JP5814062B2 - Lipid droplet / cellulite suppression device used in combination with vitamin C agent administration to target body - Google Patents
Lipid droplet / cellulite suppression device used in combination with vitamin C agent administration to target body Download PDFInfo
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Description
本発明は高周波温熱による細胞死の最小限抑制、好ましくは97%以上の細胞生存率をもたらす安全性の高いセルライト/脂質代謝の改善を図ることができる脂肪滴・セルライト抑制装置およびに脂質抑制効果増強薬、セルライト予防治療・脂肪滴抑制・脂質代謝改善用の温熱療法向けプロビタミンC製剤に関する。 The present invention provides a lipid droplet / cellulite suppressing apparatus capable of improving cellulite / lipid metabolism with high safety that can minimize cell death due to high-frequency heat, preferably a cell survival rate of 97% or more, and a lipid suppressing effect. The present invention relates to a provitamin C preparation for hyperthermia for potentiating drugs, cellulite prevention treatment, lipid droplet suppression and lipid metabolism improvement.
従来、脂肪滴に係わる組織や細胞の好ましくない発現とその影響を抑制することのできる新しい組成物とその薬剤に関するものが知られている。 2. Description of the Related Art Conventionally, a new composition capable of suppressing undesired expression of tissues and cells related to lipid droplets and its effect and a drug for the same have been known.
従来、脂肪滴形成抑制剤として、水酸化フラーレン類および有機化合物により修飾もしくは包接されたフラーレン複合体のうち1種以上を有効成分として含有していることを特徴とする脂肪滴形成抑制剤(請求項1)の発明がある(特許文献1:特開2008−222645)。
同文献1には、請求項2以下に次の通り記載されている。水酸化フラーレン類は、水酸基を1分子当り30以上40以下で結合しているフラーレンまたはその誘導体であることを特徴とする請求項1に記載の脂肪滴形成抑制剤(請求項2)。フラーレン複合体は、フラーレンおよびフラーレン誘導体のうちの1種以上と、有機オリゴマー、有機ポリマー、シクロデキストリンおよびクラウンエーテルとその類縁化合物のうちの1種以上の有機化合物との複合体であることを特徴とする請求項1に記載の脂肪滴形成抑制剤(請求項3)。γ−シクロデキストリンにより1.5〜2.0mol/mol比で包接されているフラーレン複合体であることを特徴とする請求項3に記載の脂肪滴形成抑制剤(請求項4)。請求項1から4のうちのいずれかの抑制剤からなることを特徴とする脂肪滴由来の細胞傷害の防御剤(請求項5)。請求項1から4のうちのいずれかの抑制剤からなることを特徴とする脂肪細胞分化抑制剤(請求項6)。
Conventionally, as a lipid droplet formation inhibitor, a lipid droplet formation inhibitor comprising one or more fullerene complexes modified or clathrated with a fullerene hydroxide and an organic compound as an active ingredient ( There is an invention of claim 1 (Patent Document 1: Japanese Patent Laid-Open No. 2008-222645).
The same document 1 describes the following in claims 2 and below. 2. The lipid droplet formation inhibitor according to claim 1, wherein the hydroxylated fullerene is a fullerene having a hydroxyl group bonded at 30 to 40 per molecule or a derivative thereof (claim 2). The fullerene complex is a complex of one or more of fullerenes and fullerene derivatives and one or more organic compounds of organic oligomers, organic polymers, cyclodextrins and crown ethers and related compounds. The lipid droplet formation inhibitor according to claim 1 (claim 3). 4. The lipid droplet formation inhibitor according to claim 3, which is a fullerene complex included in a ratio of 1.5 to 2.0 mol / mol with γ-cyclodextrin (claim 4). A protective agent for cytotoxicity derived from lipid droplets, comprising the inhibitor according to any one of claims 1 to 4 (claim 5). An adipocyte differentiation inhibitor comprising the inhibitor according to any one of claims 1 to 4 (claim 6).
上記特許文献1は、従来のレチノイドに代わり得る高活性の脂肪滴形成の抑制が実現され、これによって、脂肪滴由来の細胞傷害の発生が効果的に防御され、脂肪細胞そのものの分化が抑制されることになる旨の記載がされ、スキンケア、化粧品、皮膚活性剤等として有用である旨の記載がされている。
しかし、脂肪滴形成抑制剤を配合したスキンケア、化粧品等が市販されたとしてもこれだけでは脂肪滴形成抑制やセルライト抑制に十分な効果が期待できるものであるとは言えない。
Patent Document 1 realizes suppression of formation of highly active lipid droplets that can replace conventional retinoids, thereby effectively preventing the occurrence of cell damage derived from lipid droplets and suppressing differentiation of fat cells themselves. It is described that it is useful as skin care, cosmetics, skin active agents and the like.
However, even if skin care, cosmetics, etc. containing a lipid droplet formation inhibitor are marketed, it cannot be said that this alone can be expected to have a sufficient effect for suppressing lipid droplet formation and cellulite.
また、従来の高周波温熱機器では、被施術者が温熱に耐えられる範囲での高い温度設定をすることでより温熱効果が得られるとされていたが被施術者にとっては温熱による苦痛は避けがたいものであった。 In addition, with conventional high-frequency thermal equipment, it has been said that a higher thermal effect can be obtained by setting a high temperature within the range where the patient can withstand the heat, but it is difficult for the patient to suffer from the heat. It was a thing.
さらに、温熱が脂肪滴・セルライト(皮膚表面凸凹脂肪塊)を抑制するらしいことは、従来は経験的に漠然と知られているに過ぎなかったが、その具体的な温熱条件も解明されてこなかったし、その解明のために人体それ自体で解析することは、統計学的に充分に多数の症例数を確保することも困難な上に、対照群と試験群との間で人体特性条件を同一に揃えることも容易ではなく、さらに、その人体においてセルライトや脂肪滴の増減を解析することも精度と感度の観点から困難であった。 Furthermore, the fact that heat seems to suppress fat droplets and cellulite (uneven fat mass on the skin surface) has been vaguely known from experience, but the specific heat conditions have not been elucidated. However, analyzing the human body itself for the purpose of elucidating it is difficult to ensure a statistically sufficient number of cases, and the human body characteristic conditions are the same between the control group and the test group. In addition, it is difficult to analyze the increase and decrease of cellulite and fat droplets in the human body from the viewpoint of accuracy and sensitivity.
本発明は上記の点に鑑みてなされたもので、その目的とするところは、プロビタミンC剤(体内に吸収された後、化学変化を起こしてビタミンCになるものをいう。前駆体ともいう。)と高周波温熱装置を組み合わせることにより、脂肪滴抑制効果・セルライト抑制の大幅な向上を可能ならしめるとともに温熱による細胞死の最小限の抑制を図り、かつ被施術者にとって温熱による苦痛を感じることが無く穏やかな温熱処理を行うことができる脂肪滴・セルライト抑制装置および脂質抑制効果増強薬、セルライト予防治療・脂肪滴抑制・脂質代謝改善用の温熱療法向けプロビタミンC製剤を提供することにある。 The present invention has been made in view of the above points, and the object of the present invention is a provitamin C agent (which is absorbed into the body and then undergoes a chemical change to become vitamin C. It is also called a precursor. )) And a high-frequency heating device, it is possible to greatly improve the effect of suppressing lipid droplets and cellulite, and to minimize the death of cells due to heat. Is to provide a lipid droplet / cellulite suppression device and a lipid suppression effect enhancer capable of performing mild thermothermal treatment, and a provitamin C preparation for thermotherapy for cellulite prevention treatment, lipid droplet suppression, lipid metabolism improvement .
本発明に係る標的体部へのビタミンC剤の投与手段と併用する脂肪滴・セルライト抑制装置は、ビタミンC剤の標的体部への投与手段と、被施術部位に当接するインピーダンスが1000オームの表面を人肌に直接触れないようにプラスチックでコーティングしたエレクトロードと、該エレクトロードに当接する被施術対象体の背後に設置する戻し電極を備え、かつ0.45MHzの高周波の下で、温度37〜41℃の範囲の温度調節部を備えるとともに1回当たりの温熱処理時間を1分〜30分に設定する温熱処理時間調節部を備え、合計で6時間〜1週間のインターバルを置いた温熱投与手段を備えたことを特徴とする。 The lipid droplet / cellulite suppression device used in combination with the means for administering the vitamin C agent to the target body part according to the present invention has an impedance of 1000 ohms in contact with the means for administering the vitamin C agent to the target body part and the treatment site . An electrode coated with plastic so that the surface does not directly touch human skin, a return electrode installed behind the object to be treated that contacts the electrode , and a temperature of 37 at a high frequency of 0.45 MHz. Heat treatment with a temperature control unit in the range of ˜41 ° C. and a heat treatment time adjustment unit for setting the heat treatment time per time to 1 to 30 minutes, with a total interval of 6 hours to 1 week Means are provided.
本発明に係る請求項1記載のビタミンC剤は、水素添加大豆レシチン・コレステロールから構成され、超音波装置・エクストルーダから調整されたリポソームによってアスコルビン酸のまま包含する皮膚浸透性が増強されたビタミンC剤であることを特徴とする。 Vitamin C preparation according to claim 1 of the present invention is composed of hydrogenated soybean lecithin / cholesterol, and vitamin C with enhanced skin permeability including ascorbic acid by liposomes prepared from an ultrasonic device / extruder. It is an agent .
本発明に係る請求項1又は請求項2記載の脂肪滴・セルライト抑制装置と併用して使用される標的体部へ投与されるビタミンC剤は、温熱処理においても分解せずに保持させる温熱安定化剤を含有することを特徴とする。 The vitamin C agent to be administered to the target body part used in combination with the lipid droplet / cellulite suppressing device according to claim 1 or claim 2 according to the present invention is maintained at a stable temperature without being decomposed even in a thermal treatment. It contains an agent.
本発明に係る請求項3記載の温熱安定化剤は、マニトール、コーンスターチ、キチン・キトサンからなる群から選ばれた一つ以上の物質を含有することを特徴とする。 The thermal stabilizer according to claim 3 of the present invention is characterized by containing one or more substances selected from the group consisting of mannitol, corn starch, chitin and chitosan.
温熱が脂肪滴・セルライト(皮膚表面凸凹脂肪塊)を抑制するらしいことは、従来は経験的に漠然と知られているに過ぎなかったが、その具体的な温熱条件も解明されてこなかったし、その解明のために人体それ自体で解析することは、統計学的に充分に多数の症例数を確保することも困難な上に、対照群と試験群との間で人体特性条件を同一に揃えることも容易ではなく、さらに、その人体においてセルライトや脂肪滴の増減を解析することも精度と感度の観点から困難であった。 The fact that heat seems to suppress fat droplets and cellulite (skin irregularities on the skin surface) has been vaguely known from experience, but the specific heat conditions have not been elucidated. Analyzing the human body itself for the purpose of clarifying it is difficult to ensure a statistically sufficient number of cases, and to make the human body characteristic conditions identical between the control group and the test group In addition, it is difficult to analyze the increase and decrease of cellulite and fat droplets in the human body from the viewpoint of accuracy and sensitivity.
本発明は、脂肪滴やセルライトの形成に関する中心的役割を果たす前駆脂肪細胞および/または脂肪細胞を用い、さらには、これら前駆脂肪細胞および/または脂肪細胞を組み込んだ3次元ヒト皮膚組織モデル、加えて、ヒト皮膚摘出組織片、および、ユカタン産ミニ豚皮膚摘出組織片などを用いて、各々、同一の皮膚特性条件の下に、試験群と比較対照群を操作し、統計学的に充分に多数の適用例を解析して、脂肪滴とセルライトを抑制する最適条件を求めた上で、その範囲前後に絞って臨床上の最適条件を求めた。 The present invention uses preadipocytes and / or adipocytes that play a central role in the formation of lipid droplets and cellulite, and further includes a three-dimensional human skin tissue model incorporating these preadipocytes and / or adipocytes, Using a human skin-extracted tissue piece and a Yucatan mini-pig skin-extracted tissue piece, the test group and the control group were manipulated under the same skin characteristic conditions, respectively, and statistically sufficient A number of application examples were analyzed, and the optimum conditions for suppressing lipid droplets and cellulite were determined.
その結果、温熱処理装置の単独使用よりも、温熱に加えて、ビタミンC剤を併用したことにより、相乗効果として、脂肪滴とセルライトの抑制が顕著になることが判明した。
また、温熱を一定期間のインターバルを置いて温熱処理する手段と、各温熱処理手段の適用に際して本発明で見いだした特定プロビタミンC剤を特定濃度で標的体部に投与する併用手段が脂肪滴・セルライト抑制に著効を示すことが本発明によって見いだされた。
As a result, it was found that the combination of vitamin C agent in addition to warm heat is more effective in suppressing lipid droplets and cellulite as a synergistic effect than using a single heat treatment apparatus.
Further, a means for performing heat treatment at intervals of a certain period of time, and a combination means for administering the specific provitamin C agent found in the present invention to the target body part at a specific concentration upon application of each heat treatment means include lipid droplets, It has been found by the present invention that it is highly effective in suppressing cellulite.
さらに、本発明に係る脂肪抑制効果増強薬は、上記ビタミンC剤を含有するので、セルライト予防治療・脂肪滴抑制・脂質代謝改善用の温熱向け治療に有効に機能することとなる。 Furthermore, since the fat inhibitory effect enhancer according to the present invention contains the above-mentioned vitamin C agent , it effectively functions in the treatment for heat for cellulite prevention treatment, lipid droplet suppression, and lipid metabolism improvement.
さらにまた、本発明に係るセルライト予防治療・脂肪滴抑制・脂質代謝改善用の温熱療法向けビタミンC剤は、温熱処理においても分解せずに保持させる温熱安定化剤として寄与することができる。 Furthermore, the vitamin C agent for thermotherapy for cellulite prevention treatment, lipid droplet suppression, and lipid metabolism improvement according to the present invention can contribute as a thermostabilizer that retains the thermolite without being decomposed even in the heat treatment .
そして、本発明の上記ビタミンC剤は、温熱安定化剤として、マニトール、コーンスターチ、キチン・キトサンからなる群から選ばれた一つ以上の物質を含有することによりセルライト予防治療・脂肪滴抑制・脂質代謝改善用の温熱療法向けとして好適である。 The vitamin C preparation of the present invention contains at least one substance selected from the group consisting of mannitol, corn starch, chitin and chitosan as a thermal stabilizer, thereby preventing cellulite, preventing lipid droplets, and preventing lipids. It is suitable for hyperthermia for improving metabolism.
脂肪細胞/前駆脂肪細胞での脂肪滴・セルライト抑制と細胞死防御についてについて実施した。
間葉系前駆脂肪細胞OP9を培養し通常培地からインスリン含有KSR培地に置換すると、脂肪細胞への分化が開始されるが、この時に本発明のCapacitive−Resistive Electric−Transfer装置((株)インディバ・ジャパン製品:MD530) (CRet System )の身体温熱装置を使用してLevel 5, Indicator 5, 20 secに該当する電気特性を設定した。室温26.5℃から41℃にシフトアップするが、その後に、同装置のLevel 2〜3に設定した。脂肪細胞は41℃で1 min保持した。この時に、プロビタミンC剤としてアスコルビン酸−2−O−フォスフェート ナトリウム塩(APS) を1 mM (300 ppm, 0.03wt%)の濃度で投与して一日12時間おきに2回反復して3日間実施する試験区と無投与の比較対照区とを比較した。
細胞内の脂肪滴は、オイルレッドオー色素で染色した後に10%メタノール水溶液でリンスしてリンス廃液が530 nmでの吸光度がゼロと成る3〜5回まで徹底的にリンスして、細胞外や細胞表面のオイルレッドオー色素を消滅させた後に、位相差顕微鏡で撮影すると共に、細胞内のオイルレッドオー色素を抽出して530 nmで吸光プレ−トリーダーと分光光度計で細胞内脂肪滴を定量した。細胞死と細胞生存は、各々、プロピジウムヨーダイドとカルセインアミノメトキシエステルで染色し、その蛍光度を蛍光プレートリーダーで評価した。
Lipid droplet / cellulite suppression and cell death protection in adipocytes / preadipocytes were performed.
When the mesenchymal preadipocytes OP9 are cultured and replaced with the insulin-containing KSR medium from the normal medium, differentiation into adipocytes is initiated. At this time, the Capacitive-Resistive Electric-Transfer apparatus (Indiva Corp.) Electrical characteristics corresponding to Level 5, Indicator 5, 20 sec were set using a body heat apparatus of Japan product: MD530) (CRet System). The room temperature was shifted from 26.5 ° C. to 41 ° C., after which it was set to Level 2-3 of the same apparatus. Adipocytes were kept at 41 ° C. for 1 min. At this time, ascorbic acid-2-O-phosphate sodium salt (APS) as a provitamin C agent was administered at a concentration of 1 mM (300 ppm, 0.03 wt%) and repeated twice every 12 hours a day. The test group for 3 days was compared with the non-administered control group.
Lipid droplets inside the cells are stained with oil red o dye, rinsed with 10% aqueous methanol solution, and rinsed thoroughly until the rinse waste liquid has zero absorbance at 530 nm until it reaches zero. After extinguishing the oil red o dye on the cell surface, take a picture with a phase contrast microscope, extract the oil red o dye in the cell, and remove the intracellular lipid droplets with an absorption plate reader and spectrophotometer at 530 nm. Quantified. Cell death and cell survival were stained with propidium iodide and calcein aminomethoxyester, respectively, and the fluorescence was evaluated with a fluorescence plate reader.
本発明で使用するMD530は0.45MHzの高周波温熱機器であり、施術箇所への循環を通じて人体細胞の深部加温を行う。キャパシティブ用エレクトロードとレジスティブ用エレクトロードを使用し、深部加温を行う。キャパシティブモードを採用する場合、ステンレス製のエレクロロードの円形盤の表面は人の肌に直接触れないようにプラスチックで表面をコーティングしている。エレクトロードは、細胞に高周波エネルギーを透過するコンデンサーの役割をし、施術部位を加温する。透過された高周波エネルギーは、板型戻し電極に向って透過される。他方、レジスティブモードでは、直接、施術箇所に置かれたRES用エレクトロード(ステンレス製)を通して人体細胞へ高周波電流を流す。この電流は戻し電極に電導する。高周波エネルギーが細胞内に透過されたとき、細胞が抵抗体となり組織の温度を集中的に上昇させ、深部加温が行われる。本機器では、被施術者に流れる電流の強さ、被施術者に加えられる皮相電力(キャパシティブモード)、アクティブパワー(レジスティブモード)及びエレクトロードと被施術者の抵抗を測定し、モニタリングができる。 The MD 530 used in the present invention is a 0.45 MHz high-frequency heating device that warms human body cells deeply through circulation to a treatment site. Deep heating is performed using capacitive and resistive electrodes. When the capacitive mode is adopted, the surface of the stainless steel electro-rode circular disc is coated with plastic so that it does not directly touch human skin. The electrode acts as a condenser that transmits high-frequency energy to cells, and warms the treatment site. The transmitted high frequency energy is transmitted toward the plate-type return electrode. On the other hand, in the resistive mode, a high-frequency current is passed directly to the human body cell through the RES electrode (made of stainless steel) placed at the treatment site. This current is conducted to the return electrode. When high-frequency energy is permeated into the cell, the cell becomes a resistor, and the temperature of the tissue is increased intensively, and deep heating is performed. This device can measure and monitor the intensity of current flowing to the patient, the apparent power applied to the patient (capacitive mode), active power (resistive mode), and the resistance of the electrode and the patient. .
図11はMD530のインディバCRet Systemの背面図で、1は電源スイッチ、2はヒューズホルダー、3は電源ソケット、4はアース用ジャック、5は冷却ファン、6は仕様ラベルをそれぞれ示す。図12はMD530の正面図で、7はLCD画面、8はキーパッド、9はキャパシティブ用出力、10は戻し電極用出力、11はレジスティブ用出力をそれぞれ示す。図13はMD530の前面に表示されるコントロール類の表示部の説明図で、12はスタンバイインジケータランプ、13はON/OFFキー、14はメニューキー、15はキャパシティブモードキー、レジスティブモードキー、17はリセットキー、18はスタート/ストップキー、19は時間増キー、時間減キー、21は出力増/減ダイヤルノブをそれぞれ示す。 FIG. 11 is a rear view of the MD 530 indiver CRet System, where 1 is a power switch, 2 is a fuse holder, 3 is a power socket, 4 is a grounding jack, 5 is a cooling fan, and 6 is a specification label. 12 is a front view of the MD 530, 7 is an LCD screen, 8 is a keypad, 9 is a capacitive output, 10 is a return electrode output, and 11 is a resistive output. FIG. 13 is an explanatory diagram of a display section of controls displayed on the front surface of the MD 530. 12 is a standby indicator lamp, 13 is an ON / OFF key, 14 is a menu key, 15 is a capacitive mode key, a resistive mode key, 17 Is a reset key, 18 is a start / stop key, 19 is a time increase key, a time decrease key, and 21 is an output increase / decrease dial knob.
キャパシティブモードの出力に対するインピーダンスは、皮膚組織上に置かれたエレクトロードのインピーダンス(1,000オーム)に相応するように計算されている。この方法により多容量のエネルギーの透過を実現できる。エレクトロードを通じての組織への循環高周波エネルギーの行方は、図9に示す。MD530からのエネルギーは、金属軸Aを通してBの先端(エレクトロードの最下部)へ伝わる。そのエネルギーは,Cの絶縁体部分により組織と隔てられるため低下し、エレクトロード全表面、容量的に組織に伝達される。組織Dは、合計で1,000オームとなる複数の無限大の抵抗を定着させ、そのコンデンサーの性質により第二のプレートのような働きをする。図9の点線Eが第二のプレートとして定義され、コンデンサーとしての働きがなされる。1,000オーム相当の抵抗は、実際はB点とE点の間で発生し、その部分で温度上昇が起こり、その強深度は、本機器のパワー(レベル調整に対応)・エレクトロードの大きさ・施術部位の組織の特質・施術時間により決まる。特に本発明では、被施術者が被施術者にとって温熱による苦痛を感じることが無く穏やかな温熱処理、すなわち37〜41℃の範囲の温度設定により温熱処理を行うことができる点にある。 The impedance for capacitive mode output is calculated to correspond to the impedance (1,000 ohms) of the electrode placed on the skin tissue. By this method, a large amount of energy can be transmitted. The direction of circulating high frequency energy to the tissue through the electrodes is shown in FIG. The energy from the MD 530 is transmitted to the tip of B (the lowest part of the electrode) through the metal axis A. The energy is reduced because it is separated from the tissue by the insulator portion of C, and is transferred to the tissue capacitively across the entire surface of the electrode. The tissue D fixes a plurality of infinite resistances totaling 1,000 ohms, and acts like a second plate due to the nature of the capacitor. A dotted line E in FIG. 9 is defined as the second plate, which serves as a capacitor. A resistance equivalent to 1,000 ohms is actually generated between point B and point E, and the temperature rises at that point. The depth of the resistance is the power of this device (corresponding to level adjustment) and the size of the electrode.・ It depends on the characteristics of the tissue at the treatment site and the treatment time. In particular, in the present invention, the patient does not feel pain due to the heat for the patient, and the heat treatment can be performed by a gentle heat treatment, that is, a temperature setting in a range of 37 to 41 ° C.
上記の条件下において、その結果を以下に示す。
(1)脂肪細胞への分化誘導処理によって、細胞の核の周辺に、ドーナッツ状に脂肪滴(赤く染色;細胞1個当たり約20個余り)蓄積が見られた。細胞生存率は99.3±0.4%だった。
(2)41℃でのCRet温熱装置は、プロビタミンC剤なしでも脂肪滴蓄積量を22%減少した。CRet System温熱装置の適用により脂肪滴蓄積が抑制された。細胞死は0.8±0.2%と少しあるが顕著ではなかった。
The results are shown below under the above conditions.
(1) By the differentiation induction treatment into adipocytes, accumulation of fat droplets (stained in red; about 20 per cell) was observed around the cell nucleus. Cell viability was 99.3 ± 0.4%.
(2) The CRet thermal apparatus at 41 ° C. reduced the lipid droplet accumulation amount by 22% even without the provitamin C agent. The application of the CRet System thermal apparatus suppressed the accumulation of fat droplets. Although cell death was a little 0.8 ± 0.2%, it was not remarkable.
(3)プロビタミンC剤(アスコルビン酸誘導体)APS(アスコルビン酸−2−O−フォスフェート ナトリウム塩)の300 ppm (0.03wt%, 1 mM)の濃度での上記温熱装置を適用しない場合での37℃での投与は、細胞核の周辺に脂肪滴蓄積が見られたが、脂肪滴蓄積を20%抑制した。APSまたはCRet温熱装置(1 min)適用による細胞死は0.1±0.0%と少し見られたが、無視しうるレベルであった。 (3) Provitamin C agent (ascorbic acid derivative) APS (ascorbic acid-2-O-phosphate sodium salt) 300 ppm (0.03 wt%, 1 mM) in the case where the above-mentioned thermal apparatus is not applied Administration at 37 ° C. caused lipid droplet accumulation around the cell nucleus, but suppressed lipid droplet accumulation by 20%. Cell death due to the application of APS or CRet thermostat (1 min) was slightly seen as 0.1 ± 0.0%, but was negligible.
(4)APSの300 ppm投与とCRet System温熱装置の併用は、脂肪滴蓄積を54%抑制した。アスコルビン酸誘導体APSの投与とCRet System温熱装置の併用により脂肪滴抑制効果は大幅に増大した。脂肪滴のない細胞が過半数を占めた。細胞核の周辺に脂肪滴が蓄積されたが、APS投与で細胞全体に脂肪滴蓄積が抑制され、この抑制は、CRet System温熱装置の併用で一層増大した。細胞生存率は99.0±0.6%を維持した。APSから変換されたビタミンC剤が脂肪燃焼促進剤カルニチンを合成する酵素トリメチルリシン ジオキゲナーゼ&γ−ブチロベタイン ジオキシゲナーゼを促進すると共に、CRet System温熱装置による脂肪滴流動化&分散化が奏功したことを示唆するものである。
(4) The combined use of 300 ppm of APS and the CRet System heat apparatus suppressed lipid droplet accumulation by 54%. The combined use of the ascorbic acid derivative APS and the CRet System heating device significantly increased the lipid droplet suppression effect. The majority of cells were without lipid droplets. Although lipid droplets accumulated around the cell nucleus, APS administration suppressed lipid droplet accumulation throughout the cells, and this suppression was further increased by the combined use of the CRet System thermal apparatus. Cell viability was maintained at 99.0 ± 0.6%. Vitamin C agent converted from APS promotes the enzyme trimethyl lysine dioxygenase & γ-butyrobetaine dioxygenase that synthesizes the fat burning accelerator carnitine, and suggests that lipid droplet fluidization and dispersion by the CRet System thermal apparatus were successful. Is.
(5)従来法として、通常の身体温熱装置で42,43,44,45℃の温度4点で各々60分間の処理による温熱単独を行なう比較対照例を試験した結果、脂肪滴抑制は、各温度で、8.9,11.6,10.4,5.2%の抑制であり、細胞死が各々、18.1±4.7,27.0±5.9,41.3±11.0,61.8±5.5%だった。過酷な温熱装置の単独使用は細胞傷害と細胞生存の指標であるサクシネートデヒドロゲナーゼを主体とするミトコンドリア脱水素酵素の活性低下をもたらすと共に、透過型電子顕微鏡による細胞形態では、活発な細胞代謝の指標となるマイクロビラスの消失、細胞膜の破綻、シュリンキッジ(細胞萎縮)、ピクノーシス(核凝縮)、カリオレキシス(核断片化)が認められた。これらは従来の過酷な温熱装置の適用が細胞死を広範囲に惹起させるという犠牲の下に施行されていた可能性を示唆するものである。 (5) As a conventional method, as a result of testing a comparative example in which heating was carried out for 60 minutes each at four points of 42, 43, 44, and 45 ° C. with a normal body heating device, At temperature, the inhibition was 8.9, 11.6, 10.4, 5.2%, and cell death was 18.1 ± 4.7, 27.0 ± 5.9, 41.3 ± 11, respectively. 0.0, 61.8 ± 5.5%. Severe use of a hyperthermia device leads to decreased activity of mitochondrial dehydrogenase mainly composed of succinate dehydrogenase, which is an indicator of cell damage and cell survival. In addition, cell morphology by transmission electron microscopy is an indicator of active cell metabolism. Disappearance of microvillus, rupture of cell membrane, shrinkage (cell atrophy), pycnosis (nuclear condensation), and calorixis (nuclear fragmentation) were observed. These suggest the possibility that the application of the conventional severe thermal apparatus was carried out at the expense of causing cell death in a wide range.
前駆脂肪細胞を組み込んだ3次元ヒト皮膚組織モデルでの脂肪滴・セルライト抑制と細胞死防御について実施した。
ヒト皮膚繊維芽細胞OUMS−36を含むI型コラーゲンから構成される真皮、その上にIV型コラーゲン・ラミニン・エンタクチン・フィブロネクチンを含有するマトリゲルから構成される基底膜、さらにその上に、間葉系前駆脂肪細胞OP9を添加することを特徴とし、その上に、ヒト皮膚角化細胞HaCaTから構成される表皮を各々重層し、最後にエアーリフトによって角層を形成させて取得される脂肪滴形成量評価向けの3次元ヒト皮膚組織モデルを作製し、実施例1と同様な試験を行ない、脂肪滴・セルライト抑制と細胞死防御を評価し、併せて、皮膚組織片をクライオスタットで4μm厚の超薄切片を調製してオイルレッドオー色素で脂肪滴を染色した皮膚組織の断面構造において、断面積に占める脂肪滴の面積の割合をNIH−ImageとImage−Jソフトウェアで解析した。細胞死レベルはヘマトキシリンーエオシン染色によって、変質細胞の細胞総数に占める比率から、評価した。さらに、皮膚表面の凹凸形状も走査型電子顕微鏡で撮影した。皮膚表面の凹凸形状はシリコーンゴムを用いたレプリカを作製して凸凹度をラインヒストグラム化してセルライト度を評価した。
Lipid droplet / cellulite suppression and cell death protection were carried out in a 3D human skin tissue model incorporating preadipocytes.
The dermis composed of type I collagen containing human dermal fibroblasts OUMS-36, the basement membrane composed of matrigel containing type IV collagen, laminin, entactin and fibronectin, and further, the mesenchymal system The amount of lipid droplet formation obtained by adding preadipocytes OP9, on which each of the epidermis composed of human skin keratinocytes HaCaT is layered, and finally the stratum corneum is formed by air lift A three-dimensional human skin tissue model for evaluation was prepared, the same test as in Example 1 was performed, and lipid droplet / cellulite suppression and cell death protection were evaluated. In addition, the skin tissue piece was ultrathin with a thickness of 4 μm using a cryostat. In the cross-sectional structure of the skin tissue in which a slice was prepared and stained with oil red o dye, the ratio of the area of the fat drop to the cross-sectional area was expressed as NI. It was analyzed by -Image and Image-J software. The cell death level was evaluated by hematoxylin-eosin staining from the ratio of the degenerated cells to the total number of cells. Furthermore, the uneven shape of the skin surface was also photographed with a scanning electron microscope. For the uneven shape on the skin surface, a replica using silicone rubber was prepared, and the degree of cellulite was evaluated by converting the unevenness into a line histogram.
この結果、比較例として、41℃でのCRet System温熱装置の単独適用(常温からのシフトアップ34sec、設定温度到達後の温度平行時間5min、常温へ低下するシフトダウン3min14sec:以下、これらを単に「5min適用」と呼ぶ)は、プロビタミンC剤なしでも脂肪滴蓄積量を22.4±3.7%減少させた。CRet System温熱装置の単独適用による脂肪滴蓄積抑制効果が有意に示された。
もう一つの比較例として、プロビタミンC剤(アスコルビン酸誘導体)APS(アスコルビン酸−2−O−フォスフェート ナトリウム塩)の300ppm(0.03wt%)の濃度での上記温熱装置を適用しない場合での37℃での投与は、細胞核の周辺に脂肪滴蓄積が見られたが、脂肪滴蓄積を16.1±2.9%抑制した。APS単独投与またはCRet System温熱装置(5min適用)単独適用による細胞死は各々0.1±0.0%、2.2±0.3%と無視しうるレベルまたは僅少レベルであった。
As a result, as a comparative example, a single application of the CRet System heating device at 41 ° C. (shift up from room temperature 34 sec, temperature parallel time after reaching set temperature 5 min, shift down to room temperature 3 min 14 sec: 5 min application ”) reduced lipid droplet accumulation by 22.4 ± 3.7% without provitamin C agent. The effect of suppressing lipid droplet accumulation by the single application of the CRet System heating device was significantly shown.
As another comparative example, when the above-mentioned thermal apparatus at a concentration of 300 ppm (0.03 wt%) of provitamin C agent (ascorbic acid derivative) APS (ascorbic acid-2-O-phosphate sodium salt) is not applied. Administration at 37 ° C. showed lipid droplet accumulation around the cell nucleus, but suppressed lipid droplet accumulation by 16.1 ± 2.9%. Cell death due to APS alone administration or CRet System heating device (5 min application) alone was 0.1 ± 0.0% and 2.2 ± 0.3%, respectively, which were negligible or minimal.
一方、本発明では、APSの300 ppm投与とCRet System温熱装置の併用、または、温熱安定化剤としてマニトール500ppm、コーンスターチ800ppm、イカ筋(中骨)キトサン(キトサン成分83〜97%、キチン成分3〜17%;β型キチンを出発材料にして脱アセチル反応などの処理で調製したキチン・キトサン)350ppmを添加したAPSの300ppm投与とCRet System温熱装置の併用、は、脂肪滴蓄積を54.7±4.6%、または、73.3±5.8%抑制した。アスコルビン酸誘導体APSの投与とCRet温熱装置(5min適用)の併用により脂肪滴抑制効果は増大し、さらに、温熱安定化剤の添加によってこの効果が促進された。これら本発明例ではいずれも脂肪滴のほぼ検出されない細胞が過半数を占めた。細胞核の周辺に脂肪滴が蓄積されたが、APS投与で細胞全体に脂肪滴蓄積が抑制され、この抑制は、CRet System温熱装置の併用で一層増大し、さらに温熱安定化剤によって顕著になったことになる。細胞死率も各々0.3±0.0%と0.2±0.0%と無視しうるレベルだった。 On the other hand, in the present invention, 300 ppm administration of APS and CRet System heat apparatus are combined, or as a heat stabilizer, mannitol 500 ppm, corn starch 800 ppm, squid muscle (medium bone) chitosan (chitosan component 83 to 97%, chitin component 3 ˜17%; chitin / chitosan prepared by treatment such as deacetylation using β-type chitin as a starting material) 300 ppm administration of APS added with 350 ppm and combined use of CRet System thermal apparatus, 54.7 It was suppressed by ± 4.6% or 73.3 ± 5.8%. The combined use of the ascorbic acid derivative APS and the CRet heating device (5 min application) increased the lipid droplet suppression effect, and this effect was further promoted by the addition of a thermal stabilizer. In all of the examples of the present invention, the majority of the cells in which fat droplets were hardly detected accounted for. Lipid droplets accumulated around the cell nucleus, but APS administration suppressed lipid droplet accumulation throughout the cell, and this suppression was further increased by the combined use of the CRet System thermal apparatus, and was further marked by the thermal stabilizer. It will be. The cell death rates were also negligible levels of 0.3 ± 0.0% and 0.2 ± 0.0%, respectively.
改変ブロノフ拡散チェンバーに組み込んだインフォームドコンセントを提出した48歳の日本人女性の耳后下の皮膚摘出組織片での脂肪滴・セルライト抑制と細胞死防御について実施例2と同様に実施した。
脂肪滴抑制試験の終了後に当該チェンバーより単離した皮膚組織片をクライオスタットで4μm厚の超薄切片を調製し、実施例2と同様に脂肪滴・セルライト抑制と細胞死防御を評価した。
Lipid droplets / cellulite suppression and cell death protection were performed in the skin excised tissue piece under the ear of a 48-year-old Japanese woman who submitted informed consent incorporated into the modified Bronov diffusion chamber.
After completion of the lipid droplet suppression test, the skin tissue piece isolated from the chamber was prepared with an ultrathin section having a thickness of 4 μm using a cryostat, and the lipid droplet / cellulite suppression and cell death protection were evaluated in the same manner as in Example 2.
この結果、比較例として、41℃でのCRet System温熱装置の単独適用(常温からのシフトアップ33sec、設定温度到達後の温度平行時間5min、常温へ低下するシフトダウン3min27sec:以下、これらを単に「5min適用」と呼ぶ)は、プロビタミンC剤なしでも脂肪滴蓄積量を19.1±2.9%減少させた。CRet温熱装置の単独適用による脂肪滴蓄積抑制効果が有意に示された。
もう一つの比較例として、プロビタミンC剤(アスコルビン酸誘導体)APS(アスコルビン酸−2−O−フォスフェートナトリウム塩)の300ppm(0.03wt%)の濃度での上記温熱装置を適用しない場合での37℃での投与は、細胞核の周辺に脂肪滴蓄積が見られたが、脂肪滴蓄積を12.0±3.5%抑制した。APS投与またはAPSなしのCRet System温熱装置(5min適用)単独適用による細胞死は各々2.0±0.4%または6.9±0.8%と無視しうるレベルまたは僅少レベルであった。
As a result, as a comparative example, a single application of the CRet System heating device at 41 ° C. (shift up from room temperature 33 sec, temperature parallel time after reaching the set temperature 5 min, shift down to room temperature 3 min 27 sec: 5 min application)) reduced lipid droplet accumulation by 19.1 ± 2.9% even without provitamin C. The effect of suppressing lipid droplet accumulation by the single application of the CRet thermal apparatus was significantly shown.
As another comparative example, in the case where the above-mentioned heating apparatus at a concentration of 300 ppm (0.03 wt%) of provitamin C agent (ascorbic acid derivative) APS (ascorbic acid-2-O-phosphate sodium salt) is not applied. Administration at 37 ° C. caused lipid droplet accumulation around the cell nucleus, but suppressed lipid droplet accumulation by 12.0 ± 3.5%. Cell death due to APS administration or APS-free CRet System heating device (5 min application) alone was 2.0 ± 0.4% or 6.9 ± 0.8%, respectively, at negligible or negligible levels.
一方、本発明では、APSの300ppm投与とCRet System温熱装置の併用、または、温熱安定化剤としてマニトール500ppm、コーンスターチ800ppm、イカ筋(中骨)キトサン(キトサン成分83〜97%、キチン成分3〜17%;β型キチンを出発材料にして脱アセチル反応などの処理で調製したキチン・キトサン)350ppmを添加したAPSの300ppm投与とCRet System温熱装置の併用は、各々54.8±12.6%または70.4±6.3%脂肪滴蓄積を抑制した。脂肪滴抑制効果はプロビタミンCのAPSの投与とCRet System温熱装置(5min適用)の併用により明確となり、さらに温熱安定化剤を併用すると脂肪滴抑制効果は顕著になった。脂肪滴のほぼ検出されない細胞が過半数を占めた。細胞核の周辺に脂肪滴が蓄積されたが、APS投与で細胞全体に脂肪滴蓄積が抑制され、この抑制は、CRet System温熱装置の併用で一層増大したことになり温熱安定化剤による促進効果も認められた。細胞死率も各々1.1±0.2%または1.7±0.2%と無視しうるレベルだった。 On the other hand, in the present invention, 300 ppm administration of APS and CRet System thermal apparatus are combined, or as thermal stabilizer, mannitol 500 ppm, corn starch 800 ppm, squid muscle (medium bone) chitosan (chitosan component 83 to 97%, chitin component 3 to 3%) 17%; chitin / chitosan prepared by treatment such as deacetylation using β-type chitin as a starting material) 300 ppm of APS added with 350 ppm and combined use of CRet System heating device were 54.8 ± 12.6%, respectively. Alternatively, 70.4 ± 6.3% lipid droplet accumulation was suppressed. The effect of suppressing lipid droplets was clarified by the combined use of APS of provitamin C and the CRet System thermal apparatus (5 min application), and when the thermal stabilizer was used in combination, the lipid droplet suppressing effect became significant. The majority of the cells were almost undetectable lipid droplets. Lipid droplets accumulated around the cell nucleus, but APS administration suppressed lipid droplet accumulation throughout the cell, and this suppression was further increased by the combined use of the CRet System heating device, and the promotion effect by the thermal stabilizer was also increased. Admitted. The cell death rate was also negligible at 1.1 ± 0.2% or 1.7 ± 0.2%, respectively.
臨床試験での脂肪塊・セルライトはインフォームドコンセントを得た健康な26〜56歳の日本人女性24名をあらかじめ大腿最太部後側と臀部最厚部先端での皮下脂肪率をMRIで評価し、当該部の皮膚表面形状は、セルライト皮表凹凸度として、スキンキャスト皮表形状レプリカ法で評価した。
この試験前の皮下脂肪率とセルライト皮表度および平均年齢に偏りがないように24名を8名ずつ3群に分けて、毎週3回、各回、A群は、温熱安定化剤なしのAPS300ppmを太腿と臀部に1mL/cm2で塗布しCRet System温熱装置を各々10分間と15分間適用し、本発明のB群は、温熱安定化剤としてマニトール500ppm、コーンスターチ800ppm、イカ筋(中骨)キトサン(キトサン成分83〜97%、キチン成分3〜17%;β型キチンを出発材料にして脱アセチル反応などの処理で調製したキチン・キトサン)350ppmを添加したAPS300ppmを同様に塗布してCRet Systemも同様に適用し、比較対照のC群は、CRet System適用のみとして、各3週間継続して試験した。MRI画像データは0−Gaugeソフトウェアを用いて内臓脂肪との境界線をも識別し皮下脂肪を自動判別して脂肪面積を算出する計測アルゴリズムで高精度に解析した。セルライト皮表凹凸度は、太腿・臀部の各当該部を各々10cm平方、15cm平方でスキンキャストで皮表形状を複製し、キメ・シワの小(上下)振幅を平坦化してセルライトの中〜大(上下)振幅を計測対象としてImage−jソフトウェアで解析した。
Fat mass and cellulite in clinical trials were evaluated by MRI of 24 healthy Japanese women aged 26-56 years old who obtained informed consent in advance at the back of the thigh and the thickest part of the buttocks. And the skin surface shape of the said part was evaluated by the skin cast skin surface replica method as cellulite skin surface unevenness degree.
Before the test, 24 people were divided into 3 groups of 8 people each so that the subcutaneous fat rate, cellulite skin degree and average age were not biased, 3 times each week, group A was APS 300 ppm without thermal stabilizer. Was applied to the thighs and buttocks at 1 mL / cm 2 and the Cret System heating device was applied for 10 minutes and 15 minutes, respectively. Group B of the present invention was mannitol 500 ppm, corn starch 800 ppm, squid muscle (medium bone) as a thermal stabilizer. ) Chitosan (chitosan component 83 to 97%, chitin component 3 to 17%; chitin / chitosan prepared by a process such as deacetylation using β-type chitin as a starting material) APS300ppm added with 350ppm was applied in the same manner. The system was applied in the same manner, and the control group C was continuously tested for 3 weeks each as the CRet System application only. The MRI image data was analyzed with high accuracy by a measurement algorithm that uses 0-Gauge software to identify the boundary line with visceral fat, automatically discriminate subcutaneous fat, and calculate the fat area. Cellulite skin irregularities are 10 cm square and 15 cm square for each part of the thigh and buttocks, and the skin surface shape is duplicated by skin casting. Large (vertical) amplitude was analyzed with Image-j software as a measurement target.
この結果、皮下脂肪面積率は、被験者8名の平均で試験前の数値を基準にして試験後に各々、A群は84.5±7.4%、B群は68.9±4.0%、C群は92.5±6.2%であり、セルライト皮表凸凹度は、A群は73.1±4.8%、B群は44.5±2.0%、C群は88.9±9.3%と3群いずれも試験前に比較して、皮下脂肪もセルライト形状も両方とも低下し、A・B・C3群とも改善効果が有意に認められたことになるが、このうち、C群に比較してA群の改善効果が大きく、プロビタミンC剤のAPSによる併用効果が優れていることを示すと共に、A群に比較してB群は皮下脂肪もセルライト形状も両方ともより顕著な改善効果が認められ、APSの温熱に対する安定化剤の添加効果が著明なことが検証された。 As a result, the subcutaneous fat area ratio was 84.5 ± 7.4% for the A group and 68.9 ± 4.0% for the B group after the test on the basis of the values before the test on the average of the 8 subjects. , C group is 92.5 ± 6.2%, cellulite skin roughness is 73.1 ± 4.8% for group A, 44.5 ± 2.0% for group B, 88 for group C .9 ± 9.3% and all three groups compared to before the test, both subcutaneous fat and cellulite shape decreased, and the improvement effect was significantly recognized in the A, B and C3 groups. Among these, the improvement effect of A group is large compared with C group, and it shows that the combined use effect by APS of a provitamin C agent is excellent, and B group has both subcutaneous fat and cellulite shape compared with A group. In both cases, a more remarkable improvement effect was observed, and it was verified that the effect of adding the stabilizer to the heat of APS was remarkable.
本発明はエステティック美容業界及び皮膚科医療等の分野あるいは医療装置・美容装置産業等に多大の貢献することができる。 The present invention can greatly contribute to the fields of aesthetic beauty industry, dermatology, and the like, or the medical device / beauty device industry.
A 金属軸
B エレクトロード
C 絶縁体部分
E 第二のプレート
9 キャパシティブ用出力
10 戻し電極用出力
11 レジスティブ用出力
15 キャパシティブモードキー
16 レジスティブモードキー
19 時間増キー
20 時間減キー
A Metal shaft B Electrode C Insulator part E Second plate 9 Capacitive output 10 Return electrode output 11 Resistive output 15 Capacitive mode key 16 Resistive mode key 19 Time increase key 20 Time decrease key
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WO2008087803A1 (en) * | 2007-01-16 | 2008-07-24 | Hokkaido University | Liposome preparation for iontophoresis having antioxidant component encapsulated therein |
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