201038293 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種抗菌胜肽(35-胜肽)添加於所有人體用之保養品、清 潔用品'彩粧用品及面膜上，用以抑制初油酸桿菌，除可預防青春痘的用 途’對於皮濱所常I的細菌表皮荀萄球菌(Staphylococcus epidermidis)，反 皮瓌常見>的致病菌大腸桿菌(E, co丨i)、綠藏桿菌(Pseudomonas aeruginosa)與 肺炎桿菌(尺/e6咖此pnewmon/ae)，同樣可達到去菌、抗菌及降低細菌的危 害與對保養成份的干擾’使保濕、美白、甚至抗老的功效更深入更徹底， 以保持健康明亮的皮膚。 【先前技術】 查青春痘的發生是許多因素綜合作用的結果。其中除了因為皮脂 過多分泌與毛囊過度角化，造成毛孔阻塞外，毛囊内的初油酸桿菌 的增生也是引起青春痘的原因之一。因為這 些因素共同作用的結果而造成皮膚的發炎。 初油酸桿菌是一種革蘭氏陽性厭氧細菌，生存在皮膚的毛囊内， 平時雖可與皮膚和平共處，但卻也是引起青春㈣主要病賴。初油 酸桿菌繁殖後會阻塞皮脂通道，其代謝之後所產生酵素作用的結果， 會刺激毛囊角質化，並引起皮膚發炎。 抗青春痘的方法不4乎預防與治療^治療青春痘通常應依照醫師 的指示塗抹外㈣與配合口職雙f齊獨治賴式。外㈣的成分 通常為去角質侧的水楊酸與果酸、抑制皮脂腺過多分泌的維生素六 或其衍生物，以及抑制初油酸桿菌繁殖的抗生素製劑等。 雖，利用抗生素去除初油酸桿菌以治療青春痘的方法已被廣為使 用’但是過度使用抗生素已經引發初油酸桿菌產生抗藥性的問題，造 成青春痘患者即使塗抹抗生素藥f，卻依然無法阻止#春痘的產生。 品 二 另卜若&針對初’由酸桿菌的生長進行抑制，通常就能達到預防 瘦症的功效。目前市面上針對初歸««效應的洗面乳或面膜產 患通:都是添加抗氧化複合物，如過氧化苯酿或是化學殺菌成分如 氣㈣。但這些藥劑亦同樣都可能產生副作用，造成皮膚過敏或發 201038293 炎’對人體健康具有潛在的危險性。過氧化苯醯在醫療上是用來處理 普通粉刺時常見的藥物》雖然，目前沒有足夠證據顯示為致癌物品， 在低濃度下也不會有過度危害健康的症狀，但對皮膚則可能造成不適 應並產生刺激感，甚至引起過敏反應。施用於臉上做為青春痘治療藥 水，臨床案例上曾導致有人造成紅斑及水腫或發炎等症狀發生，也對 眼睛造成刺激作用。如不小心食入則能造成腹痛、噁心、嘔吐等症狀。 : 在慢性反應病情方面、長期反覆暴露在過氧化苯醯中則可能導致過敏 性皮膚疹、或是對某些人造成氣喘加重等症狀。 【發明内容】 由於針對青春痘患者所開發的醫療性化粧品或藥物大多添加抗生 〇 素，因此開發出高度去菌能力、高安全性、低敏感性且無抗藥性的其 他抗菌成分不但為消費者所期望，且具有廣大的市場性。 抗菌胜肽(antibacterial peptides)是生物體抵禦細菌的天然屏障，它 們廣泛地存在於細菌、植物與無脊椎動物的身體中，如天蠶、果绳等 昆蟲’以及青蛙、螃蟹，甚至在脊椎動物内都有發現抗菌胜肽的存在。 被發現於蠶蜗體液中的胜肽是一種很有效的抗菌胜肽，對革蘭氏陰性 細菌具有很尚的抗菌活性，並且不會對真核細胞（eukaryotic cell)造 成細胞融釋作用。研究報告指出，有超過六種以上的胜狀包含在 cecropins類中’分別為抗菌胜肽A, B，C，D，E及F，它們都是由35至 39個胺基酸片斷所組成’並且擁有極類似的序列，其中cecropin B(CB) 被認為具有最強的抗菌活性。它是由35個胺基酸片段所組成的胜肽 (亦稱35-胜肽）’且具備陽離子的特性，其特殊二度空間結構與組成胺 基酸序列中的陽離子片段，是決定殺菌能力的重要因子。經發明人多 年來致力於研究35-胜肽及其相關之各類衍生物的抗菌機制。透過抗 菌評估、序列重組、抗菌形態觀察、細胞膜與胜肽間界面作用力以及 環境中各種物理及化學因素對抗菌作用的影響所得結果，已對35-胜 肽破壞細胞膜機制有很深入的了解。根據發明人之研究及其他的學術 報導指出，胜狀的殺卤機制主要是藉著35-胜狀與細菌的細胞膜作 用來殺死細菌。當35-胜肽在接近細菌表面時，會形成特殊的二級結 構，能刺破細胞膜使膜内水分子失去平衡，膜内過高的滲透壓導致細 201038293 胞漿質的外洩，最後使細菌洞零而死亡。 t為3域㈣人駐料财會造成危#，故適合發展為新型 …、田1i作用的抗_，而55·胜肽這-類的產品還未曾發明用於添加在 化粧保養品上，可以在清潔去菌魏康前提下，達到護雜養的功效， 此為本發明之一目的。 私胜肽特別對革蘭氏陰性細菌有很高的抗菌活性，殺菌範圍也很 廣泛，包括克雷柏氏肺炎桿菌、大腸桿菌、及賴桿菌等細菌的殺菌 效能都有科學上的報$。但是目前並沒有卩3域狀來抑制初油酸桿 菌（屬革蘭氏陽性菌）生長的科學性報導，亦無用此A胜肽添加於 化妝保養品’來作為預防青春痘之應用性報導。本發明是第一次提供 〇 抗菌^55·胜肽對初油酸桿菌具有殺菌效應的證據，也因此提供此J5-胜 肽添加於化粧保養品，作為預防青春癌之可能。 【實施方式】 為對本發明所述以抗菌35-胜肽添加於化粧保養品以抑制初油酸 才干菌作為預防青春痘目的及功效進一步的了解，謹以實驗測試說明 之0 k材料與方法 1-1細菌與材料 初油酸桿菌 CPrapomlacim’ww iicwas)菌株採用 BCRC 10723 〇 (=ATCC 6919) * a RCM (Reinforced Clostridial Medium, OXOID CM 149) 液態培養基在厭氧箱(内含厭氧包AnaeroPack® . Anaero, MITSUBISHI GAS CHEMICAL CO., INC.)内以 36°C 靜置培養。 其他殺菌化學成分或市售抗痘產品： 過氧化苯酿（benzolic peroxide ) OXY10 (含 10% benzolic peroxide), The Mentholatum Co·201038293 VI. Description of the Invention: [Technical Field] The present invention relates to an antibacterial peptide (35-peptide) added to all skin care products, cleaning products, cosmetics and masks for suppressing the initial Oleic acid bacillus, in addition to the use of acne prevention, 'Staphylococcus epidermidis, which is common to Pibin, I, E. co.i, green, common pathogens Pseudomonas aeruginosa and pneumoniae (feet / e6 coffee this newcomer / ae), can also achieve sterilization, antibacterial and reduce the harm of bacteria and interference with maintenance ingredients 'to make moisturizing, whitening, and even anti-aging effect Go deeper and more thoroughly to maintain healthy and bright skin. [Prior Art] The occurrence of acne is the result of a combination of many factors. In addition to the excessive secretion of sebum and hyperkeratosis of the hair follicle, resulting in pore blockage, the proliferation of oleic acid bacteria in the hair follicle is also one of the causes of acne. The skin is inflamed as a result of the combined effects of these factors. P. eutropha is a Gram-positive anaerobic bacterium that lives in the hair follicle of the skin. Although it can coexist peacefully with the skin, it is also the main cause of youth (4). The oleic acid bacteria will block the sebaceous passage after reproduction, and the result of the enzyme produced by metabolism will stimulate the keratinization of the hair follicle and cause skin inflammation. The method of anti-acne is not to prevent and treat ^ treatment of acne usually should be applied according to the doctor's instructions (4) and with the mouth of the double-family. The components of the outer (four) are usually salicylic acid and fruit acid on the exfoliating side, vitamin VI or its derivatives which inhibit excessive secretion of sebaceous glands, and antibiotic preparations which inhibit the growth of oleic acid bacteria. Although the use of antibiotics to remove oleic acid bacteria to treat acne has been widely used 'but excessive use of antibiotics has caused the problem of resistance to oleic acid bacteria, causing acne patients to smear antibiotics f Block #春痘的产生。 Product 2 In addition, the anti-inhibition of the growth of acid bacteria can usually achieve the effect of preventing skinny. At present, the facial cleanser or mask for the initial «« effect is produced by adding antioxidant compounds such as benzene peroxide or chemical sterilization ingredients such as gas (4). However, these agents may also cause side effects, causing skin irritation or inflammation. This may pose a potential risk to human health. Phenyl peroxide is a common drug used to treat common acne. Although there is not enough evidence to show that it is a carcinogen, there is no excessive health hazard at low concentrations, but it may cause no skin. Adapts and produces a stimulating sensation and even causes an allergic reaction. Applied to the face as a acne treatment syrup, clinical cases have caused erythema and edema or inflammation and other symptoms, but also cause irritation to the eyes. If you accidentally eat it, it can cause abdominal pain, nausea, vomiting and other symptoms. : In the case of chronic reaction, long-term repeated exposure to benzoquinone may cause allergic skin rash or symptoms such as increased asthma in some people. SUMMARY OF THE INVENTION Since most of the medical cosmetics or drugs developed for acne patients are added with antibiotics, other antibacterial ingredients with high degumming ability, high safety, low sensitivity and no drug resistance have been developed not only for consumers. Expected, and has a large market. Antibacterial peptides are natural barriers against bacteria. They are widely found in bacteria, plants and invertebrates, such as insects such as silkworms and fruit ropes, as well as frogs, crabs and even vertebrates. The presence of antibacterial peptides has been found in the interior. The peptide found in the silkworm snail fluid is a very effective antibacterial peptide, has a very strong antibacterial activity against Gram-negative bacteria, and does not cause cell-melting effect on eukaryotic cells. According to the study, more than six winners are included in the cecropins category 'antibacterial peptides A, B, C, D, E and F, which are composed of 35 to 39 amino acid fragments'. And has a very similar sequence, of which cecropin B (CB) is considered to have the strongest antibacterial activity. It is a peptide consisting of 35 amino acid fragments (also known as 35-peptide) and has cationic properties. Its special second-degree structure and cationic fragments in the amino acid sequence determine the bactericidal ability. An important factor. The inventors have been working for many years to study the antibacterial mechanism of 35-peptide and its related derivatives. Through the results of antibacterial evaluation, sequence recombination, antibacterial morphology observation, interfacial interaction between cell membrane and peptide, and the effects of various physical and chemical factors on the antibacterial effect in the environment, the mechanism of disruption of cell membrane by 35-peptide has been well understood. According to the inventor's research and other academic reports, the halogen-killing mechanism of the win is mainly used to kill bacteria by the 35-win and bacterial cell membranes. When the 35-peptide is close to the surface of the bacteria, it will form a special secondary structure, which can pierce the cell membrane and make the water molecules in the membrane out of balance. The excessive osmotic pressure in the membrane leads to the leakage of the fine cytoplasm of 201038293, and finally The bacteria hole died and died. t is a domain of 3 (4) people who are responsible for the financial crisis, so it is suitable for the development of new..., the resistance of the field 1i, and the product of the 55-peptide has not been invented for the addition of makeup products. It can be achieved under the premise of cleaning and removing Weikang, which is an object of the invention. The proprietary peptides have high antibacterial activity against Gram-negative bacteria, and the range of sterilization is also wide. The bactericidal efficacy of bacteria such as Klebsiella pneumoniae, Escherichia coli, and Lactobacillus has been scientifically reported. However, there is currently no scientific report on the growth of P. oleifera (a gram-positive bacterium), and it is not useful to add this A-peptide to cosmetic care products as an application report for the prevention of acne. The present invention is the first to provide evidence that the antibacterial compound 55 has a bactericidal effect on oleic acid bacteria, and thus provides the J5-peptide added to the cosmetic skin care product as a possibility of preventing juvenile cancer. [Embodiment] In order to further understand the purpose and efficacy of the antibacterial 35-peptide added to the cosmetic skin care product according to the present invention to inhibit the priming oleic acid bacteria, the experimental results indicate that the 0 k material and method 1 -1 Bacterial and material strains of P. primata CPrapomlacim'ww iicwas) using BCRC 10723 〇 (=ATCC 6919) * a RCM (Reinforced Clostridial Medium, OXOID CM 149) Liquid medium in anaerobic tank (containing AnaeroPack® anaerobic package) Anaero, MITSUBISHI GAS CHEMICAL CO., INC.) was statically cultured at 36 °C. Other bactericidal chemical ingredients or commercially available acne products: benzolic peroxide OXY10 (containing 10% benzolic peroxide), The Mentholatum Co·
Stiemycin™(含 2¾ Erythromycin 於酒精溶液中)，Stiefel Laboratories Pte Ltd 201038293 1-2抗菌活性分析 靜置培養於液態RCM培養基約2-'3天的菌液(菌數達108)，作系列稀 釋後’取含有約103菌數的稀釋液1〇μ1，加入200μ1含有胜肽或測試樣品 的無菌水中’靜置30分鐘後將溶液均勻塗抹於RCM固態培養基上(添加 1.5% DifcoTM Agar)，經36°C厭氧培養5天後，計數培養基上的菌落數(第 二圖）。 2.結果輿討論 ^ 2·1以-胜肽對初油酸桿菌具有優良的殺菌效應 如第三圖的抗菌測試所示’ 200μ1中含有〇.〇2pg(濃度=〇.l 的 CB(35-胜肽）’在30分鐘内足以將起始菌數1498的初油酸桿菌殺死5〇0/。 以上(存活菌數剩下577)。當CB的浪度增加時，初油酸桿菌的存活數具有 繼續下降的趨勢(第三圖）。0.1 pg/ml的CB的濃度為0.0026 μΜ，足以顯示 CB對於初油酸桿菌的優良殺菌效應。 ϋ胜肽（CB)與市售抗痘產品及抗氧化複合物的殺菌效果比較 Q 因為目前市面上針對初油酸桿菌殺菌效應的洗面乳或面膜產品，通常 都是添加抗氧化複合物’如過氧化苯醯或是添加抗生素，因此我們分別以 濃度各5%與10%的過氧化苯醯、添加1〇%過氧化苯醯的市售乳膏〇χγι〇 (濃度各為16%與50%)、以及添加抗生素與酒精成份的StiemydnTM (濃度 各為50%與100%)塗液作測試比對(第四圖）。結果顯示〇 251 25 μ§Λη1的 CB對菌數約1000的初油酸桿菌，在3〇分鐘内的殺菌效果和上述的葯品 同樣優良。 2-3 35-胜肽(CB)添加於化粧保養品的濃度(以面膜為例） 201038293 針對一般人臉上的細菌教(105-106)，以及配合敷面膜的時間15_2〇分 鐘’設計出最適當的35-胜肽添加濃度、以去除臉上大部分的細菌。若是 作為預防青春痘的用途，用在臉上初油酸桿菌尚未增生時，其菌數勢必低 於臉上總細菌數。若以35-胜肽添加於化粧保養品(例如面膜)以抑制初油酸 桿菌的增生’來達到預防青春痘的目的，其濃度若足以殺死菌數1〇5_1〇6的 初油酸桿菌，便能達到預防的功效。 以第三圖與第四圖的測試結果，0.2ml濃度為0.25-1.25 pg/ml的&胜 ¢) 肽在30分鐘内可以有效殺死多於菌數1000的初油酸桿菌。以面膜為例， 若含有20ml精華液，這樣的胜肽濃度(0.25-1.25 pg/ml)其去菌效應可達 105-106個細菌’也就能達到以抑制初油酸桿菌來預防青春痘。 實驗結果證實，35-胜肽對於這種皮膚毛孔内的厭氧細菌，依然具有優 良的殺菌效果。而天然35_胜肽’此抗菌胜肽所特別具有的高安全性與高 穩定性，將特別適合添加於臉部化妝品之内，它將可讓消費者在進行臉部 肌膚保養與美白抗皱的同時，亦能藉由所添加的天然抗菌胜肽成分發揮去 〇 菌效用，進而抑制住初油酸桿菌的生長，達到預防痘痘的發生。 【圖式簡單說明】 第一圖：35-胜肽的電腦模擬結構圖 第一圖：3_5-驗(CB)對於初油酸桿菌的殺菌能力測試（實驗流程示意圖）。 取含有約1〇3菌數的稀釋液，加入200μ1含有35-胜肽或測試樣 品的無菌水中，靜置30分鐘後將溶液均勻塗抹於RCM固態培養 基上，經36t厭氧培養5天後，計數培養基上的菌落數。 第三圖：35-胜肽(CB)對於初油酸桿菌的殺菌效應。 201038293 200μΙ中含有〇.〇2pg (濃度=0.1 gg/m!)的35_胜肽，在3〇分鐘 内足以將初油酸桿菌殺死50%k以上。當35-胜肽的濃度增加時，初 油酸桿菌的存活數具有繼續下降的趨勢。 第四圖：35-胜狀(CB)與市售抗痘產品及抗氧化複合物對於初油酸桿菌的殺 菌效果比較。 比較過氧化苯醯、市售乳膏OXY10、以及添加抗生素與酒精 成份的Stiemycin™，35·胜肽對初油酸桿菌具有等同的殺菌效果。 【主要元件符號說明】StiemycinTM (containing 23⁄4 Erythromycin in alcohol solution), Stiefel Laboratories Pte Ltd 201038293 1-2 Antimicrobial Activity Analysis After standing in liquid RCM medium for about 2- to 3 days of bacterial solution (number of bacteria up to 108), after serial dilution ' Take 1 μμ1 of the dilution containing about 103 bacteria, add 200 μl of sterile water containing the peptide or test sample. After standing for 30 minutes, spread the solution evenly on RCM solid medium (add 1.5% DifcoTM Agar), through 36 After 5 days of anaerobic culture at °C, the number of colonies on the medium was counted (second image). 2. Results 舆 Discussion ^ 2·1 has excellent bactericidal effect on oleic acid bacteria as shown in the antibacterial test of the third figure '200μ1 contains 〇.〇2pg (concentration = 〇.l of CB (35 -Peptide)' is sufficient to kill 5 〇0/ of the initial bacterial count of 1498 in 30 minutes. Above (the remaining number of viable bacteria is 577). When the wave of CB increases, oleic acid bacillus The number of survivors has a tendency to continue to decline (figure 3). The concentration of CB at 0.1 pg/ml is 0.0026 μΜ, which is sufficient to show the excellent bactericidal effect of CB on oleic acid bacillus. ϋ peptide (CB) and commercially available acne Comparison of bactericidal effects of products and antioxidant complexes Q Because the current facial cleanser or mask products for the bactericidal effect of oleic acid bacteria are usually added with antioxidant complexes such as benzoquinone or antibiotics, we Commercially available creams 〇χγι〇 (16% and 50% each) with 5% and 10% benzoyl peroxide and 〇1% benzoquinone, respectively, and StiemydnTM with antibiotics and alcohol (The concentration is 50% and 100% each) the coating liquid is tested for comparison (fourth figure). The result shows 〇251 2 The CB of 5 μ§Λη1 has the same bactericidal effect as the above-mentioned drugs in the bacillus bacillus with a bacterial count of about 1000. 2-3 35-peptide (CB) is added to the concentration of the cosmetic care product ( Taking the mask as an example) 201038293 Designed the most appropriate 35-peptide addition concentration for the bacteria teaching (105-106) on the face of the general person and the time of applying the mask for 15_2 minutes to remove most of the bacteria on the face. If it is used for the prevention of acne, when the oleic acid bacillus has not yet proliferated on the face, the number of bacteria is bound to be lower than the total number of bacteria on the face. If 35-peptide is added to the cosmetic care products (such as mask) to suppress the initial The proliferation of oleic acid bacillus can achieve the purpose of preventing acne, and if the concentration is sufficient to kill the oleic acid bacteria of 1〇5_1〇6, the preventive effect can be achieved. The test of the third and fourth figures As a result, 0.2 ml of a & peptide having a concentration of 0.25 to 1.25 pg/ml can effectively kill more than 1000 bacillus eutrophicum in 30 minutes. Taking the mask as an example, if the concentration of the peptide (0.25-1.25 pg/ml) has a peptide concentration of 0.25-1.25 pg/ml, the bacteriostatic effect can reach 105-106 bacteria, which can also be achieved to inhibit the oleic acid bacillus to prevent acne. . The experimental results confirmed that the 35-peptide still has an excellent bactericidal effect on the anaerobic bacteria in the pores of the skin. The natural 35_ peptide 'this antibacterial peptide has a special high safety and high stability, it will be especially suitable for adding to the face cosmetics, it will allow consumers to carry out facial skin care and whitening and anti-wrinkle. At the same time, it is also possible to exert the antibacterial effect by the added natural antibacterial peptide component, thereby inhibiting the growth of the oleic acid bacillus and preventing the occurrence of acne. [Simple diagram of the diagram] The first picture: computer simulation structure diagram of 35-peptide. First diagram: 3_5-test (CB) bactericidal ability test for oleic acid bacteria (schematic flow diagram). Take a dilution containing about 1〇3 bacteria, add 200μ1 of sterile water containing 35-peptide or test sample, let stand for 30 minutes, then apply the solution evenly on RCM solid medium, after anaerobic culture for 36 days, Count the number of colonies on the medium. Figure 3: Sterilization effect of 35-peptide (CB) on oleic acid bacteria. 201038293 The 35-peptide containing 〇.〇2pg (concentration = 0.1 gg/m!) in 200μΙ is sufficient to kill 50% k or more of oleic acid bacteria in 3 minutes. As the concentration of the 35-peptide increases, the number of survival of the eutrophic acid bacillus continues to decline. Figure 4: Comparison of the bactericidal effect of 35-win (CB) and commercially available anti-acne products and antioxidant complexes on oleic acid bacteria. Comparing benzoquinone, commercially available cream OXY10, and StiemycinTM with antibiotics and alcohol, 35 peptides have equivalent bactericidal effects on oleic acid. [Main component symbol description]