JP6516534B2 - Method of evaluating or selecting anti-inflammatory agent - Google Patents
Method of evaluating or selecting anti-inflammatory agent Download PDFInfo
- Publication number
- JP6516534B2 JP6516534B2 JP2015072719A JP2015072719A JP6516534B2 JP 6516534 B2 JP6516534 B2 JP 6516534B2 JP 2015072719 A JP2015072719 A JP 2015072719A JP 2015072719 A JP2015072719 A JP 2015072719A JP 6516534 B2 JP6516534 B2 JP 6516534B2
- Authority
- JP
- Japan
- Prior art keywords
- added
- cells
- inflammatory agent
- test
- spc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Description
本発明は、抗炎症剤の新規な評価または選択方法を提供する。 The present invention provides novel methods for the evaluation or selection of anti-inflammatory agents.
炎症は、種々の外因性または内因性の組織障害刺激に対する生体の応答である。感染などにより生体組織の一部が障害されると、組織からは様々な生体反応修飾物質が産生・放出され、炎症が生じる。初期にはＴＮＦ−αやＩＬ−１などの炎症性サイトカインが炎症部位で産生され、炎症部位より産生されるＩＬ−８などの走化因子によって、単球やリンパ球、好中球が炎症組織に浸潤する。 Inflammation is the body's response to a variety of exogenous or endogenous tissue damage stimuli. When a part of the living tissue is damaged due to infection etc., various biological response modifiers are produced and released from the tissue, resulting in inflammation. Initially, inflammatory cytokines such as TNF-α and IL-1 are produced at the site of inflammation, and chemotactic factors such as IL-8 produced from the site of inflammation cause monocytes, lymphocytes, and neutrophils to become inflamed tissues. Infiltrate.
炎症性疾患の治療には様々な抗炎症剤が用いられるが、種々の炎症性メディエーターの産生するものとしては、いまだに決め手となるようなものはない。その理由の一つとしては、上述したように炎症反応には多数の遺伝子産物が関与するため、サイトカイン産生をブロックするだけでは不十分と考えられることである。 Although various anti-inflammatory agents are used for the treatment of inflammatory diseases, none of the products producing various inflammatory mediators have yet to be determined. One of the reasons is that, as described above, since a large number of gene products are involved in the inflammatory reaction, it is considered that simply blocking cytokine production is insufficient.
一方、最近では表皮細胞に刺激物質を接触させることで、ＬｅｕｋｏｔｒｉｅｎｅＢ４、ＮＯ等の炎症性の物質を産生することが知られている（非特許文献１）。よって、表皮細胞を刺激することで発赤、熱感、膨張、疼痛などの炎症が発症することが想起される。また、皮膚に対する刺激としては紫外線や熱、乾燥などの日常的な刺激があり、表皮細胞による炎症性物質の発現、産生抑制を評価することで、皮膚刺激に起因する炎症に有効な抗炎症剤を選択することが期待されている。実際、表皮細胞に紫外線を照射し、表皮細胞が分泌するマクロファージ遊走阻止因子（ＭＩＦ）の抑制が評価されている（特許文献１）。しかし、表皮細胞に刺激物質を接触させることに起因して表皮細胞が産生する炎症性物質を抑制する評価方法は確立されていない。 On the other hand, recently, it is known that an inflammatory substance such as Leukotriene B4 or NO is produced by bringing epidermal cells into contact with a stimulatory substance (Non-patent Document 1). Therefore, it is recalled that inflammation such as redness, heat sensation, swelling, and pain develops by stimulating epidermal cells. In addition, there are daily stimuli such as ultraviolet rays, heat and dryness as irritation to the skin, and by evaluating expression and inhibition of production of inflammatory substances by epidermal cells, an anti-inflammatory agent effective for inflammation caused by skin irritation. It is expected to choose. In fact, suppression of macrophage migration inhibitory factor (MIF), which is produced by irradiating epidermal cells with ultraviolet light and secreted by epidermal cells, has been evaluated (Patent Document 1). However, an evaluation method for suppressing an inflammatory substance produced by epidermal cells due to contacting the epidermal cells with a stimulatory substance has not been established.
従って、本発明の課題は、表皮細胞の炎症性物質産生を増加させる刺激物質、刺激物質を表皮細胞に接触させることにより増加する炎症性物質を明確にすること。及び表皮細胞への刺激と炎症の相関関係を示し、これによって皮膚の刺激に起因する炎症に有効な抗炎症剤の評価を確立する。 Therefore, the object of the present invention is to define a stimulator which increases the inflammatory substance production of epidermal cells, and an inflammatory substance which is increased by bringing the stimulator into contact with the epidermal cells. And the correlation between the stimulation to the epidermal cells and the inflammation, thereby establishing the evaluation of the anti-inflammatory agent effective for the inflammation caused by the skin stimulation.
本発明者らは、皮膚刺激によることを想定した刺激物質を用いて表皮細胞に添加、産生が促進された炎症性物質の測定、さらに抗炎症効果が知られているレモングラス抽出物を添加することによって、炎症性物質の産生抑制を確認し、皮膚の刺激に起因する炎症に有効な抗炎症剤の評価方法を見出した。 The present inventors add to epidermal cells using a stimulant assumed to be caused by skin irritation, measure an inflammatory substance whose production is promoted, and add a lemongrass extract known to have an anti-inflammatory effect. As a result, they confirmed the suppression of the production of inflammatory substances and found a method for evaluating anti-inflammatory agents effective for inflammation caused by skin irritation.
刺激物質としてはＳＰＣ、ＳｕｂｓｔａｎｃｅＰ、ＩＦＮγを想定し、用いた。ＳＰＣ（スフィンゴシルホスフォリルコリン）とは自己の細胞や組織が壊れることにより外部に放出され、自然免疫を活性化させる分子の総称ＤＡＭＰｓ（ダメージ関連分子パターン）の一例であり、アトピー性皮膚炎患者の肌で濃度が高いことが報告されている。 As stimulants, SPC, SubstanceP and IFNγ were assumed and used. SPC (Sphingosyl phosphoryl choline) is an example of a generic DAMPs (damage related molecular pattern) of molecules that are released to the outside by breaking self cells and tissues and activate innate immunity, and a patient with atopic dermatitis It is reported that the concentration is high in the skin of
ＳｕｂｓｔａｎｃｅＰとは１１個のアミノ酸からなる神経ペプチドで、一次知覚神経の神経伝達物質であり、主として痛覚情報伝達物質として知られている。ＳｕｂｓｔａｎｃｅＰは末梢神経に含有されており、神経終末から遊離され、放出されたＳｕｂｓｔａｎｃｅＰは血管拡張や血漿蛋白の漏出をもたらしたり、紅斑や浮腫を形成したり、肥満細胞の脱顆粒を促進して、ヒスタミンやロイコトリエンなどを遊離させ、一次刺激を引き起こす要因ともなる。ＳｕｂｓｔａｎｃｅＰについても表皮細胞から産生されることが知られている。 SubstanceP is a neuropeptide consisting of 11 amino acids and is a neurotransmitter of primary sensory nerves, and is mainly known as a pain signal transmitter. SubstanceP is contained in peripheral nerves, and released from nerve terminals, SubstanceP released causes vasodilation, leakage of plasma proteins, forms erythema and edema, promotes degranulation of mast cells, It releases histamine and leukotriene, etc., and is a factor that causes primary stimulation. Substance P is also known to be produced from epidermal cells.
ＩＦＮγは、Ｔｈ１細胞等が分泌するサイトカインであって、抗ウイルス作用を賦与したり、免疫系の活性化および調整に関わる作用を有することが知られている。Ｔｈ１細胞は樹状細胞等の真菌刺激を受けたときなどに産生されることが知られている。 IFNγ is a cytokine secreted by Th1 cells and the like, and is known to impart an antiviral action and to have an action related to activation and regulation of the immune system. Th1 cells are known to be produced upon stimulation with fungi such as dendritic cells.
本発明者らは、ＳＰＣ及びＩＦＮγ、ＳｕｂｓｔａｎｃｅＰが表皮細胞の刺激物質となり炎症性物質を増加させること、及び当該表皮細胞が上記刺激物質により、炎症性の物質であるＩＬ−８、ＮＯ、ＮＧＦβの産生亢進、ＩＬ−８遺伝子、ＮＧＦβ遺伝子、ＳｕｎｓｔａｎｃｅＰ遺伝子の発現が促進されることを見出した。これにより、上記の刺激物質と炎症に相関関係が確認され、皮膚の刺激に起因する炎症に有効な抗炎症剤の評価、選択方法を見出した。 The present inventors have found that SPC and IFNγ, Substance P become stimulators of epidermal cells to increase inflammatory substances, and the epidermal cells are inflammatory substances such as IL-8, NO, NGF beta It was found that expression of enhanced production, IL-8 gene, NGFβ gene and Sunstance P gene was promoted. This confirmed the correlation between the above-mentioned stimulants and inflammation, and found a method for evaluating and selecting an anti-inflammatory agent effective for inflammation caused by skin irritation.
よって、本発明によれば、皮膚刺激に起因する炎症に有効な、抗炎症剤を簡単に評価し選択することが可能となる。 Therefore, according to the present invention, it is possible to easily evaluate and select an anti-inflammatory agent effective for inflammation caused by skin irritation.
本発明の方法は、以下の工程（Ａ）〜（Ｄ）により行われる。
（Ａ）表皮細胞に刺激物質を添加、さらに抗炎症剤を添加する工程、
（Ｂ）当該表皮細胞において発現、産生した炎症性の物質を測定する工程、
（Ｃ）（Ｂ）において算出された炎症性の物質の発現量または産生量を、表皮細胞に抗炎症剤を添加した場合と、添加していない場合で比較する工程。
（Ｄ）上記（Ｃ）の結果に基づいて、皮膚に刺激を与えた場合に有効な抗炎症剤の評価または選択を行う工程。
The method of the present invention is carried out by the following steps (A) to (D).
(A) adding a stimulator to epidermal cells and further adding an anti-inflammatory agent,
(B) measuring an inflammatory substance expressed and produced in the epidermal cells,
(C) A step of comparing the expression amount or production amount of the inflammatory substance calculated in (B) in the case where an anti-inflammatory agent is added to epidermal cells and in the case where it is not added.
(D) A step of evaluating or selecting an anti-inflammatory agent effective when the skin is stimulated based on the result of the above (C).
なお、本発明における表皮細胞とは、表皮を構成する細胞であればよく、表皮ケラチノサイト、ランゲルハンス細胞、メラノサイト等が挙げられる。特に、皮膚の外側の表皮を大部分を構成する表皮ケラチノサイトが好ましい。 The epidermal cells in the present invention may be any cells constituting the epidermis, and include epidermal keratinocytes, Langerhans cells, melanocytes and the like. In particular, epidermal keratinocytes that make up the majority of the outer epidermis of the skin are preferred.
また、本発明における抗炎症剤としては、広く皮膚に対して適用する物質や組成物であれば問題ないが、防腐剤、界面活性剤、色素沈着抑制剤、チロシナーゼ活性阻害剤、メラノサイトメラニン生成抑制剤、保湿剤、細胞賦活剤／代謝活性化剤、抗酸化剤、活性酸素消去剤／ラジカル生成抑制剤、脂肪代謝促進剤、収斂剤、抗炎症剤／インターロイキン産生抑制剤／消炎剤、抗脂漏剤、抗菌剤／抗ウイルス剤、血流促進剤／血管刺激剤、抗アンドロゲン剤、構造タンパク質分解酵素（エラスターゼ、コラゲナーゼ、ケラチンプロテアーゼ、セリンプロテアーゼ、インテグリン分解酵素、インボルクリン分解酵素、フィラグリン分解酵素、ラミニン分解酵素、フィブロネクチン分解酵素、プロテオグリカン分解酵素等）活性阻害剤／構造タンパク質分解酵素発現抑制剤、構造タンパク質合成促進剤、ムコ多糖類（ヒアルロン酸又はその塩、コンドロイチン硫酸、プロテオグリカン等）分解酵素阻害剤、ムコ多糖類合成促進剤、細胞間脂質生成促進剤／細胞間脂質状態改善剤、角質溶解剤／角層剥離促進剤、プラスミノーゲンアクチベーター拮抗阻害剤、メイラード反応阻害剤、テストステロン５αレダクターゼ活性阻害剤、有臭物質消去剤等の有効成分や、その他、医薬品、医薬部外品又は化粧料の形態を形成する上で使用が好まれる植物系原料、動物系原料、微生物系原料、その他天然物原料等を由来とするエキスや代謝物等成分、又は種々の化合物等が挙げられる。 Moreover, as the anti-inflammatory agent in the present invention, there is no problem as long as it is a substance or composition widely applied to the skin, but preservatives, surfactants, pigmentation inhibitors, tyrosinase activity inhibitors, melanocyte melanin formation suppression Agents, moisturizers, cell activators / metabolic activators, antioxidants, active oxygen scavenging agents / radical generation inhibitors, fat metabolism promoters, astringents, anti-inflammatory agents / interleukin production inhibitors / anti-inflammatory agents, anti-inflammatory agents Seborrhea agent, antibacterial agent / antiviral agent, blood flow promoter / vasostimulant, antiandrogen agent, structural protein degradation enzyme (elastase, collagenase, keratin protease, serine protease, integrin degradation enzyme, involucrin degradation enzyme, filaggrin degradation enzyme , Laminin degrading enzyme, fibronectin degrading enzyme, proteoglycan degrading enzyme etc) activity inhibitor / structure tamper Protein degradation inhibitors, structural protein synthesis promoters, mucopolysaccharides (hyaluronic acid or its salts, chondroitin sulfate, proteoglycans etc) degradation enzyme inhibitors, mucopolysaccharide synthesis promoters, intercellular lipogenesis promoter / intercellular Active ingredients such as lipid condition improver, keratolytic agent / keratinocyte peel promoter, plasminogen activator antagonistic inhibitor, Maillard reaction inhibitor, testosterone 5α reductase activity inhibitor, odorant substance eliminator, and other pharmaceuticals , Extracts or metabolites derived from plant-based materials, animal-based materials, microbial-based materials, other natural-materials, etc. which are preferred for use in forming quasi drugs or cosmetics, or various components such as extracts or metabolites Compounds etc. may be mentioned.
また、本発明で用いる刺激物質としては体内に存在し、表皮細胞へ炎症に起因する刺激を与えることが予測される物質なら特に限定されない。例としては、ＴＮＦ−α、ＩＦＮ−γ、ＳＰＣ、ＳｕｂｓｔａｎｃｅＰ、ＬＰＳ、ＩＬ−１、ＴＳＬＰ等が挙げられる。刺激物質を培地に添加した後は、室温で１２時間〜７２時間程度行うのが好ましい。 Further, the stimulant used in the present invention is not particularly limited as long as it is a substance which is present in the body and which is expected to give epidermal cells a stimulus due to inflammation. Examples include TNF-α, IFN-γ, SPC, SubstanceP, LPS, IL-1, TSLP and the like. After the stimulus substance is added to the culture medium, it is preferably performed at room temperature for about 12 hours to 72 hours.
このようにして評価、選択された抗炎症剤は皮膚刺激に伴う炎症の治療剤または予防剤としての提供が可能になり、皮膚刺激の関与が推定される、乾癬や紫外線による皮膚炎症、蕁麻疹、慢性関節リウマチ、潰瘍性大腸炎、気管支炎、接触性皮膚炎、アトピー性皮膚炎等の治療または予防にも使用することが可能となる。よって、本発明の方法により選択された抗炎症剤を上記目的で医薬品、医薬部外品、化粧品に有効成分として配合することが可能となる。 The anti-inflammatory agent evaluated and selected in this way can be provided as a therapeutic agent or preventive agent for inflammation associated with skin irritation, and the involvement of skin irritation is presumed, skin inflammation due to psoriasis or ultraviolet light, urticaria It can also be used for the treatment or prevention of rheumatoid arthritis, ulcerative colitis, bronchitis, contact dermatitis, atopic dermatitis and the like. Therefore, it becomes possible to mix the anti-inflammatory agent selected by the method of the present invention as an active ingredient in medicines, quasi-drugs and cosmetics for the above purpose.
（試験条件）
以下の試験１〜９を行った。試験１〜８について、細胞は市販の正常ヒト表皮角化細胞（クラボウ）を、細胞培養培地は前培養培地としてＥｐｉＬｉｆｅ ＫＧ２（クラボウ）培地を使用し、本試験用培地として前培養培地からｈＥＧＦ、ハイドロコルチーゾン、ＢＰＥを除いたものを使用した。前培養、本試験培養ともに、メーカーの説明書に従い行った。尚、抗炎症剤としてはレモングラス抽出物（一丸ファルコス社製）を使用した。また、試験１〜８の結果についてはいずれもコントロールを１００として相対値を示した。
(Test conditions)
The following tests 1 to 9 were performed. For tests 1 to 8, the cells were commercially available normal human epidermal keratinocytes (Kurabo), and the cell culture medium was EpiLife KG2 (Kurabo) as the pre-culture medium, and hEGF from the pre-culture medium as the medium for this test. It used what except hydrocortisone and BPE. Both pre-culture and main test culture were performed according to the manufacturer's instructions. As an anti-inflammatory agent, lemongrass extract (manufactured by Ichimaru Falcos Co., Ltd.) was used. Moreover, all showed the relative value by setting 100 as a control about the result of test 1-8.
（試験１）ＳＰＣ添加によるＩＬ−８遺伝子発現 (Test 1) IL-8 gene expression by SPC addition
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態になるまで前培養した。その後、本試験用培地に置換し、２４時間培養した。翌日、新たな本試験用の培地に置換し、抗炎症剤を培養液中に添加した。細胞を２４時間培養したのち、ＳＰＣ１０μＭを正常ヒト表皮角化細胞に添加し、４．５時間培養後、ｔｏｔａｌ ＲＮＡを調整し、Ｒｅａｌ−ｔｉｍｅ ＰＣＲ法にてＩＬ−８のｍＲＮＡ相対発現量を測定した。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded in culture plates and precultured to 100% confluence. Thereafter, the medium for the test was replaced and cultured for 24 hours. The next day, the medium for the new main test was replaced, and an anti-inflammatory agent was added to the medium. After culturing the cells for 24 hours, 10 μM of SPC is added to normal human epidermal keratinocytes, and after culturing for 4.5 hours, the total RNA is adjusted, and the relative expression level of IL-8 is measured by Real-time PCR method did. The final concentration of the antiinflammatory agent is 0.1%.
（試験結果）
結果は図１に示した通り、正常ヒト表皮角化細胞に、ＳＰＣを添加するとＩＬ−８遺伝子の発現が亢進され、抗炎症剤の添加によるＩＬ−８遺伝子の発現抑制が確認できた。
(Test results)
As shown in FIG. 1, when SPC was added to normal human epidermal keratinocytes, the expression of IL-8 gene was enhanced, and the suppression of IL-8 gene expression by the addition of an anti-inflammatory agent could be confirmed.
（試験２）ＳＰＣ添加によるＩＬ−８産生 (Test 2) IL-8 production by SPC addition
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態になるまで前培養した。その後、本試験用培地に置換し、２４時間培養した。翌日、新たな本試験用の培地に置換し、抗炎症剤を培養液中に添加した。細胞を２４時間培養したのち、ＳＰＣ１０μＭを正常ヒト表皮角化細胞に添加し、さらに２４時間培養した。培養終了後上清を回収し、上清中のＨｕｍａｎ ＩＬ−８ ＥＬＩＳＡ Ｋｉｔ（Ｔｈｅｒｍｏ♯ＥＨ２ＩＬ８）を用いて測定した。上清回収後の細胞（起痒物質添加時間を２４時間経過した細胞）は、Ｃｅｌｌｃｏｕｎｔｉｎｇ ｋｉｔ−８（ＤＯＪＩＮＤＯ）を用い、細胞活性量の測定に供した。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded in culture plates and precultured to 100% confluence. Thereafter, the medium for the test was replaced and cultured for 24 hours. The next day, the medium for the new main test was replaced, and an anti-inflammatory agent was added to the medium. After culturing the cells for 24 hours, 10 μM of SPC was added to normal human epidermal keratinocytes and cultured for further 24 hours. After the culture was completed, the supernatant was collected and measured using Human IL-8 ELISA Kit (Thermo # EH2IL8) in the supernatant. The cells after collection of the supernatant (cells for which the starting time for the addition of the stimulant substance has been 24 hours) were subjected to measurement of the amount of cell activity using Cellcounting kit-8 (DOJINDO). The final concentration of the antiinflammatory agent is 0.1%.
（試験結果）
結果は図２、図３、図４に示した通り、正常ヒト表皮角化細胞に対し、ＳＰＣを添加させるとＩＬ−８の産生が亢進され、抗炎症剤の添加によるＩＬ−８の産生抑制が確認できた。
(Test results)
As shown in FIG. 2, FIG. 3 and FIG. 4, when SPC is added to normal human epidermal keratinocytes, IL-8 production is enhanced and IL-8 production is suppressed by the addition of an anti-inflammatory agent Was confirmed.
（試験３）ＳＰＣ添加によるＮＯ産生 (Test 3) NO production by SPC addition
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態になるまで前培養した。その後、本試験用の培地に置換し、２４時間培養した。その後、再度、本試験用の培地に置換し、抗炎症剤を培養液中に添加し、２４時間培養した。翌日、本試験用の培地に置換しＳＰＣ１０μＭを添加し、２０分培養した。培養終了後上清を回収し、そのＮＯ濃度をＮｉｔｒａｔｅ／Ｎｉｔｒｉｔｅ Ｆｌｕｏｒｏｍｅｔｒｉｃ Ａｓｓａｙ Ｋｉｔ（Ｃａｙｍａｎ♯７８００５１）にて測定した。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded in culture plates and precultured to 100% confluence. Thereafter, the medium was replaced with the medium for the test and cultured for 24 hours. Thereafter, the medium for the test was replaced again, and the anti-inflammatory agent was added to the culture solution and cultured for 24 hours. The next day, the medium for the test was replaced with 10 μM of SPC, and the cells were cultured for 20 minutes. After completion of the culture, the supernatant was recovered, and the NO concentration was measured with Nitrate / Nitrite Fluorometric Assay Kit (Cayman # 780051). The final concentration of the antiinflammatory agent is 0.1%.
（試験結果）
結果は図５に示した通り、正常ヒト表皮角化細胞に対し、ＳＰＣを添加させるとＮＯの産生が亢進され、抗炎症剤の添加によるＮＯの産生抑制が確認できた。
(Test results)
As shown in FIG. 5, when SPC was added to normal human epidermal keratinocytes, the production of NO was enhanced, and it was confirmed that the production of NO was suppressed by the addition of an anti-inflammatory agent.
（試験４）ＳＰＣ添加によるＮＧＦβ遺伝子発現 (Test 4) NGF beta gene expression by SPC addition
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態になるまで前培養した。その後、本試験用の培地に置換し、２４時間培養した。翌日、新たな本試験用の培地に置換し、抗炎症剤を培養液中に添加した。細胞を２４時間培養したのち、ＳＰＣ１０μＭを正常ヒト表皮角化細胞に添加し、４．５時間培養後、ｔｏｔａｌ ＲＮＡを調整し、Ｒｅａｌ−ｔｉｍｅ ＰＣＲ法を行い、ＮＧＦ−β遺伝子の発現量をもとめた。尚、同時に、Ｃｅｌｌｃｏｕｎｔｉｎｇ ｋｉｔ−８（ＤＯＪＩＮＤＯ）を用い、細胞活性量を測定した。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded in culture plates and precultured to 100% confluence. Thereafter, the medium was replaced with the medium for the test and cultured for 24 hours. The next day, the medium for the new main test was replaced, and an anti-inflammatory agent was added to the medium. After culturing the cells for 24 hours, 10 μM of SPC is added to normal human epidermal keratinocytes, and after culturing for 4.5 hours, total RNA is adjusted, and Real-time PCR is performed to obtain the expression level of NGF-β gene. The At the same time, the amount of cellular activity was measured using Cellcounting kit-8 (DOJINDO). The final concentration of the antiinflammatory agent is 0.1%.
（試験結果）
結果は図６に示した通り、正常ヒト表皮角化細胞に対し、ＳＰＣを添加させるとＮＧＦβ遺伝子の発現が亢進され、抗炎症剤の添加によるＮＧＦβ遺伝子の発現抑制が確認できた。
(Test results)
As shown in FIG. 6, when SPC was added to normal human epidermal keratinocytes, the expression of the NGFβ gene was enhanced, and the suppression of the expression of the NGFβ gene by the addition of an anti-inflammatory agent was confirmed.
（試験５）ＩＦＮγ、ＳｕｂｓｔａｎｃｅＰの併用によるＮＧＦβ遺伝子発現 (Test 5) NGFβ gene expression by combination of IFNγ and SubstanceP
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態になるまで前培養した。その後、本試験用の培地に置換し、２４時間培養した。翌日、新たな本試験用の培地に置換し、抗炎症剤を培養液中に添加した。細胞を３時間培養したのち、ＳｕｂｓｔａｎｃｅＰの終濃度が１００μＭになるように正常ヒト表皮角化細胞に添加し、１０分間室温にて静置した。その後、ＩＦＮ γ ２０ｎｇ／ｍＬをウェルに加え、５時間 ５％ＣＯ ２ 、３７℃の条件で培養した。５時間培養後、ｔｏｔａｌ ＲＮＡを調整し、Ｒｅａｌ−ｔｉｍｅ ＰＣＲ法にてＮＧＦβ遺伝子の発現量をもとめた。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded in culture plates and precultured to 100% confluence. Thereafter, the medium was replaced with the medium for the test and cultured for 24 hours. The next day, the medium for the new main test was replaced, and an anti-inflammatory agent was added to the medium. The cells were cultured for 3 hours, then added to normal human epidermal keratinocytes so that the final concentration of SubstanceP was 100 μM, and allowed to stand at room temperature for 10 minutes. Thereafter,
（試験結果）
結果は図７に示した通り、正常ヒト表皮角化細胞に対し、ＩＦＮγ、ＳｕｂｓｔａｎｃｅＰを添加させるとＮＧＦβ遺伝子の発現が亢進され、抗炎症剤の添加によるＮＧＦβの遺伝子発現抑制が確認された。
(Test results)
As shown in FIG. 7, when IFNγ and SubstanceP were added to normal human epidermal keratinocytes, the expression of the NGFβ gene was enhanced, and the suppression of the gene expression of NGFβ by the addition of an anti-inflammatory agent was confirmed.
（試験６）ＩＦＮγ、ＳＰＣの併用によるＳｕｂｓｔａｎｃｅＰ遺伝子発現 (Test 6) SubstanceP gene expression by combination of IFNγ and SPC
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態まで培養した後、本試験用の培地に置換し、２４時間培養した。翌日、新たな本試験用の培地に置換し、抗炎症剤を培養液中に添加した。細胞を３時間培養したのち、ＳＰＣ１０μＭを正常ヒト表皮角化細胞に添加し、１０分間室温にて静置した。その後、ＩＦＮ γ ２０ｎｇ／ｍＬをウェルに加え、５時間 ５％ＣＯ ２ 、３７℃の条件で培養した。５時間培養後、ｔｏｔａｌ ＲＮＡを調整し、Ｒｅａｌ−ｔｉｍｅ ＰＣＲ法にてＳｕｂｓｔａｎｃｅＰ遺伝子の発現量をもとめた。また同時に、Ｃｅｌｌｃｏｕｎｔｉｎｇ ｋｉｔ−８（ＤＯＪＩＮＤＯ）を用い、細胞活性量を測定した。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded on a culture plate, cultured to 100% confluence, then replaced with the medium for this test, and cultured for 24 hours. The next day, the medium for the new main test was replaced, and an anti-inflammatory agent was added to the medium. After culturing the cells for 3 hours, 10 μM of SPC was added to normal human epidermal keratinocytes and allowed to stand at room temperature for 10 minutes. Thereafter,
（試験結果）
結果は図１０に示した通り、正常ヒト表皮角化細胞に対し、ＩＦＮγ、ＳＰＣを添加させるとＳｕｂｓｔａｎｃｅＰの遺伝子発現が亢進され、抗炎症剤の添加によるＳｕｂｓｔａｎｃｅＰの遺伝子発現抑制が確認された。
(Test results)
As shown in FIG. 10 , when IFNγ and SPC were added to normal human epidermal keratinocytes, gene expression of SubstanceP was enhanced, and gene expression suppression of SubstanceP by addition of an anti-inflammatory agent was confirmed.
（試験７）ＩＦＮγ、ＳＰＣの併用によるＮＧＦβ産生 (Test 7) NGFβ production by combined use of IFNγ and SPC
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態になるまで前培養した。その後、本試験用の培地に置換し、２４時間培養した。翌日、新たな本試験用の培地に置換し、抗炎症剤を培養液中に添加した。細胞を３時間培養したのち、ＳＰＣ１０μＭを正常ヒト表皮角化細胞に添加し、１０分間室温にて静置した。その後、ＩＦＮ γ ２０ｎｇ／ｍＬをウェル加え、４８時間 ５％ＣＯ ２ 、３７℃の条件で培養した。培養後、上清を回収しＢｅｔａ Ｎｅｒｖｅ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ Ｈｕｍａｎ ＥＬＩＳＡ Ｋｉｔ（ａｂｃａｍ♯ａｂ９９９８６）にて測定した。上清を回収後の細胞（起痒物質添加時間を２４時間経過した細胞）は、Ｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ−８（ＤＯＪＩＮＤＯ）を用い、細胞活性量を測定した。結果は、細胞あたりのＮＧＦβ産生量を以下に記した。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded in culture plates and precultured to 100% confluence. Thereafter, the medium was replaced with the medium for the test and cultured for 24 hours. The next day, the medium for the new main test was replaced, and an anti-inflammatory agent was added to the medium. After culturing the cells for 3 hours, 10 μM of SPC was added to normal human epidermal keratinocytes and allowed to stand at room temperature for 10 minutes. Thereafter,
（試験結果）
結果は図９に示した通り、正常ヒト表皮角化細胞に対し、ＩＦＮγ、ＳＰＣを添加するとＮＧＦβを産生が亢進され、抗炎症剤の添加によるＮＧＦβの産生抑制が確認された。
(Test results)
As shown in FIG. 9, when IFNγ and SPC were added to normal human epidermal keratinocytes, NGFβ production was enhanced, and it was confirmed that NGFβ production was suppressed by the addition of an anti-inflammatory agent.
（試験８）ＩＦＮγ、ＳｕｂｓｔａｎｃｅＰの併用によるＮＧＦβ産生 (Test 8) NGFβ production by combination of IFNγ and SubstanceP
（試験方法）
正常ヒト表皮角化細胞を、培養プレートに播種し、１００％コンフルエント状態になるまで前培養した。その後、本試験用の培地に置換し、２４時間培養した。翌日、新たな本試験用の培地に置換し、抗炎症剤を培養液中に添加した。細胞を３時間培養したのち、ｓｕｂｓｔａｎｃｅＰ１００μＭ を正常ヒト表皮角化細胞に添加し、１０分間室温にて静置した。その後、ＩＦＮ γ ２０ｎｇ／ｍＬをウェルに加え、４８時間 ５％ＣＯ ２ 、３７℃の条件で培養した。培養後、上清を回収しＢｅｔａ Ｎｅｒｖｅ Ｇｒｏｗｔｈ Ｆａｃｔｏｒ Ｈｕｍａｎ ＥＬＩＳＡ Ｋｉｔ（ａｂｃａｍ♯ａｂ９９９８６）にて測定した。上清を回収後の細胞（起痒物質添加時間を２４時間経過した細胞）は、Ｃｅｌｌ ｃｏｕｎｔｉｎｇ ｋｉｔ−８（ＤＯＪＩＮＤＯ）を用い、細胞活性量を測定した。結果は、細胞あたりのＮＧＦβ産生量を以下に記した。尚、抗炎症剤の終濃度は０．１％である。
(Test method)
Normal human epidermal keratinocytes were seeded in culture plates and precultured to 100% confluence. Thereafter, the medium was replaced with the medium for the test and cultured for 24 hours. The next day, the medium for the new main test was replaced, and an anti-inflammatory agent was added to the medium. After culturing the cells for 3 hours, 100 μM of substanceP was added to normal human epidermal keratinocytes and allowed to stand at room temperature for 10 minutes. Thereafter,
（試験結果）
結果は図８に示した通り、正常ヒト表皮角化細胞に対し、ＩＦＮγ、ＳｕｂｓｔａｎｃｅＰを添加するとＮＧＦβの産生が亢進され、抗炎症剤の添加によるＮＧＦβの産生抑制が確認された。
(Test results)
As shown in FIG. 8 , when IFNγ and SubstanceP were added to normal human epidermal keratinocytes, the production of NGFβ was enhanced, and the inhibition of NGFβ production by the addition of an anti-inflammatory agent was confirmed.
（試験９）皮膚疾患改善効果 (Test 9) Skin disease improvement effect
（試験方法）
敏感肌の自覚があり、アトピー性皮膚炎や乾燥等によるかゆみを訴える成人男女１２名に対して、下記処方のクリームと、レモングラス抽出物を含まない下記処方のクリームと同じ組成のクリームを対照品として、かゆみの患部に１日２回塗布し１ヶ月間の使用試験を行った。なお、被験者は６人づつに分け、それぞれ異なるクリームを塗布した。
(Test method)
For 12 adult men and women who have sensitivity to sensitive skin and complain of itching due to atopic dermatitis or dryness, the cream with the following formulation and the cream with the same composition as the cream with the following formulation without lemongrass extract are compared As a product, it was applied twice a day to the affected area of itching and the use test was conducted for 1 month. The subjects were divided into six persons and each applied a different cream.
（処方）クリーム 重量％
１．レモングラス抽出物（一丸ファルコス社製） １．０
２．４−ｔｅｒｔ−ブチル−４’−メトキシベンゾイルメタン ０．３０
３．テトラ２−ヘキシルデカン酸アスコルビル １．０
４．濃グリセリン ３．０
５．ミツロウ １．５
６．スクラワン ８．０
７．メチルポリシロキサン ０．３０
８．デカメチルシクロペンタシロキサン ４．０
９．パルミチン酸セチル ５．０
１０．トリオクタノイン ６．０
１１．ベヘニルアルコール １．０
１２．ステアリン酸 ２．５
１３．キサンタンガム（２％水溶液） １０．０
１４．メチルパラベン 適量
１５．プロピルパラベン 適量
１６．フェノキシエタノール ０．２０
１７．精製水 全量で１００にする
(Prescription) cream weight%
1. Lemongrass extract (manufactured by Ichimaru Falcos) 1.0
2.4-tert-butyl-4'-methoxybenzoylmethane 0.30
3. Ascorbyl tetra 2-hexyldecanoate 1.0
4. Concentrated glycerin 3.0
5. Bee wax 1.5
6. Skrawan 8.0
7. Methylpolysiloxane 0.30
8. Decamethylcyclopentasiloxane 4.0
9. Cetyl palmitate 5.0
10. Trioctanoin 6.0
11. Behenyl alcohol 1.0
12. Stearic acid 2.5
13. Xanthan gum (2% aqueous solution) 10.0
14. Methyl paraben amount 15. Propyl paraben Dosage 16. Phenoxyethanol 0.20
17. Make the total amount of purified
有 効：肌のかゆみ又は敏感肌、アトピー性皮膚炎、乾燥等の皮膚疾患が改善された。
やや有効：肌のかゆみ又は敏感肌、アトピー性皮膚炎、乾燥等の皮膚疾患がやや改善された。
無 効：使用前と変化なし。
Slightly effective: Itchy skin or sensitive skin, atopic dermatitis, skin diseases such as dryness are slightly improved.
Ineffective: No change before use.
試験結果より、レモングラス抽出物を含有したクリームは肌のかゆみや敏感肌、アトピー性皮膚炎、乾燥等の皮膚疾患に対する改善効果を有しており、皮膚への刺激による炎症を評価した試験１〜８の結果と相関があると認められる。よって、試験１〜８の表皮細胞への刺激に起因する炎症を評価することによって、皮膚刺激に起因する炎症に有効な抗炎症剤を評価、選択することができる。 According to the test results, cream containing lemongrass extract has an improvement effect on skin diseases such as itchy skin, sensitive skin, atopic dermatitis, dryness, etc. It is recognized that there is a correlation with the results of. Therefore, by evaluating the inflammation caused by the stimulation to epidermal cells of Tests 1 to 8, an anti-inflammatory agent effective for the inflammation caused by the skin stimulation can be evaluated and selected.
Claims (1)
以下の（Ａ）〜（Ｄ）の工程を含む方法、
（Ａ）表皮細胞にＳＰＣ及びＩＮＦγを添加、さらに抗炎症剤を添加する工程、
（Ｂ）当該表皮細胞において発現又は産生するＮＧＦβの発現量又は産生量を測定する工程、
（Ｃ）（Ｂ）において算出されたＮＧＦβの発現量または産生量を、表皮細胞に抗炎症剤を添加した場合と、添加していない場合と、で比較する工程。
（Ｄ）上記（Ｃ）の結果に基づいて、皮膚に刺激を与えた場合に有効な抗炎症剤の評価又は選択を行う工程。 A method for evaluating or selecting an anti-inflammatory agent effective for inflammation caused by SPC and IFNγ , comprising:
A method comprising the following steps (A) to (D):
(A) adding SPC and IFNγ to epidermal cells and further adding an anti-inflammatory agent,
(B) measuring the amount of expression or production of NGF beta expressed or produced in the epidermal cells,
(C) A step of comparing the expression amount or production amount of NGFβ calculated in (B) between the case where an anti-inflammatory agent is added to epidermal cells and the case where it is not added.
(D) A step of evaluating or selecting an anti-inflammatory agent effective when the skin is stimulated based on the result of the above (C).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015072719A JP6516534B2 (en) | 2015-03-31 | 2015-03-31 | Method of evaluating or selecting anti-inflammatory agent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2015072719A JP6516534B2 (en) | 2015-03-31 | 2015-03-31 | Method of evaluating or selecting anti-inflammatory agent |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2016191673A JP2016191673A (en) | 2016-11-10 |
| JP6516534B2 true JP6516534B2 (en) | 2019-05-22 |
Family
ID=57246460
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2015072719A Expired - Fee Related JP6516534B2 (en) | 2015-03-31 | 2015-03-31 | Method of evaluating or selecting anti-inflammatory agent |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6516534B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020075872A (en) * | 2018-11-05 | 2020-05-21 | 共栄化学工業株式会社 | Skin external preparation and screening method |
| KR102465347B1 (en) * | 2020-10-07 | 2022-11-14 | 정읍시 | A composition for anti-inflammation activity comprising Chrysanthemum zawadskii leaf and stem |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3860956B2 (en) * | 2000-08-23 | 2006-12-20 | 株式会社資生堂 | Method and apparatus for measuring skin surface condition |
| JP4477285B2 (en) * | 2002-03-06 | 2010-06-09 | ピアス株式会社 | Anti-inflammatory agent, PGE2 production inhibitor, IL-1α production inhibitor and IL-6 production inhibitor |
| EP2885637B1 (en) * | 2012-08-15 | 2016-11-30 | The Procter & Gamble Company | Use of human ex vivo skin model in a method of identifying modulators of skin inflammation |
-
2015
- 2015-03-31 JP JP2015072719A patent/JP6516534B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2016191673A (en) | 2016-11-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Misery et al. | Sensitive skin | |
| US10052297B2 (en) | L-serine to be used as a drug for preventing and/or treating an inflammatory response of the skin | |
| Roure et al. | Evaluation of the efficacy of a topical cosmetic slimming product combining tetrahydroxypropyl ethylenediamine, caffeine, carnitine, forskolin and retinol, in vitro, ex vivo and in vivo studies | |
| JP5524483B2 (en) | Moisturizing skin external preparation composition containing gallocatechin gallate as an active ingredient | |
| JP2016508158A (en) | Soothing cosmetic composition based on salicylic acid | |
| TW201501726A (en) | Use of caffeamide derivative | |
| JP6516534B2 (en) | Method of evaluating or selecting anti-inflammatory agent | |
| CN114917178A (en) | Soothing and repairing composition suitable for dry skin in desert and application thereof | |
| US20160084822A1 (en) | Use of biomarkers for evaluating the effectiveness of active ingredients | |
| Bernhofer et al. | The influence of the response of skin equivalent systems to topically applied consumer products by epithelial--mesenchymal interactions | |
| Diaz et al. | Amino acid complex (AAComplex) benefits in cosmetic products: In vitro and in vivo clinical studies | |
| Lodén | Do moisturizers work? | |
| KR20200104238A (en) | Composition for treating allergic skin disease or skin pruritis comprising colchicine | |
| Pedretti et al. | Effects of topical boswellic acid on photo and age-damaged skin: clinical, biophysical, and echographic evaluations in a double-blind, randomized, split-face study | |
| AU2020445229B2 (en) | Functional food composition and cosmetic composition for improving immune function and improving skin condition containing galacto-oligosaccharide or galacto-oligosaccharide and collagen tripeptide | |
| EP4238549A1 (en) | Anti-aging composition, skin care product and cosmetic | |
| Jang et al. | Epidermal skin barrier | |
| CN104039337A (en) | Sedative for skin to respond to external stimuli and method used for tranquilizing respond | |
| Harding et al. | Determination of chemical irritation potential using a defined gene signature set on tissue-engineered human skin equivalents | |
| Guerra-Tapia et al. | [Translated article] Sensitive Scalp: Diagnosis and Practical Management | |
| KR101081178B1 (en) | Cosmetic composition for inflammation in skin containing Citrullis Vulgaris extract | |
| CN1520804A (en) | Thermal shock protein generation promotion component and skin beauty and white applicat composition | |
| CN117815216A (en) | Anti-photoaging and/or dermis pigmentation inhibitor | |
| KR20170107331A (en) | Cosmetic compositions for regulating collagen and anti-wrinkle | |
| Al-juhaishi et al. | Effects of estradiol on imiquimod-induced model of psoriasis in mice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20171130 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20180731 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20180814 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20181001 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20190409 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20190416 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6516534 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| LAPS | Cancellation because of no payment of annual fees |