JP6607676B2 - Trpv4活性化剤 - Google Patents
Trpv4活性化剤 Download PDFInfo
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- JP6607676B2 JP6607676B2 JP2015014057A JP2015014057A JP6607676B2 JP 6607676 B2 JP6607676 B2 JP 6607676B2 JP 2015014057 A JP2015014057 A JP 2015014057A JP 2015014057 A JP2015014057 A JP 2015014057A JP 6607676 B2 JP6607676 B2 JP 6607676B2
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- trpv4
- extract
- mangosteen
- hypotension
- activator
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Description
一方、マンゴスチン抽出物は、ストレスによる肌荒れの予防改善剤などに用いることができることが報告されている(特許文献3参照)。しかし、マンゴスチン抽出物がTRPV4の活性化作用を有することや、低血圧の予防又は改善及び自律神経の調整に有用であることは知られていない。
また本発明は、TRPV4活性化剤の効能を生かし、またその投与の手段としての、低血圧の予防又は改善剤及び自律神経調整剤の提供を課題とする。
また本発明は、マンゴスチン抽出物を有効成分とする、低血圧の予防又は改善剤に関する。
さらに本発明は、マンゴスチン抽出物を有効成分とする、自律神経調整剤に関する。
また本発明の低血圧の予防又は改善剤は、TRPV4チャネルを活性化して、低血圧を予防又は改善することができる。
さらに本発明の自律神経調整剤は、TRPV4チャネルを活性化して、自律神経を調整することができる。
前述のようにTRPV4の生理的役割の1つとして、TRPV4の活性化を介して交感神経活動の亢進が起こり、血圧が上昇することが知られている。したがって、TRPV4を活性化することにより、交感神経活動の過剰な低下を防止して自律神経乱れを改善することができ、ひいては低血圧も予防又は改善することができる。
また、本明細書における「自律神経の調整」とは、交感神経と副交感神経とのバランスを整えることを指す。具体的には、交感神経活動の過剰な低下を防止することを指す。
また、本明細書において「改善」とは、疾患、症状若しくは状態の好転、疾患、症状若しくは状態の悪化の防止若しくは遅延、又は疾患、症状若しくは状態の進行の逆転、防止若しくは遅延をいう。
さらに本明細書において「非治療的」とは、医療行為、すなわち治療による人体への処置行為を含まない概念である。
マンゴスチン抽出物の製造には、マンゴスチン任意の部分が使用可能であり、根、塊根、根茎、幹、枝、茎、葉(葉身、葉柄等)、樹皮、樹液、樹脂、花(花弁、子房等)、果実(成熟果実、未熟果実等)、果皮、種子等を用いることができる。また、これらの部位を複数組み合わせて用いてもよい。なかでも、本発明に用いるマンゴスチン抽出物は、マンゴスチンの果皮の抽出物であることが好ましい。
マンゴスチン抽出物の調製には、マンゴスチンをそのまま又は乾燥粉砕して用いることができる。また、マンゴスチンの水蒸気蒸留物又は圧搾物を用いることもでき、これらは精油等より精製したものを用いることもでき、また市販品を利用することもできる。マンゴスチン、又はその水蒸気蒸留物若しくは圧搾物は、いずれかを単独で、又は2種以上を組み合わせて使用してもよい。
本発明において抽出物とは、前記のような抽出方法で得られた各種溶剤抽出液、その希釈液、その濃縮液、その精製画分、その乾燥末又はその転溶液を含むものである。
その他の化粧料組成物に配合可能な成分としては、例えば、防腐剤(エチルパラベン、ブチルパラベン等)、消炎剤(例えば、グリチルリチン酸誘導体、グリチルレチン酸誘導体、サリチル酸誘導体、ヒノキチオール、酸化亜鉛、アラントイン等)、美白剤(例えば、アスコルビン酸及びその誘導体、胎盤抽出物、ユキノシタ抽出物、アルブチン等)、各種抽出物(例えば、オウバク、オウレン、シコン、シャクヤク、センブリ、バーチ、セージ、ビワ、ニンジン、アロエ、ゼニアオイ、アイリス、ブドウ、ヨクイニン、ヘチマ、ユリ、サフラン、センキュウ、ショウキュウ、オトギリソウ、オノニス、ニンニク、トウガラシ、チンピ、トウキ、海藻等)、賦活剤(例えば、ローヤルゼリー、感光素、コレステロール誘導体等)、血行促進剤(例えば、ノニル酸ワレニルアミド、ニコチン酸ベンジルエステル、ニコチン酸β−ブトキシエチルエステル、カプサイシン、ジンゲロン、カンタリスチンキ、イクタモール、タンニン酸、α−ボルネオール、ニコチン酸トコフェロール、イノシトールヘキサニコチネート、シクランデレート、シンナリジン、トラゾリン、アセチルコリン、ベラパミル、セファランチン、γ−オリザノール等)、抗脂漏剤(例えば、硫黄、チアントール等)、抗炎症剤(例えば、トラネキサム酸、チオタウリン、ヒポタウリン等)及び殺菌剤(例えば、トリクロサン、塩化セチルピリジニウム、チモール類、塩化ベンザルコニウム等)等が挙げられる。
例えば、本発明のTRPV4活性化剤、低血圧の予防又は改善剤、及び自律神経調整剤の総量中、前記有効成分の含有量は0.00001質量%以上が好ましく、0.0001質量%以上がより好ましく、2質量%以下が好ましく、0.2質量%以下がより好ましく、0.00001〜2質量%が好ましく、0.0001〜0.2質量%がより好ましい。
また、前記有効成分の投与又は摂取は、全身への投与又は摂取でもよいし、局所への投与又は摂取でもよい。
<3>TRPV4を活性化することで低血圧を予防若しくは改善、又は自律神経を調整する、前記<1>項に記載の低血圧の予防若しくは改善剤、又は自律神経調整剤。
<4>前記マンゴスチン抽出物がマンゴスチンの果皮の抽出物である、前記<1>〜<3>のいずれか1項に記載のTRPV4活性化剤、低血圧の予防若しくは改善剤、又は自律神経調整剤。
<5>前記マンゴスチン抽出物が、エタノール水溶液を抽出溶媒としてマンゴスチンを抽出して得られた、前記<1>〜<4>のいずれか1項に記載のTRPV4活性化剤、低血圧の予防若しくは改善剤、又は自律神経調整剤。
<6>前記TRPV4活性化剤、低血圧の予防若しくは改善剤、又は自律神経調整剤の総量中、前記有効成分の含有量が、0.00001質量%以上、好ましくは0.0001質量%以上、であり、2質量%以下、好ましくは0.2質量%以下、である、前記<1>〜<5>のいずれか1項に記載のTRPV4活性化剤、低血圧の予防若しくは改善剤、又は自律神経調整剤。
<8>TRPV4活性化剤、低血圧の予防若しくは改善剤、又は自律神経調整剤の製造のための、マンゴスチン抽出物の使用。
<9>マンゴスチン抽出物を、TRPV4活性化剤、低血圧の予防若しくは改善剤、又は自律神経調整剤として使用する方法。
<10>マンゴスチン抽出物を適用する、TRPV4活性化方法、低血圧の予防若しくは改善方法、又は自律神経の調整方法。
<11>前記TRPV4が、ヒト由来のTRPV4である、前記<7>〜<10>のいずれか1項に記載の使用又は方法。
<12>TRPV4を活性化することで低血圧を予防若しくは改善、又は自律神経を調整する、前記<7>〜<11>のいずれか1項に記載の使用又は方法。
<13>マンゴスチン抽出物を低血圧の予防若しくは改善、又は自律神経の調整を所望するヒトに適用する、前記<7>〜<12>のいずれか1項に記載の方法。
<14>低血圧又は自律神経の乱れが惹起された条件下で適用する、前記<7>〜<13>のいずれか1項に記載の方法。
<15>前記マンゴスチン抽出物がマンゴスチンの果皮の抽出物である、前記<7>〜<14>のいずれか1項に記載の使用又は方法。
<16>前記マンゴスチン抽出物が、エタノール水溶液を抽出溶媒としてマンゴスチンを抽出して得られた、前記<7>〜<15>のいずれか1項に記載の使用又は方法。
<17>前記TRPV4活性化剤、低血圧の予防若しくは改善剤、又は自律神経調整剤の総量中、マンゴスチン抽出物の含有量が、0.00001質量%以上、好ましくは0.0001質量%以上、であり、2質量%以下、好ましくは0.2質量%以下、である、前記<7>〜<16>のいずれか1項に記載の使用又は方法。
<19>TRPV4活性化薬、低血圧の予防若しくは改善薬、又は自律神経調整薬の製造のための、マンゴスチン抽出物の使用。
<20>TRPV4の活性化、低血圧の予防若しくは改善、又は自律神経の調整の非治療的な処置方法のために用いる、マンゴスチン抽出物の使用。
<21>前記TRPV4が、ヒト由来のTRPV4である、前記<18>〜<20>のいずれか1項に記載の抽出物又は使用。
<22>TRPV4を活性化することで低血圧を予防若しくは改善、又は自律神経を調整する、前記<18>〜<21>のいずれか1項に記載の抽出物又は使用。
<23>マンゴスチン抽出物を低血圧の予防若しくは改善、又は自律神経の調整を所望するヒトに適用する、前記<18>〜<22>のいずれか1項に記載の抽出物又は使用。
<24>マンゴスチン抽出物を低血圧又は自律神経の乱れが惹起された条件下で適用する、前記<18>〜<23>のいずれか1項に記載の抽出物又は使用。
<25>マンゴスチン抽出物を医薬組成物又は化粧料組成物の形態で適用する、前記<18>〜<24>のいずれか1項に記載の抽出物又は使用。
<26>マンゴスチン抽出物を食品、飲料、又は飼料の形態で適用する、前記<18>〜<24>のいずれか1項に記載の抽出物又は使用。
<27>前記マンゴスチン抽出物がマンゴスチンの果皮の抽出物である、前記<18>〜<26>のいずれか1項に記載の使用又は方法。
<28>前記マンゴスチン抽出物が、エタノール水溶液を抽出溶媒としてマンゴスチンを抽出して得られた、前記<18>〜<27>のいずれか1項に記載の使用又は方法。
<29>マンゴスチン抽出物の含有量が、0.00001質量%以上、好ましくは0.0001質量%以上、であり、2質量%以下、好ましくは0.2質量%以下、である、前記<18>〜<28>のいずれか1項に記載の使用。
<31>前記TRPV4が、ヒト由来のTRPV4である、前記<30>項に記載の方法。
<32>TRPV4を活性化することで低血圧を予防若しくは改善、又は自律神経を調整する、前記<30>又は<31>項に記載の方法。
<33>マンゴスチン抽出物を低血圧の予防若しくは改善、又は自律神経の調整を所望するヒトに適用する、前記<30>〜<32>のいずれか1項に記載の方法。
<34>低血圧又は自律神経の乱れが惹起された条件下で適用する、前記<30>〜<33>のいずれか1項に記載の方法。
<35>前記マンゴスチン抽出物がマンゴスチンの果皮の抽出物である、前記<30>〜<34>のいずれか1項に記載の方法。
<36>前記マンゴスチン抽出物が、エタノール水溶液を抽出溶媒としてマンゴスチンを抽出して得られた、前記<30>〜<35>のいずれか1項に記載の方法。
<37>マンゴスチン抽出物の有効量が、1日あたり、体重1kgあたり、0.1mg以上、好ましくは0.5mg以上、であり、20mg以下、好ましくは5mg以下、である、前記<30>〜<36>のいずれか1項に記載の方法。
ヒト十二指腸由来細胞株(Hutu-80細胞、American Type Culture Collectionより購入)から抽出したtotalRNAを逆転写して得られたcDNAを鋳型にして、公開されているヒトTRPV4遺伝子配列を参考に合成した、下記に示す塩基配列で表されるオリゴヌクレオチドからなるプライマーセットを用いて、下記の条件下でポリメラーゼ連鎖反応(PCR)を行った。
フォワードプライマー;5’-CACCATGGCGGATTCCAGCGAAGGCCC-3’(配列番号1)
リバースプライマー;5’-CTAGAGCGGGGCGTCATCAGTCC-3’(配列番号2)
a)PCR溶液組成
cDNA(Template) 15μL
5×PrimeStar GXL Buffer 10μL
dNTPs mixture(2.5mM) 4μL
PrimeStar GXL DNA Polymerase(タカラバイオ) 1μL
Forward Primer(10μM) 1μL
Reverse Primer(10μM) 1μL
Water 18μL
b)温度とサイクル条件
95℃ 2min
↓
98℃ 10sec 33cycles
70℃ 2min
10%牛胎児血清を含むDMEM/F12培地(インビトロジェン社製)を用いて、HEK293細胞(American Type Culture Collectionより購入)の培養を行った。HEK293細胞をT-25細胞培養用フラスコに2×105cells/Flaskで播種した。培養3日後、試験例1で作製したヒトTRPV4遺伝子発現ベクター(3μg)をTransIT-293(Mirus社製)を用いて細胞にトランスフェクションし1日培養した。これとは別に、対照としてLacZ発現ベクター(3μg)をTransIT-293(Mirus)を用いて細胞にトランスフェクションし1日培養した。Detachin(Genlantis社製)で細胞をはがし、96well Optical bottom plate(Nunc社製)に10%牛胎児血清を含むDMEM/F12培地で1.66×104cells/90μL/wellの細胞密度で播き、さらに1日培養した。
細胞内Ca2+流入活性の測定はCalcium Kit II-fluo 4(商品名、DOJINDO社製)を用いて行った。Fluo4-AMを含有したLoading bufferを試験例2で作製した細胞に90μL/well添加し、37℃で1時間インキュベートした。その後、37℃で蛍光プレートリーダーFDSS3000(商品名、浜松ホトニクス社製)を用いて蛍光強度(励起波長:488nm、蛍光波長:524nm)を2秒毎に測定した。測定開始30秒後にTRPV4作動薬であるGSK1016790A(Sigma社製、10nM(終濃度1nM))と、TRPV4特異的阻害薬であるHC067047(Sigma社製、100μM(終濃度10μM))、及び下記に示す検体溶液(終濃度の10倍濃度)を20μL/well添加し、300秒まで2秒毎に蛍光強度変化を測定した。
GSK1016790A(Sigma社製、TRPV4作動薬)及びHC067047(Sigma社製、TRPV4特異的阻害薬)をジメチルスルオキシド(DMSO、DOJINDO社製)で溶解し、使用した。
マンゴスチン抽出物:マンゴスチン(製品名:マンゴスチンα20、マンゴスチンの果皮のエタノール抽出物、日本新薬社製)を溶媒(エタノール特級、和光純薬工業社製)で溶解し、使用した。
比較例として用いたローズマリー(Rosmarinus officinalis)抽出物: RM-21Bベース(三菱化学フーズ社製)を99.5v/v%エタノール水で溶解したものを用いた。
比較例として用いたセイヨウネズ(Juniperus communis)抽出物:ジュニパーベリー(Juniper berry、新和物産より入手)40gに50%エタノール水溶液 400mlを加え、室温、静置条件下で15日間抽出を行い、その後ろ過することにより調製した。
比較例として用いたオレンジピール抽出物:オレンジピール(ビター)(栃本天海堂より入手)10gに50%エタノール水溶液 100mlを加え、室温、静置条件下で7日間抽出を行い、その後ろ過することにより得た。
TRPV4活性は測定開始後50〜200秒で評価した。
検体のTRPV4活性は、蛍光プレートリーダーFDSS3000から得られる蛍光強度(F)を検体添加前の蛍光強度(F0)で除算した強度比(F/F0)により算出した。
その結果を表1に示す。
Claims (3)
- マンゴスチン(Garcinia mangostana)抽出物を有効成分とする、TRPV4活性化剤。
- マンゴスチン(Garcinia mangostana)抽出物を有効成分とする、交感神経調整剤。
- マンゴスチン(Garcinia mangostana)抽出物を有効成分とする、交感神経調整用飲食品組成物。
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