JP3805975B2 - Wound healing agent - Google Patents

Description

【００２５】
実施例２
[細胞遊走性試験２]
本実験では上記方法とは別法のトランスウエルチャンバーを用いた方法により細胞の遊走、運動性を評価した。CCD45SK正常ヒト皮膚繊維芽細胞（大日本製薬株式会社製）は10％のFBS、0.1％NEAA含有MEM培地にて、37℃、5％炭酸ガス存在下でコンフルエントになるまで培養した。その後、細胞回収し1×10⁵ cells/mlの懸濁液を8μmのポアサイズのトランスウエルチャンバー（NUNC社製）に150μl添加し、dbcAMP及び各々被験試料（100μg／ml）を添加した。トランスウエルチャンバーは10％FBS、500μl MEMまたはアスコルビン酸200μMを分注した24 wellプレート（IWAKI）上で48時間インキュベーションし、その後トランスウエルチャンバー中の細胞を綿棒で除去し、裏面に遊走した細胞数をDiff-quik（国際試薬株式会社製）試薬で染色後光学顕微鏡下で計測した。結果を表−２に示す。平均分子量1300（表−2；No.2）の分解物に細胞遊走促進効果が高いことがわかる。また、アスコルビン酸投与によって、より優れた効果を示した（表−２；No.4）。
【００２６】
【表２】

【００２７】
処方例１
[錠剤の製造]
製造例１で得たゼラチン分解物中、平均分子量1300の画分を用いて常法に従って、下記組成の錠剤を製造した。
（組成） （配合；重量％）
ゼラチン分解物（平均分子量1300） 15
乳糖 63
コーンスターチ 20
グァーガム 1.0
L−アスコルビン酸 1.0
【００２８】
処方例２
[ジュースの製造]
製造例１で得たゼラチン分解物中、平均分子量1300の画分を用いて常法に従って、下記組成のジュースを製造した。
（組成） （配合；重量％）
果糖ブトウ糖液糖 5.00
クエン酸 10.4
L−アスコルビン酸 0.20
香料 0.02
色素 0.10
ゼラチン分解物（平均分子量1300） 1.00
水 83.28
【００２９】
処方例３
[スキンケアー製品（ハンドクリーム）の製造]
製造例1で得たゼラチン分解物中、平均分子量1300の画分を用いて常法に従って、下記組成のハンドクリームを製造した。
（組成） （配合；重量％）
イソステアリン酸イソプロピル 8.0
ホホバ油 6.0
セタノール 8.0
ステアリルアルコール 2.0
ポリオキシエチレンラウリルエーテル 1.5
プロピレングリコール 6.0
ソルビトール 1.0
パラベン 0.4
ゼラチン分解物（平均分子量1300） 0.8
ビタミンE 0.5
L−アスコルビン酸 1.0
香料 0.1
精製水 64.7
【００３０】
【発明の効果】
以上から明らかのように平均分子量500以上2500未満の領域からなるコラーゲン及び／またはゼラチン分解物は、細胞遊走促進効果を有し、更にアスコルビン酸及び／またはその誘導体を組み合わせることにより、より優れた効果を示すことから、それらを含む経口用及び皮膚外用の創傷治癒剤として有効である。
【図面の簡単な説明】
【図１】図１はコラゲナーゼで分解したゼラチンのLH−20のクロマトグラフで、製造例１で調製された各画分を示したものである。
【図２】図２はペプシンで分解したゼラチンのLH−20のクロマトグラフであり、製造例２で調製された画分を示したものである。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a wound healing agent for oral or external use.
[0002]
[Prior art]
Collagen occupies about 1/3 of in vivo protein and is present in a large amount in blood vessels, skin, and bones, and plays an important role in the formation and construction of these tissues as a scaffold for cells. Collagen was thought to be a protein with low nutritional value because it is hardly degraded by digestive enzymes, but it promotes metabolism by ingesting collagen (Japanese Patent Laid-Open No. 7-278012), and the diameter of hair becomes thicker (Nutrition Reports International, 13 , 579, 1976) and its use as a drug for treating arthropathy (Japanese Patent Laid-Open No. 63-39821) has been reported, and its effectiveness has been reviewed. Collagen and gelatin are attracting attention as medical materials because of their cell support function. For example, hemostatic agents, artificial blood vessels, anti-adhesion membranes, contact lenses for continuous wear, etc., and development for many uses is being promoted. In particular, the use as artificial skin for wounds such as cuts, stab wounds, split wounds, scissors wounds, lacerations, and wounds has attracted attention (Japanese Patent Laid-Open No. 5-184662).
[0003]
However, its use is a function that protects the skin damage from dryness and plays a role as a scaffold for self-organization invasion, and is different from a function that promotes active cell migration. In addition, it has been proposed that collagen, gelatin, and degradation products thereof are blended with urea that promotes skin regeneration such as bactericidal action, wound healing action, cell activation action, etc. (Japanese Patent Laid-Open No. Sho 61-33105). Is intended to stabilize urea in an aqueous solution, and is not a wound healing agent having a function of promoting active cell migration.
[0004]
[Problems to be solved by the invention]
The present invention has been made in view of the above circumstances, and provides an effective and safe wound healing agent that promotes the regeneration of skin against injury such as a wound, which does not cause side effects even when used continuously. That is the issue.
[0005]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned objects, the present inventors have found that a degradation product of collagen or gelatin having a specific molecular weight has a wound healing effect, and based on this finding, the present invention has been completed. It was.
[0006]
That is, the present invention
1. A wound healing agent comprising a degradation product of collagen and / or gelatin and having an average molecular weight of 500 or more and less than 2500,
2. The wound healing agent according to 1 above, comprising one or more of ascorbic acid and derivatives thereof,
3. The wound healing agent according to 1 or 2 above, which is for oral use,
4). The wound healing agent according to 1 or 2 above, which is for external use on the skin,
5). The wound healing agent according to the above 1, 2 or 3, wherein the polypeptide content is 0.01 to 50% by weight based on the total amount of the composition,
6). 5. The wound healing agent according to the above 1, 2 or 4, wherein the polypeptide content is 0.001 to 20% by weight based on the total amount of the composition.
7). 4. The wound healing agent according to 2 or 3 above, wherein the polypeptide content is 0.01 to 50% by weight and the content of ascorbic acid and its derivative is 0.001 to 80% by weight based on the total amount of the composition. ,
8). 5. The wound healing agent according to 2 or 4 above, wherein the polypeptide content is 0.001 to 20% by weight and the content of ascorbic acid and derivatives thereof is 0.001 to 60% by weight with respect to the total amount of the composition. ,
It is.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
Collagen used as a raw material for obtaining the collagen or gelatin degradation product of the present invention is extracted from connective tissue such as skin, bones and tendons of animals such as cows, pigs and fish, or heat-denatured collagen. Anything such as gelatin can be used. A collagen or gelatin solution is prepared by heating and dissolving in water of about 2 to 40 times, preferably about 5 to 10 times the weight of these raw materials. The water to be used may be tap water, pure water, distilled water or the like.
[0008]
These hydrolysiss to obtain collagen or gelatin degradation products can be performed using enzymes such as proteolytic enzymes. The enzyme used for preparing the hydrolyzate is not particularly limited, and trypsin, chymotrypsin, subtilisin, elastase, proline-specific protease, protease produced by Streptococcus microorganisms, papain, pepsin, thermolysin, etc. can be used. . In addition, carboxypeptidase Y, a protease produced by Aspergillus, a protease produced by a microorganism in the genus Streptomyces, a protease produced by a microorganism of the genus Rhizopus, a protease produced by a lactic acid bacterium, etc. can be used as an exopeptidase, preferably Collagenase derived from bacteria such as Clostridium or Streptomyces, actinomycetes or fungi is preferable. Moreover, even if it is what was produced in the other microbial cell by the gene recombination technique and has the similar substrate specificity, it can also be fermented by these microorganisms without a problem. Further, a plurality of enzymes may be mixed and used.
[0009]
The amount of enzyme used for hydrolysis is about 0.01% to 10%, preferably about 1% by weight with respect to the raw material, the temperature condition is room temperature to 55 ° C, preferably 37 ° C to 40 ° C, and the reaction time is 1 to 1%. Treat for 24 hours, preferably 1-4 hours. The pH condition is adjusted to the optimum pH before adding the enzyme. In the case of collagenase, pH 6-8 is suitable. After completion of hydrolysis, the enzyme is deactivated by heating, and after cooling, filtration, desalting, concentration, and drying may be performed as necessary.
[0010]
By separating according to molecular weight, a degradation product of collagen and gelatin, which are polypeptides having an average molecular weight used in the present invention, can be obtained. Separation by molecular weight can be preferably performed by gel filtration. The detection wavelength of the molecular weight can be determined using a UV wavelength of 210 to 290 nm, preferably 210 to 220 nm.
[0011]
A polypeptide having an average molecular weight of 500 or more and less than 2500 is used as a degradation product of collagen or gelatin. Such a polypeptide has a polypeptide having an average molecular weight of 500 or more and less than 2500 as its main constituent. More preferably, it is a polypeptide having an average molecular weight of 1000 to 1500. In particular, polypeptides having an average molecular weight of 1100 to 1300 are preferred.
[0012]
Moreover, L-ascorbic acid can be mentioned as an ascorbic acid used by this invention, The derivative | guide_body can mention the ester or salt. As more specific examples, esters include L-ascorbic acid monostearate, L-ascorbic acid monopalmitate, ascorbic acid monoalkyl esters such as L-ascorbic acid monooleate, L-ascorbic acid monophosphate, L Ascorbic acid monoester derivatives such as ascorbic acid-2-sulfuric acid, ascorbic acid dialkyl esters such as L-ascorbic acid distearate, L-ascorbic acid dipalmitate, L-ascorbic acid dioleate, ascorbic acid such as L-ascorbic acid diphosphate Ascorbic acid diester derivatives, L-ascorbic acid tristearate, L-ascorbic acid tripalmitate, ascorbic acid trialkyl esters such as L-ascorbic acid trioleate, and ascorbyl such as L-ascorbic acid triphosphate It can be mentioned acid triester derivatives such as, but not limited thereto. Examples of the salt include salts of ascorbic acid and various bases. For example, an alkali metal salt (for example, sodium salt), an alkaline earth metal salt (for example, calcium salt, magnesium salt, etc.), etc. can be mentioned, but it is not limited to these.
[0013]
The polypeptide of the present invention has an excellent cell migration promoting function and can be used as a wound healing agent for oral or external use. Moreover, the more superior effect is shown by using together ascorbic acid and / or its derivative (s) described above.
[0014]
The collagen and gelatin degradation product of the present invention is useful for injuries such as wounds such as cuts, stab wounds, split wounds, fold wounds, tears, and wounds. By promoting the cell migration action of 3 ′, 5′-cyclic adenosine monophosphate (cAMP), which is a mediator of wound healing, cells are quickly brought to the wound site. It also enhances cell activation effects such as collagen production, cell proliferation and differentiation, and promotes skin regeneration. Therefore, the present invention provides an excellent wound healing agent.
[0015]
The “oral” wound healing agent of the present invention can be used as food, medicine and the like. You may use as a dietary supplement, functional food, health food, food for specified health, etc. As a form, it can be made into a dosage form such as powder, powder, granule, tablet, capsule and liquid which can be taken orally. Moreover, as a foodstuff, it can also mix | blend with drinks, such as juice. In the case of oral use, the addition amount of the polypeptide is preferably 0.01 to 50% by weight, particularly preferably 15% by weight or more and 50% by weight or less, based on the total amount. Moreover, when it contains ascorbic acid or its derivative (s), the content thereof is preferably 0.001 to 80% by weight, particularly preferably 0.1% by weight or more and 80% by weight or less. Although there is no restriction | limiting in particular about the intake as a foodstuff and a pharmaceutical, About 0.03-3g / day is preferable.
[0016]
In addition, the “external skin” wound healing agent of the present invention can be a product included in cosmetics, quasi-drugs, pharmaceuticals and the like referred to in the Pharmaceutical Affairs Law. Used in skin care products, hair care products, ointments and the like. The dosage form is not particularly limited, but the dosage form is an aqueous system, a solubilization system, an emulsification system, a powder system, an oil liquid system, a gel system, an ointment system, an aerosol system, a water-oil two-layer system, a water-oil- A wide range of dosage forms are possible, such as a powder three-layer system. The polypeptide content in the composition for external use of the present invention is about 0.001 to 20% by weight, preferably 0.1 to 10% by weight, based on the total weight. Moreover, when it contains ascorbic acid or its derivative (s), the content is preferably 0.001 to 60% by weight, particularly preferably 0.001 to 10% by weight. The amount to be applied externally for skin is appropriately selected depending on the difference in symptoms, but is usually in the range of about 0.00001 to 0.5 g as the amount of polypeptide per day. Of course, it may be administered 1 to 4 times per day.
Oral preparations and external preparations for skin can be produced by a formulation method generally used in the fields of food, medicine and cosmetics.
The collagen and gelatin degradation product of the present invention can be said to have no problem in toxicity due to the properties of collagen and gelatin.
[0017]
As described above, the present invention is an oral or external wound healing agent characterized by comprising a degradation product of collagen and / or gelatin and having an average molecular weight of 500 or more and less than 2500, Combined with the previously reported cell activation activity of ascorbic acid and its derivatives (Ayumi of Medicine, June issue, 39-44, 1996), a superior wound healing effect is expressed. That is, by promoting cell migration against wounds such as wounds, stab wounds, split wounds, wounds, lacerations, and wounds, the cells are quickly concentrated in the wound, and collagen production, cell proliferation, differentiation, etc. It is an excellent wound healing agent that promotes skin regeneration by enhancing cell activation.
[0018]
【Example】
Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
[0019]
Production Example 1
[Collagenase digestion degradation product]
As collagen protein, 30 g of gelatin prepared from bovine dermis was dissolved by heating in 300 ml of distilled water. Collagenase type I (manufactured by Worthington Biochemical Corp) (300 mg) was added, and the mixture was adjusted to pH 7.5 with aqueous ammonia and allowed to stand at 37 ° C. for 1 hour. After completion of the reaction, the reaction solution was heated at 100 ° C. for 3 minutes to inactivate the enzyme, and then sterile filtered through a 0.45 μm filter.
[0020]
This filtrate was subjected to gel filtration with Sephadex LH-20 (Pharmacia) equilibrated with distilled water, fractionated into 6 fractions, and each was freeze-dried. The results of gel filtration chromatography are shown in FIG. Each peak was tested using Superdex Peptide HR10 / 30 (Pharmacia) and the average molecular weight was determined using a 0.1M sodium phosphate buffer (pH7.2) eluate containing 0.3M NaCl. The samples were 10000, 2500, 1300, 1030, 490, 320, respectively. Each fraction was used in the test of Example 1. The fractions 1300 and 320 were also used in the test of Example 2.
[0021]
Production Example 2
[Pepsin digestion degradation product]
As collagen protein, 30 g of gelatin prepared from bovine dermis was dissolved by heating in 300 ml of distilled water. 300 mg of pepsin (Biozyme Lab., LTD) was added, the pH was adjusted to 2.0 with dilute hydrochloric acid, and the mixture was allowed to stand at 37 ° C. for 1 hour. After completion of the reaction, the solution was re-adjusted to pH 7 with an aqueous sodium hydroxide solution, reacted by heating at 100 ° C. for 3 minutes to inactivate pepsin, and then sterile filtered through a 0.45 μm filter.
[0022]
This filtrate was subjected to gel filtration using Sephadex LH-20 (Pharmacia) equilibrated with distilled water. When the average molecular weight of this product was measured, it was widely distributed as 20000-60. Of this fraction, 12000 fractions were lyophilized and used in the test of Example 1. The results of gel filtration chromatography are shown in FIG.
[0023]
Example 1
[Cell migration test 1]
CCD45SK normal human skin fibroblasts (Dainippon Pharmaceutical Co., Ltd.) in 10% non-immobilized fetal bovine serum (hereinafter abbreviated as FBS), 0.1% non-essential amino acids (hereinafter abbreviated as NEAA) in MEM medium The cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas. The confluent cells were replaced with a medium containing 0.5% FBS, 0.1% NEAA, and after acclimatization for 24 hours, the medium was removed by suction, and a part of the cell layer was detached with a cell scraper to create a wound. The wounded cells are washed with PBS, preincubated with 1μM N ⁶ , 2'-O-dibutyryl adenosine 3 ': 5'-cyclic monophosphate (hereinafter abbreviated as dbcAMP) as an initiator of cell migration for 24 hours, The test sample was added at 10 μg / ml (or ascorbic acid; 200 μM), and the cell migration promoting effect was examined. After 48 hours, the cells were fixed with PBS containing 10% formalin, stained with hematoxylin, and the number of migrated cells was measured by photography. The results are shown in Table-1. From Table 1, it can be seen that the cell migration promoting effect is high with the addition of degradation products having average molecular weights of 1030 and 1300 (Table 1; No. 4, 5). Moreover, the more superior effect was shown by ascorbic acid administration (Table-1; No. 9).
[0024]
[Table 1]

[0025]
Example 2
[Cell migration test 2]
In this experiment, cell migration and motility were evaluated by a method using a transwell chamber different from the above method. CCD45SK normal human skin fibroblasts (Dainippon Pharmaceutical Co., Ltd.) were cultured in MEM medium containing 10% FBS and 0.1% NEAA until they became confluent in the presence of 5% carbon dioxide at 37 ° C. Thereafter, the cells were collected and 150 μl of a suspension of 1 × 10 ⁵ cells / ml was added to an 8 μm pore-sized transwell chamber (NUNC), and dbcAMP and each test sample (100 μg / ml) were added. The transwell chamber was incubated on a 24-well plate (IWAKI) dispensed with 10% FBS, 500 μl MEM or ascorbic acid 200 μM for 48 hours, after which the cells in the transwell chamber were removed with a cotton swab, and the number of cells that had migrated to the back surface Was stained with a Diff-quik reagent (made by Kokusai Reagent Co., Ltd.) and measured under an optical microscope. The results are shown in Table-2. It can be seen that degradation products with an average molecular weight of 1300 (Table-2; No. 2) have a high effect of promoting cell migration. Moreover, the more superior effect was shown by ascorbic acid administration (Table-2; No. 4).
[0026]
[Table 2]

[0027]
Formulation Example 1
[Manufacture of tablets]
Using the fraction having an average molecular weight of 1300 in the gelatin degradation product obtained in Production Example 1, tablets having the following composition were produced.
(Composition) (Formulation; wt%)
Gelatin degradation product (average molecular weight 1300) 15
Lactose 63
Cornstarch 20
Guar gum 1.0
L-ascorbic acid 1.0
[0028]
Formulation example 2
[Manufacture of juice]
In the gelatin degradation product obtained in Production Example 1, a juice having the following composition was produced according to a conventional method using a fraction having an average molecular weight of 1300.
(Composition) (Formulation; wt%)
Fructose butter sugar liquid sugar 5.00
Citric acid 10.4
L-ascorbic acid 0.20
Perfume 0.02
Dye 0.10
Gelatin degradation product (average molecular weight 1300) 1.00
Wed 83.28
[0029]
Formulation Example 3
[Manufacture of skin care products (hand cream)]
A hand cream having the following composition was produced according to a conventional method using a fraction having an average molecular weight of 1300 in the gelatin degradation product obtained in Production Example 1.
(Composition) (Formulation; wt%)
Isopropyl isostearate 8.0
Jojoba oil 6.0
Cetanol 8.0
Stearyl alcohol 2.0
Polyoxyethylene lauryl ether 1.5
Propylene glycol 6.0
Sorbitol 1.0
Paraben 0.4
Gelatin degradation product (average molecular weight 1300) 0.8
Vitamin E 0.5
L-ascorbic acid 1.0
Fragrance 0.1
Purified water 64.7
[0030]
【The invention's effect】
As is apparent from the above, collagen and / or gelatin degradation products consisting of a region having an average molecular weight of 500 or more and less than 2500 have a cell migration promoting effect, and more excellent effects by combining ascorbic acid and / or its derivatives. Therefore, it is effective as a wound healing agent for oral and external use containing them.
[Brief description of the drawings]
FIG. 1 is a chromatograph of gelatin LH-20 decomposed with collagenase, showing each fraction prepared in Production Example 1. FIG.
FIG. 2 is a chromatograph of gelatin LH-20 decomposed with pepsin, showing the fraction prepared in Production Example 2.

Claims (1)

Collagenase- treated degradation product of collagen and / or gelatin, which contains a polypeptide having an average molecular weight of 1,300 and one or more of ascorbic acid and its derivatives, and promotes fibroblast migration at the wound site A wound healing agent characterized by inducing .

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