經濟部智慧財產局員工消費合作社印製 1313177 A7 B7 五、發明說明（丨） 本發明係關於自天然物中萃取美白化妝品原料，尤其是 關於自檸檬皮中利用丙二醇（Propylene glycol)萃取出含酪胺 酸酵素(Tyrosinase)抑制劑之混合物。 人體皮膚表現之黑色，係因黑色素細胞合成不同型態不同 量的黑色素以抵抗日光的傷害。黑色素在黑色素體 (Melanosome)中合成堆積，人體的膚色就是由Melanosome的 大小型態、顏色、分佈情形決定。黑色素的生成機制不會因人 種不同而有所不同，所以世界不同人種皮膚沉澱黑色素的現 象，都可以用黑色素的生成機制來解釋。黑色素細胞中的酪胺 酸酵素活性決定了黑色素的生成速度。此酵素可將細胞內酪胺 酸(Tyrosine)轉換成L-3,4雙羥基苯丙胺基(L-DOPA， L-dihydroxy phenylalanine)，再形成多巴色素(Dopachrome)， 最後形成黑色素(Melanin)，如圖一所示。 若要抑制黑色素生成，就必須抑制酪胺酸被酪胺酸酵素轉 換的反應，習知美白成分對苯二酚(Hydroquinone)便是替代酪 胺酸被酪胺酸酵素氧化。由於Hydroquinone易朿[]激皮膚，於 是常以Hydroquinone衍生物如熊果素(Arbutin)來減少刺激。 Arbutin缺點有單價高具光敏性，顏色變深，配方中不能存在 離子性成分。習知的美白成分有以下缺點： 1.對光線不穩定有褐變(如Arbutin，麹酸Kojic acid)黃變現象 (抗壞血酸Ascorbic acid及其衍生物）：美白產品常在用了 一段時間變色如變黃變黑，造成產品穩定性不佳。 2·對酸鹼値有很大的限制：如果酸在pH=2.3以下才能發揮最 3 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） ------------------η---t?----1---- (請先閲讀背面之注意事項再填寫本頁) 經濟部智慧財產局員Η消費合作社印製 1313177 A7 _ B7 五、發明說明（V) 大的效果，Arbutin也必須在酸性範圍(愈酸愈好，通常在 PH<4)，當然相對刺激也大，而且耐酸性化妝品原料有限。 3.爲非競爭性抑制劑··美白成分通常結構與酪胺酸相似，而 達到競爭性抑制效果(如Arbutin)，濃度愈高，效果愈好， 刺激愈大，產品也更易變色。 因此業界有需求尋找新的美白成分’使用上安全並能克服 習知美白成分的缺點。 本發明的目的爲提供一種自檸檬皮中萃取出含酪胺酸酵 素抑制劑之混合物的方法，該萃取係使用丙二醇。 本發明的另一個目的爲提供一種自檸檬皮萃取，經部分純 化所得之含酪胺酸酵素抑制劑之美白混合物’含有一種蛋白質 或胜肽。雖然此一蛋白質爲酪胺酸酵素抑制劑’但其他成份或 有加成效果，或有抗老化效果，不需過度純化因而相對成本 低。本發明所提供之美白混合物所具有的其他優點如下： 1. 其可作用pH爲7-9，不需在酸性範圍中使用’相對上對皮 膚刺激性低且無須特殊配方或原料，只要在製備時注意在 45°C以下再加入，不要引起蛋白質變性即可。 2. 其爲非競爭性抑制劑，無須高濃度即可有效抑制黑色素生 成，若與Arbutin相比，Arbutin之原料價格約爲NT$70,000， 且一般建議用量約需7%，相對上本美白混合物對酪胺酸酵 素的抑制效果更佳且所需濃度低。 3. 含該美白混合物之化妝品無須使用特殊遮光容器，不因光 線使化妝品變色。 4 ------------------—tr--------- (請先閱讀背面之注意事項再填寫本頁) 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） A7 1313177 五、發明說明（7)) 4. 含S亥美白混合物之化妝品無使用上之限制’大多數美白化 妝品都是在白天防曬晚上美白，早晚均可使用。 5. 含該美白混合物之化妝品溫和，不刺激不易引起過敏反應。 6. 其在25°C左右可發揮最大活性，即使37°C(人體體溫)下仍 能對酪胺酸酵素達近80%左右之抑制效果，適合開發成美 白化妝品。 7. 使用短時間內即有抑制效果，作用一小時即可對酪胺酸酵 素達90%以上抑制效果。 圖式之簡單說明 圖一：黑色素生成機制。 圖二：美白混合物於不同pH値下對酪胺酸酵素抑制效果。 圖三：美白混合物對酪胺酸酵素的抑制型式之雙倒數作圖。 圖四··美白混合物在不同波長(nm)下之吸收(ABS，Ministry of Economic Affairs, Intellectual Property Bureau, Staff and Consumer Cooperatives, Printing 1313177 A7 B7 V. INSTRUCTIONS (丨) The present invention relates to the extraction of whitening cosmetic raw materials from natural materials, in particular, the extraction of cheese containing propylene glycol (Propylene glycol) from lemon peel. A mixture of inhibitors of Tyrosinase. The blackness of the human skin is due to the synthesis of different types of melanin by melanocytes to protect against sunlight. Melanin is synthesized and accumulated in the melanosome. The skin color of the human body is determined by the large and small states, colors and distribution of the Melanosome. The mechanism of melanin production does not vary from person to person, so the phenomenon of melanin precipitation in different skin types in the world can be explained by the mechanism of melanin production. The activity of tyrosinase in melanocytes determines the rate of melanin production. This enzyme converts intracellular Tyrosine into L-3, L-dihydroxy phenylalanine, then forms Dopachrome, and finally forms Melanin. As shown in Figure 1. In order to inhibit melanin production, it is necessary to inhibit the reaction of tyrosine to be converted by tyrosinase. The conventional whitening component Hydroquinone is an alternative to tyrosine oxidized by tyrosinase. Since Hydroquinone is prone to irritating the skin, Hydroquinone derivatives such as Arbutin are often used to reduce irritation. The disadvantage of Arbutin is that the unit price is highly photosensitive, the color is deep, and there is no ionic component in the formulation. Conventional whitening ingredients have the following disadvantages: 1. Browning (such as Arbutin, Kojic acid) yellowing (ascorbic acid and its derivatives) is unstable to light: whitening products are often used for a period of time like color Turning yellow to black, resulting in poor product stability. 2. There are great restrictions on acid and alkali: if the acid is below pH=2.3, it can be used for the most 3 paper scales. It is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) -------- ----------η---t?----1---- (Please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau Η Consumer Cooperative Print 1313177 A7 _ B7 V. Inventive Note (V) The big effect, Arbutin must also be in the acidic range (the better the acid, usually at pH < 4), of course, the relative stimulation is also large, and the acid-resistant cosmetic raw materials are limited. 3. Non-competitive inhibitors · Whitening ingredients usually have a structure similar to tyrosine, and achieve competitive inhibition effects (such as Arbutin). The higher the concentration, the better the effect, the greater the stimulation, and the more susceptible the product is. Therefore, there is a need in the industry to find new whitening ingredients that are safe to use and overcome the shortcomings of conventional whitening ingredients. SUMMARY OF THE INVENTION An object of the present invention is to provide a method for extracting a mixture containing a tyrosinase inhibitor from lemon peel using propylene glycol. Another object of the present invention is to provide a whitening mixture containing a tyrosinase inhibitor obtained by partial purification from a lemon peel, which contains a protein or a peptide. Although this protein is a tyrosinase inhibitor', other ingredients may have an additive effect or have an anti-aging effect, which does not require excessive purification and is relatively low in cost. The other advantages of the whitening mixture provided by the present invention are as follows: 1. It has a working pH of 7-9, and does not need to be used in the acidic range. ' Relatively low to skin irritation and no special formulation or raw material, as long as it is prepared Please pay attention to add below 45 °C, do not cause protein denaturation. 2. It is a non-competitive inhibitor, which can effectively inhibit melanin production without high concentration. If compared with Arbutin, the raw material price of Arbutin is about NT$70,000, and the recommended dosage is about 7%, which is relative to the whitening mixture. Tyrosinase has a better inhibitory effect and a lower concentration. 3. Cosmetics containing this whitening mixture do not require the use of special light-shielding containers, and do not discolor the cosmetics due to light. 4 -------------------tr--------- (Please read the notes on the back and fill out this page) This paper scale applies to Chinese national standards. (CNS) A4 specification (210 X 297 mm) A7 1313177 V. Description of invention (7)) 4. No restrictions on the use of cosmetics containing S-Hai whitening mixture. Most whitening cosmetics are whitening at night during the day, whitening sooner or later. Can be used. 5. The cosmetic containing the whitening mixture is mild and does not irritate and is not susceptible to allergic reactions. 6. It can exert maximum activity at around 25 °C, and it can inhibit the tyrosinase by nearly 80% even at 37 °C (body temperature), and it is suitable for the development of whitening cosmetics. 7. It can inhibit the tyrosinase by more than 90% in one hour. A brief description of the schema Figure 1: Melanogenesis mechanism. Figure 2: Inhibition of tyrosinase by whitening mixture at different pH. Figure 3: Double reciprocal plot of the inhibitory version of the whitening mixture on tyrosic acid. Figure 4. Absorption of whitening mixtures at different wavelengths (nm) (ABS,
Absorption)。（A)用80%丙二醇爲樣品做測量；⑼以美 白混合物爲樣品做測量。 圖五：美白混合物抑制酪胺酸酵素生成〇-qUin〇ne(A)用80% 丙二醇爲樣品做測量(B)用50μ1之美白混合物爲樣品做 測量(C)用ΙΟΟμΙ之美白混合物爲樣品做測量 本發明所提供之方法如下。取新鮮檸檬皮，將其切碎，再 加入丙二醇與其混合。其混合比例爲：每10〇g碎檸檬皮加入 100-1000ml之丙二醇。將該混合液充分混合，利用超音波處 理1〜59秒以打破細胞壁，再以微波照射進行殺菌，然後將混 5 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公髮） ------------------^--"------ (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 ILV »»3- 1313177 A7 B7 五、發明說明（(\ ) 合液進行離心分離’取上層液即爲本發明之含酪胺酸酵素抑制 劑之美白混合物。若取lOOg打碎之檸檬皮，依上述方法進行 萃取，可得丙二醇體積用量70-90%之美白混合物。 萃取出的美白混合物具有以下特性： 1. 在25°C有最高抑制酪胺酸酵素活性。 2. 最適 pH 7-9。 3. 具光穩定性。 4. 爲非競爭性抑制劑。 酪胺酸酵素抑制活性之測定方法：一般分光光度儀於 475nm下測吸光値’分另[|測含有及不含本發明美白混合物之實 驗組及對照組之吸光値’計算吸光値減少百分比，以得到抑制 活性之比率。抑制百分比率高，表示美白效率佳。實驗組(含 抑制劑反應活性)及對照組(不含抑制劑反應活性)的樣品組成 如下： 0)實驗組：35μ1依本發明萃取之含酪胺酸酵素抑制劑之美白 混合物’ 15μ1(15ιιώ)酪胺酸酵素溶液及450μ1之16.7mM 磷酸鉀(Potassium Phosphate)緩衝溶液 pH 6·5 包含 3.4mM L-DOPA 及 0.5% Triton X-100，於 475 nm 下測吸光値。 ⑻對照組：35μ1 80%丙二醇，15μ1(15ιιώ)酪胺酸酵素溶液 及45〇μ1之16.7mM磷酸鉀緩衝溶液pH 6.5包含3.4mM L-DOPA 及 0.5 %Triton X-100，於 475nm 下測吸光値。 6 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） (請先閲讀背面之注意事項再填寫本頁) .0——r 訂--------- 經濟部智慧財產局員工消費合作社印製 Va 經濟部智慧財產局員工消費合作社印製 1313177 A7 B7 五、發明說明（彳） 抑制百分比之計算公式： 抑制百分比(％)=[ 1 -(實驗組之吸光値)/ (對照組之吸光値)] X 100% 其中的DOPA及1000 unit/ml酪胺酸酵素溶液(存於 16.7mM磷酸鉀緩衝溶液)購自美國SIGMA公司。 1單位(1 unit)的酪胺酸酵素的定義爲包含L-DOPA反應混 合物0.5ml(其pH値爲6·5)，在25°C時每單位酵素使得a475吸 光値每分鐘增加0.00卜 實施例1 取新鮮檸檬’將其去籽，去果肉’再將檸檬皮切碎。取100g 該碎檸檬皮與400 ml丙二醇充分混合，利用超音波處理20 秒，再用微波處理三分鐘。再將混合溶液進行離心分離，取得 350 ml上層液，爲美白混合物(其即爲含酪胺酸酵素抑制劑之 混合物），該美白混合物係存於80%(80 wt %)之丙二醇溶液 中。 實施例2 :不同作用時間之抑制百分比之測定 以實施例1所得到的美白混合物爲含酪胺酸酵素抑制劑之 混合物，以配製實驗組樣品，及對照組樣品(參酪胺酸酵素抑 制活性之測定方法所述之樣品組成）。 先將15μ1酪胺酸酵素溶液和35μ1美白混合物混合’ ^ 25〇C分別靜置0，1，2，...，6小時。再將上述靜置之混合物 分別加入450μ1的L-DOPA受質(Substrate)以開始反應。故可 7 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------------tl — t·· — — I---- (請先閱讀背面之注意事項再填寫本頁) 1313177 A7 B7 五、發明說明（V)得到不同時間之抑制百分比如下 作用時間(小時） 抑制百分比(％) __ 0 34.0±0.8 1 93.5±0.8 2 95.4 ±0.9 3 84.6 ±0.6 4 84.2 ±0.8 5 85.7±1.6 6 86.2 ±0.4 --- C請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 眚施例3 :熱穩定性之測定以實施例1所得之混合物爲含抑制劑之美白混合物以配製 實驗組樣品，對照組之樣品配製如前述。將樣品在25°c，37°c 及50°C三溫度下，測量吸光値以計算出抑制百分比。先將15μ1酪胺酸酵素溶液和35μ1美白混合物混合，分別 在各個溫度靜置1小時。再將上述混合溶液加入450μ1的 L-DOPA受質(Substrate)以進行反應。 故得到在25〇C，37°C及50°C之溫度下，測得抑制百分比 分別爲90%，79%及69% ° 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） — 一 tr--------- --Γ 1313177Absorption). (A) Measurement with 80% propylene glycol as a sample; (9) Measurement with a whitening mixture as a sample. Figure 5: Whitening mixture inhibits tyrosinase production 〇-qUin〇ne (A) is measured with 80% propylene glycol as a sample (B) 50 μl whitening mixture is used as a sample (C) ΙΟΟμΙ whitening mixture is used as a sample The method of measuring the present invention is as follows. Take fresh lemon peel, chop it, and mix with propylene glycol. The mixing ratio is: 100-1000 ml of propylene glycol is added per 10 gram of crushed lemon peel. The mixture is thoroughly mixed, ultrasonically treated for 1 to 59 seconds to break the cell wall, and then sterilized by microwave irradiation, and then the mixed paper size is applied to the Chinese National Standard (CNS) A4 specification (210 X 297 mil) - -----------------^--"------ (Please read the notes on the back and fill out this page.) Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumption Cooperatives ILV »»3- 1313177 A7 B7 V. Description of the invention ((\) Centrifugation with liquid mixture' Take the upper layer liquid as the whitening mixture containing the tyrosinase inhibitor of the present invention. If you take 100g of crushed lemon peel According to the above method, a whitening mixture of propylene glycol in a volume of 70-90% can be obtained. The extracted whitening mixture has the following characteristics: 1. The highest inhibition of tyrosinase activity at 25 ° C. 2. Optimal pH 7- 9. 3. Light stability 4. Is a non-competitive inhibitor. Determination of tyrosinase inhibitory activity: General spectrophotometer at 475nm to measure absorbance 分 ' separate [| test with and without the present invention Absorbance 实验 of the experimental group and the control group of the whitening mixture Ratio to obtain the ratio of inhibitory activity. The high percentage inhibition rate indicates good whitening efficiency. The sample composition of the experimental group (containing inhibitor activity) and the control group (without inhibitor activity) is as follows: 0) Experimental group: 35 μ1 The whitening mixture containing the tyrosinase inhibitor according to the present invention '15μ1 (15ιιώ) tyrosinase solution and 450μ1 of 16.7mM potassium phosphate (Potassium Phosphate) buffer solution pH 6·5 contains 3.4mM L-DOPA and 0.5 % Triton X-100, measured at 475 nm. (8) Control group: 35μ1 80% propylene glycol, 15μ1 (15ιιώ) tyrosinase solution and 45〇μ16.7 mM potassium phosphate buffer solution pH 6.5 containing 3.4mM L-DOPA and 0.5% Triton X-100, absorbance at 475nm value. 6 This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) (please read the notes on the back and fill out this page). 0——r Order--------- Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Va Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1313177 A7 B7 V. Invention Description (彳) Calculation formula for percent inhibition: percent inhibition (%) = [ 1 - (absorbance of experimental group) ) / (absorbance of the control group)] X 100% Among them, DOPA and 1000 unit/ml tyrosinase solution (stored in 16.7 mM potassium phosphate buffer solution) were purchased from SIGMA, USA. One unit (1 unit) of tyrosinase is defined as containing 0.5 ml of L-DOPA reaction mixture (pH 6 is 6.5). At 25 ° C, each unit of enzyme makes a475 absorbance 增加 increase by 0.00 per minute. Example 1 Take a fresh lemon 'to remove the seeds, go to the flesh' and then chop the lemon peel. 100 g of the crushed lemon peel was thoroughly mixed with 400 ml of propylene glycol, ultrasonically treated for 20 seconds, and microwaved for three minutes. Further, the mixed solution was centrifuged to obtain 350 ml of the supernatant liquid, which was a whitening mixture (which is a mixture containing a tyrosinase inhibitor), and the whitening mixture was stored in an 80% (80 wt%) propylene glycol solution. Example 2: Determination of percentage inhibition of different action times The whitening mixture obtained in Example 1 was a mixture containing a tyrosinase inhibitor to prepare an experimental group sample, and a control sample (paratyrosinase inhibitory activity) The sample composition described in the measurement method). The 15 μl tyrosinase solution and the 35 μl whitening mixture were first mixed with '^25〇C, respectively, and allowed to stand for 0, 1, 2, ..., for 6 hours. The above-mentioned still mixture was separately added to 450 μl of L-DOPA Substrate to start the reaction. Therefore, the paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -----------------tl — t·· — — I--- - (Please read the notes on the back and fill out this page) 1313177 A7 B7 V. INSTRUCTIONS (V) Percentage of inhibition at different times as follows (hours) Percent inhibition (%) __ 0 34.0 ± 0.8 1 93.5 ± 0.8 2 95.4 ±0.9 3 84.6 ±0.6 4 84.2 ±0.8 5 85.7±1.6 6 86.2 ±0.4 --- C Please read the notes on the back and fill out this page. Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Cooperatives, Inc. : Determination of Thermal Stability The mixture obtained in Example 1 was an inhibitor-containing whitening mixture to prepare an experimental group sample, and the control sample was prepared as described above. The absorbance was measured at three temperatures of 25 ° C, 37 ° C and 50 ° C to calculate the percent inhibition. First, a 15 μl tyrosinase solution and a 35 μl whitening mixture were mixed and allowed to stand at each temperature for 1 hour. The above mixed solution was further added to 450 μl of L-DOPA Substrate to carry out the reaction. Therefore, the percentages of inhibition measured at 25 ° C, 37 ° C and 50 ° C are 90%, 79% and 69% respectively. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 PCT) — a tr--------- --Γ 1313177
經濟部智慧財產局員工消費合作社印製V 五、發姐ilM ( ^ )—__— __溫度(°C)__迎制百分比(％) 25 90.36 37 79.30 50 69.10 實施例4 :不同pH値的測定 I. 實驗組樣品組成如下： 1. 300μ1之0.1M緩衝溶液混合物似CH3COONa， NaH2P04，Na2B407 及 Tris_HCl 製成數個 pH 値：4，5， 6,7,8,9)包含0.5%1^〇11又-100; 2. 35μ1之美白混合物； 3. 15μ1(15 unit)之 1000 unit/ml 酪胺酸酵素溶液； 4. 150μ1之10 mM L-DOPA(以16.7mM磷酸鉀緩衝溶液配製）。II. 對照組樣品組成如下： 1· 300μ1之0.1M緩衝溶液混合物似CH3COONa， NaH2P04 ’ Na2B407 及 Tris-HCl 製成數個 pH 値：4，5， 6 5 7 5 8 5 0.5% Triton X-1〇〇 ； 2· 35μ1 之 80%丙二醇； 3· 15μ1(15 unit)之 1000 unit/ml 酪胺酸酵素溶液； 9 本紙張尺度適用中關家標準（CNS)A4規格（210 X 297公釐) " ' .一___J________*411^______訂_________· (請先閱讀背面之注意事項再填寫本頁) -l·. 經濟部智慧財產局員工消費合作社印製 1313177 A7 B7 五、發明說明（8") 4. 150μ1之10 mM L-DOPA(以16.7m]V[磷酸鉀緩衝溶液配 製）。Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing V V. Sister ilM ( ^ ) — __ — __ temperature (°C) __ percentage of welcome (%) 25 90.36 37 79.30 50 69.10 Example 4: different pH値Determination I. The experimental group sample composition is as follows: 1. 300μ1 of 0.1M buffer solution mixture like CH3COONa, NaH2P04, Na2B407 and Tris_HCl made several pH 値: 4,5, 6,7,8,9) contains 0.5%1 ^〇11 again-100; 2. 35μ1 whitening mixture; 3. 15μ1 (15 unit) 1000 unit/ml tyrosinase solution; 4. 150μ1 10 mM L-DOPA (prepared with 16.7mM potassium phosphate buffer solution ). II. The composition of the control sample is as follows: 1· 300μ1 of 0.1M buffer solution mixture like CH3COONa, NaH2P04 'Na2B407 and Tris-HCl made several pH 値: 4,5, 6 5 7 5 8 5 0.5% Triton X-1 〇〇; 2· 35μ1 of 80% propylene glycol; 3·15μ1 (15 unit) of 1000 unit/ml tyrosine solution; 9 This paper size applies to the National Standard (CNS) A4 specification (210 X 297 mm) " ' . A ___J________ * 411 ^ ______ order _________ · (Please read the note on the back and then fill out this page) -l·. Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 1313177 A7 B7 V. Invention Description (8") 4. 150 μl of 10 mM L-DOPA (prepared with 16.7 m] V [potassium phosphate buffer solution).
先將15μ1酪胺酸酵素溶液和35μ1美白混合物及300μ1緩 衝液混合，在25°C靜置一小時。將150μ1之l〇mM L-DOPA 受質加進上述混合溶液以進行反應。故得到在不同pH値下所 測得之抑制百分比如下： pH 抑制百分比(％) 4 - 5 29·0±3.6 6 17.4±2.1 7 60.0±4.4 8 60.8±3.4 9 47.1±1.1 並作圖如圖二。 實施例5 :美白混合物抑制作用型式之分析 I. 實驗組樣品組成如下： 1. 450 μΐ 之 16.7 mM 磷酸鉀緩衝液(PH6.5)包含 0.1，0.35 0.75，1.5，3 或 6 mM L-DOPA 及 0.5% Triton X-100 ; 2. 15或35 μΐ之美白混合物； 3. 15 μΐ之1000 units/ml酪胺酸酵素溶液。 II. 對照組樣品組成如下： 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） -----------—i丨订· (請先閱讀背面之注意事項再填寫本頁)First, a 15 μl tyrosinase solution and a 35 μl whitening mixture and 300 μl of a buffer were mixed and allowed to stand at 25 ° C for one hour. 150 μl of 1 mM L-DOPA substrate was added to the above mixed solution to carry out a reaction. Therefore, the percentage of inhibition measured at different pH 如下 is as follows: pH inhibition percentage (%) 4 - 5 29·0 ± 3.6 6 17.4 ± 2.1 7 60.0 ± 4.4 8 60.8 ± 3.4 9 47.1 ± 1.1 two. Example 5: Analysis of the whitening mixture inhibition pattern I. The experimental group sample composition is as follows: 1. 450 μΐ of 16.7 mM potassium phosphate buffer (pH 6.5) containing 0.1, 0.35 0.75, 1.5, 3 or 6 mM L-DOPA And 0.5% Triton X-100; 2. 15 or 35 μΐ whitening mixture; 3. 15 μΐ of 1000 units/ml tyrosinase solution. II. The composition of the control sample is as follows: The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -----------—i丨定· (Please read the back of the note first) Please fill out this page again)
I L·I L·
II A7 B7 1313177 五、發明說明) 1. 450 μΐ 之 16.7 mM 磷酸鉀緩衝液(pH6.5)包含 0.1，0.35， 0.75，1.5，3 或 6mML-DOPA 及 0.5%TritonX-100 ; 2. 15 或 35 μΐ 之 80 %丙二醇； 4. 15 μ1(15 units)之 1000 units/ml 酪胺酸酵素溶液。 先將15μ1酪胺酸酵素溶液和15或35μ1美白混合物混合在 25°C靜置一小時。將上述混合物加至450μ1之L-DOPA受質以 進行反應。故得到以下之反應數據： 起始速度(Vi) 起始速度(Vi) l/Vi l/Vi 受晳濃度 一 當沒有抑制劑萃當美白混合物當沒有抑制濟!(萃當美白混合物 [S] 取物存在 二35μ1 取物存在 =35μ1 (mM) (A〇.D.475/min) (Δ O.D.475/min) (min/Δ O.D.475) (min/Δ O.D.475) 0.10 0.008 0.0007 124.40 1471.35 0.35 0.018 0.0013 54.17 746.55 0.75 0.033 0.0025 30.68 403.71 1.50 0.033 0.0030 30.62 329.92 3.00 0.043 0.0061 23.13 165.08 6.00 0.048 0.0075 20.92 133.87 根據以上數據以l/Vi對1/[S]作圖得到圖三所示之雙倒數 作圖。由圖可知美白混合物對酪胺酸酵素的作用型式屬於非競 爭性抑制劑。 實施例ό :美白混合物與Arbutin比較 I.實驗組樣品： 11 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） :!-------參·!备---------0 (請先閱讀背面之注意事項再填寫本頁) 經濟部智慧財產局員工消費合作社印製 1313177 A7 B7 五、發明說明（θ ) 1. 450 μΐ之16.7 mM磷酸鉀緩衝液(pH 6.5)包含3.4 mM L-DOPA 及 0.5% Triton X-100 ; (請先閱讀背面之注意事項再填寫本頁) 2. 15 μΐ之各種抑制劑： Α.美白混合物 B. 5 mg/ml Arbutin(在 80%丙二醇中）。 3.35 μ1(35 units)之 1000 units/ml 酪胺酸酵素溶液。 II.對照組樣品： 1. 450 μΐ之16.7 mM磷酸鉀緩衝液(ρΗ6·5)包含3.4 mM L-DOPA 及 0.5% Triton X-100 ; 2. 15 μΐ 之 80%丙二醇； 3. 35 μ1(35 units)之 1000 units/ml 酪胺酸酵素溶液。II A7 B7 1313177 V. INSTRUCTIONS) 1. 450 μΐ of 16.7 mM potassium phosphate buffer (pH 6.5) contains 0.1, 0.35, 0.75, 1.5, 3 or 6mML-DOPA and 0.5% TritonX-100; 2. 15 or 35 μΐ of 80% propylene glycol; 4. 15 μl (15 units) of 1000 units/ml tyrosinase solution. The 15 μl tyrosinase solution and the 15 or 35 μ1 whitening mixture were first mixed and allowed to stand at 25 ° C for one hour. The above mixture was added to 450 μl of L-DOPA substrate to carry out the reaction. Therefore, the following reaction data is obtained: Starting speed (Vi) Starting speed (Vi) l/Vi l/Vi Clear concentration When there is no inhibitor, when whitening the mixture, there is no inhibition! (Extraction whitening mixture [S] The presence of two 35μ1 is present in the presence of =35μ1 (mM) (A〇.D.475/min) (Δ OD475/min) (min/Δ OD475) (min/Δ OD475) 0.10 0.008 0.0007 124.40 1471.35 0.35 0.018 0.0013 54.17 746.55 0.75 0.033 0.0025 30.68 403.71 1.50 0.033 0.0030 30.62 329.92 3.00 0.043 0.0061 23.13 165.08 6.00 0.048 0.0075 20.92 133.87 Based on the above data, the double reciprocal plot shown in Figure 3 is obtained by plotting l/Vi versus 1/[S]. It can be seen from the figure that the effect of the whitening mixture on tyrosine is a non-competitive inhibitor. Example ό: Whitening mixture compared with Arbutin I. Experimental group sample: 11 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) :!------- 参·!备---------0 (Please read the notes on the back and fill out this page) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative 1313177 A7 B7 V. Description of the invention (θ) 1. 450 μΐ of 16.7 mM phosphorus Potassium acid buffer (pH 6.5) contains 3.4 mM L-DOPA and 0.5% Triton X-100; (Please read the note on the back and fill out this page) 2. Various inhibitors of 15 μΐ: Α. Whitening mixture B. 5 mg/ml Arbutin (in 80% propylene glycol) 3.35 μl (35 units) of 1000 units/ml tyrosinase solution II. Control sample: 1. 450 μΐ of 16.7 mM potassium phosphate buffer (ρΗ6· 5) Contains 3.4 mM L-DOPA and 0.5% Triton X-100; 2. 15 μΐ 80% propylene glycol; 3. 35 μ1 (35 units) 1000 units/ml tyrosinase solution.
先將35μ1酪胺酸酵素溶液和15μ1美白混合物或Arbutin 在25°C混合，靜置一小時。將上述混合物加至450μ1之L-DOPA 受質以進行反應。故得到以下抑制百分比： 樣品 抑制百分比(％) 美白混合物 90.3 ±0.3 Arbutin 32±1 經濟部智慧財產局員工消費合作社印製 實施例7 : 取80%丙二醇當做Blank，測量200-600 nm之吸收(ABS ’ Absorption)，得到圖四 A。 將美白混合物稀釋100倍，測量200-600 nm之吸收，得 到圖四B。 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐） A7 B7 1313177 五、發明說明u ) 比較圖四A，圖四B ’可知美白混合物含有三種主成分， 其中一成分爲蛋白質或胜狀，因在280 nm顯示有吸收。 (請先閱讀背面之注意事項再填寫本頁) 實施例8 :美白混合物抑制酪胺酸酵素生成O-quinone I. 實驗組樣品： 1. 450 μΐ之16.7 mM磷酸鉀緩衝液(pH 6.5)包含3.4 mM L-DOPA 及 0.5% Triton X-100 ; 2.50或100 μΐ之美白混合物； 3. 15 μ1(15 units)之 1000 units/ml 酪胺酸酵素溶液。 II. 對照組樣品： 1. 450 μΐ之16.7 mM磷酸鉀緩衝液(pH6_5)包含3.4 mM L-DOPA 及 0.5% Triton X-100 ; 2. 10 μΐ 之 80%丙二醇； 3. 15 μ1(15 units)之 1000 units/ml 酷胺酸酵素溶液。 將對照組之各樣品溶液在25°C混合，靜置30分，測量 200-600 nm之吸收，得到圖五A。將實驗組之各樣品溶液在 25°C混合，靜置30分，測量200-600 nm之吸收，以50及ΙΟΟμΙ 之美白混合物爲樣品分別得到圖五Β及C。 經濟部智慧財產局員工消費合作社印製 因O-quinone在波長475nm處有較高之吸光値，比較圖五 A，B及C，波長475nm處之吸光値會隨美白混合物之加入量 增加而減少，由此可知美白混合物能抑制酪胺酸酵素生成 O-quinone ° 13 本紙張尺度適用中國國家標準（CNS)A4規格（210 X 297公釐）The 35 μl tyrosinase solution was first mixed with a 15 μl whitening mixture or Arbutin at 25 ° C and allowed to stand for one hour. The above mixture was added to a 450 μl L-DOPA substrate to carry out the reaction. Therefore, the following percentage of inhibition was obtained: % inhibition of sample (%) Whitening mixture 90.3 ± 0.3 Arbutin 32±1 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed Example 7: Take 80% propylene glycol as Blank, measure absorption at 200-600 nm ( ABS 'Absorption', get Figure 4A. The whitening mixture was diluted 100 times and the absorption at 200-600 nm was measured to obtain Figure 4B. This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) A7 B7 1313177 V. Invention description u) Compare Figure 4A, Figure 4B' to know that the whitening mixture contains three main components, one of which is protein. Or win, because there is absorption at 280 nm. (Please read the notes on the back and fill out this page.) Example 8: Whitening mixture inhibits tyrosinase production O-quinone I. Experimental group samples: 1. 450 μΐ of 16.7 mM potassium phosphate buffer (pH 6.5) contains 3.4 mM L-DOPA and 0.5% Triton X-100; 2.50 or 100 μΐ whitening mixture; 3. 15 μ1 (15 units) of 1000 units/ml tyrosinase solution. II. Control sample: 1. 450 μL of 16.7 mM potassium phosphate buffer (pH 6_5) containing 3.4 mM L-DOPA and 0.5% Triton X-100; 2. 10 μΐ 80% propylene glycol; 3. 15 μ1 (15 units ) 1000 units / ml carbamic acid enzyme solution. Each sample solution of the control group was mixed at 25 ° C, allowed to stand for 30 minutes, and the absorption at 200-600 nm was measured to obtain Figure 5A. Each sample solution of the experimental group was mixed at 25 ° C, allowed to stand for 30 minutes, and the absorption of 200-600 nm was measured, and the whitening mixture of 50 and ΙΟΟμΙ was used as a sample to obtain Fig. 5 and C, respectively. The Ministry of Economic Affairs' Intellectual Property Bureau employee consumption cooperative printed because O-quinone has a high absorption 波长 at a wavelength of 475nm. Comparing Figure 5A, B and C, the absorption 値 at 475nm will decrease with the addition of whitening mixture. Thus, it can be seen that the whitening mixture can inhibit the production of tyrosinase O-quinone ° 13 This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)