TWI469800B - A skin whitening purplish anthocyanin gel and its preparation method - Google Patents

Description

具皮膚美白之紫米花青素凝膠劑及其製備方法Skin whitening purple rice anthocyanin gel and preparation method thereof

本發明係提供一種具皮膚美白之紫米花青素凝膠劑，尤指其技術上提供一種從紫米中濃縮、酸化脫醣所萃取之花青素萃取物所製作之凝膠劑，具有抑制黑色素形成及美白之功效者。The present invention provides a skin whitening purple flower anthocyanin gel, in particular to a gel agent prepared by extracting an anthocyanin extract extracted from purple rice and acidifying and degumming, which has the inhibition Melanin formation and whitening effect.

人體皮膚黝黑的過程主要為黑色素的產生與沉積。黑色素的化學生成主要來自DOPA遇到自由基等氧化作用而開始聚合反應而產生。在最新的研究指出，哺乳類動物的免疫系統中，黑色素細胞並非單純僅產生黑色素的功能而已，黑色素細胞同時也是一種巨噬細胞，而且可以受到發炎反應的調控，因此黑色素的產生與免疫系統的反應有關，當氧化性傷害產生時，也會造成皮膚黑色素的產生，這主要的黑色素產生機制是來自於多巴胺於細胞中接觸到自由基或是具有高能的氧化性物質，可以透過酪氨酸的共同作用而產生聚合反應，進一步合成產生黑色素。又類鐸受體(toll-like receptors，TLRs)原本是人體免疫的第一道防線的接收器，用來抵抗致病原與產生免疫反應，而人類黑色素細胞目前已經被證實會表現類鐸受體(TLRs)的種類多達9種，因此推測可能有某些類鐸受體(TLRs)與黑色素的合成、沉積有關，故透過抑制部份類鐸受體過度表現，也是另一個可以達到使皮膚美白的策略。有鑑於此，發明人本於多年從事相關研究與設計經驗，針對上述之目標，詳加設計與審慎評估後，終得一確具實用性之本發明。The process of darkening human skin is mainly the production and deposition of melanin. The chemical formation of melanin is mainly caused by the initiation of polymerization by DOPA in the presence of an oxidation reaction such as a radical. In the latest research, it is pointed out that in the immune system of mammals, melanocytes are not only melanin-producing functions, melanocytes are also macrophages, and can be regulated by inflammatory reactions, so melanin production and immune system response Related to, when oxidative damage occurs, it also causes the production of melanin in the skin. This main melanin production mechanism is derived from the contact of dopamine in cells with free radicals or high-energy oxidizing substances, which can pass through tyrosine. The reaction produces a polymerization reaction and further synthesis produces melanin. Toll-like receptors (TLRs) were originally the first line of defense for human immunity, used to fight pathogens and produce immune responses, and human melanocytes have now been shown to exhibit sputum-like receptors. There are up to nine species of TLRs, so it is speculated that some types of terpenoid receptors (TLRs) may be involved in the synthesis and deposition of melanin. Therefore, by inhibiting the excessive expression of some terpenoid receptors, another can be achieved. Skin whitening strategy. In view of this, the inventor has been engaged in relevant research and design experience for many years, and after detailed design and careful evaluation of the above objectives, the invention has finally become practical.

黑色素的產生與免疫系統的反應有關，當氧化性傷害產生時，也會造成皮膚黑色素的產生；又類鐸受體(toll-like receptors，TLRs)原本是人體免疫的第一道防線的接收器，用來抵抗致病原與產生免疫反應，而人類黑色素細胞目前已經被證實會表現類鐸受體(TLRs)的種類多達9種，因此推測可能有某些類鐸受體(TLRs)與黑色素的合成、沉積有關，故透過抑制部份類鐸受體過度表現，也是另一個可以達到使皮膚美白的策略。The production of melanin is related to the reaction of the immune system. When oxidative damage occurs, it also causes the production of melanin in the skin. The toll-like receptors (TLRs) are the first line of defense for human immunity. Used to fight pathogens and produce immune responses, and human melanocytes have been shown to exhibit up to 9 species of terpenoid receptors (TLRs), so it is speculated that there may be certain terpenoid receptors (TLRs) and The synthesis and deposition of melanin are related to each other, so by inhibiting the excessive expression of some of the terpenoid receptors, it is another strategy that can achieve skin whitening.

為達到上述之目標，本發明提供一種具皮膚美白之紫米花青素凝膠劑，該凝膠劑主要含有從紫米中濃縮、酸化脫醣所萃取之花青素萃取物。其製備方法包含以下步驟：步驟一：取1kg紫米，加入2 L甲醇浸泡24小時濃縮為浸膏；步驟二：加入pH 3.2醋酸水溶液至該浸膏中後加熱至80℃，並使其反應10小時；步驟三：使用乙酸乙酯萃取收集有機層的紫色溶液，濃縮後再冷凍乾燥成粉末，該粉末可使用二次水溶解後形成紫米花青素萃取物(0.3g/ml)；步驟四：將該紫米花青素萃取物利用濾膜過濾後保存於4℃備用；步驟五：另取1.5g殺菌後的水溶性高分子膠(carbopol)加入900ml殺菌後的二次去離子水中，使用0.1N的氫氧化鈉水溶液調整酸鹼值至中性，再加入5ml已滅菌的甘油；及步驟六：加入步驟四所獲得之紫米花青素萃取物5ml後，再使用超音波均質震盪即完成紫米花青素凝膠的製備。In order to achieve the above object, the present invention provides a skin whitening purple anthocyanin gel which mainly comprises an anthocyanin extract extracted from a purple rice and concentrated by acidification and de-sugarization. The preparation method comprises the following steps: Step 1: Take 1kg of purple rice, add 2 L of methanol to soak for 24 hours and concentrate to extract; Step 2: add pH 3.2 acetic acid aqueous solution to the extract, heat to 80 ° C, and react 10 hours; Step 3: Extracting the purple solution of the organic layer with ethyl acetate, concentrating and lyophilizing to a powder, which can be dissolved in secondary water to form an extract of purple anthocyanin (0.3 g/ml); 4: The purple anthocyanin extract is filtered through a filter and stored at 4 ° C for use; Step 5: 1.5 g of the sterilized water-soluble polymer glue (carbopol) is added to 900 ml of sterilized secondary deionized water. Use 0.1N sodium hydroxide solution to adjust the pH to neutral, then add 5ml of sterilized glycerin; and step 6: add 5ml of the purple anthocyanin extract obtained in step 4, then use ultrasonic homogenization shock The preparation of the purple rice anthocyanin gel was completed.

本發明所製備之紫米花青素萃取物可預防部分的類鐸受體過度表現，且紫米花青素具有抗氧化的功能，不但可以抑制酪氨酸酶，可有效阻斷透過氧化性機制黑素色的產生，進而達到抑制非氧化性機制黑色素產生的功能。將該紫米花青素萃取物製作為凝膠劑後，塗擦在人體上連續六週不但不會影響皮膚的正常生理機能，且在塗擦第二週就已經確實產生具有美白的功效。再者，紫米花青素凝膠劑中所含的花青素本身具有抗發炎的功能，因此不會使血色素於皮膚真皮層沉積，可避免發炎反應而導致血色素沉積於皮膚真皮層，達到良好之美白功效。The purple anthocyanin extract prepared by the invention can prevent partial overexpression of the terpenoid receptor, and the purple anthocyanins have anti-oxidation function, can not only inhibit tyrosinase, but also effectively block the oxidative mechanism black. The production of plain color, in turn, achieves the function of inhibiting the production of melanin by a non-oxidative mechanism. After the purple rice anthocyanin extract is made into a gel, rubbing on the human body for six weeks does not affect the normal physiological function of the skin, and the whitening effect is indeed produced in the second week of rubbing. Furthermore, the anthocyanins contained in the purple rice anthocyanin gel have an anti-inflammatory function, so that the hemoglobin is not deposited on the dermis layer of the skin, and the inflammatory reaction is prevented to cause hemoglobin deposition on the dermis layer of the skin, achieving good results. Whitening effect.

有關本發明所採用之技術、手段及其功效，茲舉一較佳實施例並配合圖式詳細說明於后，相信本發明上述之目的、構造及特徵，當可由之得一深入而具體的瞭解。The above-mentioned objects, structures and features of the present invention will be described in detail with reference to the preferred embodiments of the present invention. .

花青素(Anthocyanidins)是一種水溶性植物色素，分子結構上的分類屬於類黃酮的一種，並具有可以提供抗氧 化功能的官能基。花青素具有比維生素E高五十倍、比維生素C高二十倍的抗氧化能力，而且還可抵抗自由基對人體細胞的破壞，降低低密度膽固醇(LDL)及減少心血管疾病。為達到抑制部份類鐸受體過度表現，進而達到皮膚美白的目的，本發明首先使用具有抗氧化、抗發炎功能之花青素來證實花青素對於類鐸受體是否能產生抑制的功能，進而使小鼠黑色素瘤細胞的黑色素不易沉積於皮膚的功效。紫米果皮部份含有大量的花青素。本發明使用甲醇萃取紫米，經過濃縮、酸化脫醣、冷凍乾燥的程序製備花青素萃取物保存液(詳細之萃取步驟說明如後)，再利用HPLC測定紫米的結果發現含有cyanidin花青素，請參閱第一圖所示為紫米花青素的HPLC分析圖譜，結果發現，其中主要含有兩種型態的花青素，其中一種是cyanidin-3-glucoside，另外一種是peonidin-3-glucoside，這兩種成分又以cyanidin-3-glucoside含量較高，為紫米所萃取出來的花青素主要成分，與先前文獻相比較，未經酸化脫醣處理的紫米花青素粗萃物含有較多的cyanidin-3-glucoside及少量的peonidin-3-glucoside。經酸化脫醣處理的cyanidin花青素成份穩定，水溶性佳，在水溶液中不易水解變質，但仍需避光低溫保存，經由測試的實驗結果如下表1及第二圖所示，表1顯示：紫米花青素對於酪氨酸酶的抑制能力與減少小鼠黑色素瘤細胞B16的黑色素分泌能力隨著劑 量濃度上升而增加，又第二圖為經過紫米花青素處理與未經過處理的黑色素瘤細胞類鐸受體表現量，經過花青素處理後的B16細胞中，TLR2、TLR7的表現量有下降的趨勢，其餘的類鐸受體則不受影響。Anthocyanidins are water-soluble plant pigments. The molecular structure is classified into flavonoids and has antioxidant properties. Functionalized functional groups. Anthocyanins are 50 times more potent than vitamin E and 20 times more potent than vitamin C. They also resist free radical damage to human cells, lower low density cholesterol (LDL) and reduce cardiovascular disease. In order to achieve the purpose of inhibiting the excessive expression of some of the terpenoid receptors and thereby achieving skin whitening, the present invention firstly uses anthocyanins having anti-oxidation and anti-inflammatory functions to confirm whether anthocyanins can inhibit the steroid-like receptors. Furthermore, the melanin of mouse melanoma cells is not easily deposited on the skin. The purple rice peel contains a large amount of anthocyanins. The invention extracts purple rice by using methanol, and prepares an anthocyanin extract preservation solution by a process of concentration, acidification, de-sugarization and freeze-drying (detailed extraction steps are as follows), and then using HPLC to determine the purple rice, the result is found to contain cyanidin cyanine For the prime, please refer to the HPLC analysis of the purple anthocyanins shown in the first figure. It was found that there are mainly two types of anthocyanins, one of which is cyanidin-3-glucoside and the other one is pionidin-3- Glucose, which is a high content of cyanidin-3-glucoside, which is the main component of anthocyanin extracted from purple rice. Compared with the previous literature, the crude extract of purple anthocyanin without acidification and de-sugar treatment Contains more cyanidin-3-glucoside and a small amount of pionidin-3-glucoside. The acidated and defatted cyanidin anthocyanin has stable components, good water solubility, and is not easily hydrolyzed and deteriorated in aqueous solution, but it still needs to be stored in the dark at low temperature. The experimental results of the test are shown in Table 1 and Figure 2 below. Table 1 shows : The ability of purple rice anthocyanins to inhibit tyrosinase and decrease the melanin secretion ability of mouse melanoma cells B16 The amount of TAR2 and TLR7 was expressed in the B16 cells treated with anthocyanin after the anthocyanin treatment and the untreated melanoma cell type receptors were observed. The downward trend, the remaining terpenoid receptors are not affected.

再者，請參閱第三圖所示，本發明亦針對紫米花青素對於黑色素瘤細胞的毒性測試。測試結果顯示，花青素劑量在200μ g/ml的濃度之下經過24小時的培養對於B16細胞並未產生明顯的毒性。Furthermore, please refer to the third figure, the present invention is also directed to the toxicity test of purple anthocyanins for melanoma cells. The test results showed that 24 hours after a dose of anthocyanin for B16 cells did not produce significant toxicity at concentrations below 200 μ g / ml of.

綜上所述，紫米萃取的cyanidin花青素有效抑制酪氨酸酶(tyrosinase)的作用，同時也能抑制B16細胞的黑色素產生。由於紫米花青素cyanidin抑制黑色素的效果佳，因此具有很好的潛力可以進一步開發成美白保養品的主要成分。In summary, the cyanidin anthocyanin extracted by purple rice effectively inhibits the action of tyrosinase and also inhibits melanin production in B16 cells. Since the purple anthocyanin cyanidin has a good effect of inhibiting melanin, it has a good potential to be further developed into a main component of whitening skin care products.

由於紫米花青素的穩定性，因此加入以水合高分子聚合膠與甘油製備的凝膠也具有良好的穩定性，可以提供保 溼、抗氧化、美白的能力，故本發明提供一種具皮膚美白之紫米花青素凝膠劑之製備方法，在製備凝膠劑前，首先應取得花青素萃取物，再製作成凝膠劑，詳細步驟為：步驟一：取1kg紫米，加入2L甲醇浸泡24小時濃縮為浸膏；步驟二：加入pH 3.2醋酸水溶液至該浸膏中後加熱至80℃，並使其反應10小時；步驟三：使用乙酸乙酯萃取收集有機層的紫色溶液，濃縮後再冷凍乾燥成粉末並保存於-20℃，該粉末可使用二次水溶解後形成紫米花青素萃取物(0.3g/ml)；步驟四：將該紫米花青素萃取物利用0.45μ M濾膜過濾後保存於4℃備用；步驟五：另取1.5g的水溶性高分子膠(carbopol)溶解於5ml的乙醇中殺菌後，加入900ml殺菌後的二次去離子水中，使用0.1N的氫氧化鈉水溶液調整酸鹼值至7.0，再加入5ml已滅菌的甘油；及步驟六：加入步驟四所獲得之紫米花青素萃取物5ml後，再使用超音波均質震盪30分鐘後即完成紫米花青素凝膠的製備。Due to the stability of the purple anthocyanins, the gel prepared by adding the hydrated polymer gel and glycerin also has good stability and can provide moisturizing, anti-oxidation and whitening ability, so the present invention provides a skin whitening The preparation method of the purple rice anthocyanin gel, before the preparation of the gel, the anthocyanin extract should be obtained first, and then the gel is prepared. The detailed steps are as follows: Step 1: Take 1kg of purple rice, add 2L methanol to soak Concentrate to extract in 24 hours; Step 2: Add pH 3.2 acetic acid aqueous solution to the extract, heat to 80 ° C, and let it react for 10 hours; Step 3: Extract the purple layer of the organic layer with ethyl acetate, concentrate, and concentrate Then freeze-dried into a powder and stored at -20 ° C, the powder can be dissolved in secondary water to form an extract of purple anthocyanin (0.3 g / ml); Step 4: the purple anthocyanin extract using 0.45 μ M The filter membrane is filtered and stored at 4 ° C for use; Step 5: Another 1.5 g of water-soluble polymer glue (carbopol) is dissolved in 5 ml of ethanol, and then added to 900 ml of sterilized secondary deionized water, using 0.1 N. Sodium hydroxide The solution adjusts the pH to 7.0, and then adds 5 ml of sterilized glycerin; and step 6: after adding 5 ml of the purple anthocyanin extract obtained in the fourth step, and then using ultrasonic homogenization for 30 minutes, the purple anthocyanin is completed. Preparation of the gel.

製備完成後，本發明使用該紫米花青素凝膠對於人體皮膚於日常生活中的保濕與美白效果進行評估實驗，將該紫米花青素凝膠塗擦於人體3個不同的皮膚區域，經過六 週的連續塗擦後，使用儀器測定皮膚黑色素含量，最後收集測得數據統計比較經過六週美白測試的差異如第四圖所示：連續六週塗擦紫米花青素凝膠的皮膚黑色素含量有明顯的下降(*=P<0.05,**=P<0.01)，六週平均的皮膚血黑素含量與尚未塗擦紫米花青素凝膠的皮膚有明顯的差異，第二週的皮膚黑色素含量已經開始有下降，第六週的皮膚黑色素含量與尚未塗擦紫米花青素凝膠的皮膚相比較，黑色素含量下降12%，尤其在第二週的黑色素含量已下降10%，並且持續維持了五週並無上升或是回復的趨勢，直到第六週因為測試組別的所有塗擦紫米花青素凝膠皮膚黑色素含量均明顯下降，使誤差值變小，且不影響皮膚的正常生理機能，達到良好之美白功效。After the preparation, the present invention uses the purple rice anthocyanin gel to evaluate the moisturizing and whitening effect of human skin in daily life, and applies the purple rice anthocyanin gel to three different skin areas of the human body, after six After continuous application of the week, the instrument used to determine the skin melanin content, and finally collected the measured data. The difference between the six-week whitening test was as shown in the fourth figure: the skin melanin content of the purple-flowered anthocyanin gel was observed for six weeks. The decrease (*=P<0.05, **=P<0.01), the average skin melanin content of the six weeks is significantly different from the skin of the unwashed purple anthocyanin gel, and the skin melanin content of the second week has been The beginning of the decline, the skin melanin content in the sixth week compared with the skin that has not been rubbed with the purple anthocyanin gel, the melanin content decreased by 12%, especially in the second week, the melanin content has decreased by 10%, and continued for five weeks. There was no tendency to rise or revert. Until the sixth week, the melanin content of all the rubbed anthocyanin gels in the test group decreased significantly, which made the error value smaller and did not affect the normal physiological function of the skin. Whitening effect.

前文係針對本發明之較佳實施例為本發明之技術特徵進行具體之說明；惟，熟悉此項技術之人士當可在不脫離本發明之精神與原則下對本發明進行變更與修改，而該等變更與修改，皆應涵蓋於如下申請專利範圍所界定之範疇中。The present invention has been described with reference to the preferred embodiments of the present invention. However, those skilled in the art can change and modify the present invention without departing from the spirit and scope of the invention. Such changes and modifications shall be covered in the scope defined by the following patent application.

第一圖：紫米花青素的HPLC分析圖譜。First: HPLC analysis of purple anthocyanins.

第二圖：經過紫米花青素處理與未經過處理的黑色素瘤細胞類鐸受體表現量。Figure 2: Expression of sputum receptors in melanoma cells treated with purple anthocyanins and untreated.

第三圖：紫米花青素對於黑色素瘤細胞的毒性測試圖。Figure 3: Toxicity test of purple anthocyanins for melanoma cells.

第四圖：連續六週塗擦紫米花青素凝膠的皮膚黑色素含量有明顯的下降(*=P<0.05,**=P<0.01)。Figure 4: There was a significant decrease in melanin content in the skin of the purple anthocyanin gel for six weeks (*=P<0.05, **=P<0.01).

Claims (7)

一種具皮膚美白之紫米花青素凝膠劑之製備方法，包含以下步驟：步驟一：取1kg紫米，加入2L甲醇浸泡24小時濃縮為浸膏；步驟二：加入pH 3.2醋酸水溶液至該浸膏中後加熱至80℃，並使其反應10小時；步驟三：使用乙酸乙酯萃取收集有機層的紫色溶液，濃縮後再冷凍乾燥成粉末，該粉末可使用二次水溶解後形成紫米花青素萃取物(0.3g/ml)；步驟四：將該紫米花青素萃取物利用濾膜過濾後保存於4℃備用；步驟五：另取1.5g殺菌後的水溶性高分子膠(carbopol)加入900ml殺菌後的二次去離子水中，使用0.1N的氫氧化鈉水溶液調整酸鹼值至中性，再加入5ml已滅菌的甘油；及步驟六：加入步驟四所獲得之紫米花青素萃取物5ml後，再使用超音波均值震盪即完成紫米花青素凝膠的製備。 A method for preparing a skin whitening purple flower anthocyanin gel comprises the following steps: Step 1: taking 1kg of purple rice, adding 2L methanol for 24 hours and concentrating into an extract; Step 2: adding pH 3.2 acetic acid aqueous solution to the dip After the paste is heated to 80 ° C, and allowed to react for 10 hours; Step 3: extraction of the purple layer of the organic layer with ethyl acetate extraction, concentration and then freeze-dried into a powder, which can be dissolved in secondary water to form purple rice Anthocyanin extract (0.3g/ml); Step 4: The purple anthocyanin extract is filtered through a filter and stored at 4 ° C for use; Step 5: Another 1.5 g of sterilized water-soluble polymer glue (carbopol) Add 900ml of sterilized secondary deionized water, adjust the pH value to neutral with 0.1N sodium hydroxide solution, and then add 5ml of sterilized glycerin; and Step 6: Add the purple broccoli obtained in step 4 After 5 ml of the extract, the preparation of the purple anthocyanin gel was completed by ultrasonic mean shaking. 如申請專利範圍第1項所述之具皮膚美白之紫米花青素凝膠劑之製備方法，其中該步驟三之冷凍乾燥後之粉末之保存溫度為-20℃。 The method for preparing a skin whitening purple anthocyanin gel according to the first aspect of the invention, wherein the storage temperature of the powder after the freeze drying in the third step is -20 °C. 如申請專利範圍第1項所述之具皮膚美白之紫米花青素凝膠劑之製備方法，其中該步驟四所使用之濾膜為0.45μM濾膜。 The method for preparing a skin whitening purple anthocyanin gel according to claim 1, wherein the filter used in the step 4 is a 0.45 μM filter. 如申請專利範圍第1項所述之具皮膚美白之紫米花青素凝膠劑之 製備方法，其中該步驟五所述之水溶性高分子膠之殺菌方法為：取1.5g的水溶性高分子膠(carbopol)溶解於5ml的乙醇中殺菌。 A skin whitening purple flower anthocyanin gel as described in claim 1 The preparation method, wherein the water-soluble polymer glue of the step 5 is sterilized by dissolving 1.5 g of a water-soluble polymer glue (carbopol) in 5 ml of ethanol for sterilization. 如申請專利範圍第1項所述之具皮膚美白之紫米花青素凝膠劑之製備方法，其中該步驟六中使用超音波均值震盪時間為30分鐘。 The method for preparing a skin whitening purple flower anthocyanin gel according to claim 1, wherein the ultrasonic mean shaking time is 30 minutes in the step 6. 一種紫米花青素凝膠劑用以製備抑制類鐸受體(toll-like receptor，TLR)表現的醫藥組合物之用途，其中該紫米花青素凝膠劑包含一紫米花青素萃取物，而該紫米花青素萃取物包含cyanidin-3-glucoside及peonidin-3-glucoside，其中該類鐸受體為TLR2及TLR7。 A use of a purple rice anthocyanin gel for preparing a pharmaceutical composition for inhibiting the expression of a toll-like receptor (TLR), wherein the purple anthocyanin gel comprises a purple anthocyanin extract, The purple anthocyanin extract comprises cyanidin-3-glucoside and peonidin-3-glucoside, wherein the purine receptors are TLR2 and TLR7. 如申請專利範圍第6項所述之用途，其中該醫藥組合物進一步抑制黑色素形成，其係透過該抑制類鐸受體之路徑。 The use according to claim 6, wherein the pharmaceutical composition further inhibits melanin formation by passing through the pathway of inhibiting the steroid receptor.