JP7463429B2 - 口腔用組成物 - Google Patents
口腔用組成物 Download PDFInfo
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- JP7463429B2 JP7463429B2 JP2022072979A JP2022072979A JP7463429B2 JP 7463429 B2 JP7463429 B2 JP 7463429B2 JP 2022072979 A JP2022072979 A JP 2022072979A JP 2022072979 A JP2022072979 A JP 2022072979A JP 7463429 B2 JP7463429 B2 JP 7463429B2
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Description
項1.
(A)ヒアルロン酸、ヒアルロン酸の塩、クマザサ抽出物、及びゼニアオイ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、歯周組織再生促進及び/又は修復促進用組成物。
項2.
(A)ヒアルロン酸、ヒアルロン酸の塩、クマザサ抽出物、及びゼニアオイ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、歯肉細胞活性化及び/又は修復促進用組成物。
項3.
(A)ヒアルロン酸、ヒアルロン酸の塩、クマザサ抽出物、及びゼニアオイ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、歯槽膿漏予防及び/又は治療用組成物。
項4.
(A)ヒアルロン酸、ヒアルロン酸の塩、クマザサ抽出物、及びゼニアオイ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、口腔における抗炎症用組成物。
項5.
(A)ヒアルロン酸、ヒアルロン酸の塩、クマザサ抽出物、及びゼニアオイ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、口腔におけるバリア機能向上用組成物。
項6.
(A)ヒアルロン酸、ヒアルロン酸の塩、クマザサ抽出物、及びゼニアオイ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、口腔における抗菌活性促進用組成物。
項7.
前記(A)成分が、ヒアルロン酸及び/又はその塩である、項1~6のいずれか1項に記載の組成物。
本発明の歯周組織再生及び/又は修復促進用組成物、歯肉細胞活性化及び/又は修復促進用組成物、歯槽膿漏予防及び/又は治療用組成物並びに、口腔における抗炎症用組成物、バリア機能向上用組成物、及び抗菌活性促進用組成物は、
(A)ヒアルロン酸、ヒアルロン酸の塩、クマザサ抽出物、及びゼニアオイ抽出物からなる群より選択される少なくとも1種以上を含有する。
(A)成分のうち、ヒアルロン酸としては、公知の物質を用いることができ、ヒアルロン酸、その誘導体、及びそれらの塩が挙げられる。ヒアルロン酸は、酸性ムコ多糖類の一種であり、グルクロン酸とN-アセチルグルコサミンの二糖を構成単位として含む多糖類である。ヒアルロン酸は、鶏冠や、サメの皮、臍帯、眼球、皮膚、及び軟骨などの動物組織、ストレプトコッカス属微生物等のヒアルロン酸生産微生物、動物細胞若しくは植物細胞の培養物から抽出、回収することができる。また、市販品を購入することもできる。
本明細書で、歯周病又は歯周病変とは、歯肉(いわゆる、歯茎)、セメント質、歯根膜及び歯槽骨よりなる歯周組織の健全な状態が損なわれた状態を指し、細菌の感染によって引き起こされうる。
[適用部位]
歯周病は口腔内の歯の一部又は全般的に起こりうることが知られているが、本発明の組成物は、口腔内の局所又は全体のいずれにも用いることができる。
本発明の組成物が適用される対象者の年齢層は、特に制限されないが、歯槽膿漏を有する、又は有するおそれがある者が加齢とともに増加するという観点から、好ましくは成人、より好ましくは30歳以上、さらに好ましくは中年齢者(45歳以上)から高年齢者(55歳以上)である。
本発明の組成物のpHは、(A)成分の種類、他の配合成分の種類及び含有量、製剤形態、使用方法等に応じて適宜設定され、生理学的又は薬学的に許容できる範囲であれば制限されないが、使用感を良好とする観点から、好ましくはpH3~9、より好ましくは4~8.5、更により好ましくは4.5~7.5であり、また一方で歯の溶解(酸蝕)のリスクを減らす観点からは、pH5.5~6.0とすることが好ましい。
本発明の組成物の剤形は医薬品、医薬部外品、化粧品として公知の形態であれば、特に限定されず、例えば液剤、フォーム(泡)剤、クリーム剤、ペースト剤、スプレー剤、パスタ剤、ジェル剤、フィルム剤、錠剤(トローチなど)などが挙げられるが、少量の組成物を長時間滞留させて、歯周組織や歯周病の原因菌に作用させる観点から、好ましくは液剤、フォーム剤、クリーム剤、ペースト剤又はスプレー剤であり、より好ましくは液剤、フォーム剤、クリーム剤又はペースト剤である。
ヒト口腔ケラチノサイト(上皮細胞)は、Human Oral Keratinocyte:HOK(クラボウ社製、ロットNo.12685)を用いた。ヒト口腔ケラチノサイトの培養は、特に記載がない限りにおいて、培地はOral keratinocyte medium(Sciencell, No.2611)を使用し、37℃、5%炭酸ガス及び95%空気の環境下で行った。
24well plate(Cell Bind、Corning社製)にヒト口腔ケラチノサイト(上皮細胞)を細胞数32000cells/wellで播種した。24時間培養後、表1に示す各成分をそれぞれの濃度にて溶解させた培地に交換し培養した。
翌日、大腸菌由来リポ多糖(Lipopolysaccharides from Escherichia coli:LPS、Sigma社製)と表1に示す各成分をそれぞれの濃度にて溶解させた培地に交換し、さらに6時間培養した。また、バックグラウンドの遺伝子発現量を算出するために、LPS及び各成分を添加しない培地で処理した群も設けた。培養後、PBS(-)で2回洗浄し、RNeasy(登録商標) Mini Kit(Qiagen社製)を用いてRNAを抽出したのち、TOYOBO ReverTra Ace qPCR RT Master Mix with gDNA Remover(TOYOBO社製)を用いてcDNAを作製した。作製したcDNAをPremix Ex Taq を用いてqRT-PCRにより解析した。なお、Taqman ProbeはApplied Biosystems社より購入した、GAPDH(Hs02758991_g1)、IL-8(Hs00174103_m1)、IL-1A(Hs00174092_m1)を用いた。
<発現低減率(%)>
=(1-((各実施例における遺伝子発現量-比較例1‐1における遺伝子発現量)/
(比較例1‐2における遺伝子発現量-比較例1‐1における遺伝子発現量)))×100
96well plate(Cell Bind, Corning社製)にヒト口腔ケラチノサイトを細胞数5600cells/wellで播種した。24時間培養後、表2に示す各成分をそれぞれの濃度にて溶解させた培地に交換し培養した。
翌日、LPS100μgと表2に示す各成分をそれぞれの濃度にて溶解させた培地に交換し、さらに一日培養した。培養後、培地上清(ELISA用サンプル)を回収し、Human IL-1 alpha/IL-1F1 DuoSet ELISA(R&D Systems社製)にてIL-1αの発現量を測定した。また、上清を除去した培養プレートをPBSで2回洗浄し、Hoechst 33342を培地に1000倍希釈し、well内培地と置換した。10分培養後、Image Xpress((Molecular Devices社)で画像撮影した(16視野/well)。機器の専用の細胞カウントプログラム(MetaXpress)で解析し、細胞数を測定した。ELISAで得られる各群のIL-1αの発現量を、Hoechst染色によって得られた細胞数の値で割って、細胞数あたりのIL-1αタンパク質発現量を求めた。そして、LPSや各成分を添加しない培地のみで処理したとき(比較例2-1)の細胞当たりIL-1αタンパク質発現量を1とし、LPSや各成分を添加した場合の相対タンパク質発現量を算出した。
<IL-1α発現低減率(%)>
=(1-((各実施例におけるタンパク質発現量-比較例2-1におけるタンパク質発現量)/(比較例2‐2におけるタンパク質発現量-比較例2‐1におけるタンパク質発現量)))×100
24well plate(Cell Bind、Corning社製)にヒト口腔ケラチノサイトを細胞数32000cells/wellで播種した。24時間培養後、表3に示す各成分をそれぞれの濃度にて溶解させた培地に交換し、6時間培養した。培養後、PBS(-)で2回洗浄し、RNeasy Mini Kit(Qiagen社製)を用いてRNAを抽出したのち、TOYOBO ReverTra Ace qPCR RT Master Mix with gDNA Remover(TOYOBO社製)を用いてcDNAを作製した。作製したcDNAをPremix Ex Taq(タカラバイオ社製)を用いてqRT-PCRにより解析した。なお、Taqman ProbeはApplied Biosystems社より購入した、GAPDH(Hs02758991_g1)、Defensin-1(Hs00608345_m1)を用いた。
<β‐Defensin-1発現促進率(%)>
=((各実施例における遺伝子発現量/比較例3-1における遺伝子発現量)-1)×100
24well plate(Cell Bind、Corning社製)にヒト口腔ケラチノサイトを細胞数32000cells/wellで播種した。24時間培養後、表4に示す各成分をそれぞれの濃度にて溶解させた培地に交換し、6時間培養した。培養後、PBS(-)で2回洗浄し、RNeasy Mini Kit(Qiagen社製)を用いてRNAを抽出したのち、TOYOBO ReverTra Ace qPCR RT Master Mix with gDNA Remover(TOYOBO社製)を用いてcDNAを作製した。作製したcDNAをPremix Ex Taq(タカラバイオ社製)を用いてqRT-PCRにより解析した。なお、Taqman ProbeはApplied Biosystemより購入した、GAPDH(Hs02758991_g1)、Claudin-1(Hs00221623_m1)を用いた。
<Claudin‐1発現促進率(%)>
=((各実施例における遺伝子発現量/比較例4-1における遺伝子発現量)-1)×100
12well plate(12mm Transwell(登録商標) with 0.4μm Pore Polyester Membrane Insert, Sterile、Corning社製)のインサートにヒト口腔ケラチノサイトを細胞数96000cells/wellで播種した。3日培養後、TNFα及びヒアルロン酸ナトリウムを表5の濃度で溶解させた分化誘導培地(2mM Ca2+添加Oral Keratinocyte medium)に交換し、5日間培養した。その後、インサートの電気抵抗値(TER値)をMillicell(登録商標)ERS-2 Voltohmmeter(Millipore社製)により測定した。
<バリア機能改善率(%)>
=((実施例5‐1におけるTER値-比較例5‐1におけるTER値)/(コントロールにおけるTER値-比較例5‐1におけるTER値))×100
96well plate(Cell Bind, Corning社)にヒト歯根膜線維芽細胞を細胞数3000cells/wellとなるよう播種した。6時間培養後、ウシ胎仔血清(FBS)(MP Biomedicals、ロットNo.A15035)濃度を10%から0.2%に置き変えて一晩培養した。細胞播種から24時間後にヒアルロン酸ナトリウム、クマザサ抽出物、ゼニアオイ抽出物を表6に示す濃度にて溶解させた0.2%FBS含有培地に交換し、48時間から72時間培養した。培養後、血清を含有しない培地で1回洗浄し、Cell Counting Kit-8(CCK-8、Dojindo)により細胞数測定を行った。
<CCK-8による細胞増殖促進率(%)>
=((各実施例における細胞数/比較例6-1における細胞数)-1)×100
24well plate(Cell Bind、Corning社製)にヒト口腔ケラチノサイトを細胞数32000cells/wellで播種した。24時間培養後、表7-aおよびbに示す各成分をそれぞれの濃度にて溶解させた培地に交換し、6時間培養した。培養後、PBS(-)で2回洗浄し、RNeasay Mini Kit(Qiagen社製)を用いてRNAを抽出したのち、TOYOBO ReverTra Ace qPCR RT Master Mix with gDNA Remover(TOYOBO社製)を用いてcDNAを作製した。作製したcDNAをPremix Ex Taq(タカラバイオ社製)を用いてqRT-PCRにより解析した。なお、Taqman ProbeはApplied Biosystemより購入した、GAPDH(Hs02758991_g1)、HAS1(Hs00758053_m1)、HAS3(Hs00193435_m1)を用いた。
<発現促進率(%)>
=((各実施例における遺伝子発現量/比較例7-1又は8-1における遺伝子発現量)-1)×100
アラントイン 0.1質量%
セチルピリジニウム塩化物水和物 0.02質量%
リン酸水素ナトリウム水和物 0.04質量%
グリチルリチン酸二カリウム 0.1質量%
2-アルキル-N-カルボキシメチル-
N-ヒドロキシエチルイミダゾリニウムベタイン 0.5質量%
ポリオキシエチレン硬化ヒマシ油 1質量%
ヒアルロン酸ナトリウム 0.001質量%
プロピレングリコール 10質量%
クエン酸 適量
フェノキシエタノール 0.2質量%
エデト酸2ナトリウム 0.01質量%
香料 0.1質量%
水 残部
合計 100質量%
塩化ベンゼトニウム 0.01質量%
ポリオキシエチレン硬化ヒマシ油 2質量%
濃グリセリン 5質量%
エタノール 10質量%
キシリトール 0.5質量%
クマザサエキス(固形分換算として) 0.001質量%
クエン酸 適量
パラベン 0.1質量%
エデト酸2ナトリウム 0.01質量%
メントール 0.01質量%
香料 0.02質量%
水 残部
合計 100質量%
酢酸トコフェロール 0.08質量%
アラントイン 0.05質量%
セチルピリジニウム塩化物水和物 0.04質量%
グリチルリチン酸二カリウム 0.2質量%
流動パラフィン 12質量%
モノステアリン酸ソルビタン 1質量%
ポリソルベート60 2質量%
ヒアルロン酸ナトリウム 0.001質量%
クマザサ抽出物(固形分換算として) 0.00001質量%
パルミチン酸デキストリン 0.3質量%
カルボキシビニルポリマー 1.1質量%
セタノール 5質量%
ラウリル硫酸ナトリウム 2質量%
1,3-ブチレングリコール 5質量%
アルギニン 適量
エデト酸2ナトリウム 0.02質量%
パラベン 0.05質量%
香料 0.2質量%
水 残部
合計 100質量%
マクロゴール400 1質量%
ベンザルコニウム塩化物 0.01質量%
ラウリル硫酸ナトリウム 2質量%
ピロリン酸カルシウム 40質量%
濃グリセリン 15質量%
ゼニアオイ抽出物 (固形分換算として) 0.001質量%
アルギン酸ナトリウム 0.2質量%
カラギーナン 1質量%
クエン酸 適量
フェノキシエタノール 0.3質量%
香料 0.02質量%
水 残部
合計 100質量%
Claims (5)
- クマザサ抽出物を含有することを特徴とする、歯根膜線維芽細胞の増殖促進用組成物。
- ゼニアオイ抽出物を含有することを特徴とする、口腔ケラチノサイトの抗炎症用組成物。
- ゼニアオイ抽出物及びクマザサ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、口腔ケラチノサイトのバリア機能向上用組成物。
- ゼニアオイ抽出物を含有することを特徴とする、口腔ケラチノサイトの抗菌活性促進用組成物。
- ゼニアオイ抽出物及びクマザサ抽出物からなる群より選択される少なくとも1種以上を含有することを特徴とする、口腔ケラチノサイトのヒアルロン酸産生促進用組成物。
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