JP2023517117A - 皮膚潰瘍の治療または改善のための竜眼肉含有混合生薬抽出物を含む局所用組成物およびその使用 - Google Patents
皮膚潰瘍の治療または改善のための竜眼肉含有混合生薬抽出物を含む局所用組成物およびその使用 Download PDFInfo
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Abstract
Description
好ましい脂溶性重合体は、ポリビニルピロリドン-エイコセン共重合体、ポリビニルピロリドン-ヘキサデセン共重合体、ニトロセルロース、デキストリン脂肪酸エステル、シリコーンポリマーなどを含んでもよい。
乾燥竜眼肉(Longanae Arillus)(Buyoung Yakup Co.Ltd.)20g、乾燥藁本(Ligustici Tenuissimi Rhizoma)(Buyoung Yakup Co.Ltd.)20gおよび乾燥オンジ(Polygalae radix)(Buyoung Yakup Co.Ltd.)20gを小片に切断した後、6倍体積(v/w)の20%エタノール水溶液と混合し、その混合物を90±5℃で3日間還流抽出した。抽出物をろ紙(孔径10μm未満)を通して濾過させ、残渣を除去した後、残りの残渣を4倍体積(v/w)の20%エタノール水溶液でさらに2回抽出し、抽出物をろ紙(孔径、10μm未満)を通して濾過した。
実施例1に開示されたものとは異なる混合比および異なる溶媒を用いたことを除いては、全ての手順が実施例1と同様であり、竜眼肉(LA)、藁本(LT)およびオンジ(PR)の様々な本発明の混合抽出物、すなわち、本発明の混合抽出物(2)ないし本発明の混合抽出物(6)を得た。これを以下の実験にて試験試料として使用する。
本発明の抽出物の抗炎症活性を決定するために、文献(Jeong et al.,2019,J.Invest.Dermatol.,May;139 (5):pp1098-1109)に記載された手順に従って、HaCaT細胞を用いた以下のサイトカイン発現抑制試験を行った。
本発明の抽出物の促進活性を決定するために、文献(Lee et al.,2020,Int J Mol Sci.2020 Jan 5;21(1):343)に記載された手順に従って、HaCaT細胞を用いた以下の細胞増殖試験を行った。
本発明の抽出物の回復活性を決定するために、文献(Na et al.,2016,J Invest Dermatol.2016 Apr;136(4):847-858)に記載された手順に従って、HaCaT細胞を用いた以下の細胞創傷試験を行った。
本発明の抽出物の慢性潰瘍に対する治療効果を確認するために、参考文献(Long M, Rojo de la Vega M, Wen Q, Bharara M, Jiang T, Zhang R, Zhou S, Wong PK, Wondrak GT, Zheng H, Zhang DD (2016) An Essential Role of NRF2 in Diabetic Wound Healing.Diabetes. 65:780-793)に開示された方法に従って、マウスを用いた動物モデル試験を行った。
8週齢のC57BL雄マウス(230g、Daehanbiolink Co.Ltd)を、温度および湿度が22±1℃および50±5%に維持され、12時間および夜間毎に光を調節するケージで飼育した。
慢性潰瘍に対する本発明の抽出物の治療効果を確認するために、上記4-1段階で調製した糖尿病誘発マウスを用いた動物モデル試験を参考文献(Long M, Rojo de la Vega M, Wen Q, Bharara M, Jiang T, Zhang R, Zhou S, Wong PK, Wondrak GT, Zheng H, Zhang DD (2016) An Essential Role of NRF2 in Diabetic Wound Healing. Diabetes.65:780-793)に記載の方法に従って行った。
皮膚創傷の治癒率=(N日/0日)×100
図3に示すように、陰性対照群がDIWで治療を受けた場合に比べて糖尿病誘導群の治癒率が減少したのに対し、試験試料群の治癒率は、糖尿病誘導群および陰性対照群に比べて大幅に増加した。
本発明の抽出物の皮膚創傷の回復効果を確認するために、上記4-1段階で調製した糖尿病誘発マウスを対象とした試験試料群と対照群との組織学的差異を、参考文献(Long M, Rojo de la Vega M, Wen Q, Bharara M, Jiang T, Zhang R, Zhou S, Wong PK, Wondrak GT, Zheng H, Zhang DD (2016) An Essential Role of NRF2 in Diabetic Wound Healing.Diabetes.65:780-793)に開示された方法に従って4-3段階の試験結果に基づいて行った。
8週齢のC57BL雄マウス(230g、Daehanbiolink Co.Ltd)を上記実験例4-1段階にて調製した(a)対照群と(b)糖尿病誘発マウス群に分類し、300μlのAvertin(25mg/mL T48402、Sigma-Aldrich、USA)を腹腔内注射してマウスを麻酔させた。
図4に示すように、興味深いことに、本発明の混合抽出物で処理した試験試料群では、対照群からは観察されなかった肉芽組織が観察された。肉芽組織は、創傷治癒の増殖中に形成された多数の新しい血管、線維芽細胞および成長因子などの細胞からなる(Grotendorst GR, Martin GR, Pencev D, Sodek J, Harvey AK (1985) Stimulation of granulation tissue formation by platelet-derived growth factor in normal and diabetic rats.The journal of clinical investigation.76:2323-2329.)。
成長因子に関与するプロ炎症性サイトカインの発現に本発明の抽出物が及ぼす抑制効果を確認するために、試験動物を用いたサイトカイン発現に対する以下の抑制試験を、文献(Long M, Rojo de la Vega M, Wen Q, Bharara M, Jiang T, Zhang R, Zhou S, Wong PK, Wondrak GT, Zheng H, Zhang DD (2016) An Essential Role of NRF2 in Diabetic Wound Healing.Diabetes.65:780-793.)に開示された手順に従って行った。
7日目に、外科用はさみ(PF-24.10、Professional、Parkistan)を用いて創傷の両側で3mmだけ間隔をおいて、マウスの創傷皮膚組織を得た。皮膚組織を液体窒素(Dongas,Korea)で凍結させ、全RNAを抽出した。トリRNA試薬(FATR001、Favorgen,Taiwan)を凍結皮膚組織に添加し、ビーズ(D1031-05、Bedbug、USA)を用いて破砕した。
相対的定量=(1/2)^(所望の遺伝子Ct-18S Ct)
7日目に外科用はさみ(PF-24.10,Professional,Parkistan)を用いて創傷の両側から3mmだけ間隔をおいてマウスの創傷皮膚組織を採取した。皮膚組織をPBS溶液に添加し、4℃の振とう機で一晩洗浄した。皮膚組織をRIPA緩衝液(自己製造、0.1%SDS、0.5%デオキシコール酸ナトリウム、1%Triton X-100、2mM EDTA、50mM Tris-HCl(pH8.0)、150mM NaCl)に入れ、氷冷で30分間インキュベートした。
平均および標準誤差は、実験で得られた試験結果から計算した。有意差検定はt検定を用いて分析し、有意水準(P-値)は、P≦0.05=*、P≦0.01=**、およびP≦0.001=***で表した。
以下、賦形剤の剤形化方法および種類について説明するが、本発明がこれに限定されるものではない。代表的な調製例は、以下の通り説明する。
実施例の抽出物(WIN-1001X) 1.00%
グリセロール 3.00%
エタノール 1.00%
プロピレングリコール 0.10%
香料 微量
蒸留水 100%以下
実施例の抽出物(WIN-1002X) 3.00%
L-アスコルビン酸-2-リン酸マグネシウム 1.00%
可溶性コラーゲン(1%溶液) 1.00%
クエン酸ナトリウム 0.10%
1,3-ブチレングリコール 3.00%
蒸留水 100%以下
実施例の抽出物(WIN-1003X) 3.00%
ポリエチレングリコモノステアレート 2.00%
モノステアリン酸グリセリン 1.00%
セチルアルコール 4.00%
スクアレン 6.00%
トリ2-グリセリルエチルヘキサノエート 6.00%
スフィンゴ-糖脂質 1.00%
1,3-ブチレングリコール 3.00%
蒸留水 100%以下
実施例の抽出物(WIN-1004X) 5.00%
ポリビニルアルコール 13.00%
L-アスコルビン酸-2-リン酸マグネシウム 1.00%
ラウロイルヒドロキシプロリン 1.00%
可溶性コラーゲン(1%溶液) 2.00%
1,3-ブチレングリコール 3.00%
エタノール 5.00%
蒸留水 100%以下
糖 20g
フルクトース 20g
レモン風味 最適量
蒸留水 100ml
実施例の抽出物(WIN-1005X) 2.00%
ヒドロキシエチレンセルロース(2%溶液) 12.00%
キサンタンガム(2%溶液)2.00%
1,3-ブチレングリコール3.00%
グリセリン濃度 4.00%
ヒアルロン酸ナトリウム 5.00%
蒸留水 100ml
Claims (10)
- 皮膚潰瘍を治療および改善する竜眼肉、藁本およびオンジの混合生薬抽出物を有効成分として含む、局所用医薬組成物。
- 前記混合生薬抽出物は、(a)0.01-100:0.01-100:0.01-100重量部(w/w)の範囲の竜眼肉、藁本およびオンジの乾燥重量(w/w)を基準とした混合比を有する竜眼肉、藁本およびオンジの混合生薬抽出物、または(b)0.01-100:0.01-100:0.01-100重量部(w/w)の範囲の竜眼肉、藁本およびオンジの乾燥重量(w/w)を基準とした混合比を有する竜眼肉、藁本およびオンジの各抽出物の混合物であることを特徴とする、請求項1に記載の局所用医薬組成物。
- 前記抽出物は、水、C1-C4低級アルキルアルコール、例えば、メタノール、エタノール、プロパノール、ブタノール、など、アセトン、酢酸エチル、クロロホルム、ヘキサン、ブチレングリコール、プロピレングリコールまたはグリセリンから選択される1つ以上の溶媒で抽出されることを特徴とする、請求項1に記載の局所用医薬組成物。
- 前記皮膚潰瘍が、褥瘡または糖尿病性潰瘍から選択されることを特徴とする、請求項1に記載の局所用医薬組成物。
- 竜眼肉、藁本およびオンジの混合生薬抽出物およびその薬学的に許容される担体の有効量を前記哺乳動物に局所的に投与する段階を含む、哺乳動物の皮膚潰瘍を治療または改善する方法。
- 有効成分であって、ヒトを含む哺乳動物の皮膚潰瘍を治療または改善するために使用される局所用製剤を調製するための竜眼肉、藁本およびオンジの混合生薬抽出物の使用。
- 皮膚潰瘍を治療および改善するのに有効な量で竜眼肉、藁本およびオンジの混合生薬抽出物を有効成分として含む、化粧料組成物。
- 前記抽出物は、水、C1-C4低級アルキルアルコール、例えば、メタノール、エタノール、プロパノール、ブタノール、など、アセトン、酢酸エチル、クロロホルム、ヘキサン、ブチレングリコール、プロピレングリコールまたはグリセリンから選択される1つ以上の溶媒で抽出されることを特徴とする、請求項7に記載の化粧料組成物。
- 前記皮膚潰瘍は、褥瘡または糖尿病性潰瘍から選択されることを特徴とする、請求項7に記載の化粧料組成物。
- 前記組成物は、化粧水、スキンソフナー、スキントナー、収斂剤、ローション、ミルクローション、モイスチャーローション、栄養ローション、マッサージクリーム、栄養クリーム、水分クリーム、ハンドクリーム、ファンデーション、エッセンス、栄養エッセンス、パック、クレンジングフォーム、クレンジングローション、クレンジングクリーム、ボディローション、ボディクレンザー、トリートメント、美容液などから選択された形態であることを特徴とする、請求項7に記載の化粧料組成物。
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