TW201228676A - Pharmaceutical composition for skin whitening - Google Patents

Abstract

The present invention discloses a pharmaceutical composition for skin whitening, which contains an effective amount of melanogenesis inhibitor selected from the following groups: Homochlorcyclizine, Danazol, and their combinations. The present invention also discloses the uses of the pharmaceutical composition in the preparation of a skin whitening products. Pharmaceutical composition of the present invention may further contain adjuvants commonly used in cosmetics or dermatology fields, such as: hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, screening agents, pigments, chelating agents, odor absorbers, and dyes, etc.

Description

201228676 六、發明說明： 【發明所屬之技術領域】 本發明疋有關於-種用於皮膚美白的藥學組成物，以 及該藥學組成物在製備一皮膚美白產品上的用途。 【先前技術】 目前醫療臨床及化妝品市場上所使用的皮膚美白藥效 成分’大多具有生理毒性，例如對苯二盼（HydR)quin〇ne) 、熊果素（Arbutin)與麴酸（K〇jic八⑽等，在使用上有 其限制❿王球皮膚美白醫療及化妝品市場又極其龐大， 因此各大醫學研究單位與化妝品公司莫不積極從事新一代 皮膚美白藥效成份的研究開發。 然而’每一個冑藥開發必經相_的人體生理吸收盘代 謝、誘導突變性、致癌性、急毒性等安全性試驗，所需的 費用與時程之成本十分龐大，甚至高於當初新藥的有效性 試驗開發成本。因此，如何能夠避開龐大成本需求及十分 費時的人體安全性測試，也就是，只需針對某一化合物進 订皮膚美白有效性試驗，例如體外細胞黑色素生成抑制試 驗等，而於證實該化合物具有人體皮膚美白療效之後，便 可很快實際應用於產業界中，是相關業界努力方向。 有蓉於此，舊藥新用是一種可快速達到上述目的之方 二二於市售之學名藥都已經通過人體試驗，可使用於人 體治療疾病。因此，口 i脸、工# ，、要將這些學名藥進行完整的 白有效性試驗，確認其具有皮膚美白藥效，即可报快實p 應用於皮膚美白臨床醫療與美白化妝品產業界t q㈣ 201228676 新藥一樣重新進行安全性試驗。 已知’同氣克利定（Homochlorcyclizine，以下簡稱HC) 是一種經衛生署核定且醫界常用之H1抗組織胺藥劑，它主 要被用來治療過敏以及由感冒所引起的不適症狀。另外， 單那若（Danazol，以下簡稱Dz)是一種合成的17 α-乙炔基 睪固酮（17 a -ethinyl testosterone)，它是 FDA (U.S. Food and201228676 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a pharmaceutical composition for skin whitening, and the use of the pharmaceutical composition for preparing a skin whitening product. [Prior Art] At present, the skin whitening medicinal ingredients used in the medical clinical and cosmetic market are mostly physiologically toxic, such as HydR quin〇ne, Arbutin and citric acid (K〇jic VIII). (10) Etc., there are restrictions on its use. The skin whitening medical and cosmetic market is extremely large. Therefore, major medical research units and cosmetics companies are not actively engaged in the research and development of a new generation of skin whitening medicinal ingredients. However, 'every 胄The development of drugs must be based on the human body's physiological absorption disk metabolism, induced mutation, carcinogenicity, acute toxicity and other safety tests, the cost and time cost of the process is very large, even higher than the effectiveness of the original drug development test cost Therefore, how to avoid the huge cost requirements and the very time-consuming human safety test, that is, only to test the skin whitening effectiveness test for a certain compound, such as in vitro cell melanogenesis inhibition test, etc., and confirm the compound After having the skin whitening effect of human body, it can be quickly applied to the industry, which is related industry. The direction of hard work. There is a new use of old medicine, which is a kind of prescription that can quickly achieve the above purpose. The commercially available scientific name medicines have passed the human test and can be used for human body treatment of diseases. Therefore, mouth i face, work# , to carry out a complete whiteness test of these generic drugs, confirm that they have skin whitening efficacy, can report fast p applied to skin whitening clinical medical and whitening cosmetics industry t q (4) 201228676 new drug re-testing safety Homochlorcyclizine (H) is a H1 antihistamine agent approved by the Department of Health and commonly used in the medical profession. It is mainly used to treat allergies and discomfort caused by colds. Danazol (hereinafter referred to as Dz) is a synthetic 17 α-ethinyl testosterone, which is the FDA (US Food and

Drug Administration)在1970年初期時即認可的一種用於治 療子宮内膜異位（endometriosis)的第一線用藥。但是迄今尚 未有關於HC與Dz具有皮膚美白功效的相關研究報導被提 出。 經研究’申請人意外地發現單那若與同氣克利定具有 抑制黑色素生成的能力，因而被預期可供用於製造皮膚美 白產品（skin whitening products)，例如化妝品與醫藥品。 【發明内容】 於是，在第一個方面，本發明提供一種用於皮膚美白 的藥學組成物，其包含有一有效量之選自下列群組中的藥 物：同氣克利定（H〇m〇Chl0rCyCiizine)、單那若（Danaz〇1)，以 及它們的組合。 在第二個方面，本發明提供一種如上所述的藥學組成 物供用於製備一皮膚美白產品的用途。 【實施方式】 有關本發明之前述及其他技術内容、特點與功效，在 以下配合參考圖式之一個較佳實施例的詳細說明中將可 清楚的呈現。 201228676 要被瞭解的是：除非另外有所定義，在本文中所使用 的所有技術性與科學術語具有熟悉本發明所屬技藝的人士 所共同瞭解的意義。 為了開發具有皮膚美白效用的新產品，申請人嘗試從 般醫院所使用的學名藥當中挑選了 201種藥物作為篩選 試驗的標的藥物。接著，中請人利Μ B16黑色素腫瘤細 胞與斑馬魚活體胚胎來進行皮膚美白藥效成份的篩選研究 。經由實驗結果發現，在受測試的2〇1種藥物中同氯克 利定（Homochlorcyclizine)以及單那若（Danaz〇i)具有抑制黑 色素生成的能力，亦即具有黑色素生成抑制劑 (melanogenesis inhibitor)之活性。 於是，本發明提供一種用於皮膚美白的藥學組成物， 其包含有一有效量之選自於下列群組中的黑色素生成抑制 劑.同氣克利定（Homochlorcyclizine)、單那若（Danazol)， 以及它們的組合。 依據本發明的藥學組成物可被應用於製造下列任一種 產品：皮膚美白用化妝品以及皮膚美白用醫藥品。 依據本發明的藥學組成物可與一皮膚外用劑合併使用 。如本文中所用的’ “皮膚外用劑”此術語意指一通常在化妝 品或醫樂品中被使用的外用成份，包括，但不限於：其他 的美白劑、保濕劑 '抗氧化劑、紫外線吸收劑、介面活性 劑、增稠劑、色料以及皮膚營養劑等等。 根據本發明的藥學組成物亦可進一步包含有在化妝品 或皮膚科領域中經常被使用的佐劑，例如：親水性或親脂 201228676 性的膠凝劑（gelling agents)、親水性或親脂性的活性劑 (active agents)、保存劑（preserving agents)、抗氧化劑 (antioxidants)、溶劑、香料（fragrances)、填料（fillers)、遮 蔽劑（screening agents)、色料、螯合劑（chelating agents)、 氣味吸收劑（odor absorbers)以及染料等。各種佐劑是以該領 域照慣例所考慮的數量而被使用。 依據本發明的藥學組成物可以被製造成任一種可接受 的藥物或化妝品形式，包括，但不限於：水性溶液、水-醇 溶液或油性溶液、呈水包油型（〇il-in-water)或油包水型 (water-in-oil)或複合型之乳劑（emulsions)、水性或油性凝膠 、乳霜（cream)、油膏（ointment)、乳（milk)、乳液（lotion)、 乳漿（serum)、軟膏（paste)、泡沫（foam)或分散液（dispersion) 等等。 特別地，依據本發明的藥學組成物可以被製成下列任 一種化妝品形式：化妝水（tonic water)、唇彩（lip colors)、 粉底（foundations)、膚乳（milk)、面霜（cream)、面膜（masks) 、凝膠（gel)、氣霧（aerosol)、乳狀的乳液（milky lotions)、慕 斯（mousse)、分散液（dispersions)、乳霜（cream)、衛浴用水 液（toilet waters)、貼布（packs)與清潔劑（cleansings)，以及卸 妝用的清潔乳、洗面皂（wash soap)等等。 依據本發明的藥學組成物亦可進一步包含有其他已知 對於美白有幫助的美白劑或其他活性成分，例如：酪胺酸 酵素抑制劑（諸如維生素C、熊果素、麴酸、槲皮酮以及兒 茶素等）、抗痘劑、抗菌劑、鎮痛劑、麻醉劑、抗皮膚發炎 201228676 劑、止癢劑、抗發炎劑、抗過角化劑（antihyperkeratolytic agents)、抗乾皮膚劑（anti-dry skin agents)、抗汗劑 (antipsoriatic agents)、抗老化劑、抗敵劑（antiwrinkle 、. agents)、抗皮脂溢出劑（antiseborrheic agents)、美黑劑（self tanning agents) 、 傷口 治療劑 （wound-healing agents) 、 皮質 類固醇（corticosteroids)或激素（hormones)等等。 【實施方式】 較佳實施例之詳細說明 # 本發明將就下面的實施例來做進一步說明，但應瞭解 . 的是，該等實施例僅是供例示說明用，而不應被解釋為本 發明的實施上的限制。 【實施例-活體外試驗】 A.生物材料 鼠 B16 黑色素瘤細胞（Mouse B16 melanoma cells，4A5) 是購自食品工業發展研究所的生物資源保存及研究中心 (BCRC of FIRDI)。 ^ 這些鼠B16黑色素瘤細胞是培養在含有1 〇% (v/v) fetal bovine serum 的 Dulbecco’s modified Eagle’s medium (DMEM)中，並培養於24-well平盤上，每一平盤含有約 5xl05個細胞，並將該等平盤置於含5% C02之37°C培養箱 . 中培養。經過一天（24小時）的培養後，進行分組： 1. 控制組1 :完全未以刺激藥物和黑色素抑制劑處理。 2. 控制組2 :僅以特定濃度之刺激藥物處理。 3. 實驗組：加入特定濃度的刺激藥物與不同濃度之黑 201228676 色素抑制劑。 4. 對照組1 :加入特定濃度之刺激藥物與特定濃度之熊 果素（arbutin，目前常見之美白藥物）。 5. 對照組2 :加入特定濃度之刺激藥物與特定濃度之抗 組織胺藥劑。 前述刺激藥物為 10 nM a-MSH(a-melanocyte stimulating hormone)或 100 μΜ IBMX(3-isobutyl-methylxanthine)，其中，α-MSH是黑色素刺激生成荷爾蒙 ，可提高細胞之酪胺酸酶活性，而增加黑色素細胞之黑色 素生成量。而IBMX可藉由活化cAMP，提高酪胺酸酶活性 ，增加黑色素細胞之黑色素生成量。前述黑色素抑制劑分 別為HC與Dz。前述抗組織胺藥劑包含兩種H1抗組織胺藥 劑，分別為hydroxyzine與cetirizine，及兩種H2抗組織胺 藥劑，分別為cimetidine與ranitidine。完成上述分組處理 後，各組細胞再繼續培養兩天（48小時），然後取出測定黑色 素含量（melanin content) 0 B.黑色素生成抑制試驗 在鼠B16黑色素瘤細胞經過前述兩天（48小時）的培養 後，取出鼠B16黑色素瘤細胞並以PBS沖洗兩次，以數位 相機對這些球狀鼠B16黑色素瘤細胞進行攝影，然後，將 這些鼠B16黑色素瘤細胞置於溶解缓衝溶液（lysis buffer)( 包含 20 mM sodium phosphate (pH 6.8) and 1% Triton X-100) 中，並置入Dounce homogenizer中並進行30次衝程，將細 胞破壞並均質化，然後，對均質化後之物質進行離心作業 201228676 (15000xg，15 min)。最後取出離心液中之黑色素粒（meianin pellets)溶解在含有 20% DMSO 的 1 N NaOH (95。〇 中 1 小時，並測定其在490 nm的吸光值，並以標準的合成黑色 素來測定這些鼠B16黑色素瘤細胞之黑色素粒的黑色素含 量。 而上層懸浮液中的蛋白質含量是使用Bradf〇rd assay進 行測疋，並以牛血清白蛋白（b〇vine serum aibumin, BSA)作 為蛋白質標準品，前述黑色素含量會依據測定之蛋白質含 置進行校準（adjusted)。 其測試結果如圖1、2所示，鼠B16黑色素瘤細胞經過 cx-MSH或IBMX的刺激後，其黑色素含量會明顯增加，然 而，當鼠B16黑色素瘤細胞同時以HC &a_MSH進行處理 或同時以HC及IBMX時，黑色素量會明顯降低，且當hc 濃度達到4 μΜ時，黑色素含量會降低到近似未經刺 激的細胞’顯示H C對於㈣U B i 6黑色素瘤細胞的黑色素 生成有正相關。 同樣的田同時以Dz對鼠b 16黑色素瘤細胞進行處理 時’黑色素量都會明顯降低’且當Dz濃度達2〇 _時，鼠 黑色素瘤細胞的黑、&素含#已遠低於未經a_MSH或 IBMA刺激時的程度，顯示Dz對於抑制黑色素具有極佳之 效果。 由上述結果發現’原本作為抗組織胺藥物的HC與原本 作為治療子宮内膜異位藥4㈣Dz，對於黑色素瘤細胞的黑 色素生成具有抑制功效，故本案發明人推測其應可作為— 201228676 種黑色素抑制劑，而可用於皮膚美白之用途。 為進一步確認HC與Dz可用以作為黑色素抑制劑，並 了解其而抑制黑色素細胞之黑色素生成的作用機轉，乃繼 續進行細胞毒性試驗、細胞酪胺酸酶活性與酪胺酸酶蛋白 質表現...等試驗。 C. MTT assy 以MTT assy測試HC與Dz分別對鼠B16黑色素瘤細 胞存活率（viability)的影響。當鼠B16黑色素瘤細胞完成上 述分組後之兩天（48小時）之培養，將原培養液換掉，改用1 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide)溶液混合於 phosphate-buffered saline (PBS)進行培養2小時，然後，再將培養液換掉，改 加入 DMSO(dimethyl sulfoxide，Sigma Chemical Co.)，接著 ，以分光光度計（spectrophotometer)測定溶出的formazan結 晶在570 nm的吸光值。 D. 與市面常見之美白藥物（熊果素）的黑色素生成抑制 效果進行比較 比照上述『A.生物材料/黑色素生成抑制試驗』之實驗 方式，進行HC、Dz與熊果素分別對於黑色素瘤細胞之黑色 素生成的抑制實驗 E. 量測細胞路胺酸酵（cellular tyrosinase)活性與黑色 素想的路胺酸梅（melanosomal tyrosinase)活性 1.測定細胞酪胺酸酶活性： 分別將經過刺激藥物及HC、Dz或熊果素等黑色素抑制 10 201228676 劑處理過的鼠B16黑色素瘤細胞、經過刺激藥物但未經黑 色素抑制劑處理的鼠B16黑色素瘤細胞，及完全未經刺激 藥物與黑色素抑制劑處理之鼠B16黑色素瘤細胞，分別置 於 20 mM sodium phosphate (pH 6.8)、1% Triton X-100 與 1 mM PMSF構成之4 °C試劑中，然後，以Dounce homogenizer進行30次衝程，破壞細胞並均質化。接著， 加入清潔劑（detergen)，解開鍵結於黑色素體（melanosome) 的赂胺酸酶（membrane-bound tyrosinase)，然後將溶出物 進行離心作業（15000xg，15分鐘）。取出離心上層懸浮液作 為接下來使用之第一酵素來源，此上層懸浮液中的蛋白質 含量是以Bradford assay進行測定，並以BSA作為待白質 標準品。 酪胺酸酶活性測試方式如下： 製備1 ml的反應混合物，此反應混合物包含50 mM phosphate buffer (pH 6.8)、2.5 mM L-dopa，及 500 Kg 的蛋 白質（取自前述第一酵素來源），將此反應混合物於37 °C環 境下培養15分鐘。接著，測定此反應混合物在475 nm的 吸光值，藉以測定出dopachrome生成量。 一個單位的酪胺酸酶活性是定義為：在1分鐘内產生1 μ mole dopachrome所需的總酵素量，而本反應中的 dopachrome 量是以 Lambert-Beer Law 進行计算’ dopachrome 的克分子消失係數（molar extinction coefficient) 為3600 M·1.cm_1，並以此反應中的蛋白質含量來將酷胺酸 酶活性標準化。 201228676 2.測定黑色素體的酪胺酸酶活性： 製備第一酵素來源’取出經過刺激藥物與經過hc或 Dz等黑色素抑制劑處理過的鼠Bi6黑色素瘤細胞、經過刺 激藥物但未經黑色素抑制劑處理的鼠B16黑色素瘤細胞， 及元全未經刺激樂物與黑色素抑制劑處理之鼠B16專色素 瘤細胞’並分別以PBS沖洗兩次。然後，將這些鼠b丨6黑Drug Administration) A first-line medication approved for the treatment of endometriosis in the early 1970s. However, relevant research reports on the skin whitening effect of HC and Dz have not been reported so far. According to the study, the applicant unexpectedly discovered that the combination of succinyl and dexamethasone has the ability to inhibit melanin production and is therefore expected to be useful for the manufacture of skin whitening products such as cosmetics and pharmaceuticals. SUMMARY OF THE INVENTION Accordingly, in a first aspect, the present invention provides a pharmaceutical composition for skin whitening comprising an effective amount of a drug selected from the group consisting of homogenidine (H〇m〇Chl0rCyCiizine) ), Danaruo (Danaz〇1), and combinations thereof. In a second aspect, the invention provides a use of a pharmaceutical composition as described above for the preparation of a skin whitening product. The above and other technical contents, features, and advantages of the present invention will be apparent from the following detailed description of the preferred embodiments. 201228676 It is to be understood that all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of the invention, unless otherwise defined. In order to develop a new product with skin whitening effect, the applicant tried to select 201 drugs from the generic drugs used in general hospitals as the target drugs for screening tests. Next, the B16 melanoma tumor cells and the zebrafish living embryos were used to screen the skin whitening ingredients. Through experimental results, it was found that Homochlorcyclizine and Danaz〇i have the ability to inhibit melanin production, that is, melanogenesis inhibitor, in the two drugs tested. active. Accordingly, the present invention provides a pharmaceutical composition for skin whitening comprising an effective amount of a melanin production inhibitor selected from the group consisting of Homochlorcyclizine, Danazol, and Their combination. The pharmaceutical composition according to the present invention can be applied to manufacture any of the following products: cosmetics for skin whitening and medicines for skin whitening. The pharmaceutical composition according to the present invention can be used in combination with a skin external preparation. The term 'skin external preparation' as used herein means a topical ingredient that is usually used in cosmetics or medical treatments, including, but not limited to, other whitening agents, moisturizers, antioxidants, ultraviolet absorbers. , surfactants, thickeners, colorants, and skin nutrients. The pharmaceutical composition according to the present invention may further comprise an adjuvant which is often used in the field of cosmetics or dermatology, for example, hydrophilic or lipophilic 201228676 gelling agents, hydrophilic or lipophilic Active agents, preserving agents, antioxidants, solvents, fragrances, fillers, screening agents, colorants, chelating agents, odors Odor absorbers, dyes, etc. Various adjuvants are used in the quantity considered by the practice in this field. The pharmaceutical composition according to the present invention may be manufactured into any acceptable pharmaceutical or cosmetic form including, but not limited to, an aqueous solution, a water-alcohol solution or an oily solution, in the form of an oil-in-water (〇il-in-water). ) or water-in-oil or composite emulsions, aqueous or oily gels, creams, ointments, milks, lotions, Serum, paste, foam or dispersion, and the like. In particular, the pharmaceutical composition according to the present invention may be formulated into any of the following cosmetic forms: tonic water, lip colors, foundations, milk, cream, mask (masks), gels, aerosols, milky lotions, mousse, dispersions, creams, toilet waters , packs and cleansings, as well as cleansing milk for cleansing, wash soap, and the like. The pharmaceutical composition according to the present invention may further comprise other whitening agents or other active ingredients known to be useful for whitening, such as tyrosinase inhibitors (such as vitamin C, arbutin, niacin, quercetin, and children). Tea, etc.), anti-acne agents, antibacterial agents, analgesics, anesthetics, anti-skin inflammation 201228676 agents, antipruritic agents, anti-inflammatory agents, antihyperkeratolytic agents, anti-dry skin agents (anti-dry skin) Agents, antipsoriatic agents, anti-aging agents, anti-wrinkle agents, agents, anti-seborborheic agents, self-tanning agents, wound-healing agents Agents), corticosteroids or hormones, etc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be further described with respect to the following embodiments, but it should be understood that these embodiments are for illustrative purposes only and should not be construed as Limitations on the implementation of the invention. [Example - In vitro test] A. Biomaterials Mouse B16 melanoma cells (4A5) were purchased from the Center for Bioresource Conservation and Research (BCRC of FIRDI). ^ These murine B16 melanoma cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 1% (v/v) fetal bovine serum and cultured on 24-well plates, each containing approximately 5 x 105 cells. And the plates were placed in a 37 ° C incubator containing 5% C02. After one day (24 hours) of incubation, groupings were performed: 1. Control group 1: Not treated with stimulating drugs and melanin inhibitors at all. 2. Control group 2: Only treated with a specific concentration of stimulating drug. 3. Experimental group: Add a specific concentration of stimulating drugs with different concentrations of black 201228676 pigment inhibitor. 4. Control group 1: Add a specific concentration of stimulant and a specific concentration of arbutin (current common whitening drug). 5. Control group 2: Add a specific concentration of stimulating drug to a specific concentration of antihistamine agent. The aforementioned stimulating drug is 10 nM a-MSH (a-melanocyte stimulating hormone) or 100 μΜ IBMX (3-isobutyl-methylxanthine), wherein α-MSH is melanin-stimulated to generate hormones, which can increase the tyrosinase activity of the cells, and Increase the amount of melanin produced by melanocytes. IBMX can increase tyrosinase activity and increase melanin production by melanocytes by activating cAMP. The aforementioned melanin inhibitors are HC and Dz, respectively. The aforementioned antihistamine agent comprises two H1 antihistamine agents, namely, hydroxyzine and cetirizine, and two H2 antihistamine agents, respectively cimetidine and ranitidine. After the above grouping treatment was completed, each group of cells was further cultured for two days (48 hours), and then the melanin content was measured. The B. melanin production inhibition test was carried out in the mouse B16 melanoma cells for the first two days (48 hours). After the culture, the mouse B16 melanoma cells were taken out and washed twice with PBS, and these spherical mouse B16 melanoma cells were photographed with a digital camera, and then these mouse B16 melanoma cells were placed in a lysis buffer. (Including 20 mM sodium phosphate (pH 6.8) and 1% Triton X-100), placed in Dounce homogenizer and subjected to 30 strokes, disrupted and homogenized the cells, and then centrifuged the homogenized material. 201228676 (15000xg, 15 min). Finally, the meinin pellets in the centrate were taken out and dissolved in 1 N NaOH (95% hydrazine) containing 20% DMSO for 1 hour, and their absorbance at 490 nm was measured, and the mice were determined by standard synthetic melanin. The melanin content of the melanin particles of B16 melanoma cells. The protein content in the supernatant suspension was measured by Bradf〇rd assay, and b〇vine serum aibumin (BSA) was used as a protein standard. The melanin content will be adjusted according to the measured protein content. The test results are shown in Figure 1 and 2. The murine content of murine B16 melanoma cells after stimulation with cx-MSH or IBMX will increase significantly. When murine B16 melanoma cells were treated with HC & a_MSH or both HC and IBMX, the amount of melanin was significantly reduced, and when hc concentration reached 4 μΜ, the melanin content decreased to approximately unstimulated cells. HC has a positive correlation with melanin production in (iv) U B i 6 melanoma cells. The same field is also treated with Dz on murine b 16 melanoma cells. When the 'melanin amount is significantly reduced' and when the Dz concentration reaches 2〇_, the black and & prime content of murine melanoma cells is much lower than that without a_MSH or IBMA stimulation, indicating that Dz has inhibition of melanin. Excellent results. From the above results, it was found that 'HC originally used as an antihistamine drug and originally used as a treatment for endometriosis 4 (4) Dz have inhibitory effects on melanoma production of melanoma cells, so the inventor of the present invention speculated that it should be — 201228676 melanin inhibitors, which can be used for skin whitening. To further confirm that HC and Dz can be used as melanin inhibitors, and to understand the effects of inhibiting the melanin production of melanocytes, continue the cytotoxicity test, Cell tyrosinase activity and tyrosinase protein expression. C. MTT assy The effect of HC and Dz on the viability of murine B16 melanoma cells was tested by MTT assy. The cells were cultured for two days (48 hours) after the above grouping, and the original culture solution was replaced with 1 mg/ml MTT (3-(4,5-dimethylth). Iazol-2-yl)-2,5-diphenyltetrazolium bromide) was mixed with phosphate-buffered saline (PBS) for 2 hours. Then, the culture solution was replaced and DMSO (dimethyl sulfoxide, Sigma Chemical Co.) was added. Next, the absorbance of the dissolved formazan crystal at 570 nm was measured with a spectrophotometer. D. Comparison of the melanin production inhibitory effect of the common whitening drug (arbutin) in the market. According to the above-mentioned "A. Biomaterial/melanin production inhibition test", the melanin production of melanoma cells by HC, Dz and arbutin was performed. Inhibition Experiment E. Measurement of cellular tyrosinase activity and melanoma melrosinase activity 1. Determination of tyrosinase activity: respectively, stimulating drugs and HC, Dz or arbutin Melanin inhibits 10 201228676 treated mouse B16 melanoma cells, murine B16 melanoma cells treated with stimulating drugs but not treated with melanin inhibitors, and murine B16 melanoma cells treated with completely unstimulated drugs and melanin inhibitors, The cells were placed in a 4 °C reagent consisting of 20 mM sodium phosphate (pH 6.8), 1% Triton X-100 and 1 mM PMSF, respectively, and then subjected to 30 strokes with a Dounce homogenizer to disrupt the cells and homogenize. Next, a detergent was added to release the membrane-bound tyrosinase bound to the melanosome, and the extract was centrifuged (15000 x g, 15 minutes). The centrifuged supernatant suspension was taken as the first source of enzyme for subsequent use. The protein content of the supernatant was determined by the Bradford assay and BSA was used as the standard for white matter. The tyrosinase activity was tested as follows: Prepare 1 ml of the reaction mixture containing 50 mM phosphate buffer (pH 6.8), 2.5 mM L-dopa, and 500 Kg of protein (taken from the first source of the aforementioned enzyme). The reaction mixture was incubated at 37 ° C for 15 minutes. Next, the absorbance of the reaction mixture at 475 nm was measured to determine the amount of dopachrome produced. One unit of tyrosinase activity is defined as the total amount of enzyme required to produce 1 μ mole of dopachrome in 1 minute, and the amount of dopachrome in this reaction is calculated by Lambert-Beer Law's molar disappearance coefficient of dopachrome The (molar extinction coefficient) was 3600 M·1.cm_1, and the glutamate activity was normalized by the protein content in the reaction. 201228676 2. Determination of tyrosinase activity of melanosome: Preparation of first enzyme source 'Removal of stimulating drug and murine Bi6 melanoma cells treated with melanin inhibitors such as hc or Dz, stimulating drugs but no melanin inhibitor The treated mouse B16 melanoma cells, and the murine B16-specific pigmented tumor cells treated with the melanin inhibitor and the melanin inhibitor were washed twice with PBS. Then, these mice b丨6 black

色素瘤細胞浸入 20 mM sodium phosphate (pH 6.8)與 1 mM PMSF混合構成之4。(：試劑中，並在無清潔劑的情況下以 Dounce homogenizer進行50次衝程，破壞細胞並均質化。 接著’將溶出物進行離心作業（l〇〇〇Xg，15分鐘），而使得溶 出物中之核酸（nuclei)與未破之細胞被沉澱，然後，取出離 心上層懸浮液，並對此上層懸浮液進行第二次離心（4〇〇〇〇xg ，30分鐘，4 Y )，以獲得充滿黑色素體之粒子 (melanosome-enriched pellet)。然後，再將此獲得之粒子 (pellet)重新懸浮於含有1 mM PMSF之〇 5 ml的2〇 mM sodium phosphate (pH 6.8)中’作為第二酵素來源。The pigmentoma cells were immersed in 20 mM sodium phosphate (pH 6.8) mixed with 1 mM PMSF to form 4. (: In the reagent, and without the detergent, 50 strokes were performed with Dounce homogenizer to destroy the cells and homogenize. Then 'the extract was centrifuged (10 μg, 15 minutes) to make the dissolution The nucleic acid (nuclei) and the unbroken cells were precipitated, and then the supernatant suspension was taken out, and the supernatant was subjected to a second centrifugation (4 〇〇〇〇 x g, 30 minutes, 4 Y) to obtain It is filled with melanosome-enriched pellet. Then, the obtained pellet is resuspended in 5 ml of 2 mM sodium phosphate (pH 6.8) containing 1 mM PMSF as the second enzyme. source.

製備lml反應混合物，此反應混合物包含5〇 mM phosphate buffer (pH 6.8)、2.5 mM L-d〇pa，及 70 蛋白 質（取自第二酵素來源）。並在3：rc環境下，每分鐘量測一 次此反應混合物在475 nm的吸光值，以動態監測 Dopachrome的生成。其黑色素體的酪胺酸酶活性單位如前 所述。 F.量測無細胞（ceii_free)路胺睃梅活性 製備第三酵素來源，其製備方法如前述第一酵素來源 12 201228676 ’但不同的是’這裡是所採用的細胞是未經刺激藥物與黑 色素抑制劑處理的鼠B16黑色素瘤細胞。One ml of the reaction mixture was prepared. This reaction mixture contained 5 mM phosphate buffer (pH 6.8), 2.5 mM L-d〇pa, and 70 protein (from the second enzyme source). The absorbance at 475 nm of this reaction mixture was measured every minute in a 3:rc environment to dynamically monitor the formation of Dopachrome. The melanosome tyrosinase activity unit of the melanosome is as described above. F. Measuring the cell-free (ceii_free) amygdalin activity to prepare a third enzyme source, the preparation method is as described above for the first enzyme source 12 201228676 'but the difference is 'here the cells used are unstimulated drugs and melanin Inhibitor-treated murine B16 melanoma cells.

製備1 ml的反應混合物，此反應混合物包含50 mM phosphate buffer (pH 6.8)、2.5 mM L-do_pa、預定測試濃度的 黑色素抑制劑，及500 gg的蛋白質（取自第三酵素來源）， 將此反應混合物在37 °C環境培養15分鐘，並量測反應混 合物在475 nm的吸光值，以測定dopachrome的生成，測定 HC與Dz等黑色素抑制劑對無細胞系統之酪胺酸酶活性的 抑制功效。相對的酪胺酸酶活性為含有黑色素抑制劑之反 應混合物的酵素活性除以無黑色素刺激藥物之反應混合物 的酵素活性。 G. Western blot analysis 取出前述已培養兩天之鼠B16黑色素瘤細胞，並以4°C PBS沖洗三次，再將鼠B16黑色素瘤細胞並溶解於含有 protease inhibitors cocktail (Abeam, Cambridge, UK)之 4°C lysis buffer (含有 20 mM sodium phosphate (pH 6.8)、1% Triton X-100、1 mM PMSF，及 1 mM EDTA)中，製成細胞 溶解液。然後，以Bradford assay來測定蛋白質含量，並以 BSA為標準品。 自前述細胞溶解液取出100 Kg的蛋白質，並以SDS-PAGE (SDS-polyacrylamide gel electrophoresis)進行分離。 接著，將凝膠中的蛋白質區帶轉移到polyvinyl difluoride (PVDF)膜（MP Biomedicals Co.，Irvine，CA，USA)上，並以 含有 5。/。non-fat skim milk(脫脂牛奶）的 TBS-T buffer 對 13 201228676 PVDF膜進行封阻處理（blocking)。 接著將PVDF膜浸於含有一級抗體之溶液中，在此採用 之一級抗體分別為兔子多株抗體（rabbit polyclonal antibodies)與小鼠單株抗-β-肌動蛋白抗體（mouse monoclonal anti-p-actin antibodies) > 使 rabbit polyclonal antibodies 與赂 胺酸酶形成抗原-抗體複合體，使mouse monoclonal anti-β-actin antibodies與β-肌動蛋白（β-actin)(作為内部控制組）形 成抗原-抗體複合體。接下來，再以horseradish peroxidase-conjugated secondary antibody(結合有 辣根過 氧化的 二次抗 體）漂洗此PVDF膜，接上二次抗體。再以Amersham ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) 檢測所有鍵結的抗體，並以 densitometer system GS-700(Bio-Rad, CA，USA)對每一條區帶的訊號強度進行定量 ，並以内部控制組訊號進行標準化（normalize)。 H. RT-PCR (Reverse-transcription polymerase chain reaction) 以 RNeasyTMmini Kit (Qiagen，CA，USA)萃取出所有區 帶的RNA，並以OD26〇/OD28。ratio評估所有區帶之RNA樣 本的品質。 將各區帶的RNA樣品分別置於含有oligo(dT) primers (MD Bio. Co·，Taipei，Taiwan)與反轉錄酶（Roche Molecular Biochemicals，Mannheim, Germany)的 42 0C 環境進行 90 分 鐘的反轉錄。接著，以 PCR Sprint Thermal Cycler HBSP02110 (Thermo Fisher Scientific Inc·，IL，USA)對所得 14 201228676 之cDNA以PCR進行倍增，PCR之單一循環條件依序為： 94 °C進行5分鐘；94 °C進行30秒；56。(：進行30秒， 及72 °C進行1分鐘，共進行19次循環。接著，在72 °C 進行 10 分鐘的 elongation .cycle。 在此RT-PCR中，供用於小鼠B16黑色素瘤細胞之 tyrosinase的基因表現的引子對為： 前向引子F1 : 5'-ggccagctttcaggcagaggt-3f(序列辨識編號：1) # 反向引子R1 : . 5’-tggtgcttcatgggcaaaatc-3'(序列辨識編號：2) 另外，供用於小鼠 GAPDH (mouse glyceraldehyde-3-phosphate dehydrogenase)之基因表現[作為内部對照組 (internal control)]的引子對為： 前向引子F2 : 5’-accacagtccatgccatcac-3’（序列辨識編號：3) 反向引子R2 : • 5f-tccaccaccctgttgctgta-3f (序列辨識編號：4) 接著，將RT-PCR產物在含有2% agarose gel的TBE buffer中進行膠電泳（工作電壓100 V，40分鐘），然後，以 上述western blot analysis對每一個區帶的信號強度進行定 - 量，並以内部控制組（internal control)進行標準化。 I.統計分析 本研究之每一組實驗皆會進行三次取平均，實驗數據 是以平均值土標準偏差（S.D.)來表示（統計顯著性，；7<〇.〇5) 15 201228676 。所有的數據是藉由Student，sitest進行統進分析。 【試驗結果】 接著，即針對上述各種檢測之結果分別說明如下： A.黑色素抑制劑對細胞存活率的影響 在進-步研究黑色素抑制劑對鼠Bl6黑色素瘤細胞之 黑色素生成的抑制機制前，須先敎黑色素抑制劑不會對 鼠删黑色素瘤細胞產生毒性之濃度，本實施例是以ΜΗ assay測定經過黑色素抑制劑處理過之細胞的存活力，並以 市面常見之熊果素（Arbutin)作為對照組。 如圖3所示’ 4_HC並不會對鼠Bi6黑色素瘤細胞 產生細胞毒性(eyt〇t〇xieity)，而HC對鼠B i 6黑色素瘤細胞 之細胞毒性的ic5。值為14.5 μΜ，為了忽略Hc會產生的細 胞毒性之影響，在本案中所使用之HC最高濃度為$ 如圖4所示’20_Dz並不會對鼠B16黑色素瘤細胞 產生細胞毒性，而且Dz對鼠B16黑色素瘤細胞的之細胞毒 性的1C5。值為70 μΜ，為了忽略Dz會產生的細胞毒性之影 響，在本案中所使用之Dz最高濃度為2〇μΜ。 Β.黑色素抑制刻對黑色素生成的影響 為了測定黑色素抑制劑對鼠Β16黑色素瘤細胞的存活 率影響，需測定每一種黑色素抑制劑處理的這些鼠Β丨6黑 色素瘤細胞的黑色素量。 其測試結果如圖5所示，鼠B16黑色素瘤細胞經過α_ MSH的刺激後，其黑色素含量會明顯增加，然而，當鼠 Β16黑色素瘤細胞同時以Hc &a_MSH進行處理時，黑色 16 201228676 素量會明顯降低，且當HC濃度達到4 μΜ時，黑色素含量 會降低到近似未經a_MSH刺激的細胞’顯示HC對於抑制 鼠B16黑色素瘤細胞的黑色素生成有正相關，且達到IC50 的漢度值為4.6 μΜ，比較熊果素與HC之抑制功效時，2 μΜ HC的抑制效果即已優於100 μΜ的熊果素。此外，本 案另以ΙΒΜΧ刺激鼠Β16黑色素瘤細胞，並測定HC對 ΙΒΜΧ處理之鼠Β16黑色素瘤細胞之黑色素生成抑制效果， 結果顯示HC也可抑制經ΙΒΜΧ處理之鼠Β16黑色素瘤細胞 的黑色素生成。 如圖6所示，此外，由於HC為一種Η1抗組織胺藥劑 ’為確認其它抗組織胺藥劑對於受a_MSH刺激之鼠β 16黑 色素瘤細胞是否也會具有抑制黑色素生成的功效，本發明 以多種抗組織胺藥劑進行上述實驗，抗組織胺藥劑包含兩 種H1抗組織胺藥劑，分別為hydr0XyZine與cetirizine，及 兩種H2抗組織胺藥劑’分別為cimetidine與ranitidine，由 於實驗方式與上述HC之實驗方式相同，因此不再詳述，其 測试結果顯示，沒有一種抗組織胺藥劑對於a_MSH刺激之 鼠B16黑色素瘤細胞的黑色素生成具有抑制效果，即便抗 ，，且織胺藥劑濃度已提咼至20 μΜ，再者，H1抗組織胺藥劑 也不會降低HC抑制鼠Β16黑色素瘤細胞之黑色素生成的功 效。由此更進一步證實，HC抑制黑色素生成之功效是一般 抗組織胺藥劑所沒有的。Prepare 1 ml of the reaction mixture containing 50 mM phosphate buffer (pH 6.8), 2.5 mM L-do_pa, a predetermined test concentration of melanin inhibitor, and 500 gg of protein (from the third enzyme source). The reaction mixture was incubated at 37 ° C for 15 minutes, and the absorbance of the reaction mixture at 475 nm was measured to determine the formation of dopachrome, and the inhibitory effect of melanin inhibitors such as HC and Dz on the tyrosinase activity of the cell-free system was determined. . The relative tyrosinase activity is the enzyme activity of the reaction mixture containing the melanin inhibitor divided by the enzyme activity of the reaction mixture without the melanin-stimulated drug. G. Western blot analysis The above-mentioned two-day cultured mouse B16 melanoma cells were removed and washed three times with 4° C. PBS, and then the mouse B16 melanoma cells were dissolved in a vaccine containing cocktail inhibitors (Abeam, Cambridge, UK). A cell lysate was prepared in a °C lysis buffer containing 20 mM sodium phosphate (pH 6.8), 1% Triton X-100, 1 mM PMSF, and 1 mM EDTA. The protein content was then determined by the Bradford assay and BSA was used as a standard. 100 Kg of protein was taken out from the above cell lysate and separated by SDS-PAGE (SDS-polyacrylamide gel electrophoresis). Next, the protein band in the gel was transferred to a polyvinyl difluoride (PVDF) membrane (MP Biomedicals Co., Irvine, CA, USA) and contained 5. /. The TBS-T buffer of non-fat skim milk was blocked by the 13 201228676 PVDF membrane. Next, the PVDF membrane is immersed in a solution containing a primary antibody, wherein the primary antibody is a rabbit polyclonal antibody and a mouse monoclonal anti-p-actin antibody (mouse monoclonal anti-p- Actin antibodies) > The rabbit polyclonal antibodies form an antigen-antibody complex with the glutamate, allowing mouse monoclonal anti-β-actin antibodies and β-actin (as an internal control group) to form antigens - Antibody complex. Next, the PVDF membrane was rinsed with a horseradish peroxidase-conjugated secondary antibody (secondary antibody combined with horseradish peroxidation) and secondary antibody was attached. All bound antibodies were detected by Amersham ECL system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and the signal intensity of each zone was quantified using a densitometer system GS-700 (Bio-Rad, CA, USA). It is normalized by the internal control group signal. H. RT-PCR (Reverse-transcription polymerase chain reaction) All regions of RNA were extracted with RNeasyTM mini Kit (Qiagen, CA, USA) at OD26〇/OD28. Ratio evaluates the quality of RNA samples in all zones. RNA samples from each zone were separately placed in a 42 0C environment containing oligo(dT) primers (MD Bio. Co., Taipei, Taiwan) and reverse transcriptase (Roche Molecular Biochemicals, Mannheim, Germany) for 90 minutes of reverse transcription. . Next, the cDNA of 14 201228676 was multiplied by PCR using PCR Sprint Thermal Cycler HBSP02110 (Thermo Fisher Scientific Inc., IL, USA). The single cycle conditions of PCR were: 94 ° C for 5 minutes; 94 ° C for 94 ° C 30 seconds; 56. (: 30 seconds, and 1 hour at 72 °C for a total of 19 cycles. Next, a 10 minute elongation.cycle was performed at 72 ° C. In this RT-PCR, it was used for mouse B16 melanoma cells. The primer pair for the gene expression of tyrosinase is: forward primer F1 : 5'-ggccagctttcaggcagaggt-3f (sequence identification number: 1) # reverse primer R1 : . 5'-tggtgcttcatgggcaaaatc-3' (sequence identification number: 2) The gene expression for the expression of mouse GAPDH (mouse glyceraldehyde-3-phosphate dehydrogenase) [as internal control] is: forward primer F2: 5'-accacagtccatgccatcac-3' (sequence identification number: 3 Reverse primer R2: • 5f-tccaccaccctgttgctgta-3f (SEQ ID NO: 4) Next, the RT-PCR product was subjected to gel electrophoresis in a TBE buffer containing 2% agarose gel (operating voltage 100 V, 40 minutes), then The signal intensity of each zone was quantified by the above western blot analysis and normalized by internal control. I. Statistical analysis Each set of experiments in this study will be performed in three Averaged, the experimental data is expressed as the mean soil standard deviation (SD) (statistical significance,; 7 < 〇. 〇 5) 15 201228676. All data is analyzed by Student, sitest. Next, the results of the above various tests are described as follows: A. Effect of melanin inhibitor on cell viability Before further research on the inhibitory mechanism of melanin inhibitor on melanin production in murine Bl6 melanoma cells, The melanin inhibitor does not cause toxicity to the melanoma cells of the mouse. In this example, the viability of the cells treated with the melanin inhibitor is determined by the sputum assay, and the commercially available Arbutin is used as a control group. Figure 3 shows that '4_HC does not produce cytotoxicity (eyt〇t〇xieity) on murine Bi6 melanoma cells, while HC has an ic5 value of 14.5 μΜ for cytotoxicity of murine B i 6 melanoma cells, in order to ignore Hc The resulting cytotoxicity effect, the highest concentration of HC used in this case is $20_Dz as shown in Figure 4 and does not produce cells in murine B16 melanoma cells. Toxic, and the cytotoxicity of Dz on murine B16 melanoma cells is 1C5. The value was 70 μΜ, and the highest concentration of Dz used in this case was 2 μμΜ in order to ignore the effect of cytotoxicity caused by Dz. Β. Effect of melanin inhibition on melanin production To determine the effect of melanin inhibitors on the viability of sputum 16 melanoma cells, the amount of melanin in these melamine 6 melanoma cells treated with each melanin inhibitor was determined. The test results are shown in Figure 5. The murine content of murine B16 melanoma cells was significantly increased after stimulation with α_MSH. However, when the sputum 16 melanoma cells were simultaneously treated with Hc & a_MSH, black 16 201228676 The amount will be significantly reduced, and when the HC concentration reaches 4 μΜ, the melanin content will decrease to approximately cells that are not stimulated by a_MSH. 'Showing that HC has a positive correlation with the melanin production of murine B16 melanoma cells, and reaches the IC50 Hanta value. For 4.6 μΜ, the inhibitory effect of 2 μΜ HC was better than 100 μΜ of arbutin when comparing the inhibitory effects of arbutin and HC. In addition, in this case, sputum sputum 16 melanoma cells were stimulated by sputum, and the melanin production inhibitory effect of HC on sputum-treated sputum 16 melanoma cells was measured. The results showed that HC also inhibited melanin production by sputum-treated sputum 16 melanoma cells. As shown in Fig. 6, in addition, since HC is a Η1 antihistamine agent, in order to confirm whether other antihistamine agents also have an effect of inhibiting melanin production in aβHH-stimulated rat β 16 melanoma cells, the present invention is various. The anti-histamine agent was subjected to the above experiment. The antihistamine agent contained two H1 antihistamine agents, respectively hydr0XyZine and cetirizine, and two H2 antihistamine agents, respectively cimetidine and ranitidine, due to the experimental method and the above HC experiment. The method is the same, so it will not be described in detail. The test results show that no antihistamine agent has an inhibitory effect on melanin production of a_MSH-stimulated mouse B16 melanoma cells. Even if it is resistant, the concentration of the lysine agent has been improved. 20 μΜ, in addition, the H1 antihistamine agent did not reduce the efficacy of HC inhibiting melanin production by sputum 16 melanoma cells. It was further confirmed that the effect of HC inhibiting melanin production is not found in general antihistamine agents.

Dz抑制黑色素生成之功效的測試結果如圖7所示，鼠 B16黑色素瘤細胞經過a_MSH或ΐΒΜχ的刺激後，其黑色 17 201228676 素含量都會明顯增加’然而當同時以Dz對鼠Bi6黑色素瘤 細胞進行處理時，黑色素量都會明顯降低，且當濃度達 2〇 μΜ時’ ^ Bl6黑色素瘤細胞的黑色素含量已遠低於未 經α-MSH或IBMA刺激時的程度，顯示Dz對於抑制黑色素 具有極佳之效果，達到ICsQ的濃度值為9·3 μΜ，且相較於 熊果素，5 μΜ Dz的抑制效果即已大幅優於1〇〇 μΜ的熊果 素。 ·、 C.黑色素抑制劑對於酪胺酸酶活性的影響 因為酪胺酸酶是黑色素生成之酵素中的主要關鍵角色 ，所以目前已被探討之黑色素抑制劑抑制黑色素生成的主 要機制，都是藉由降低細胞的酪胺酸酶活性來達成。因此 本發明必須確認所採用之兩種黑色素抑制劑之抑制功效是 否也是藉由降低細胞的酪胺酸酶活性來達成。 如圖8所示，經α-MSH處理後之細胞路胺酸酶活性會 明顯提高，但是細胞酪胺酸酶經〇1_崖311刺激提高之活性， 並不會因為有HC的處理而降低。為更進一步探討，本發明 進一步自經過HC處理之鼠b 16黑色素瘤細胞與未經過 處理之鼠B16黑色素瘤細胞分離取出黑色素體 (melanosomes) ’並研究這兩種黑色素體之酪胺酸酶活性的 動力學，其結果如圖9所示，與前述細胞酪胺酸酶活性的 測試結果是一致的，相較於未經H(：處理之鼠B16黑色素瘤 細胞，HC處理後之黑色素體的酪胺酸酶活性並未降低。 接著’ HC對無細胞系統（ceu_free SyStem)之赂胺酸酶活 性的抑制測試結果如圖10所示，其測試結果顯示HC也無 18 201228676 法在無細胞系統下’抑制鼠B16黑色素瘤細胞之酪胺酸酶 活性，相反的，kojic acid(麴酸，一種常見的酪胺酸酶抑制 劑）對酪胺酸酶活性出現顯著的抑制作用。 也就是說，HC雖然可將a_MSH刺激後之鼠B16累色 素瘤細胞的黑色素量降低至未受α-MSH刺激前的等級，但 卻不會降低細胞路胺酸酶的活性，也不會降的黑色素體之 酪胺酸酶的活性’在無細胞系統中，也同樣無法降低路胺 酸酶活性，證實HC抑制細胞之黑色素生成的機制完全不同 於現有黑色素抑制劑。 接著，探討Dz對酪胺酸酶活性的影響，其測試結果如 圖11 ' 12所示，經過Dz處理後，雖然細胞酪胺酸酶活性 會明顯降低，但在無細胞系統下，Dz也無法直接抑制鼠 B16黑色素瘤細胞之路胺酸酶的活性。 D.黑色素抑制劑對酪胺酸酶蛋白質（tyr〇sinase pr〇tein) 與酪胺酸酶蛋白質之mRNA的影樂 為了進一步証實前述黑色素抑制劑不會降低細胞酪胺 酸酶活性，本發明以Western blotting，來確定未經黑色素 抑制劑處理與經過黑色素抑制劑處理之鼠B16黑色素瘤細 胞中的酷胺酸酶蛋白質表現量與酪胺酸酶蛋白質之mRNA 表現量。 如圖13、14所示’鼠b 16黑色素瘤細胞在經過a_MSH 刺激後，路胺酸酶蛋白質表現量（如圖13)與其mRNA表現 量（如圖14)皆明顯增加，但此增加情況並未因為HC的處理 而下降。由此可進一步證實HC完全不會降低細胞酪胺酸酶 19 201228676 活性。 曰DZ對於赂胺酸酶蛋白f與酷胺酸酶蛋白質表現 里的：K式’‘果分別如圖15、16所示’經過&處理後，酪 胺酸酶蛋白質增加的情況都會下降，尤其當Dz提高至20 _時，其下降幅度更為明顯。然而，酪胺酸酶蛋白質 :RNA表現量卻未因為加人2〇幽Dz而下降。此說明Dz 是在未影響路胺酸酶蛋白f mRNA表現量的情況下，藉由 調降酪胺酸酶蛋白質表現量’而相對抑制細胞酪胺酸酶活 性，進而抑制黑色素生成。 【資施例-活艟内試驗】 —透過上述活體外黑、色素瘤細胞之黑色素抑制試驗，證 貫本案所使用《HC肖Dz對鼠黑色素瘤細胞之黑色素生成 具有極佳之功效，並進—步確認HC與Dz抑制黑色素生成 之作用機轉，2 HC肖Dz抑制黑色素生成之效果大幅優於 市面常用之美白藥物（熊果素）。 為更進一步證實HC與DZ對於對活體内細胞之黑色素 抑制是否具有功效，本發明乃更進一步以斑馬魚進行活體 測試，藉由觀察斑馬魚體表黑色紋路的表現量與其後續生 長發育情況，來評估此兩種藥物對活體内黑色素細胞之黑 色素生成抑制的功效。 A·生物材料 本案所使用之活體為斑馬魚（刀7·0 Ab strain ' ，斑馬魚成魚是購買自一般水族館。飼養條件固定如 下.溫度28.5t，曰照/無光比例為14小時/1〇小時 20 201228676 ’日夜循環。以購自水族館的活小蝦進行餵養，每曰 固定餵食三次’斑馬魚胚胎是收取自斑馬魚之自然排 卵。 B.測定黑色素抑制劑對斑馬魚之黑色素生成的影響 將收取之受精印在24孔培養皿_以胚胎培養基 進行培養’每孔加入2 m L培養基及16〜1 8個胚胎。 在胚胎發育至第9小時時，於不同測試條件中，將溶 於DMSO的測試藥品（分別為HC與Dz)加入胚胎培養 基中（DMSO最終濃度為0.1% )，控制組則僅加入 DMSO，繼續培養至第48小時，每24小時重新置換 新的培養基。收取胚胎樣品時’觀察並記錄培養基中 胚胎存活率及有無不正常型態之胚胎發育，若有不正 常型態之胚胎發育，則利用解刮顯微鏡進行觀察記錄 ，並以未經黑色素抑制劑處理之斑馬魚胚胎進行對照比較 ，觀察斑馬魚胚胎之黑色素表現量。在本發明中，所使用 之HC濃度為12·5 μΜ，使用之Dz濃度為6 25 μΜ。 【試驗結果】 如圖17、18所示’經過HC或Dz處理後之斑馬魚胚胎 身上顯示的黑色素生成量明顯降低’且就發育型態方面： 行觀察，、經HC肖Dz處理後之斑馬魚胚胎的發育並無出現 異狀’證實所加入之HC濃度（12_5陶)與Dz濃度^㈣) 並不會影響斑馬魚胚胎之發育，其結果與上Uc、Dz分別 對鼠B16黑色素瘤細胞的黑色素生成抑制效果相符。 综上所述，證實HC不會抑制細胞路胺酸酶、黑色素體 21 201228676 的酷胺酸酶，及無細胞系統（cell-free system)的鼠類路胺酸 酶的活性，且western blotting與RT-pcr也分別證實，經 過HC處理過之鼠B16黑色素瘤細胞中的路胺酸酶蛋白質與 其mRNA表現量並未被往下調降，這些結果說明HC抑制 鼠B16黑色素瘤細胞之黑色素生成的作用，並不會影響赂 胺酸酶之活性，相對證實HC抑制黑色素生成的作用機制並 非經由降低鼠B16黑色素瘤細胞的赂胺酸酶活性。配合以 其它抗組織胺藥劑進行試驗後，也證實HC抑制B16黑色素 瘤細胞的黑色素生成作用，是透過組織胺受器拮抗作用 (histamine receptor antagonism)以外的機制。 相反的’ DC則是透過降低細胞路胺酸酶活性的方式， 來抑制鼠B16黑色素瘤細胞的黑色素生成，但DC並不會影 響酪胺酸酶蛋白之mRNA表現量。 因此，申請人證實在HC與Dz的無毒性濃度範圍内， HC與Dz對於黑色素的生成有很強的抑制效果且Re與The test results of Dz inhibiting the production of melanin are shown in Fig. 7. When the mouse B16 melanoma cells were stimulated by a_MSH or sputum, the content of black 17 201228676 was significantly increased'. However, when Dz was used to treat mouse Bi6 melanoma cells simultaneously. At the time of treatment, the amount of melanin was significantly reduced, and when the concentration reached 2〇μΜ, the melanin content of '^ Bl6 melanoma cells was much lower than that without α-MSH or IBMA stimulation, indicating that Dz is excellent for inhibiting melanin. The effect is that the concentration of ICsQ is 9·3 μΜ, and the inhibitory effect of 5 μΜ Dz is much better than that of 1 μ〇〇 of arbutin compared to arbutin. ·, C. The effect of melanin inhibitors on tyrosinase activity Because tyrosinase is a major key player in melanin-producing enzymes, the main mechanism by which melanin inhibitors have been investigated to inhibit melanin production are This is achieved by reducing the tyrosinase activity of the cells. Therefore, the present invention must confirm whether the inhibitory efficacy of the two melanin inhibitors used is also achieved by lowering the tyrosinase activity of the cells. As shown in Fig. 8, the cell glutaminase activity after α-MSH treatment was significantly increased, but the cell tyrosinase stimulated the activity by 〇1_崖311 and was not lowered by the treatment with HC. . For further investigation, the present invention further separates melanosomes from the treated rat b 16 melanoma cells and the untreated mouse B16 melanoma cells and studies the tyrosinase activities of the two melanosomes. The kinetics, the results are shown in Figure 9, consistent with the test results of the aforementioned cellular tyrosinase activity, compared to the non-H (: treated mouse B16 melanoma cells, HC treated melanosomes The tyrosinase activity was not reduced. The results of the inhibition test of the 'ciclease activity of the cell-free system (ceu_free SyStem) are shown in Figure 10. The test results show that HC also has no 18 201228676 method in the cell-free system. Under the 'inhibition of tyrosinase activity of murine B16 melanoma cells, conversely, kokutic acid (an acid, a common tyrosinase inhibitor) significantly inhibited tyrosinase activity. Although HC can reduce the amount of melanin in mouse B16 dermal pigmentoma cells after a_MSH stimulation to a level not before α-MSH stimulation, it does not decrease the activity of cellular glutaminase, nor does it decrease. The activity of melanosome tyrosinase in the cell-free system also failed to reduce the activity of the glutaminase, confirming that the mechanism of melanin production by HC-inhibiting cells is completely different from the existing melanin inhibitor. Next, the discussion of Dz on tyramine The effect of acidase activity, the test results are shown in Figure 11 '12. After Dz treatment, although the cell tyrosinase activity is significantly reduced, but in the cell-free system, Dz can not directly inhibit the mouse B16 melanoma cells. The activity of the lysinase D. The melanin inhibitor on the tyrosinase protein (tyr〇sinase pr〇tein) and the mRNA of the tyrosinase protein in order to further confirm that the aforementioned melanin inhibitor does not reduce the cell tyrosin Aminotinase activity, the present invention uses Western blotting to determine the expression of glutaminase protein and mRNA expression of tyrosinase protein in murine B16 melanoma cells treated with melanin inhibitors and melanin inhibitors. As shown in Figures 13 and 14, the expression of the glutaminase protein (Figure 13) and its mRNA expression after stimulation with a_MSH in murine b 16 melanoma cells The amount (Figure 14) was significantly increased, but this increase was not decreased by the treatment of HC. It was further confirmed that HC did not reduce the activity of cell tyrosinase 19 201228676. 曰DZ for the citase protein The expression of f and tyrosinase protein: K-type 'fruits as shown in Figures 15 and 16 respectively, after the & treatment, the increase in tyrosinase protein will decrease, especially when Dz is increased to 20 _ The decline was more pronounced. However, the tyrosinase protein: RNA expression did not decrease due to the addition of 2 〇 Dz. This shows that Dz inhibits the activity of the tyrosinase protein mRNA and inhibits the activity of the tyrosinase protein, thereby inhibiting the production of melanin by inhibiting the activity of the tyrosinase protein. [Supplementary Example - Intravital Test] - Through the above-mentioned melanin inhibition test of in vitro black and pigmentoma cells, it is proved that the use of HC Xiao Dz has excellent effects on melanin production of murine melanoma cells. Steps to confirm the effect of HC and Dz inhibition of melanin production, 2 HC Xiao Dz inhibition of melanin production is much better than the commonly used whitening drugs (arbutin). In order to further confirm whether HC and DZ have efficacy for melanin inhibition of cells in vivo, the present invention further tests the zebrafish in vivo by observing the expression of the black lines on the surface of the zebrafish and its subsequent growth and development. The efficacy of these two drugs on the inhibition of melanin production by melanocytes in vivo was evaluated. A. Biomaterials The living body used in this case is zebrafish (knife 7·0 Ab strain ', zebrafish adult fish is purchased from the general aquarium. The feeding conditions are fixed as follows. Temperature 28.5t, illuminating/matte ratio is 14 hours/1 〇 20 20 201228676 'Day and night cycle. Feeding with live shrimp from the aquarium, fixed feeding three times per ' zebrafish embryos are naturally ovulated from zebrafish. B. Determination of melanin production by melanin inhibitors on zebrafish The effects will be collected in a 24-well culture dish _ cultured in embryo culture medium. 2 m L medium and 16 to 18 embryos per well. When the embryo develops to the 9th hour, it will dissolve under different test conditions. The test drugs in DMSO (HC and Dz, respectively) were added to the embryo culture medium (final concentration of DMSO was 0.1%), and the control group was only added with DMSO, and the culture was continued until the 48th hour, and the new medium was replaced every 24 hours. During the sample, 'observe and record the embryo survival rate in the medium and the embryo development with or without abnormal pattern. If there is an abnormal type of embryo development, use a scratch-off microscope. The melatonin expression of the zebrafish embryo was observed by comparison with the zebrafish embryos which were not treated with melanin inhibitor. In the present invention, the HC concentration used was 12.5 μΜ, and the Dz concentration was 6 25 μΜ [Test Results] As shown in Figures 17 and 18, 'the amount of melanin produced by zebrafish embryos treated with HC or Dz is significantly reduced' and in terms of developmental pattern: observation, HC Xiao Dz treatment The development of the zebrafish embryo did not appear abnormal. 'Confirmed that the added HC concentration (12_5 pottery) and Dz concentration ^ (4)) did not affect the development of the zebrafish embryo, and the results were the same as the upper Uc, Dz and the mouse B16. The melanoma production of melanoma cells is consistent with the inhibitory effect. In summary, it was confirmed that HC does not inhibit the activity of cellular glutaminase, melanosome 21 201228676 uranylase, and cell-free system of murine lutylase, and western blotting and RT-pcr also confirmed that the expression of glutaminase protein and its mRNA in HC-treated mouse B16 melanoma cells were not down-regulated. These results indicate that HC inhibits melanin production in murine B16 melanoma cells. It does not affect the activity of the glutaminase, and it is relatively confirmed that the mechanism of action of HC inhibiting melanin production is not to reduce the glandular enzyme activity of murine B16 melanoma cells. In combination with other antihistamine agents, it was also confirmed that HC inhibits the melanin production of B16 melanoma cells by a mechanism other than histamine receptor antagonism. The opposite 'DC' inhibits melanin production in murine B16 melanoma cells by reducing cellular altaminase activity, but DC does not affect the mRNA expression of tyrosinase protein. Therefore, Applicants have confirmed that HC and Dz have a strong inhibitory effect on the production of melanin in the non-toxic concentration range of HC and Dz, and Re and

Dz分別對經過a_MSH或ΙΒΜχ刺激之細胞的黑色素生成的 抑制功效，都遠優於熊果素（常見的美白藥物卜在斑馬魚胚The inhibitory effect of Dz on melanin production by a_MSH or sputum-stimulated cells is much better than that of arbutin (a common whitening drug in zebrafish embryo).

胎之活體試驗方面，也證實在無細胞毒性濃度範圍内，HC 與Dz皆不會影響斑馬魚胚胎之發育，且可明顯降低斑馬魚 胚胎身上的黑色素表現量。因此，Dz與HC確實可進一步 應用於製成抑制黑色素細胞之黑色素生成的藥學組合物， 並可用於開發成具有皮膚美白功效之化妝保養品，確實可 達到本發明之目的。 惟以上所述者’僅為本發明之較佳實施例而已，當不 22 201228676 能以此限定本發明實施之範圍，即大凡依本發明申請專利 範圍及發明說明内容所作之簡單的等效變化與修飾，皆仍 屬本發明專利涵蓋之範圍内。 【圖式簡單說明】 圖1顯示HC分別對a_MSH與IBMX處理之鼠B16專 色素瘤細胞的黑色素生成量的影響； 圖2顯示Dz分別對a_MSH與ΙΒΜχ處理之鼠奪 色素瘤細胞的黑色素生成量的影響； 圖3顯示HC對鼠B16黑色素瘤細胞之存活率的影響； 圖4顯不Dz對鼠B16黑色素瘤細胞之存活率的影響； 圖5(a)顯示HC與熊果素分別對a_MSH處理之鼠 黑色素瘤細胞經均質化處理與離心後所取出離心液中之黑 色素粒（melanin pellets);圖5(b)顯示HC與熊果素分別對a MSH處理之鼠B16黑色素瘤細胞的黑色素生成量的影響·， 圖6顯示HC與多種抗組織胺藥劑分別對a_MSH處理 之鼠B16黑色素瘤細胞的黑色素生成量的影響； 圖7(a)顯示Dz與熊果素分別對理之鼠Bi6累 色素瘤細胞經均質化處理與離心後所取出離心液中之黑色 素粒（melanin pellets);圖7(b)顯示Dz與熊果素分別對α_ MSH處理之鼠Β16黑色素瘤細胞的黑色素生成量的影響； 圖8顯示經過HC處理與未經過HC處理之鼠B1 6累色 素瘤細胞的絡胺酸酶活性變化； 圖9顯示經過HC處理與未經過HC處理之鼠B16黑色 素瘤細胞之黑色素體的路胺酸酶活性隨時間變化曲線； 23 201228676 圖10顯示HC對無細胞系統之路胺酸酶活性的影響； 圖11顯示經過Dz處理與未經過dz處理之鼠b 16黑色 素瘤細胞的酪胺酸酶活性變化； 圖12顯示Dz對無細胞系統之鼠B16黑色素瘤細胞的 酪胺酸酶活性的影響； 圖13(a)顯示鼠B16黑色素瘤細胞經a_MSH與HC處理 後，經Western blot analysis之酪胺酸酶蛋白質區帶影像； 圖13(b)顯示圖13(a)之各區帶之酪胺酸酶蛋白質表現強度； 圖14(a)顯示鼠B16黑色素瘤細胞與HC處理 後’經Western blot analysis之之酪胺酸酶蛋白質mRNA區 帶影像，圖14(b)顯示圖14(a)之各區帶的赂胺酸酶蛋白質 mRNA表現強度，並以GAPDH訊號強度為參考基準； 圖15(a)顯示鼠B16黑色素瘤細胞經01_]^311與Dz處理 後’經Western blot analysis之酪胺酸酶蛋白質區帶影像； 圖15(b)顯不圖15(a)之各區帶的酿胺酸酶蛋白質表現強度 圖16(a)顯示鼠B16黑色素瘤細胞與Dz處理 後，經Western blot analysis之之酪胺酸酶蛋白質mRNA區 帶影像；圖16(b)顯示圖16(a)之各區帶的酪胺酸酶蛋白質 mRNA表現強度，並以GAPDH訊號強度為參考基準； 圖17顯示經HC處理與未經HC處理之斑馬魚胚胎身 上之黑色素生成量的表現；及 圖18顯示經Dz處理與未經Dz處理之斑馬魚胚胎身上 之黑色素生成量的表現。 24 201228676 【主要元件符號說明】 無In vivo testing of the fetus, it was also confirmed that HC and Dz did not affect the development of zebrafish embryos in the range of no cytotoxicity, and significantly reduced the melanin expression in zebrafish embryos. Therefore, Dz and HC can be further applied to a pharmaceutical composition for inhibiting melanin production by melanocytes, and can be used for development as a cosmetic skin care product having skin whitening effect, and it is indeed possible to attain the object of the present invention. However, the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the practice of the present invention, that is, the simple equivalent variation of the scope of the invention and the description of the invention. And modifications are still within the scope of the invention patent. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the effect of HC on the melanin production of a BMS-specific pigmentoma cells treated with a_MSH and IBMX, respectively. Figure 2 shows the amount of melanin produced by Dz for a_MSH and sputum-treated murine melanoma cells, respectively. Figure 3 shows the effect of HC on the survival rate of murine B16 melanoma cells; Figure 4 shows the effect of Dz on the survival rate of murine B16 melanoma cells; Figure 5 (a) shows that HC and arbutin are treated separately for a_MSH Melanin pellets in the centrifugation of murine melanoma cells after homogenization and centrifugation; Figure 5(b) shows the effect of HC and arbutin on melanin production in a MSH-treated mouse B16 melanoma cells, respectively. · Figure 6 shows the effect of HC and various antihistamines on melanin production in a_MSH treated mouse B16 melanoma cells; Figure 7 (a) shows that Dz and arbutin are homogenized to Bi6 pigmented melanoma cells. Melanin pellets in the centrifugation solution after centrifugation and centrifugation; Figure 7(b) shows the amount of melanin produced by Dz and arbutin in α_MSH-treated sputum 16 melanoma cells, respectively. Effect; Figure 8 shows changes in tyrosinase activity of murine B16 melanoma cells after HC treatment and without HC treatment; Figure 9 shows melanosomes of HC-treated and untreated HC-treated B16 melanoma cells Luminase activity as a function of time; 23 201228676 Figure 10 shows the effect of HC on the aluonase activity of the cell-free system; Figure 11 shows the tyramine of the mouse b 16 melanoma cells treated with Dz and without dz treatment Changes in acidase activity; Figure 12 shows the effect of Dz on the tyrosinase activity of murine B16 melanoma cells in a cell-free system; Figure 13 (a) shows that B16 melanoma cells were treated with a-MSH and HC and analyzed by Western blot. The tyrosinase protein band image; Figure 13 (b) shows the intensity of tyrosinase protein expression in each zone of Figure 13 (a); Figure 14 (a) shows the mouse B16 melanoma cells treated with HC Western blot analysis of the mRNA region of the tyrosinase protein, Figure 14 (b) shows the intensity of the mRNA expression of the glutaminase protein in each region of Figure 14 (a), based on the intensity of the GAPDH signal; Figure 15 (a) shows the mouse B16 black The tumor cells were imaged by Western blot analysis of the tyrosinase protein band after treatment with 01_]^311 and Dz; Figure 15(b) shows the intensity of the tyrosinase protein in each zone of Figure 15(a) Figure 16 (a) shows the tyrosinase protein mRNA band image of the mouse B16 melanoma cells treated with Dz after Western blot analysis; Figure 16 (b) shows the tyramine of each zone of Figure 16 (a) The performance of the acidase protein mRNA is based on the intensity of the GAPDH signal; Figure 17 shows the expression of melanin production in HC-treated and non-HC-treated zebrafish embryos; and Figure 18 shows Dz treatment and no Dz The expression of melanin production in the treated zebrafish embryos. 24 201228676 [Explanation of main component symbols]

25 201228676 序列表 <110>國立臺南大學 <120> —種用於皮膚美白的藥學組成物 <130> SP-15247 <160〉 4 <170> Patent In version 3.5 <210> 1 <211〉 21 <212> DNA <213>人工的序列 <220> <223>用於小鼠酪胺酸酶基因的前向引子F1 <400> 1 ggccagcttt caggcagagg ΐ <210> 2 <211> 21 <212> DNA <213>人工的序列 <220> <223>用於小鼠酪胺酸酶基因的反向引子R1 <400> 2 201228676 tggtgct tea tgggcaaaat c 2125 201228676 Sequence Listing <110> National Tainan University <120> A pharmaceutical composition for skin whitening <130> SP-15247 <160> 4 <170> Patent In version 3.5 <210><211> 21 <212> DNA <213> Artificial sequence <220><223> Forward primer for mouse tyrosinase gene F1 <400> 1 ggccagcttt caggcagagg ΐ <210&gt 2 <211> 21 <212> DNA <213> Artificial sequence <220><223> Reverse primer for mouse tyrosinase gene R1 <400> 2 201228676 tggtgct tea tgggcaaaat c 21

<210> 3 <211〉 20 <212> DNA <213>人工的序列 <220> <223>用於小鼠GAPDH基因的前向引子F2 <400〉 3 accacagtcc atgccatcac 20<210> 3 <211>20 <212> DNA <213> Artificial sequence <220><223> Forward primer for mouse GAPDH gene F2 <400> 3 accacagtcc atgccatcac 20

<210> 4 <211〉20 <212〉 DNA <213>人工的序列 <220> <223〉用於小鼠GAPDH基因的反向引子R2 <400> 4 tccaccaccc tgt tgctgta 20 2<210> 4 <211>20 <212> DNA <213> Artificial sequence <220><223> Reverse primer for mouse GAPDH gene R2 <400> 4 tccaccaccc tgt tgctgta 20 2

Claims (1)

201228676 七、申凊專利範圍·· i 一種用於皮膚美白的藥學組成物，其包含有一有效量之 選自下列群組中的藥物：同氣克利定（Homochlorcyclizine) 、單那若（Danazol)，以及它們的組合。 2. 一種如申請專利範圍第丨 皮膚美白產品的用途。的樂予組成物供用於製備一201228676 VII. Application scope of patents·· i A pharmaceutical composition for skin whitening comprising an effective amount of a drug selected from the group consisting of Homochlorcyclizine and Danazol. And their combination. 2. A use of a skin whitening product as claimed in the patent application. Le composition for use in preparation