JP2016191673A - Method of evaluating or selecting anti-inflammatory agents - Google Patents
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Abstract
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 a novel method for evaluating or selecting 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 various exogenous or endogenous tissue damage stimuli. When a part of a biological tissue is damaged due to infection or the like, various biological response modifiers are produced and released from the tissue, and inflammation occurs. Initially, inflammatory cytokines such as TNF-α and IL-1 are produced at the inflammatory site, and monocytes, lymphocytes, and neutrophils are inflamed by chemotactic factors such as IL-8 produced from the inflammatory site. 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。 Various anti-inflammatory agents are used for the treatment of inflammatory diseases, but there are still no decisive factors for the production of various inflammatory mediators. One reason for this is that, as described above, many gene products are involved in the inflammatory response, and it is considered insufficient to simply block cytokine production.
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 inflammatory substances such as Leukotriene B4 and NO are produced by bringing a stimulating substance into contact with epidermal cells (Non-patent Document 1). Thus, it is recalled that inflammation such as redness, heat, swelling, and pain develops by stimulating epidermal cells. In addition, there are daily stimuli such as ultraviolet rays, heat, and dryness as stimuli to the skin, and anti-inflammatory agents effective for inflammation caused by skin irritation are evaluated by evaluating the expression and suppression of production of inflammatory substances by epidermal cells. Is expected to choose. In fact, suppression of macrophage migration inhibitory factor (MIF) secreted by epidermal cells by irradiating the epidermal cells with ultraviolet rays has been evaluated (Patent Document 1). However, an evaluation method for suppressing inflammatory substances produced by epidermal cells due to contact of stimulating substances with epidermal cells 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。 Accordingly, an object of the present invention is to clarify a stimulating substance that increases production of inflammatory substances in epidermal cells, and an inflammatory substance that increases by bringing the stimulating substance into contact with epidermal cells. And shows a correlation between inflammation on epidermal cells and inflammation, thereby establishing an evaluation of anti-inflammatory agents effective for inflammation caused by skin irritation.
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 the epidermal cells using a stimulating substance assumed to be due to skin irritation, measure the inflammatory substance whose production has been promoted, and add a lemongrass extract known to have an anti-inflammatory effect Thus, the suppression of production of inflammatory substances was confirmed, and a method for evaluating an anti-inflammatory agent effective for inflammation caused by skin irritation was found.
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 the stimulating substance, SPC, Substance P, and IFNγ were assumed and used. SPC (sphingosylphosphorylcholine) is an example of a generic name of DAMPs (damage-related molecular patterns) of molecules that are released to the outside by the destruction of their own cells and tissues and activate innate immunity. Patients with atopic dermatitis Concentrations are reported to be high in the skin.
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, is a neurotransmitter of primary sensory nerves, and is known mainly as a pain signal transmitter. SubstanceP is contained in peripheral nerves, released from nerve endings, and released SubstanceP causes vasodilation and leakage of plasma proteins, forms erythema and edema, promotes degranulation of mast cells, Histamine, leukotriene, etc. are liberated and it becomes a factor causing primary stimulation. SubstanceP 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 have an antiviral action and an action related to activation and regulation of the immune system. It is known that Th1 cells are produced when receiving fungal stimulation 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, IFNγ, and Substance P become epidermal cell stimulants and increase inflammatory substances, and that the epidermal cells are inflammatory substances such as IL-8, NO, and NGFβ by the stimulants. It has been found that enhanced production, IL-8 gene, NGFβ gene, and expression of the ResistanceP gene are promoted. Thus, a correlation between the stimulant and inflammation was confirmed, and a method for evaluating and selecting an anti-inflammatory agent effective for inflammation caused by skin irritation was found.
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 performed by the following steps (A) to (D).
(A) a step of adding a stimulating substance to the epidermal cells, and further adding an anti-inflammatory agent;
(B) a step of measuring an inflammatory substance expressed and produced in the epidermal cells,
(C) A step of comparing the expression level or production level of the inflammatory substance calculated in (B) between when the anti-inflammatory agent is added to the epidermal cells and when 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 (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。 In addition, the epidermal cell in this invention should just be a cell which comprises an epidermis, and an epidermis keratinocyte, a Langerhans cell, a melanocyte, etc. are mentioned. In particular, epidermal keratinocytes that constitute most of the epidermis outside 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。 In addition, the anti-inflammatory agent in the present invention is not a problem as long as it is a substance or composition that is widely applied to the skin, but preservatives, surfactants, pigmentation inhibitors, tyrosinase activity inhibitors, melanocyte melanin production inhibition Agents, moisturizers, cell activators / metabolic activators, antioxidants, active oxygen scavengers / radical production inhibitors, fat metabolism promoters, astringents, anti-inflammatory agents / interleukin production inhibitors / anti-inflammatory agents, anti-inflammatory agents Seborrheic agent, antibacterial / antiviral agent, blood flow promoter / vascular stimulant, antiandrogen, structural proteolytic enzyme (elastase, collagenase, keratin protease, serine protease, integrin degrading enzyme, involucrin degrading enzyme, filaggrin degrading enzyme , Laminin degrading enzyme, fibronectin degrading enzyme, proteoglycan degrading enzyme, etc.) activity inhibitor / structural tamper Pyrolytic enzyme expression inhibitor, structural protein synthesis promoter, mucopolysaccharide (hyaluronic acid or its salts, chondroitin sulfate, proteoglycan, etc.) degrading enzyme inhibitor, mucopolysaccharide synthesis promoter, intercellular adipogenesis promoter / between cells Active ingredients such as lipid condition improvers, keratolytic agents / stratum exfoliation accelerators, plasminogen activator antagonist inhibitors, Maillard reaction inhibitors, testosterone 5α reductase activity inhibitors, odorant elimination agents, and other pharmaceuticals Ingredients such as extracts and metabolites derived from plant-based raw materials, animal-based raw materials, microbial-based raw materials, and other natural product raw materials that are preferred for use in forming quasi-drugs or cosmetics, Compounds and the like.
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。 In addition, the stimulating substance used in the present invention is not particularly limited as long as it is a substance that exists in the body and is predicted to give a stimulus due to inflammation to epidermal cells. Examples include TNF-α, IFN-γ, SPC, SubstanceP, LPS, IL-1, TSLP, and the like. After the stimulating substance is added to the medium, it is preferably performed at room temperature for about 12 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 or preventive agent for inflammation associated with skin irritation, and it is estimated that skin irritation is involved. Psoriasis, UV-induced skin inflammation, 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 | blend the anti-inflammatory agent selected by the method of this invention as an active ingredient in a pharmaceutical, a quasi-drug, and cosmetics for the said objective.
（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 1-8, the cells used were commercially available normal human epidermal keratinocytes (Kurabo), the cell culture medium used EpiLife KG2 (Kurabo) medium as the preculture medium, and hEGF from the preculture medium as the test medium. The thing except hydrocortisone and BPE was used. Both pre-culture and main test culture were performed according to the manufacturer's instructions. In addition, lemongrass extract (Ichimaru Falcos) was used as an anti-inflammatory agent. Moreover, about the result of Tests 1-8, all set the control as 100 and showed the relative value.
（試験１）ＳＰＣ添加によるＩＬ−８遺伝子発現 (Test 1) IL-8 gene expression by adding 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 on culture plates and pre-cultured until 100% confluent. Thereafter, the medium was replaced with the test medium and cultured for 24 hours. The next day, the medium was replaced with a new medium for this test, and an anti-inflammatory agent was added to the culture solution. After culturing the cells for 24 hours, add 10 μM SPC to normal human epidermal keratinocytes, and after culturing for 4.5 hours, adjust the total RNA and measure the relative expression level of IL-8 mRNA by Real-time PCR method did. The final concentration of the anti-inflammatory 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 a result, 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 was confirmed.
（試験２）ＳＰＣ添加によるＩＬ−８産生 (Test 2) IL-8 production by adding 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 on culture plates and pre-cultured until 100% confluent. Thereafter, the medium was replaced with the test medium and cultured for 24 hours. The next day, the medium was replaced with a new medium for this test, and an anti-inflammatory agent was added to the culture solution. After culturing the cells for 24 hours, 10 μM SPC was added to normal human epidermal keratinocytes, and further cultured for 24 hours. After completion of the culture, the supernatant was collected and measured using the Human IL-8 ELISA Kit (Thermo # EH2IL8) in the supernatant. Cells after collecting the supernatant (cells for which 24 hours had passed after the addition of the starting material) were subjected to measurement of the amount of cell activity using Cellcounting kit-8 (DOJINDO). The final concentration of the anti-inflammatory 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, the results show that when SPC is added to normal human epidermal keratinocytes, the production of IL-8 is enhanced, and the production of IL-8 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 adding 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。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 on culture plates and pre-cultured until 100% confluent. Thereafter, the medium was replaced with the medium for this test and cultured for 24 hours. Thereafter, the medium was replaced again with the medium for this test, and an anti-inflammatory agent was added to the culture solution, followed by culturing for 24 hours. The next day, the medium was replaced with the medium for this test, and 10 μM SPC was added, followed by incubation for 20 minutes. After completion of the culture, the supernatant was collected, and its NO concentration was measured with a Nitrate / Nitrite Fluorometric Assay Kit (Cayman # 780051). The final concentration of the anti-inflammatory 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 a result, as shown in FIG. 5, when SPC was added to normal human epidermal keratinocytes, NO production was enhanced, and suppression of NO production by adding an anti-inflammatory agent was confirmed.
（試験４）ＳＰＣ添加によるＮＧＦβ遺伝子発現 (Test 4) NGFβ gene expression by adding 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 on culture plates and pre-cultured until 100% confluent. Thereafter, the medium was replaced with the medium for this test and cultured for 24 hours. The next day, the medium was replaced with a new medium for this test, and an anti-inflammatory agent was added to the culture solution. After culturing the cells for 24 hours, 10 μM SPC is added to normal human epidermal keratinocytes. After culturing for 4.5 hours, total RNA is prepared, and Real-time PCR is performed to determine the expression level of NGF-β gene. It was. At the same time, the amount of cell activity was measured using Cellcounting kit-8 (DOJINDO). The final concentration of the anti-inflammatory 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 a result, as shown in FIG. 6, when SPC was added to normal human epidermal keratinocytes, the expression of the NGFβ gene was enhanced, and it was confirmed that the expression of the NGFβ gene was suppressed by the addition of the anti-inflammatory agent.
（試験５）ＩＦＮγ、ＳｕｂｓｔａｎｃｅＰの併用によるＮＧＦβ遺伝子発現 (Test 5) NGFβ gene expression by combined use 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％ＣＯ２、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 on culture plates and pre-cultured until 100% confluent. Thereafter, the medium was replaced with the medium for this test and cultured for 24 hours. The next day, the medium was replaced with a new medium for this test, and an anti-inflammatory agent was added to the culture solution. After culturing the cells for 3 hours, the cells were 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 a result, 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 NGFβ gene expression 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 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％ＣＯ２、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 and cultured to 100% confluence, then replaced with the medium for this test, and cultured for 24 hours. The next day, the medium was replaced with a new medium for this test, and an anti-inflammatory agent was added to the culture solution. After culturing the cells for 3 hours, 10 μM SPC was added to normal human epidermal keratinocytes and allowed to stand for 10 minutes at room temperature. 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、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 a result, as shown in FIG. 8, when IFNγ and SPC were added to normal human epidermal keratinocytes, the gene expression of Substance P was enhanced, and suppression of gene expression of Substance P by the 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％ＣＯ２、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 on culture plates and pre-cultured until 100% confluent. Thereafter, the medium was replaced with the medium for this test and cultured for 24 hours. The next day, the medium was replaced with a new medium for this test, and an anti-inflammatory agent was added to the culture solution. After culturing the cells for 3 hours, 10 μM SPC was added to normal human epidermal keratinocytes and allowed to stand for 10 minutes at room temperature. Thereafter, 20 ng / mL of IFNγ was added to the wells and cultured for 48 hours under conditions of 5% CO 2 and 37 ° C. After the culture, the supernatant was collected and measured by Beta Nerve Growth Factor Human ELISA Kit (abcam # ab99986). Cells after recovering the supernatant (cells after 24 hours of addition of the causative agent) were measured for cell activity using Cell counting kit-8 (DOJINDO). As a result, the amount of NGFβ produced per cell is shown below. The final concentration of the anti-inflammatory agent is 0.1%.
（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 a result, as shown in FIG. 9, when IFNγ and SPC were added to normal human epidermal keratinocytes, production of NGFβ was enhanced, and suppression of NGFβ production by addition of an anti-inflammatory agent was confirmed.
（試験８）ＩＦＮγ、ＳｕｂｓｔａｎｃｅＰの併用によるＮＧＦβ産生 (Test 8) NGFβ production by combined use 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％ＣＯ２、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 on culture plates and pre-cultured until 100% confluent. Thereafter, the medium was replaced with the medium for this test and cultured for 24 hours. The next day, the medium was replaced with a new medium for this test, and an anti-inflammatory agent was added to the culture solution. After culturing the cells for 3 hours, substanceP100 μM was added to normal human epidermal keratinocytes and allowed to stand at room temperature for 10 minutes. Thereafter, 20 ng / mL of IFNγ was added to the wells, and cultured for 48 hours under conditions of 5% CO 2 and 37 ° C. After the culture, the supernatant was collected and measured by Beta Nerve Growth Factor Human ELISA Kit (abcam # ab99986). Cells after recovering the supernatant (cells after 24 hours of addition of the causative agent) were measured for cell activity using Cell counting kit-8 (DOJINDO). As a result, the amount of NGFβ produced per cell is shown below. The final concentration of the anti-inflammatory agent is 0.1%.
（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、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 a result, as shown in FIG. 10, when IFNγ and SubstanceP were added to normal human epidermal keratinocytes, the production of NGFβ was enhanced, and the suppression 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)
Contrasting creams with the following formula and creams with the same composition as the following formula without lemongrass extract for 12 adult men and women who are aware of sensitive skin and complain of itching due to atopic dermatitis or dryness. As a product, it was applied to the affected area of itching twice a day, and a one month use test was conducted. In addition, the test subject was divided into six persons, and different creams were applied.
（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 (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. Beeswax 1.5
6). Scrawan 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 Methylparaben adequate amount15. Propylparaben adequate amount 16. Phenoxyethanol 0.20
17. Purify water to 100
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: Skin itch or sensitive skin, atopic dermatitis, skin diseases such as dryness were slightly improved.
Invalid: 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。 From the test results, the cream containing lemongrass extract has an improvement effect on skin diseases such as itchy skin, sensitive skin, atopic dermatitis, and dryness. Test 1 was evaluated for inflammation caused by irritation to the skin. It is recognized that there is a correlation with the result of ˜8. Therefore, an anti-inflammatory agent effective for inflammation caused by skin irritation can be evaluated and selected by evaluating inflammation caused by stimulation of epidermal cells in tests 1 to 8.
Claims (2)
（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 evaluation or selection method according to claim 1 includes the following steps (A) to (D): (A) adding a stimulating substance to epidermal cells, and further adding an anti-inflammatory agent;
(B) a step of measuring an inflammatory substance expressed and produced in the epidermal cells,
(C) A step of comparing the expression level or production level of the inflammatory substance calculated in (B) between when the anti-inflammatory agent is added to the epidermal cells and when 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 (C).
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| JP2020075872A (en) * | 2018-11-05 | 2020-05-21 | 共栄化学工業株式会社 | Skin external preparation and screening method |
| KR20220046737A (en) * | 2020-10-07 | 2022-04-15 | 정읍시 | A composition for anti-inflammation activity comprising Chrysanthemum zawadskii leaf and stem |
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| JP2020075872A (en) * | 2018-11-05 | 2020-05-21 | 共栄化学工業株式会社 | Skin external preparation and screening method |
| KR20220046737A (en) * | 2020-10-07 | 2022-04-15 | 정읍시 | A composition for anti-inflammation activity comprising Chrysanthemum zawadskii leaf and stem |
| KR102465347B1 (en) | 2020-10-07 | 2022-11-14 | 정읍시 | A composition for anti-inflammation activity comprising Chrysanthemum zawadskii leaf and stem |
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