JP7453783B2 - 成分含量が変化した緑茶抽出物を含む皮膚美白用又は皮膚しわ予防又は改善用組成物 - Google Patents
成分含量が変化した緑茶抽出物を含む皮膚美白用又は皮膚しわ予防又は改善用組成物 Download PDFInfo
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- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
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- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
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Description
他の具現例として、前記抽出物は、メラニン生成を抑制することができ、また他の具現例として、前記抽出物は、チロシナーゼ(tyrosinase)の活性を抑制することができる。すなわち、前記抽出物又は組成物は、メラニン生成を抑制し又はチロシナーゼの活性を抑制して皮膚美白効果を示すことができる。
100gの緑茶(Camellia sinensis、済州島のオソルロック農場)に50%エタノール1000mlを加え、60℃で1時間還流撹拌した。前記試料の温度を室温に下げ、ろ過して得た溶液を減圧蒸留して、濃い褐色粉末の一般告示型緑茶抽出物(GT-LE-35CAT、試料1)23gを収得した(収率23%)。
前記実施例1の試料1及び試料2の緑茶抽出物が老化したヒト皮膚線維芽細胞において皮膚組織分解酵素のMMP-1生成抑制に及ぼす影響、EGCGとGCGの重量比、及びGCGの含量によるMMP-1生成抑制効能を確認するために、MMP-1抑制活性評価を行った。具体的に、下記の段階にて行った。
ヒト真皮線維芽細胞(normal human dermal fibroblast、NHDF;Lonza、Switzerland)を10%ウシ血清(fetal bovin serum)を含むDMEM培地(Dulbecco’s modified Eagle’s Medium、Gibco 1210-0038)で培養し、培養はいずれも37℃、5% CO2培養器で行った。継代培養(sub culture)の回数が6~9回の細胞を若い細胞とし、継代培養の際に継代毎に細胞の数を数えて、全細胞数の1/5程度をさらに新たに培養する方式で37回の継代培養を行い、老化した真皮線維芽細胞を得た。老化した真皮線維芽細胞に対し、EGCG:GCGの割合、GCGの濃度に応じてEGCG、GCGを処理し、又は試料1又は試料2を処理した。
MMP-1の生成量を確認するために、ELISA(enzyme-linked immunosorbent assay)法を用いた(Amersham、USA)。前記1-1.段階で試料を処理した細胞の培養液を回収し、1000xgで遠心分離して上澄液を分離した後、培養液をMMP-1抗体がコートされた96-ウェルプレートに入れて反応させた後、二次抗体(horse reddish peroxidase、HRP)を常温で1時間反応させ、100μLのPBSで洗浄した。TMB substrate solution(Pierce、USA)を用い、反応後の490nm波長における吸光度を測定して発色程度を確認することでMMP-1の生成量を確認し、MMP-1抑制活性(%)は、下記の式にて求めた。
MMP-1抑制活性(%)=(試料添加群のO.D. at 490nm/試料無添加群のO.D. at 490nm)×100
Lonza社から購入のMelanocyte細胞株(皮膚細胞種)を96ウェルプレート(well plate、FALCON)にウェル当たり1×105ずつシードし(seeding)、37℃、5% CO2インキュベーターで24時間培養した後、前記実施例1の高温処理緑茶抽出物(試料2)を10ppm及び100ppm処理し、48時間さらに培養した。
細胞生存率(%)=(試料処理群吸光度-反応試薬だけの吸光度)/(無処理群吸光度-反応試薬だけの吸光度)×100
メラニン生成が過度のヒト黒色腫細胞のMNT-1細胞(human melanoma cell line、Lonza、SWISS)を6ウェルプレートに1×106cell/wellでシートし(seeding)、37℃、5% CO2条件下で24間培養した後、前記実施例1の試料1と試料2をそれぞれ10ppm及び100ppm処理し、37℃、5% CO2インキュベーターで48時間培養した。48時間培養された前記細胞に破砕液(Lysis Buffer;1% NP-40、50mM Tris-HCL;pH7.5、150mM NaCl)を500μl処理した後、セルスクレーター(Cell scraper)を用いてマイクロチューブ(microtube)に集めた後、13,000rpmで15分間遠心分離(Eppendorf、centrifuge 5415R、ドイツ)した。次いで、沈殿物を分離し、1N 水酸化ナトリウム50μlを処理して沈殿物を溶解させた後、メラニン色素に特異的な450nm波長における吸光度を測定し、下記の式にてメラニンの量を全タンパク質の量に対する値に補正した。このとき、対照群(control)としては、試料を処理していないMNT-1細胞を用いて、その結果を図4に示した。
メラニンの量(%)=吸光度/全タンパク質の量
前記実験例2に係る細胞溶解物10μgにDOPA(ジヒドロキシフェニルアラニン;dihydroxyphenylalanine)10mM(同量10μg)を添加し、チロシナーゼの作用によって生成されたドーパクロム(dopachrome)の生成量を、490nmにおける吸光度を比較して測定した。チロシナーゼ活性を非処理群の吸光度に対する比率にて求め、図5に示した。
前記実施例1に係る試料2を150mg準備し、ラクトース440mg、とうもろこし澱粉430mg及びステアリン酸マグネシウム2mgを混合して軟質カプセル充填液を製造した。そして、該充填液とは別途に、ゼラチン66重量部、グリセリン24重量部及びソルビトール液10重量部の割合で軟質カプセルシートを製造し、前記充填液を充填させて、軟質カプセルを製造した。
前記実施例1に係る試料2を150mg準備し、ビタミンE 15mg、ビタミンC 15mg、ガラクトオリゴ糖250mg、乳糖60mg及び麦芽糖140mgを混合してから、流動層乾燥幾を用いて顆粒化した後、糖エステル(sugar ester)8mgを添加した。この組成物を通常の方法にて打錠して、錠剤を製造した。
前記実施例1に係る試料2を80mg準備し、ビタミンE 9mg、ビタミンC 9mg、ブドウ糖10g、クエン酸0.6g、及び液状オリゴ糖25gを混合した後、精製水400mlを加えて瓶に充填した。瓶に充填した後、30℃で4~5秒間殺菌して、ドリンク剤を製造した。
前記実施例1に係る試料2を150mg準備し、ビタミンE 9mg、ビタミンC 9mg、無水結晶ブドウ糖250mg及び澱粉550mgを混合した後、流動層造粒幾を用いて顆粒に成形した後、包に充填して、顆粒剤を製造した。
前記実施例1に係る試料2を150mg準備し、ビタミン混合物(ビタミンAアセテート70μg、ビタミンE 1.0mg、ビタミンB1 0.13mg、ビタミンB2 0.15mg、ビタミンB6 0.5mg、ビタミンB12 0.2μg、ビタミンC 10mg、ビオチン10μg、ニコチン酸アミド1.7mg、葉酸50μg)と無気質混合物(硫酸第一鉄1.75mg、酸化亜鉛0.82mg、炭酸マグネシウム25.3mg、第一リン酸カリウム15mg、第二リン酸カルシウム55mg、クエン酸カリウム90mg、炭酸カルシウム100mg、塩化マグネシウム24.8mg)を組み合わせて、健康食品を製造した。
前記実施例1に係る試料2を50mg準備し、クエン酸1000mg、オリゴ糖100g、梅濃縮液2g、タウリン1g、残量して精製水を添加して、900mLの健康飲料を製造した。
前記実施例1に係る試料2を10mg準備し、マルトデキストリンを混合してから精製水15mlを加え、アンプル瓶に充填した後、殺菌して、液状アンプル剤を製造した。
Claims (15)
- 抽出物の総重量を基準に、4~15重量%の(-)-ガロカテキンガレート((-)-gallocatechin gallate、GCG)及び4~15重量%の(-)-エピガロカテキンガレート((-)-epigallocatechin gallate、EGCG)を含む緑茶抽出物を有効成分として含み、
前記抽出物中のEGCG、エピガロカテキン(epigallocatechin、EGC)、(-)エピカテキン((-)epicatechin、EC)、エピカテキン3-O-ガレート(epicatechin 3-O-gallate、ECG)、GCG、ガロカテキン(gallocatechin、GC)、カテキン(catechin、C)及びカテキンガレート(catechin gallate、CG)の総含量は、前記抽出物の総重量を基準に19~30重量%の範囲であり、
前記抽出物中のEGCG、EGC、EC及びECGの総含量は、前記抽出物の総重量を基準に20重量%以下である、
皮膚しわ予防又は改善用組成物。 - 前記抽出物中のGCG:EGCGの重量比は、1:0.5~2の範囲である、請求項1に記載の組成物。
- 前記抽出物は、水及びC1~C4のアルコールのいずれか一方以上によって1回以上抽出した抽出物である、請求項1または2に記載の組成物。
- 当該組成物中の前記抽出物の含量は、当該組成物の全重量に対し、1重量%~100重量%の範囲である、請求項1~3のいずれか一項に記載の組成物。
- 前記有効成分の投与量は、5mg/kg/日~1000mg/kg/日の範囲である、請求項1~4のいずれか一項に記載の組成物。
- 前記抽出物は、MMP-1(matrix metalloproteinase-1)の発現を抑制する、請求項1~5のいずれか一項に記載の組成物。
- 当該組成物は、食品、薬学又は化粧料組成物である、請求項1~6のいずれか一項に記載の組成物。
- 抽出物の総重量を基準に、4~15重量%の(-)-ガロカテキンガレート((-)-gallocatechin gallate、GCG)及び4~15重量%の(-)-エピガロカテキンガレート((-)-epigallocatechin gallate、EGCG)を含む緑茶抽出物を有効成分として含み、
前記抽出物中のEGCG、エピガロカテキン(epigallocatechin、EGC)、(-)エピカテキン((-)epicatechin、EC)、エピカテキン3-O-ガレート(epicatechin 3-O-gallate、ECG)、GCG、ガロカテキン(gallocatechin、GC)、カテキン(catechin、C)及びカテキンガレート(catechin gallate、CG)の総含量は、前記抽出物の総重量を基準に、19~30重量%の範囲であり、
前記抽出物中のEGCG、EGC、EC及びECGの総含量は、前記抽出物の総重量を基準に20重量%以下である、
皮膚美白用組成物。 - 前記抽出物中のGCG:EGCGの重量比は、1:0.5~2の範囲である、請求項8に記載の組成物。
- 前記抽出物は、水及びC1~C4のアルコールのいずれか一方以上によって1回以上抽出した抽出物である、請求項8または9に記載の組成物。
- 当該組成物中の前記抽出物の含量は、当該組成物の全重量に対し、1重量%~100重量%の範囲である、請求項8~10のいずれか一項に記載の組成物。
- 前記有効成分の投与量は、5mg/kg/日~1000mg/kg/日の範囲である、請求項8~11のいずれか一項に記載の組成物。
- 前記抽出物は、メラニン生成を抑制する、請求項8~12のいずれか一項に記載の組成物。
- 前記抽出物は、チロシナーゼ(tyrosinase)の活性を抑制する、請求項8~13のいずれか一項に記載の組成物。
- 当該組成物は、食品、薬学又は化粧料組成物である、請求項8~14のいずれか一項に記載の組成物。
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