JP6929954B2 - モリンガ葉抽出物及び杜仲抽出複合物の抗菌、抗酸化、抗炎症、歯槽骨消失の抑制及び再生による歯肉炎及び歯周炎の改善方法 - Google Patents
モリンガ葉抽出物及び杜仲抽出複合物の抗菌、抗酸化、抗炎症、歯槽骨消失の抑制及び再生による歯肉炎及び歯周炎の改善方法 Download PDFInfo
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Description
本発明による組成物には、モリンガ抽出物及び杜仲抽出物を有効成分として含む。
S1)ザクロの皮と種を取り除いてザクロ果肉のみを収得する段階と、
S2)ザクロ果肉を100〜105℃で50〜80秒間殺菌した後、48〜55℃に冷却する段階と、
S3)冷却したザクロ果肉に48〜55℃ででん粉解酵素を処理する段階と、
S4)ザクロ果肉分解物を順次に70〜100℃及び400〜850mbarで加温加圧して2回以上加熱濃縮し、40〜80℃及び100〜350mbarで減温減圧して1回以上加熱濃縮する段階
選択的に、S1段階とS2段階との間、S3段階とS4段階との間、及びS4段階後のうちいずれか一つ以上でろ過段階をさらに含み得る。
本発明は、ザクロ皮と種を含まないザクロ果肉のみを用いた抽出物を提供する。ザクロの皮と種は副作用をもたらし得、例えば、ザクロ皮に含まれた特定のアルカロイド(alkaloid)は、身体機能を低下させる作用をし、呼吸器系と筋肉に影響を及ぼし、中毒されれば、発作、痙攣、昏睡状態などが起こり得る。また、ザクロ種の抽出物を服用すれば、一部の人にとってアレルギーである舌の腫脹(tongue swelling)などの副作用を起こし得る。
ザクロ果肉は、順次に殺菌後に冷却する段階を経る。殺菌は、100〜105℃で50〜80秒間、より望ましくは55〜70秒間迅速に行うことが望ましく、48〜55℃に冷却することが望ましい。
冷却したザクロ果肉にでん粉解酵素を処理する段階を経る。望ましくは、48〜55℃で10〜60分間施し、より望ましくは48〜55℃で20〜40分間施し得る。利用可能なでん粉解酵素は特に制限されず、当業界における公知の多様なでん粉解酵素を用いることができ、例えば、ペクチナーゼ(pectinase)、プロテイナーゼ(proteinase)、アミラーゼ、セルラーゼなどを用い得るが、望ましくはペクチナーゼを用い得る。
ザクロ果肉分解物を順次に加温加圧して加熱濃縮し、減温減圧して加熱濃縮する段階を経る。
本発明は、歯周疾患の予防、改善または治療に用いるための組成物を提供する。より具体的に、本発明は、モリンガ抽出物及び杜仲抽出物を有効成分として含む歯周疾患の予防、改善または治療方法に用いるための組成物を提供する。
本発明による組成物は、歯周疾患の改善、予防及び/または治療に効果を奏する。
[1.試験物質製造]
ザクロ、桔梗、梔子、黄ごん、モリンガ、蓮の葉、ハリグワの葉、生姜、芍薬、牛膝、杜仲、レッドクローバー、蒲公英及び蒲公英根の抽出物(即ち、14種の天然物素材)を次の製造工程によって製造した。各々の原料に対し、以下の抽出条件で各二つ以上のロットを製造して抽出収率、指標成分の含量、性状などを分析した。
ザクロ濃縮物は、次のような方法で製造した。
桔梗、梔子、黄ごん、モリンガ、蓮の葉、ハリグワの葉、生姜、芍薬、牛膝、杜仲、蒲公英及び蒲公英根の抽出物を、次の方法によって製造した。
前記1.1.の製造方法によって製造されたザクロ濃縮粉末、1.2.の製造方法によって製造された14種の試料のEPDモデルに対する歯肉炎及び歯周炎の改善効果を確認した。
以下では、ザクロ濃縮粉末及びモリンガ葉を含む13種(桔梗、梔子、黄ごん、モリンガ、蓮の葉、ハリグワの葉、生姜、芍薬、牛膝、杜仲、レッドクローバー、蒲公英及び蒲公英根)の抽出物各々の歯肉炎及び歯周炎の改善効果を評価した。
以下では、ザクロ濃縮粉末及びモリンガ葉を含む13種(桔梗、梔子、黄ごん、モリンガ、蓮の葉、ハリグワの葉、生姜、芍薬、牛膝、杜仲、レッドクローバー、蒲公英及び蒲公英根)の抽出物、各々の歯肉炎及び歯周炎の改善効果を評価した。
ザクロ濃縮粉末及びモリンガ葉を含む13種(桔梗、梔子、黄ごん、モリンガ、蓮の葉、ハリグワの葉、生姜、芍薬、牛膝、杜仲、レッドクローバー、蒲公英及び蒲公英根)の抽出物を40mg/mlの濃度で滅菌蒸留水に溶解し、5ml/kgの用量(200mg/kg)で歯牙境部結紮24時間後から毎日1回ずつ10日間各々経口投与し、INDは、1mg/mlの濃度で滅菌蒸留水に溶解し、5ml/kgの用量(5mg/kg)で歯牙境部結紮24時間後から毎日1回ずつ10日間経口投与した。正常媒体対照群及びEPD(歯周疾患) 対照群においては、天然物抽出物の代わりに媒体である滅菌蒸留水のみを同一用量及び頻度で経口投与した。
本研究者などの以前の方法[Lee et al.、2014; Park et al.、2016]に従って、実験動物を10日間実験室の環境に順化させた後、げっ歯類用吸入麻酔器(Surgivet、Waukesha、WI、USA)と換気装置(Model 687、Harvard Apparatus、Cambridge、UK)を用いて3%のイソフルラン(isoflurane、Hana Pharm.Co.、Hwasung、Korea)と70%のN2O及び28.5%のO2混合ガスで全身吸入麻酔し、左側切歯の歯牙境部に3−0ナイロン縫合糸を用いて結紮を施して歯周炎及び歯肉疾患を誘発した。一方、正常媒体対照群では、切歯の歯牙境部の部分のみを確認した後、結紮しなかった。
11日間EPDを施した歯肉組織を均質化した後、ELISA kit(IL−1β rat ELISA kit、Abcam、Cambridge、UK)を用いて測定した。試験方法は、製造社の方法にしたがって行い、均質化したサンプルと標準品100uLを各ウェル(well)に添加した後、37℃培養器で2時間培養した。培養後に上澄液を除去し、各ウェルに1Xbiotinで標識された抗体(antibody)を各100uLずつ入れた後、37℃培養器で1時間培養した。各ウェルの上澄液を除去して3回洗浄した後、100uLの1Xhorseradish peroxidase−conjugated avidinを入れた後、37℃で1時間培養した。各ウェルに、3,3’,5,5’−テトラメチルベンジジンを90uL入れた後、37℃で15〜30分間培養した。各ウェルに停止液(stop solution)を50uLずつ入れた後、マイクロプレートリーダー(microplate Reader、Tecan、Mannedorf、Switzerland)で450nmの波長で吸光度を測定して結果値を計算した。
歯槽骨消失量の測定は、Crawford et al.[1987]の方法によって測定し、サンプル投与10日後(EPD11日後)ラットを犠牲させた後、歯牙と歯ぐきを最大限に摘出した。EPDを施した二つの歯牙の間の歯ぐきの端部と、切歯(incisor)の端部、即ち、歯ぐきの外に露出した歯牙の最下端部とを平行にして面積を計算してmm/ratsで表した。
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.01)歯肉組織内IL−1β含量の増加が認められたが、IND、モリンガ葉、黄ごん、レッドクローバー、牛膝、梔子、杜仲、蒲公英根、ザクロ、蒲公英、生姜、及び桔梗抽出物の投与群順にEPD対照群に比べて有意味な(p<0.01)IL−1β含量の減少が各々認められた。一方、蓮の葉、ハリグワの葉及び芍薬抽出物投与群においては、EPD対照群に比べて有意味な歯肉組織内IL−1β含量の変化は認められなかった。
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.01)歯槽骨量の減少が認められたが、IND、レッドクローバー、杜仲、モリンガ葉、牛膝及び梔子抽出物の投与群順にEPD対照群に比べて有意味な(p<0.01またはp<0.05)歯槽骨量の増加が認められた。一方、ザクロ、桔梗、黄ごん、蓮の葉、ハリグワの葉、生姜、芍薬、蒲公英及び蒲公英根の投与群においては、EPD対照群に比べて有意味な歯槽骨量の変化は認められなかった。
現在、代表的な歯肉炎及び歯周炎改善モデルに用いられている歯牙境部結紮ラット歯周疾患(EPD)を用いて、9種のモリンガ葉抽出物(MF)及び杜仲抽出物(EC)の複合組成物(MF:EC=1:1、1:2、1:4、1:6、1:8、2:1、4:1、6:1及び8:1複合組成、mg:mg)を各々滅菌蒸留水に溶解し、5ml/kgの用量(200mg/kg)で毎日1回ずつ10日間強制で経口投与し、体重、歯槽骨損失率(alveolar bone loss index)、口腔組織内の好気性生菌水、歯肉内IL−1β及び腫瘍壊死因子(tumor necrosis factor;TNF)−αの含量、脂質過酸化(malondialdehyde;MDA含量)、誘導型一酸化窒素合成酵素(inducible nitric oxide synthase;iNOS)及びミエロペルオキシダーゼ(myeloperoxidase;MPO)活性の変化を組織病理学的変化とともに観察した。本実験では、以前の報告に準して[Kang et al.、2016]、各々のMF及びEC単独組成物と比較して、同時に統計学的に有意味な(p<0.01またはp<0.05)薬効上昇を示す複合組成物を、相乗効果を示すMF:EC複合組成物として認め、non−steroid anti−inflammatory drugs(NSAIDs系抗炎症剤であるインドメタシン(IND)5mg/kgを経口投与群と各々比較評価した。
適正量のMFまたはECを滅菌蒸留水に直接溶解し、5ml/kgの用量で歯牙境部結紮24時間後から、毎日1回ずつ10日間強制で経口投与した。即ち、MF及びEC単独組成物においては、各々40mg/mlの濃度で滅菌蒸留水に溶解し、5ml/kg(200mg/kg)の用量で経口投与し、MF:ECの1:1、1:2、1:4、1:6、1:8、2:1、4:1、6:1及び8:1複合組成物においては、5mlの蒸留水にMF及びEC各々を100:100、66:134、40:160、28:172、22:178、134:66、160:40、172:28及び178:22mg:mgずつ溶解し、5ml/kg(総量200mg/kg)の用量で各々経口投与した。INDも1mg/mlの濃度で滅菌蒸留水に溶解し、5ml/kgの用量(5mg/kg)で歯牙境部結紮24時間後から毎日1回ずつ10日間経口投与し、正常媒体対照群及びEPD対照群においては、同一の投与ストレスを加えるために、天然物抽出物またはINDの代わりに、媒体である滅菌蒸留水のみを同一用量及び頻度で経口投与した。
本研究者などの以前の方法[Lee et al.、2014; Park et al.、2016]に従って、実験動物を10日間実験室の環境に順化させた後、げっ歯類用吸入麻酔器(Surgivet、Waukesha、WI、USA)と換気装置(Model 687、Harvard Apparatus、Cambridge、UK)を用いて3%のイソフルラン(isoflurane、Hana Pharm.Co.、Hwasung、Korea)と70%のN2O及び28.5%のO2混合ガスで全身吸入麻酔し、左側切歯の歯牙境部に3−0ナイロン縫合糸を用いて結紮を施して歯周炎及び歯肉疾患を誘発した。一方、正常媒体対照群では、切歯の歯牙境部の部分のみを確認した後、結紮しなかった。
増体量を測定するために実験期間における体重変化を電子はかりによって測定した。歯牙結紮1日後、試料投与初日の体重を測定し、10日後、最終サンプル投与日の体重を測定して増体量を測定した。
[歯肉組織内のIL−1βとTNF−αの変化確認]
11日間EPDを施した歯肉組織を均質化した後、ELISA kit(IL−1β rat ELISA kit、Abcam、Cambridge、UK)を用いて測定した。試験方法は、製造社の方法にしたがって行い、均質化したサンプルと標準品100uLを各ウェルに添加した後、37℃培養器で2時間培養した。培養後に上澄液を除去し、各ウェルに1Xbiotinで標識された抗体(antibody)を各100uLずつ入れた後、37℃培養器で2時間培養した。培養後、上澄液を除去し、各ウェルに1Xbiotinで標識された抗体(antibody)を各100uLずつ入れた後、37℃培養器で1時間培養した。各ウェルの上澄液を除去して3回洗浄した後、100uLの1Xhorseradish peroxidase−conjugated avidinを入れた後、37℃で1時間培養した。各ウェルに、3,3’,5,5’−テトラメチルベンジジンを90uL入れた後、37℃で15〜30分間培養した。各ウェルに停止液(stop solution)を50uLずつ入れた後、マイクロプレートリーダー(microplate Reader、Tecan、Mannedorf、Switzerland)で450nmの波長で吸光度を測定して結果値を計算した。
脂質過酸化指標であるMDAを測定するために、歯牙の結紮部位と接触している歯肉組織を切り取った後、50mMのTris−HCl、0.1mMのEGTA、1mMのフッ化フェニルメチルスルホニル(pH7.4)バッファー(buffer)に入れてホモジナイザーによって均質化した。100ulの均質化した歯肉組織に、200uLの反応混合物(8.1%(w/v)のドデシル硫酸ナトリウム、1500ulの20%(v/v)の酢酸、1500ulの0.8%(w/v)のチオバルビツール酸、700ulのDW)を添加する。95℃で1時間反応させた後、3000Xgで10分間遠心分離した後、上澄液をUV/分光光度計を用いて650nmで測定する。
破骨細胞数の測定は、TRAP染色によってTRAP陽性多核細胞の数を数えて評価した。TRAP染色は、TRAP染色キット(Sigma−Aldrich、St.Louis、MO、USA)を用いてキットに記載の染色過程を守って施した。脱パラフィンと水和過程を経たスライドを固定液(クエン酸塩溶液25ml、アセトン65ml、37%のホルムアルデヒド8ml)に30秒間浸漬した後、流れる水に10分間水洗した。37℃の蒸留水45ml、ファストガーネットGBC溶液0.5ml、亜硝酸ナトリウム溶液0.5ml、ナフトールAS−BI 0.5ml、アセテート溶液2ml、酒石酸塩溶液1mlを混合した溶液をスライドガラスに滴下した後、平均16時間程度、37℃のヒューミッドチャンバーで反応させた。その後、100%のアルコールに水洗し、対照染色のために1時間の間メチルグリーンで染色して封入した。光学顕微鏡を用いて歯槽骨の表面に沿って形成されたTRAP陽性多核の破骨細胞(核3つ以上)の数を測定した。
歯槽骨消失量の測定は、Crawford et al.[1987]の方法によって測定し、サンプル投与10日後(EPD11日後)ラットを犠牲させた後、歯牙と歯ぐきを最大限に摘出する。EPDを施行した二つの歯牙の間の歯ぐきの端部と、切歯の端部、即ち、歯ぐきの外に露出した歯牙の最下端部とを平行にして面積を計算してmm/ratsで表した。
好気性生菌数の測定のために結紮した左側切歯を抜いた後、結紮部と接触していた歯周組織を切り取った後、0.3mlのブレインハートインフュージョン培地(BHI、Becton、Dicknson and company、USA)に盛る。組織を均質化した後、1:100、そして1:1000の割合で希釈して血液寒天培地(blood agar)に接種して37℃で48時間5%のCO2条件で培養した後、コロニー数を測定して生菌数を計算した。
生菌数=コロニー数×105CFU/g tissues
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.05)体重の減少が、結紮後7日(投与開始後6日)後から認められ始め、10日間の投与期間における増体量も、正常媒体対照群に比べて有意味な(p<0.01)減少を示した。一方、MF及びEC単独組成物、MF:ECが1:1及び8:1である複合組成物投与群においては、EPD対照群に比べて有意味な(p<0.01またはp<0.05)体重の増加が投与開始8日後から認められ、MF:ECが4:1及び6:1である複合組成物投与群においては投与開始6日後から、MF:ECが1:2、1:4及び2:1である複合組成物投与群においては投与開始7日後から、MF:ECが1:6及び1:8である複合組成物投与群においては投与開始9日及び10日後から、EPD対照群に比べて有意味な(p<0.01またはp<0.05)体重の増加が各々認められ、全てのMFとEC単独及び複合組成物投与群においては、EPD対照群に比べて有意味な(p<0.01)増体量の増加が各々認められた。
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.01)歯肉組織内IL−1β含量の増加が認められたが、EC単独組成物200mg/kg投与群を含む全ての実験物質投与群においてはEPD対照群に比べて有意味な(p<0.01)IL−1β含量の減少が各々認められ、特に、MF:ECが2:1及び4:1である複合組成物投与群においては、各々のMF及びEC単独組成物投与群に比べても有意味な(p<0.01またはp<0.05)歯肉組織内IL−1β含量の減少が同順に認められた。
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.01)歯肉組織内TNF−α含量の増加が認められたが、MF:EC 1:1の複合組成物200mg/kg投与群を含む全ての実験物質投与群においては、EPD 対照群に比べて有意味な(p<0.01)TNF−α含量の減少が各々認められ、特に、MF:ECが2:1及び4:1である複合組成物投与群においては、各々のMF及びEC単独組成物投与群に比べても有意味な(p<0.01またはp<0.05)歯肉組織内TNF−α含量の減少が同順に認められた。
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.01)歯肉組織内MDA含量の増加が認められたが、MF:ECが1:2である複合組成物200mg/kg投与群を含む全ての実験物質投与群においては、EPD対照群に比べて有意味な(p<0.01)MDA含量の減少が各々認められ、特に、MF:ECが2:1及び4:1である複合組成物投与群においては、各々のMF及びEC単独組成物投与群に比べても有意味な(p<0.01またはp<0.05)歯肉組織内MDA含量の減少が同順に認められた。
破骨細胞数は、EPD対照群においては、正常媒体対照群に比べて715.63%の変化を示したが、IND、MF及びEC単独組成物、MF:ECが、1:1、1:2、1:4、1:6、1:8、2:1、4:1、6:1及び8:1である複合組成物200mg/kg投与群においては、各々EPD対照群に比べて、−50.19、−34.48、−43.68、−36.40、−43.30、−41.00、−35.63、−34.87、−63.22、−57.09、−41.76及び−34.48%の変化を示した。
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.01)歯槽骨量の減少が認められたが、MF:ECが6:1である複合組成物200mg/kg投与群を含む全ての実験物質投与群においては、EPD対照群に比べて有意味な(p<0.01)歯槽骨量の増加が認められ、特に、MF:ECが2:1及び4:1である複合組成物投与群においては、各々のMF及びEC単独組成物投与群に比べても有意味な(p<0.01またはp<0.05)歯槽骨量の増加が同順に認められた。
EPD対照群の場合、正常媒体対照群に比べて有意味な(p<0.01)口腔内頬側溝部の総好気性生菌数の増加が認められたが、EPD対照群と類似な生菌数を示したIND 5mg/kg投与群を除いた全ての実験物質投与群においては、EPD対照群に比べて有意味な(p<0.01)口腔内生菌数の減少が各々認められ、特に、MF:ECが2:1及び4:1である複合組成物投与群においては、各々のMF及びEC単独組成物投与群に比べても有意味な(p<0.01またはp<0.05)口腔内頬側溝部の総好気性生菌数の減少が同順に認められた。
Claims (7)
- モリンガ抽出物及び杜仲抽出物を有効成分として含む歯周疾患の予防または改善用の組成物であって、
前記モリンガ抽出物と杜仲抽出物との重量比(モリンガ抽出物:杜仲抽出物)が、2:1〜4:1であり、
前記モリンガ抽出物が、1.2〜5mg/gのアストラガリンを含むモリンガ葉の水抽出物であり、前記杜仲抽出物が、1.0〜15mg/gのピノレシノールジグルコシドを含む杜仲皮の水抽出物であることを特徴とする、組成物。 - 前記組成物が、食品または薬学組成物であることを特徴とする請求項1に記載の組成物。
- 前記モリンガ抽出物と杜仲抽出物との重量比(モリンガ抽出物:杜仲抽出物)が、2:1または4:1であることを特徴とする請求項1または請求項2に記載の組成物。
- 前記歯周疾患が、歯肉炎または歯周炎であることを特徴とする請求項1または請求項2に記載の組成物。
- 前記組成物には、ザクロ、桔梗、梔子、黄ごん、蓮の葉、ハリグワの葉、生姜、芍薬、牛膝、レッドクローバー、蒲公英及び蒲公英根の抽出物からなる群より選択された一つ以上の抽出物が有効成分としてさらに含まれることを特徴とする請求項1または請求項2に記載の組成物。
- 前記ザクロ抽出物には、0.5〜3mg/gのエラグ酸が含まれたことを特徴とする請求項5に記載の組成物。
- 前記桔梗、梔子、黄ごん、蓮の葉、ハリグワの葉、生姜、芍薬、牛膝、レッドクローバー、蒲公英及び蒲公英根の抽出物の抽出溶媒が、水、炭素数1〜4の低級炭化水素またはこれらの混合物であることを特徴とする請求項5に記載の組成物。
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