて加水分解することによって得てもよい。コラーゲン加水分解物に含まれるアミノ酸配列：Ｇｌｙ－Ｘ－Ｙからなるトリペプチドの種類は、加水分解の原料となるコラーゲンの種類や加水分解に使用するコラゲナーゼの種類や加水分解条件等により変化しうる。本発明のコラーゲン加水分解物は、トリペプチドＧｌｙ－Ｘ－Ｙ（Ｇｌｙ－Ｘ－Ｙはアミノ酸配列であり、Ｘ及びＹは独立にＧｌｙ以外のアミノ酸残基を示す）を、２０質量％以上を含み、より好ましくは２５質量％以上、３０質量％以上、３５質量％以上、４０質量％以上、４５質量％以上、５０質量％以上含むことができる。コラーゲン加水分解物中上記トリペプチドの含有率は、例えば２０～８０質量％、２０～７５質量％、２０～７０質量％、２０～６５質量％、２０～６０質量％、２０～５５質量％または２０～５０質量％の範囲であってもよい。コラーゲン加水分解物中のトリペプチド含有率は、例えば特許第４０９９５４１号公報に記載の方法を用いて、高めることができる。 The collagen hydrolyzate may be obtained by hydrolyzing the peptide bond of type I collagen with a bacterial collagenase that cleaves the peptide bond of type I collagen at the amino terminal of glycine. The type of tripeptide consisting of the amino acid sequence contained in the collagen hydrolyzate: Gly-XY may change depending on the type of collagen used as a raw material for hydrolysis, the type of collagenase used for hydrolysis, the hydrolysis conditions, and the like. .. The collagen hydrolyzate of the present invention contains 20% by mass or more of the tripeptide Gly-XY (Gly-XY is an amino acid sequence, and X and Y independently indicate amino acid residues other than Gly). It can be contained, more preferably 25% by mass or more, 30% by mass or more, 35% by mass or more, 40% by mass or more, 45% by mass or more, and 50% by mass or more. The content of the above tripeptide in the collagen hydrolyzate is, for example, 20 to 80% by mass, 20 to 75% by mass, 20 to 70% by mass, 20 to 65% by mass, 20 to 60% by mass, 20 to 55% by mass or. It may be in the range of 20 to 50% by mass. The tripeptide content in the collagen hydrolyzate can be increased, for example, by using the method described in Japanese Patent No. 4099541.
本発明の一態様では、トリペプチドのアミノ酸配列：Ｇｌｙ－Ｘ－Ｙにおいて、Ｘはアミノ酸残基Ｐｒｏ又はＡｌａを示し、ＹはＧｌｙ以外のアミノ酸残基を示すトリペプチドであってもよい。コラーゲンのアミノ酸組成は、グリシン（Ｇｌｙ）が全アミノ酸残基の約１／３を占め、次にプロリン（Ｐｒｏ）とアラニン（Ａｌａ）が多いため、上記アミノ酸配列の位置Ｘのアミノ酸残基は、プロリン（Ｐｒｏ）又はアラニン（Ａｌａ）であることが多い。 In one aspect of the present invention, in the amino acid sequence of tripeptide: Gly-XY, X may be a tripeptide showing an amino acid residue Pro or Ala, and Y may be a tripeptide showing an amino acid residue other than Gly. In the amino acid composition of collagen, glycine (Gly) occupies about 1/3 of all amino acid residues, followed by proline (Pro) and alanine (Ala), so that the amino acid residue at position X in the above amino acid sequence is Often proline (Pro) or alanine (Ala).
本発明のコラーゲン加水分解物は、トリペプチドＧｌｙ－Ｐｒｏ－Ｙ（Ｇｌｙ－Ｐｒｏ－Ｙはアミノ酸配列であり、ＹはＧｌｙ以外のアミノ酸残基を示す）を、１０質量％以上を含み、より好ましくは１５質量％以上、２０質量％以上、２５質量％以上、３０質量％以上、３５質量％以上、４０質量％以上含むことができる。コラーゲン加水分解物中上記トリペプチドの含有率は、例えば１０～７０質量％、１０～６５質量％、１０～６０質量％、１０～５５質量％または１０～５０質量％の範囲であってもよい。 The collagen hydrolyzate of the present invention contains 10% by mass or more of the tripeptide Gly-Pro-Y (Gly-Pro-Y is an amino acid sequence and Y indicates an amino acid residue other than Gly), which is more preferable. Can contain 15% by mass or more, 20% by mass or more, 25% by mass or more, 30% by mass or more, 35% by mass or more, and 40% by mass or more. The content of the tripeptide in the collagen hydrolyzate may be in the range of, for example, 10 to 70% by mass, 10 to 65% by mass, 10 to 60% by mass, 10 to 55% by mass, or 10 to 50% by mass. ..
本発明のコラーゲン加水分解物は、例えば、Ｇｌｙ－Ｐｒｏ－Ｈｙｐ、Ｇｌｙ－Ｌｅｕ－Ｈｙｐ、Ｇｌｙ－Ｐｒｏ－Ａｒｇ、Ｇｌｙ－Ａｌａ－Ａｒｇ、Ｇｌｙ－Ｐｒｏ－Ｓｅｒ、Ｇｌｙ－Ｐｒｏ－Ａｌａ、Ｇｌｙ－Ｐｒｏ－Ｇｌｎ、Ｇｌｙ－Ａｌａ－Ｈｙｐ、Ｇｌｙ－Ａｌａ－Ａｌａ、Ｇｌｙ－Ｓｅｒ－Ｈｙｐ、Ｇｌｙ－Ａｌａ－Ａｌａ、Ｇｌｙ－Ｐｒｏ－Ｐｒｏ、Ｇｌｙ－Ａｌａ－Ｐｒｏ、Ｇｌｙ－Ｐｒｏ－Ｖａｌ、Ｇｌｙ－Ｐｒｏ－Ｌｙｓ、Ｇｌｙ－Ｇｌｕ－Ｈｙｐ、Ｇｌｙ－Ｐｈｅ－Ｈｙｐ、Ｐｒｏ－Ｈｙｐ－Ｇｌｙ、Ａｌａ－Ｈｙｐ－Ｇｌｙ、Ｇｌｙ－Ｇｌｕ－Ａｒｇ、Ｇｌｙ－Ａｌａ－Ｌｙｓ、Ｇｌｙ－Ａｓｐ－Ａｌａ、Ｇｌｙ－Ｇｌｕ－ＡｌａおよびＧｌｙ－Ａｒｇ－Ｈｙｐなどのトリペプチド、並びにＰｒｏ－Ｈｙｐ、Ｈｙｐ－Ｇｌｙ、Ｇｌｙ－Ｐｒｏ、Ａｌａ－Ｈｙｐ、Ｐｒｏ－ＡｌａおよびＣｙｃｌｏ（Ｇｌｙ－Ｐｒｏ）などのジペプチドなどを含むことができるが、これらに限定されない。Ｃｙｃｌｏ（Ｇｌｙ－Ｐｒｏ）は、（以下ｃＧＰと記載する場合がある）は、グリシンとプロリンが縮合結合した構造を保有する環状のジペプチドである。 The collagen hydrolyzate of the present invention is, for example, Gly-Pro-Hyp, Gly-Leu-Hyp, Gly-Pro-Arg, Gly-Ala-Arg, Gly-Pro-Ser, Gly-Pro-Ala, Gly-Pro. -Gln, Gly-Ala-Hyp, Gly-Ala-Ala, Gly-Ser-Hyp, Gly-Ala-Ala, Gly-Pro-Pro, Gly-Ala-Pro, Gly-Pro-Val, Gly-Pro-Lys , Gly-Glu-Hyp, Gly-Phe-Hyp, Pro-Hyp-Gly, Ala-Hyp-Gly, Gly-Glu-Arg, Gly-Ala-Lys, Gly-Asp-Ala, Gly-Glu-Ala and Gly -Can include, but is limited to, tripeptides such as Arg-Hyp and dipeptides such as Pro-Hyp, Hyp-Gly, Gly-Pro, Ala-Hyp, Pro-Ala and Cyclo (Gly-Pro). Not done. Cyclo (Gly-Pro) (hereinafter sometimes referred to as cGP) is a cyclic dipeptide having a structure in which glycine and proline are condensed and bonded.
本発明の一態様では、本発明のコラーゲン加水分解物中、Ｇｌｙ－Ｐｒｏ－ＨｙｐおよびＧｌｙ－Ｌｅｕ－Ｈｙｐの含有率は、それぞれ１から２０質量％であり、好ましくは２から１５質量％であり、より好ましくは３から１０質量％である。本発明の一態様では、本発明のコラーゲン加水分解物中、Ｇｌｙ－Ｐｒｏ－Ａｒｇ、Ｇｌｙ－Ａｌａ－Ａｒｇ、Ｇｌｙ－Ｐｒｏ－Ｓｅｒ、Ｇｌｙ－Ｐｒｏ－Ａｌａ、Ｇｌｙ－Ｐｒｏ－Ｇｌｎ、Ｇｌｙ－Ａｌａ－Ｈｙｐ、Ｇｌｙ－Ａｌａ－Ａｌａ、Ｇｌｙ－Ｓｅｒ－Ｈｙｐ、Ｇｌｙ－Ａｌａ－Ａｌａ、Ｇｌｙ－Ｐｒｏ－ＰｒｏおよびＣｙｃｌｏ（Ｇｌｙ－Ｐｒｏ）の含有率は、それぞれ０．１から１０質量％であり、好ましくは０．２から８質量％であり、より好ましくは０．３から６質量％である。本発明の一態様では、本発明のコラーゲン加水分解物中、Ｇｌｙ－Ａｌａ－Ｐｒｏ、Ｇｌｙ－Ｐｒｏ－Ｖａｌ、Ｇｌｙ－Ｐｒｏ－Ｌｙｓ、Ｇｌｙ－Ｇｌｕ－Ｈｙｐ、Ｇｌｙ－Ｐｈｅ－Ｈｙｐ、Ｐｒｏ－Ｈｙｐ－Ｇｌｙ、Ａｌａ－Ｈｙｐ－Ｇｌｙ、Ｇｌｙ－Ｇｌｕ－Ａｒｇ、Ｇｌｙ－Ａｌａ－Ｌｙｓ、Ｇｌｙ－Ａｓｐ－Ａｌａ、Ｇｌｙ－Ｇｌｕ－ＡｌａおよびＧｌｙ－Ａｒｇ－Ｈｙｐの含有率は、それぞれ０．０１から５質量％である。 In one aspect of the present invention, the content of Gly-Pro-Hyp and Gly-Leu-Hyp in the collagen hydrolyzate of the present invention is 1 to 20% by mass, preferably 2 to 15% by mass, respectively. , More preferably 3 to 10% by mass. In one aspect of the present invention, in the collagen hydrolyzate of the present invention, Gly-Pro-Arg, Gly-Ala-Arg, Gly-Pro-Ser, Gly-Pro-Ala, Gly-Pro-Gln, Gly-Ala- The content of Hyp, Gly-Ala-Ala, Gly-Ser-Hyp, Gly-Ala-Ala, Gly-Pro-Pro and Cyclo (Gly-Pro) is 0.1 to 10% by mass, respectively, preferably 0.1 to 10% by mass. It is 0.2 to 8% by mass, more preferably 0.3 to 6% by mass. In one aspect of the present invention, in the collagen hydrolyzate of the present invention, Gly-Ala-Pro, Gly-Pro-Val, Gly-Pro-Lys, Gly-Glu-Hyp, Gly-Phe-Hyp, Pro-Hyp- The contents of Gly, Ala-Hyp-Gly, Gly-Glu-Arg, Gly-Ala-Lys, Gly-Asp-Ala, Gly-Glu-Ala and Gly-Arg-Hyp are 0.01 to 5% by mass, respectively. Is.
コラーゲン加水分解物に含まれるトリペプチド又はジペプチドの同定は、特に限定されないが、例えば、ＨＰＬＣ、ＬＣ－ＭＳ、ＬＣ－ＭＳ／ＭＳを使用して行うことができる。 The identification of the tripeptide or dipeptide contained in the collagen hydrolyzate is not particularly limited, but can be performed by using, for example, HPLC, LC-MS, LC-MS / MS.
本発明のコラーゲン加水分解物は、非抗原性および低アレルゲン性コラーゲン加水分解物であるが、さらに種々の方法による精製処理に供することもできる。（Shinmoto H. et al., 2001, Food Sci. Technol. Res., Vol 7, No. 4, 331-332）。 The collagen hydrolyzate of the present invention is a non-antigenic and low allergenic collagen hydrolyzate, but can also be subjected to purification treatment by various methods. (Shinmoto H. et al., 2001, Food Sci. Technol. Res., Vol 7, No. 4, 331-332).
コラーゲン由来の配列を保有するトリペプチド類やジペプチド類を摂取した場合、トリペプチド類やジペプチド類が高い血中移行性を示すことに加えて、摂取されたトリペプチド類の消化分解により生成した代謝物ジペプチドも高濃度で血中に出現することが報告されている（Yamamoto S. et al., 2015, Bioscience,Biotechnology, and Biochemistry, Vol. 79, No. 12, 2026-2033; Yamamoto S. et al.,2016, Biol. Pharm. Bull. 39, 428-434; Sontakke S. B. et al., 2016, J. Agric. Food Chem. 64, 7127-7133）。すなわち、コラーゲン加水分解物を摂取すると、血中代謝物としてトリペプチド類及びジペプチド類が出現し、次にそれらが組織へと移行して生理活性を発揮することで薬理作用が現れることから、はじめからトリペプチド類などを含有するコラーゲン加水分解物では、トリペプチド類及びジペプチド類の吸収性がより向上することで、より高い薬理活性が得られると考えられる。 When tripeptides and dipeptides having a sequence derived from collagen are ingested, the tripeptides and dipeptides show high blood transferability and the metabolism generated by digestion and decomposition of the ingested tripeptides. It has been reported that physical dipeptides also appear in blood at high concentrations (Yamamoto S. et al., 2015, Bioscience, Biotechnology, and Biochemistry, Vol. 79, No. 12, 2026-2033; Yamamoto S. et. al., 2016, Biol. Pharm. Bull. 39, 428-434; Sontakke S. B. et al., 2016, J. Agric. Food Chem. 64, 7127-7133). That is, when collagen hydrolyzate is ingested, tripeptides and dipeptides appear as blood metabolites, and then they migrate to tissues and exert physiological activity to exert pharmacological action. Therefore, it is considered that a collagen hydrolyzate containing tripeptides and the like can obtain higher pharmacological activity by further improving the absorbability of tripeptides and dipeptides.
更には、平均分子量５０００のゼラチン加水分解物を摂取した場合、血中には多様なトリペプチド類、あるいはジペプチド類が代謝物として出現することが報告されている（Iwai K. et al., J. Agric. Food Chem. 2005, 53, 6531-6536; Ichikawa S. et al., International Journal of Food Sciences and Nutrition, February 2010; 61(1): 52-60）。それらの代謝物はゼラチン加水分解物中の高分子画分のペプチドから生成したものであると考えられる。 Furthermore, it has been reported that various tripeptides or dipeptides appear as metabolites in the blood when a gelatin hydrolyzate having an average molecular weight of 5000 is ingested (Iwai K. et al., J). . Agric. Food Chem. 2005, 53, 6531-6536; Ichikawa S. et al., International Journal of Food Sciences and Nutrition, February 2010; 61 (1): 52-60). These metabolites are believed to be produced from the peptides of the macromolecular fraction in the gelatin hydrolyzate.
本発明の実施形態の一つにおいて、本発明の歯肉炎の発症予防剤および／または症状改善剤は、歯肉中毛細血管係蹄の拡張、屈曲および／またはねじれを予防および／または改善することにより、歯肉炎の発症を予防し、および／または症状を改善することができる。本明細書中、歯肉中毛細血管係蹄の拡張とは、毛細血管係蹄が太くなっていることを意味する。歯肉中毛細血管係蹄の拡張が予防されるとは、歯肉中の毛細血管係蹄が太くなりにくいことを意味する。歯肉中毛細血管係蹄の拡張が改善されるとは、細くなっていることを意味する。歯肉中毛細血管係蹄の屈曲とは、毛細血管が係蹄部以外の場所で折れ曲がった状態を指す。歯肉中毛細血管係蹄のねじれとは、屈曲が複数個所で連なって生じ、血管が左右に蛇行したり折れ曲がった状態を指す。本明細書中、歯肉中毛細血管係蹄の拡張、屈曲および／またはねじれは、毛細血管血流スコープを使用して、例えば１００倍から４００倍に拡大して、観察し、計測することができる。本明細書中、歯肉中毛細血管係蹄の拡張、屈曲および／またはねじれが予防および／または改善されていることは、限定されないが、例えばコラーゲン加水分解物の投与前後の歯肉中毛細血管係蹄を比較観察することによりまたは正常部位と歯肉炎発症部位の歯肉中毛細血管係蹄を比較観察することにより、判断することができる。 In one of the embodiments of the present invention, the agent for preventing the onset of periodontitis and / or the agent for improving the symptoms of the present invention prevents and / or improves the dilation, bending and / or twisting of the periodontal capillaries of the gingiva. , Can prevent the onset of periodontitis and / or ameliorate symptoms. In the present specification, the expansion of the gingival medium capillary hoof means that the capillary hoof is thickened. Preventing dilation of the gingival capillary hoof means that the capillary hoof in the gingiva is less likely to thicken. Improved dilation of the gingival capillaries means thinner. Bending of the capillaries in the gingiva refers to a state in which the capillaries are bent at a place other than the hoof. Twisting of the gingival capillaries is a state in which bending occurs in a row at multiple points and the blood vessels meander or bend from side to side. As used herein, dilation, flexion and / or twisting of the gingival capillary hoof can be observed and measured, eg, magnified 100-fold to 400-fold, using a capillary blood flow scope. .. As used herein, the prevention and / or improvement of dilation, flexion and / or twisting of the gingival capillary sac is not limited, but for example, the gingival capillary sac before and after administration of collagen hydrolyzate. Can be determined by comparative observation of the gingival capillaries at the normal site and the site of onset of gingival inflammation.
歯肉炎の代表的な所見は歯肉の発赤や腫脹、出血であるが、これは毛細血管形態そのものに変化が生じることによる。歯肉炎で最も多いプラーク性歯肉炎（単純性歯肉炎）を例に挙げると、はじめに炎症に伴って内縁上皮直下の細動静脈が拡張して血管の透過性が増し、炎症性細胞の組織内への浸潤が起こる。侵襲が長く続くと、細動脈血管への血流は開大したままとなり、細胞の代謝に直接かかわっている真性毛細血管や中心毛細血管への血流が阻害されてしまう。すると停止した循環を補完するために、内縁上皮下血管網の拡張した血管から新たな係蹄が生じる。そしてこの新生係蹄の先端は拡張、屈曲、ねじれを示す。さらに炎症が波及すると外縁上皮でも血流障害に伴う毛細血管係蹄の屈曲、蛇行、ねじれが生じる。係蹄の数も増加し、毛細血管の形態は健常の歯肉とは異なった状態へと変化する（野坂洋一郎. 歯肉の微小循環. 岩手医科大学歯学雑誌, 1978, 3.1: 40-53.）。これらの変化により歯肉に充血やうっ血が起こり、発赤や腫脹を呈する。しかも炎症部の血管は脆弱であるため、ブラッシングによる刺激で出血しやすくなる。 Typical findings of periodontitis are redness, swelling, and bleeding of the gums, which are due to changes in the morphology of the capillaries themselves. Taking plaque gingival inflammation (simple gingival inflammation), which is the most common periodontitis, as an example, first, the fibrillation vein just below the medial epithelium dilates with inflammation, increasing the permeability of blood vessels and within the tissue of inflammatory cells. Infiltration occurs. If the invasion continues for a long time, the blood flow to the arteriole blood vessels remains dilated, and the blood flow to the true capillaries and the central capillaries, which are directly involved in cell metabolism, is obstructed. A new hooves emerge from the dilated vessels of the medial subepithelial vascular network to complement the stopped circulation. And the tip of this new hoof shows expansion, bending and twisting. Furthermore, when inflammation spreads, the outer epithelium also undergoes bending, meandering, and twisting of the capillary hoof associated with impaired blood flow. The number of hoofs also increases, and the morphology of the capillaries changes to a state different from that of healthy gingiva (Yoichiro Nosaka. Microcirculation of gingiva. Iwate Medical University Dental Journal, 1978, 3.1: 40-53.). These changes cause hyperemia and congestion in the gums, resulting in redness and swelling. Moreover, since the blood vessels in the inflamed area are fragile, bleeding is likely to occur due to stimulation by brushing.
中村ら（中村治郎; 原耕二; 橋口精範. 妊婦歯肉における毛細管顕微鏡所見と歯肉嚢内滲出液の観察. 口腔病学会雑誌, 1965, 32.2: 201-208.）が歯肉炎を有する歯肉と正常歯肉の毛細血管係蹄を毛細管顕微鏡にて撮影し、太さ、長さ、単位面積中の係蹄数、吻合および屈曲の度合いと係蹄の配置の６項目を比較したところ、歯肉炎では、係蹄がより太い、より長い、係蹄数が多い、吻合や屈曲などの形態変化、係蹄の配置が不規則、という異常が高頻度で起きていた。さらに、毛細血管係蹄に強い異常があるほど出血量が増加する傾向が観られた。すなわち歯肉の出血傾向は、中村ら（1965, 上掲）の指標でも評価することができる。 Nakamura et al. (Jiro Nakamura; Koji Hara; Seiken Hashiguchi. Capillary microscopic findings and observation of intragingival exudate in pregnant women's gingiva. Journal of the Oral Disease Society, 1965, 32.2: 201-208.) The capillaries of the gingiva were photographed with a capillary microscope, and the six items of thickness, length, number of gingivals in a unit area, degree of anastomosis and flexion, and placement of the gingiva were compared. Abnormalities such as thicker and longer gingiva, large number of gingiva, morphological changes such as anastomosis and flexion, and irregular arrangement of gingiva occurred frequently. Furthermore, there was a tendency for the amount of bleeding to increase as there was a stronger abnormality in the capillary hoof. That is, the bleeding tendency of the gingiva can also be evaluated by the index of Nakamura et al. (1965, above).
後記する実施例に示すように、歯肉炎モデルのイヌに、本発明のコラーゲン加水分解物を２か月間投与したところ、投与しない群と比較して、毛細血管がより細く、短くなり、吻合や屈曲が消え、配置も適切となった。辺縁歯肉の組織染色画像観察では、本発明のコラーゲン加水分解物を投与した群では、投与しない群と比較して、炎症性細胞の浸潤がより少なく、溶血を示唆する領域が小さいことが観察された。この結果は、本発明のコラーゲン加水分解物が、歯肉中毛細血管係蹄の拡張、屈曲および／またはねじれを予防および／または改善することにより、歯肉のうっ血や充血を解消し、出血しにくい健常な歯肉に回復させる効果を有することを示唆する。 As shown in the examples described later, when the collagen hydrolyzate of the present invention was administered to a dog of a gingival inflammation model for 2 months, the capillaries became thinner and shorter, and the capillaries became thinner and shorter than those in the non-administered group. The bend disappeared and the placement was appropriate. In the tissue-stained image observation of the marginal gingiva, it was observed that the group to which the collagen hydrolyzate of the present invention was administered had less infiltration of inflammatory cells and a smaller area suggesting hemolysis than the group to which it was not administered. Was done. The results show that the collagen hydrolysate of the present invention eliminates gingival congestion and hyperemia by preventing and / or ameliorating dilation, flexion and / or twisting of the gingival capillaries, and is healthy and less prone to bleeding. It is suggested that it has a healing effect on the gingiva.
本発明の実施形態の一つにおいて、本発明の歯肉炎の発症予防剤および／または症状改善剤は、歯肉組織中コラーゲンの産生を亢進し、且つコラーゲン繊維の過形成または瘢痕化を抑制することにより、歯肉炎の発症を予防し、および／または症状を改善することができる。本明細書中、コラーゲン繊維の過形成とは、コラーゲンが過剰に産生されることを意味し、具体的には、例えば、炎症性サイトカインの過剰な産生が起こり、炎症性サイトカインにより活性化された線維芽細胞によって、コラーゲンが過剰に産生される。炎症性サイトカインの過剰な産生は、例えば細菌由来の内毒素により起こるが、これに限定されない。本明細書中、瘢痕化とは、炎症を受けた歯肉組織が、肉芽組織の形成を経て、過剰な膠原線維が蓄積した状態で不完全に修復されることを意味する。 In one of the embodiments of the present invention, the agent for preventing the onset of gingival inflammation and / or the agent for improving the symptoms of the present invention enhances the production of collagen in the gingival tissue and suppresses the hyperformation or scarring of collagen fibers. Can prevent the onset of gingival inflammation and / or ameliorate the symptoms. In the present specification, collagen fiber hyperformation means that collagen is overproduced, and specifically, for example, overproduction of inflammatory cytokines occurs and is activated by inflammatory cytokines. Fibroblasts overproduce collagen. Excessive production of inflammatory cytokines is caused, for example, by, but is not limited to, bacterial endotoxins. As used herein, scarring means that the inflamed gingival tissue is incompletely repaired with the accumulation of excess collagen fibers through the formation of granulation tissue.
歯周病には感染源、炎症、血管の形態異常などコラーゲン繊維の消失以外にも多くの要素が関わることから、コラーゲン産生を促進させることだけで健常歯肉を回復することはできないと考えられる。さらに、コラーゲン産生の過剰な促進によるコラーゲンの過形成には注意が必要である。過形成により生じた組織は従来のコラーゲン線維の配列と異なった方向に形成され、その後瘢痕化するので、コラーゲン原繊維配列にはモザイク構造が生じる。瘢痕は血管も少なく、血流が悪い上、一見して健常であってもモザイク構造の隙間からの再感染が起こりやすく、歯肉炎が再発しやすい。 Since periodontal disease involves many factors other than the disappearance of collagen fibers such as infection source, inflammation, and abnormal morphology of blood vessels, it is considered that healthy gingiva cannot be recovered only by promoting collagen production. In addition, caution should be exercised in collagen hyperplasia due to excessive promotion of collagen production. The tissue produced by hyperformation is formed in a direction different from that of the conventional collagen fiber arrangement, and then scarred, so that a mosaic structure is formed in the collagen fibrillar arrangement. Scars have few blood vessels, poor blood flow, and even if they are seemingly healthy, reinfection from the gaps in the mosaic structure is likely to occur, and gingival inflammation is likely to recur.
後記する実施例では、歯肉炎モデルのイヌに、本発明のコラーゲン加水分解物を２か月間投与したところ、投与しない群と比較して、辺縁歯肉の組織染色画像観察において、炎症性細胞の浸潤がより少なく、溶血を示唆する領域もほとんど観られず、膠原繊維が広く明瞭な領域として観察され、さらに膠原繊維の間には毛細血管が侵入している様子が観られた。これは、コラーゲン産生を促進して線維を回復させ、しかもコラーゲン繊維は瘢痕化せずに血行の良い正常な線維が再生されていることを示唆する。この結果は、本発明のコラーゲン加水分解物が、歯肉組織中コラーゲンの産生を亢進し、且つコラーゲン繊維の過形成または瘢痕化を抑制していることを示す。 In the example described later, when the collagen hydrolyzate of the present invention was administered to a dog of a gingival inflammation model for 2 months, inflammatory cells were observed in the tissue-stained image observation of the marginal gingiva as compared with the group not administered. There was less invasion, few areas suggesting hemolysis were observed, collagen fibers were observed as wide and clear areas, and capillaries were observed to invade between the collagen fibers. This suggests that collagen production is promoted to restore the fibers, and that the collagen fibers are not scarred and normal fibers with good blood circulation are regenerated. This result indicates that the collagen hydrolyzate of the present invention enhances the production of collagen in the gingival tissue and suppresses the hyperplasia or scarring of collagen fibers.
後記する実施例では、歯肉炎モデルのイヌに、本発明のコラーゲン加水分解物を２か月間投与したところ、コラーゲン線維の間には毛細血管が配しており、瘢痕化することなく正常に回復することが示唆され、さらに、歯肉への炎症細胞の浸潤が抑制されており、歯肉の毛細血管係蹄の形態も回復していた。これらの結果は、コラーゲンペプチドの摂取が単純に歯肉のコラーゲン繊維の産生を促進するだけでなく、歯肉炎の病態を改善し、なおかつ再感染が起こりにくい健常な歯肉（コラーゲン繊維）の再構築に寄与することを示している。そのため歯肉炎の再発が予防できると考えられる。よって本発明の実施形態の一つにおいて、本発明の歯肉炎の発症予防剤および／または症状改善剤は、歯肉炎の再発を予防する。また、健常な歯肉炎の再構築に寄与することにより、歯肉炎に伴う歯肉からの出血を予防および／または改善されることができる。よって、本発明の実施形態の一つにおいて、本発明の歯肉炎の発症予防剤および／または症状改善剤は、歯肉炎に伴う歯肉からの出血を予防および／または改善することができる。 In the example described later, when the collagen hydrolyzate of the present invention was administered to a dog of the gingival inflammation model for 2 months, capillaries were arranged between the collagen fibers, and the dog recovered normally without scarring. Furthermore, the infiltration of inflammatory cells into the gingiva was suppressed, and the morphology of the capillaries of the gingiva was restored. These results show that ingestion of collagen peptide not only promotes the production of collagen fiber in the gingiva, but also improves the pathophysiology of gingival inflammation and reconstructs healthy gingiva (collagen fiber) that is less likely to be re-infected. It shows that it contributes. Therefore, it is considered that the recurrence of periodontitis can be prevented. Therefore, in one of the embodiments of the present invention, the agent for preventing the onset of periodontitis and / or the agent for improving the symptoms of the present invention prevents the recurrence of periodontitis. In addition, by contributing to the reconstruction of healthy periodontitis, gingival bleeding associated with periodontitis can be prevented and / or ameliorated. Therefore, in one of the embodiments of the present invention, the onset preventive agent and / or symptom ameliorating agent for gingival inflammation of the present invention can prevent and / or improve gingival bleeding associated with gingival inflammation.
本発明の実施形態の一つにおいて、本発明の歯肉炎の発症予防剤および／または症状改善剤は、歯肉炎に伴う歯肉組織中コラーゲンの消失を抑制することができる。歯肉炎の炎症部では、炎症と同時にコラーゲン繊維の破壊・減少が起こる。まず炎症により線維芽細胞からのコラーゲンの分泌は抑制される。そして、線維芽細胞自身とマクロファージが分泌するｃｏｌｌａｇｅｎａｓｅがコラーゲン繊維を切断し、この断片を種々の分解酵素がさらに小さな断片やアミノ酸に分解する。さらに線維芽細胞とマクロファージはコラーゲン原繊維を貪食し、細胞内でも消化する（非特許文献１）。これらの作用によりコラーゲン繊維の緻密で規則的な配列が破壊されると、歯肉の機能は低下し細菌からの防御力が低下する。後記する実施例に示すように、歯肉炎モデルのイヌに、本発明のコラーゲン加水分解物を２か月間投与したところ、コラーゲン産生が促進され線維の回復がより進んでおり、しかもコラーゲン繊維は瘢痕化せずに血行の良い正常な線維が再生されていることが示唆された。さらに炎症部位からの血管の撤退が進み、炎症自体もほぼ収まっており、より回復が進んでいることが示唆された。 In one of the embodiments of the present invention, the agent for preventing the onset of gingival inflammation and / or the agent for improving the symptoms of the present invention can suppress the disappearance of collagen in the gingival tissue associated with gingival inflammation. In the inflamed area of periodontitis, collagen fibers are destroyed or reduced at the same time as inflammation. First, inflammation suppresses the secretion of collagen from fibroblasts. Then, collagenase secreted by fibroblasts themselves and macrophages cleaves collagen fibers, and various degrading enzymes decompose these fragments into smaller fragments and amino acids. Furthermore, fibroblasts and macrophages phagocytose collagen fibrils and digest them intracellularly (Non-Patent Document 1). When these actions disrupt the dense and regular arrangement of collagen fibers, the function of the gingiva is reduced and the defense against bacteria is reduced. As shown in the examples described later, when the collagen hydrolyzate of the present invention was administered to a dog of a gingival inflammation model for 2 months, collagen production was promoted and the recovery of fibers was further advanced, and the collagen fibers were scarred. It was suggested that normal fibers with good blood circulation were regenerated without being transformed. Furthermore, the withdrawal of blood vessels from the inflamed site progressed, and the inflammation itself was almost subsided, suggesting that recovery is progressing.
本発明の歯肉炎の発症予防剤および／または症状改善剤は、種々の剤型とすることができる。例えば、経口投与剤としては錠剤、顆粒剤、散剤、カプセル剤、ソフトカプセル剤等の固形剤、溶液剤、懸濁剤、乳化剤等の液剤、凍結乾燥剤等が挙げられる。局所薬としては、塗布や外用剤や噴霧剤が挙げられる。低分子ペプチドは皮膚や粘膜から吸収されるためである。本発明の歯肉炎の発症予防剤および／または症状改善剤は、コラーゲン加水分解物のほかに医薬として許容される添加物を含むことができる。医薬として許容される添加物は、例えば安定化剤、滑剤、湿潤剤、乳化剤、結合剤、ｐＨ調整剤、保存剤等である。 The onset preventive agent and / or symptom ameliorating agent for gingival inflammation of the present invention can be in various dosage forms. For example, examples of the oral administration agent include tablets, granules, powders, capsules, solid agents such as soft capsules, solutions, suspensions, liquids such as emulsifiers, freeze-drying agents and the like. Examples of topical drugs include applications, external preparations and sprays. This is because small molecule peptides are absorbed through the skin and mucous membranes. The agent for preventing the onset of gingival inflammation and / or the agent for improving the symptoms of the present invention may contain a pharmaceutically acceptable additive in addition to the collagen hydrolyzate. Pharmaceutically acceptable additives are, for example, stabilizers, lubricants, wetting agents, emulsifiers, binders, pH regulators, preservatives and the like.
本発明の歯肉炎の発症予防剤および／または症状改善剤は、平均分子量３００～９００を有するコラーゲン加水分解物を、１日につき体重１ｋｇあたり１０～１０００ｍｇ、好ましくは１０～３００ｍｇ、１日１回又は数回に分けて投与又は摂取することができる。本発明の歯肉炎の発症予防剤および／または症状改善剤は、分子量４００以下の低分子画分に相当のコラーゲン加水分解物を、１日につき体重１ｋｇあたり１０～１０００ｍｇ、好ましくは１０～３００ｍｇ、１日１回又は数回に分けて投与又は摂取することができる。投与量は、対象の年齢、体重、症状の違い、症状の程度、剤型等により適宜調節することができる。投与又は摂取の期間は、特に制限されないが、２週間以上であることが好ましく、１か月以上、または２か月以上であることがより好ましい。 The agent for preventing the onset of gingival inflammation and / or the symptom improving agent of the present invention is a collagen hydrolyzate having an average molecular weight of 300 to 900, which is 10 to 1000 mg / kg body weight per day, preferably 10 to 300 mg once a day. Alternatively, it can be administered or ingested in several divided doses. The agent for preventing the onset of gingival inflammation and / or the symptom improving agent of the present invention contains a collagen hydrolyzate corresponding to a low molecular weight fraction having a molecular weight of 400 or less, in an amount of 10 to 1000 mg, preferably 10 to 300 mg, per 1 kg of body weight per day. It can be administered or ingested once a day or in several divided doses. The dose can be appropriately adjusted according to the age, body weight, difference in symptoms, degree of symptoms, dosage form, etc. of the subject. The period of administration or ingestion is not particularly limited, but is preferably 2 weeks or longer, and more preferably 1 month or longer, or 2 months or longer.
本発明の歯肉炎の発症予防剤および／または症状改善剤は、経口摂取可能な任意の形態、（例えば、溶液、懸濁液、粉末、固体成形物など）の食品に加工することができる。任意の形態に加工された食品は、そのまま摂取してもよいし、コーヒーなど嗜好飲料やヨーグルトなど乳製品などに混ぜて摂取することもできる。 The gingival inflammation onset preventive agent and / or symptom ameliorating agent of the present invention can be processed into food in any form that can be orally ingested (for example, solution, suspension, powder, solid molded product, etc.). The food processed into any form may be ingested as it is, or may be ingested by mixing it with a favorite beverage such as coffee or a dairy product such as yogurt.
食品の例としては、本発明を限定するものではないが、錠剤、顆粒剤、カプセル剤、ソフトカプセル剤及びドリンク剤等のサプリメント、あるいは炭水化物類（パン、麺、米飯、餅など）、菓子類（クッキー、ゼリー、チョコレート、チューインガム、キャンディーなど）、乳製品（チーズ、ヨーグルト、バターなど）、粉末食品（粉末スープ、粉末ムース、粉末ゼリー、粉末甘味料）、栄養食、ダイエット食、スポーツ用栄養食や、飲料（果汁含有飲料、果汁ジュース、野菜ジュース、サイダー、ジンジャーエール、アイソトニック飲料、アミノ酸飲料、ゼリー飲料、コーヒー飲料、緑茶、紅茶、ウーロン茶、麦茶、乳飲料、乳酸菌飲料、ココア、ビール、発泡酒、第三のビール、ノンアルコール飲料、ビール風味飲料、リキュール、チューハイ、清酒、果実酒、蒸留酒）を挙げることができる。本発明の一態様において食品は、機能性表示食品又は特定保健用食品である。 Examples of foods include, but are not limited to, tablets, granules, capsules, supplements such as soft capsules and drinks, carbohydrates (bread, noodles, rice, rice cakes, etc.), confectionery (bread, noodles, rice, rice cakes, etc.). Cookies, jelly, chocolate, chewing gum, candy, etc.), dairy products (cheese, yogurt, butter, etc.), powdered foods (powdered soup, powdered mousse, powdered jelly, powdered sweeteners), nutritional foods, diet foods, nutritional foods for sports And beverages (fruit-containing beverages, fruit juice juices, vegetable juices, ciders, ginger ales, isotonic beverages, amino acid beverages, jelly beverages, coffee beverages, green tea, tea, oolong tea, wheat tea, milk beverages, lactic acid bacteria beverages, cocoa, beer, foaming Sake, third beer, non-alcoholic beverage, beer-flavored beverage, liqueur, chuhai, sake, fruit liquor, distilled liquor) can be mentioned. In one aspect of the present invention, the food is a food with functional claims or a food for specified health use.
また、本発明のコラーゲン加水分解物、あるいは本発明のコラーゲン加水分解物に含有されるトリペプチド類、ジペプチド類は生体構成成分であると共に、すでに長年の食経験があるコラーゲン加水分解物に含有されている成分であることから、安全性が高いと考えられる。 Further, the collagen hydrolyzate of the present invention, or the tripeptides and dipeptides contained in the collagen hydrolyzate of the present invention are biological constituents and are contained in the collagen hydrolyzate having many years of eating experience. It is considered to be highly safe because it is a component of collagen.
コラーゲンはグリシン（Ｇｌｙ）が３残基ごとに繰り返す配列であるので、トリペプチド代謝物としては、Ｇｌｙ－Ｘａａ－Ｙａａ、Ｘａａ－Ｙａａ－Ｇｌｙ、Ｙａａ－Ｇｌｙ－Ｙａａ（Ｘａａ、Ｙａａは任意のアミノ酸）といった配列が生成し、ジペプチド代謝物としてはＧｌｙ－Ｘａａ、Ｘａａ－Ｙａａ、Ｙａａ－Ｇｌｙといった配列が生成する。また、酵素で素材を低分子化することにより、はじめから上記の配列を含む素材も存在する。例えばコラーゲン・トリペプチド（ＣＴＰ）製品群であるＨＡＣＰ－５０、ＨＡＣＰ－０１、ＨＡＣＰ－ＴＦ、ＨＡＣＰ－ＣＦ（ゼライス株式会社）などは、Ｇｌｙ－Ｐｒｏ－ＨｙｐをはじめとするＧｌｙ－Ｘａａ－Ｙａａ配列のペプチドを合わせて５～９０％、Ｇｌｙ－Ｐｒｏ、Ｐｒｏ－Ｈｙｐ、Ｈｙｐ－ＧｌｙといったＧｌｙ－Ｘａａ、Ｘａａ－Ｙａａ、Ｙａａ－Ｇｌｙ配列を合わせて０．０１～２％含む。そのほかの製品には、Ｃｏｌｌａｇｅｎ Ｔｒｉｐｅｐ２０Ｓ（Ａｍｉｃｏｇｅｎ社）、ＧＦＦ－０１（ニッピ）、ＤＦＦ－０１（ニッピ）などがある。いずれの素材も単一の配列の低分子ペプチドのみを含有するということはなく、様々な配列のペプチド群を含有する。一般的にこれら低分子ペプチドの血中吸収性は優れており、血中濃度も摂取量依存的に上昇する。例えば、分子量４００以下の低分子ペプチドを５０％含有する素材と、分子量４００以下の低分子ペプチドをほとんど含まないコラーゲンペプチドを摂取したとき、両者の主要代謝物の血中濃度の差異は４～５倍程度である（YAMAMOTO, Shoko, et al. Absorption and urinary excretion of peptides after collagen tripeptide ingestion in humans. Biological and Pharmaceutical Bulletin, 2016, 39.3: 428-434.）。
以上のように、コラーゲンを摂取すると、高分子ペプチドの消化、あるいは低分子ペプチドの直接移行によって、血中には様々な代謝物が生じる。後記する実施例の結果から、これらのペプチドの生理活性の総合力によって歯肉のホメオスタシスが正常化され、病態を改善させると考えられる。
Since collagen is a sequence in which glycine (Gly) repeats every 3 residues, the tripeptide metabolites include Gly-Xaa-Yaa, Xaa-Yaa-Gly, and Yaa-Gly-Yaa (Xaa and Yaa are arbitrary amino acids). ) Is generated, and sequences such as Gly-Xaa, Xaa-Yaa, and Yaa-Gly are generated as dipeptide metabolites. In addition, there are materials containing the above sequences from the beginning by reducing the molecular weight of the material with an enzyme. For example, the collagen tripeptide (CTP) product group HACP-50, HACP-01, HACP-TF, HACP-CF (Zelice Co., Ltd.) and the like have Gly-Xaa-Yaa sequences including Gly-Pro-Hyp. Contains 5 to 90% of the peptides in total, and 0.01 to 2% of Gly-Xaa, Xaa-Yaa, and Yaa-Gly sequences such as Gly-Pro, Pro-Hyp, and Hyp-Gly. Other products include Collagen Tripep20S (Amicogen), GFF-01 (Nippi), DFF-01 (Nippi) and the like. Neither material contains only a single sequence of small molecule peptides, but a group of peptides of various sequences. In general, these small molecule peptides have excellent blood absorbability, and their blood concentrations also increase in an intake-dependent manner. For example, when a material containing 50% of a small molecule peptide having a molecular weight of 400 or less and a collagen peptide containing almost no small molecule peptide having a molecular weight of 400 or less are ingested, the difference in blood concentration of the main metabolites between the two is 4 to 5. It is about double (YAMAMOTO, Shoko, et al. Absorption and urinary excretion of peptides after collagen tripeptide ingestion in humans. Biological and Pharmaceutical Bulletin, 2016, 39.3: 428-434.).
As described above, when collagen is ingested, various metabolites are produced in the blood by digestion of high molecular weight peptides or direct transfer of small molecule peptides. From the results of the examples described later, it is considered that the homeostasis of the gingiva is normalized and the pathological condition is improved by the total power of the physiological activities of these peptides.
以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除および置換を行うことができる。 The embodiments of the present invention described below are for purposes of illustration only and do not limit the technical scope of the present invention. The technical scope of the invention is limited only by the description of the claims. Modifications of the present invention, for example, addition, deletion and replacement of the constituent elements of the present invention may be made on condition that the gist of the present invention is not deviated.
＜実施例１＞
（１）コラーゲン加水分解物に含まれるペプチド分析
試験物質としてコラーゲン加水分解物「ＨＡＣＰ－５０」（ゼライス株式会社にて作成）を使用した。上記コラーゲン加水分解物の平均分子量は約６００である。
ＬＣ－ＭＳ法により、上記コラーゲン加水分解物に含まれるペプチドを分析した。分析は７ロットについて独立して行った。実験条件は以下のとおりである。装置としてＡＣＱＵＩＴＹ ＵＹＰＬＣ Ｈ－Ｃｌａｓｓ Ｓｙｓｔｅｍ ｗｉｔｈ ＳＱ Ｄｅｔｅｃｔｏｒ２（ウォーターズ社）、カラムとしてＡＣＣＱ－ＴＡＧ ＵＬＴＡ Ｃ１８（ウォーターズ社、直径２．１×１００ｍｍ、粒子径１．７μｍ）を用いた。溶媒Ａに０．１％ギ酸水溶液を、溶媒Ｂとして０．１％ギ酸を含む５０％アセトニトリル水溶液を用い、カラム温度４５℃の条件で０．６ｍＬ／ｍｉｎの速度で溶媒を流した。グラジエント条件として、０～５分まで溶媒Ａを１００％、５～１０分まで溶媒Ｂを徐々に２５％まで上昇させた。これに０．１あるいは０．０２％で溶媒Ａに溶解したコラーゲン加水分解物水溶液を注入し、各トリペプチドの質量を検出することで分析した。
<Example 1>
(1) Peptide analysis contained in collagen hydrolyzate Collagen hydrolyzate "HACP-50" (prepared by Zerice Co., Ltd.) was used as a test substance. The average molecular weight of the collagen hydrolyzate is about 600.
The peptide contained in the collagen hydrolyzate was analyzed by the LC-MS method. The analysis was performed independently for 7 lots. The experimental conditions are as follows. ACQUITY UYPLC H-Class System with SQ Detector2 (Waters) was used as an apparatus, and ACCQ-TAG ULTA C18 (Waters, diameter 2.1 × 100 mm, particle diameter 1.7 μm) was used as a column. A 0.1% formic acid aqueous solution was used as the solvent A, and a 50% acetonitrile aqueous solution containing 0.1% formic acid was used as the solvent B, and the solvent was flowed at a rate of 0.6 mL / min under the condition of a column temperature of 45 ° C. As a gradient condition, the solvent A was gradually increased to 100% from 0 to 5 minutes, and the solvent B was gradually increased to 25% from 5 to 10 minutes. An aqueous solution of collagen hydrolyzate dissolved in solvent A at 0.1 or 0.02% was injected into this, and the mass of each tripeptide was detected for analysis.
ＬＣ－ＭＳ法により、表１に記載のトリペプチド類及びジペプチド類が検出された。表１の含量比（％）は、７ロットで観察された、コラーゲン加水分解物に対する各ペプチドの含量比の範囲である。
＜実施例２＞
歯肉炎モデルの作製：ビーグル犬８頭（３１～３２ヶ月齢）に対し、上顎両側の犬歯、前歯と、下顎の両側犬歯（上下合わせて６歯）にフロスを巻き付け、スーパーボンドで固定した。このまま２か月間、軟食（Ｐｅｄｉｇｒｅｅ 成犬用 ビーフ、マースジャパンリミテッド、およびＨｉｌｌ‘ｓ ＳＣＩＥＮＳ ＤＩＥＴ アダルト１歳～６歳 成犬用 チキン、日本ヒルズ・コルゲート株式会社）を与えて飼育し、歯頚部の炎症を惹起させた。
<Example 2>
Preparation of gingival inflammation model: For 8 beagle dogs (31-32 months old), floss was wrapped around the canines and anterior teeth on both sides of the maxilla and the canines on both sides of the mandible (6 teeth in total) and fixed with a super bond. For 2 months as it is, they were bred with soft food (Pedigree adult dog beef, Mars Japan Limited, and Hill's SCIENS DIET adult 1 to 6 year old chicken for adult dogs, Japan Hills Corrugate Co., Ltd.). Caused inflammation.
コラーゲン・トリペプチド タブレット（ゼライス株式会社）を動物に投与した。コラーゲン・トリペプチド タブレット（ゼライス株式会社）は、コラーゲン加水分解物として、コラーゲン・トリペプチド（ＨＡＣＰ－５０）を含む。コラーゲン・トリペプチド タブレット（ゼライス株式会社）を投与した群をＣＴＰ群と記載する。 Collagen tripeptide tablets (Zerais Co., Ltd.) were administered to animals. Collagen tripeptide tablet (Zerais Co., Ltd.) contains collagen tripeptide (HACP-50) as a collagen hydrolyzate. The group to which the collagen / tripeptide tablet (Zerais Co., Ltd.) was administered is referred to as a CTP group.
ＣＴＰの投与：上記８頭のビーグル犬を２群に分け、一方をＣＴＰ群、もう一方をコントロール群とした。ＣＴＰ群にのみに、ＣＴＰを含む錠剤（コラーゲン・トリペプチド タブレット、ゼライス株式会社）を毎日２か月間投与した。投与量はコラーゲンペプチドとして１６０ｍｇ／ｋｇ体重、（分子量４００以下の低分子画分として８０ｍｇ／ｋｇ体重）であった。なおＣＴＰ投与期間については両群とも固形餌（イヌ用 ＣＬＥＡ Ｄｏｇ Ｄｉｅｔ ＣＤ-５Ｍ（繁殖・飼育用）、日本クレア株式会社）とした。試験の全期間を通して、ブラッシング、スケーリングは実施しなかった。 Administration of CTP: The above eight beagle dogs were divided into two groups, one in the CTP group and the other in the control group. Only the CTP group was administered CTP-containing tablets (collagen tripeptide tablet, Zerice Co., Ltd.) daily for 2 months. The dose was 160 mg / kg body weight of collagen peptide (80 mg / kg body weight of a low molecular weight fraction having a molecular weight of 400 or less). Regarding the CTP administration period, solid diet (CLEA Dog Diet CD-5M for dogs (for breeding / breeding), Japan Claire Co., Ltd.) was used for both groups. No brushing or scaling was performed throughout the study.
１）毛細血管係蹄の形態の観察
中村ら（中村治郎; 原耕二; 橋口精範. 妊婦歯肉における毛細管顕微鏡所見と歯肉嚢内滲出液の観察. 口腔病学会雑誌, 1965, 32.2: 201-208.）に基づいて、毛細血管係蹄の形態を観察した。投与開始０および２ヶ月目に毛細血管血流スコープＧＯＫＯ Ｂｓｃａｎ－Ｚ（ＧＯＫＯ株式会社）を用いて辺縁歯肉の毛細血管を倍率１０５倍で撮影した。投与開始時と投与２ヶ月目の形態の６項目、太さ、長さ、数、吻合、屈曲および配置の変化を、改善：１点、変化なし：０点、悪化：－１点としてスコア化した。具体的には、太さについて、太くなると悪化、細くなると改善と判断し、長さについて、長くなると悪化、短くなると改善と判断した。数については、単位面積中の血管数が減ると悪化、増えると改善と判断した。吻合については、単位面積中の吻合部が増えると悪化、減ると改善と判断し、屈曲については、単位面積中の屈曲が増えると悪化、減ると改善と判断し、配置については血管の配置が規則的でないと悪化、規則的となると改善と判断した。配置の判断について、実際の観察では規則的な場合と不規則な場合が非常に明確であった。特に、各血管のループ間距離を観察し、距離が均等でない場合には不規則、均等である場合には規則的と判断した。これらの６項目は、画像解析・計測ソフトウェア ＷｉｎＲＯＯＦ２０１３（三谷商事株式会社）を用いて計測した。
1) Observation of the morphology of capillary hoofs Nakamura et al. (Jiro Nakamura; Koji Hara; Seiken Hashiguchi. Capillary microscopic findings and observation of exudate in the gingival sac in pregnant women's gingiva. Journal of the Oral Disease Society, 1965, 32.2: 201-208. ), The morphology of the capillary hoof was observed. At 0 and 2 months after the start of administration, the capillaries of the marginal gingiva were photographed at a magnification of 105 times using a capillary blood flow scope GOKO Bscan-Z (GOKO Co., Ltd.). 6 items of morphology at the start of administration and 2 months after administration, changes in thickness, length, number, anastomosis, flexion and placement were scored as improvement: 1 point, no change: 0 point, deterioration: -1 point. did. Specifically, it was judged that the thickness was worse when it became thicker and improved when it became thinner, and the length was judged to be worse when it became longer and improved when it became shorter. Regarding the number, it was judged that it deteriorated when the number of blood vessels in the unit area decreased, and improved when it increased. For anastomosis, it is judged that the anastomosis in the unit area is worsened and decreased, and it is judged to be improved. For flexion, it is judged that the flexion in the unit area is worsened and decreased. If it is not regular, it will be worse, and if it is regular, it will be improved. Regarding the judgment of placement, it was very clear in actual observation that there were regular cases and irregular cases. In particular, the distance between the loops of each blood vessel was observed, and it was judged that the distances were irregular when they were not even and regular when they were even. These 6 items were measured using image analysis / measurement software WinROOF2013 (Mitani Corporation).
２）辺縁歯肉の組織染色画像の評価
２か月間の投与ならびに測定の終了後、麻酔下にて辺縁歯肉を切除して回収し、ホルマリン溶液に含侵した後、辺縁歯肉の先端部の組織の切片プレパラートを作成し、これをエラスティカ・マッソン染色に供した。染色後の切片プレパラートを顕微鏡下で撮影し、歯肉の状態を目視で観察した。さらに、画像から炎症部位のみを抜き出したのち、画像解析・計測ソフトウェア ＷｉｎＲＯＯＦ２０１３（三谷商事株式会社）にて炎症組織総面積に対する膠原繊維（コラーゲン繊維）面積の比率（％）を算出した。また、膠原繊維の血行を評価するため、膠原繊維中の毛細血管断面の個数をカウントした。炎症部位の充血の状態を評価するため、赤血球が集合した１０×２０μｍ以上のサイズの領域の個数をカウントした。
2) Evaluation of tissue-stained image of marginal gingiva After 2 months of administration and measurement, marginal gingiva was excised and recovered under anesthesia, impregnated with formalin solution, and then the tip of marginal gingiva. Tissue section preparations were made and subjected to elastica-masson staining. The section preparation after staining was photographed under a microscope, and the condition of the gingiva was visually observed. Further, after extracting only the inflamed site from the image, the ratio (%) of the collagen fiber area to the total inflamed tissue area was calculated by the image analysis / measurement software WinROOF2013 (Mitani Shoji Co., Ltd.). In addition, in order to evaluate the blood circulation of collagen fibers, the number of capillary cross sections in the collagen fibers was counted. In order to evaluate the state of hyperemia at the inflamed site, the number of regions having a size of 10 × 20 μm or more in which red blood cells were aggregated was counted.
結果
１）毛細血管係蹄の形態観察の結果と考察
摂取０および２ヶ月目の代表的な毛細血管係蹄の画像を図１に示した。図１より、コントロール群では、２ヶ月間の間に毛細血管がより太く、長く、数も多くなり、吻合が増え、屈曲して蛇行やねじれを生じ、配置が乱れた様子が観察された。それに対しＣＴＰ群では、毛細血管がより細く、短くなり、吻合や屈曲が消え、配置も適切となった様子が観察された。
形態の初期値（投与開始前の形態）からの変化をスコア化した結果を表２に示し、さらにレーダーチャートとして図２に示す。
ＣＴＰ群の太さの項目が改善傾向を示していることから、拡張の予防や改善が生じることが示唆される。さらに、長さ、吻合および屈曲の項目が改善傾向の改善度から、血管の屈曲によるねじれの予防や改善が生じることが示唆される。さらに、全ての項目の総合点が改善していることは、ＣＴＰの投与によって異常な毛細血管の形成の進行が抑制され、それによって歯肉のうっ血や充血が解消し、出血しにくい健常な歯肉に回復していることを示唆する。
Results 1) Results and discussion of morphological observation of capillary hoofs Fig. 1 shows images of typical capillary hoofs at 0 and 2 months after ingestion. From FIG. 1, in the control group, it was observed that the capillaries became thicker, longer, larger in number, increased in anastomosis, bent and caused meandering and twisting, and the arrangement was disturbed during the two months. On the other hand, in the CTP group, it was observed that the capillaries became thinner and shorter, the anastomosis and flexion disappeared, and the arrangement was appropriate.
The results of scoring the changes from the initial values of the morphology (forms before the start of administration) are shown in Table 2, and further shown in FIG. 2 as a radar chart.
Since the item of the thickness of the CTP group shows an improving tendency, it is suggested that prevention or improvement of dilation occurs. Furthermore, the degree of improvement in the items of length, anastomosis, and flexion suggests that twisting due to flexion of blood vessels is prevented or improved. Furthermore, the improvement in the overall score of all items is that the administration of CTP suppresses the progression of abnormal capillarity formation, thereby eliminating gingival congestion and hyperemia, resulting in healthy gingiva that is less likely to bleed. Suggests recovery.
２）辺縁歯肉の組織染色画像の結果と考察
図３に、辺縁歯肉の代表的なエラスティカ・マッソン染色画像、ならびに画像から炎症部位のみを抜き出した解析用画像を示した。コントロール群では炎症性細胞の強い浸潤と、溶血を示唆する赤血球の集まった領域が多数観られた。膠原繊維は炎症の隙間にごく細い帯状に存在するのみであった。一方のＣＴＰ群は、炎症性細胞の浸潤がより少なく、溶血を示唆する領域もほとんど観られず、膠原繊維が広く明瞭な領域として観察された。さらに膠原繊維の間には毛細血管が侵入している様子が観られた。図４に、膠原繊維（コラーゲン繊維）の面積の割合を、図５にコラーゲン繊維における毛細血管領域の個数を、さらに図６には、炎症組織中の溶血する血管の個数を示した。
2) Results and discussion of tissue-stained images of marginal gingiva Figure 3 shows a representative elastica-masson-stained image of marginal gingiva and an analysis image in which only the inflamed site is extracted from the image. In the control group, strong infiltration of inflammatory cells and many areas of red blood cells suggesting hemolysis were observed. Collagen fibers were only present in the crevices of inflammation in the form of very thin strips. On the other hand, in the CTP group, less infiltration of inflammatory cells was observed, few regions suggesting hemolysis were observed, and collagen fibers were observed as wide and clear regions. Furthermore, it was observed that capillaries invaded between the collagen fibers. FIG. 4 shows the ratio of the area of collagen fibers (collagen fibers), FIG. 5 shows the number of capillary regions in collagen fibers, and FIG. 6 shows the number of blood vessels that hematoes in the inflamed tissue.
これらの結果によると、コントロール群では未だコラーゲン繊維が退縮した状態であり、炎症に伴う脆弱な新生血管が存在し炎症部位に炎症性細胞が供給され続けていると共に溶血が起こりやすい状態であるのに対し、ＣＴＰ群ではコラーゲン産生が促進され線維の回復がより進んでおり、しかもコラーゲン繊維は瘢痕化せずに血行の良い正常な線維が再生されていることが示唆された。さらに炎症部位からの血管の撤退が進み、炎症自体もほぼ収まっており、より回復が進んでいることが示唆された。 According to these results, in the control group, collagen fibers are still in a retracted state, fragile new blood vessels associated with inflammation are present, inflammatory cells are continuously supplied to the inflamed site, and hemolysis is likely to occur. On the other hand, in the CTP group, collagen production was promoted and the recovery of fibers was further advanced, and it was suggested that collagen fibers were not scarred and normal fibers with good blood circulation were regenerated. Furthermore, the withdrawal of blood vessels from the inflamed site progressed, and the inflammation itself was almost subsided, suggesting that recovery is progressing.
上記試験では試験期間を通してブラッシング、スケーリングなどの歯科保健的処置をしていないにも関わらず上記のような回復が観られた。すなわちコラーゲンペプチドの摂取は歯科保健行動の差異、体調・体質による口腔環境の悪化のしやすさから歯肉炎を予防や改善する有力な手段であると言える。 In the above test, the above recovery was observed even though no dental health treatment such as brushing and scaling was performed throughout the test period. In other words, it can be said that ingestion of collagen peptide is a powerful means for preventing or improving gingival inflammation due to differences in dental health behavior and the tendency of deterioration of the oral environment due to physical condition and constitution.
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