TWI664973B - Application of lotus leaf extract for the treatment of pigmentation syndrome - Google Patents
Application of lotus leaf extract for the treatment of pigmentation syndrome Download PDFInfo
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Abstract
本發明係揭露一種荷葉萃取物之用途，其係能夠有效地抑制皮膚細胞內之黑色素形成及累積，以降低皮膚細胞內黑色素之含量，達到保護皮膚細胞及維持美觀之功效，並且能夠有效地治療或改善色素沉積綜合症。 The invention discloses a use of a lotus leaf extract, which is capable of effectively inhibiting the formation and accumulation of melanin in skin cells, thereby reducing the content of melanin in skin cells, achieving the effect of protecting skin cells and maintaining aesthetics, and being capable of effectively treating Or improve pigmentation syndrome.
Description
本發明係有關於植物萃取物之第二用途，特別係指一種荷葉萃取物用於治療色素沉積綜合症之用途。 The present invention relates to a second use of a plant extract, in particular to the use of a lotus leaf extract for the treatment of pigmentation syndrome.
按，黑色素係為存在於人體皮膚或毛髮之一種蛋白質，其會影響膚色、髮色和瞳孔的顏色，並且，其主要功用在於避免皮膚受到紫外線傷害。黑色素係由存在於皮膚基底層之黑色素母細胞(melanoblast)受到紫外線之刺激所生成者。具體來說，當黑色素母細胞受刺激後，會啟動保護皮膚細胞之機制，活化酪氨酸酶，進而生成黑色素，亦即人體係透過形成黑色素之機制達到保護皮膚細胞之功效。當黑色素被形成後，一般來說，黑色素會隨著角質代謝而被去除，惟，當皮膚受損嚴重、代謝速率降低，則會無法排除過量之黑色素，導致斑點出現於皮膚上。 Pressed, melanin is a protein that exists in human skin or hair, which affects the color of skin tone, hair color and pupil, and its main function is to protect the skin from ultraviolet rays. Melanin is produced by the stimulation of ultraviolet rays by melanocytes that are present in the basal layer of the skin. Specifically, when the melanocytes are stimulated, the mechanism of protecting the skin cells is activated, and the tyrosinase is activated to generate melanin, that is, the human system achieves the effect of protecting the skin cells through the mechanism of forming melanin. When melanin is formed, in general, melanin is removed along with keratin metabolism. However, when the skin is severely damaged and the metabolic rate is lowered, excessive melanin cannot be excluded, causing spots to appear on the skin.
雖然黑色素為保護皮膚之一道重要防線，不過，基於現今審美觀之標準來說，皮膚維持無暇白皙是被認為美的。因此，許多研究皆致力於找尋有效且天然或化學合成之藥劑以改善色素沉積，然而，目前所開發出與美白相關之產品，都具有高毒性、低穩定性、皮膚滲透率不佳及活性不佳，而無法達到去除色斑之功效，以致於無法被廣泛地應用於市場上。 Although melanin is an important line of defense for protecting the skin, it is considered beautiful to maintain flawless whiteness based on the standards of today's aesthetics. Therefore, many studies have focused on finding effective and natural or chemically synthesized agents to improve pigmentation. However, the products developed with whitening have high toxicity, low stability, poor skin penetration and activity. Good, and can not achieve the effect of removing stains, so that it can not be widely used in the market.
荷葉，係為蓮之葉片，經研究指出，荷葉含有如荷葉鹼(nuciferine)、荷葉鹼(pronuciferine)、鵝掌楸鹼(liriodenine)等多種生物鹼、 類黃酮、荷葉苷(nelumboside)、槲皮素、異槲皮甙等成份。過去藥典中記載荷葉具有輕熱解毒、止血、涼血等功效，而臨床研究指出荷葉鹼能夠有效分解體內脂肪，能夠作為減重產品之有效成份，因此，近年對於荷葉功效之研究皆聚焦於降血脂、代謝症候群、抗氧化等領域，亦即目前研究中係未有指出或是揭露明確證據證實荷葉或其萃取物具有治療色素沉積綜合症之用途。 The lotus leaf is the leaf of lotus. According to the research, the lotus leaf contains various alkaloids such as nuciferine, pronuciferine and liriodenine. Flavonoids, linumboside, quercetin, isoquercetin and other ingredients. In the past Pharmacopoeia, the load leaf has the effects of light and heat detoxification, hemostasis, cooling blood, etc., and clinical research indicates that lotus leaf can effectively decompose body fat and can be used as an effective component of weight loss products. Therefore, in recent years, research on lotus leaf efficacy has focused on In the fields of blood lipids, metabolic syndrome, anti-oxidation, etc., the current research has not pointed out or revealed clear evidence to prove that lotus leaf or its extract has the purpose of treating pigmentation syndrome.
本發明之主要目的係在於提供一種荷葉萃取物之用途，其係用於治療色素沉積綜合症。 The main object of the present invention is to provide a use of a lotus leaf extract for the treatment of pigmentation syndrome.
本發明之另一目的係在於提供一種荷葉萃取物之用途，其係能夠有效地抑制皮膚細胞內之黑色素形成及累積，以降低皮膚細胞內黑色素之含量，達到保護皮膚細胞及維持美觀之功效。 Another object of the present invention is to provide a lotus leaf extract which is capable of effectively inhibiting melanin formation and accumulation in skin cells, thereby reducing the content of melanin in skin cells, thereby protecting skin cells and maintaining aesthetics.
據此，為能達成上述目的，本發明係接露一種將荷葉萃取物用於製備治療色素沉積綜合症之組合物之用途，其中色素沈積綜合症係指皮膚具有色斑之疾病。 Accordingly, in order to achieve the above object, the present invention is directed to the use of a lotus leaf extract for the preparation of a composition for treating pigmentation syndrome, wherein the pigmentation syndrome refers to a disease in which the skin has pigmentation.
其中，荷葉萃取物係將荷葉以水進行萃取所得者。 Among them, the lotus leaf extract is obtained by extracting lotus leaves with water.
其中，荷葉萃取物係能抑制與黑色素生成相關蛋白質之表現，其中與黑色素生成相關蛋白質係選自由酪氨酸酶、MITF(Microphthalmia transcription factor)、TRP1(Tyrosinase related protein-1)、CREB(cAMP responseelement-binding protein)或PKA(Protein kinase A)。 Among them, the lotus leaf extract can inhibit the expression of proteins related to melanin production, wherein the protein associated with melanin production is selected from tyrosinase, MITF (Microphthalmia transcription factor), TRP1 (Tyrosinase related protein-1), CREB (cAMP responseelement). -binding protein) or PKA (Protein kinase A).
其中，該組合物係被製備為膏狀或凝膠狀，而含有至少1%wt之荷葉萃取物，又以含有1~2%wt荷葉萃取物時具有較佳之功效。 Wherein, the composition is prepared as a paste or a gel, and contains at least 1% by weight of the lotus leaf extract, and has a better effect when it contains 1 to 2% by weight of the lotus leaf extract.
其中，荷葉萃取物係被製備為濃度至少為0.3mg/mL之溶液，而 又以當荷葉萃取物係被製備為濃度為0.3~0.5mg/mL之溶液時具有較佳之功效。 Wherein, the lotus leaf extract is prepared as a solution having a concentration of at least 0.3 mg/mL, and Further, when the lotus leaf extract is prepared as a solution having a concentration of 0.3 to 0.5 mg/mL, it has a better effect.
本發明之另一實施例係揭露一種組合物，其係包含有荷葉萃取物、水、丙二醇及凝膠，其中荷葉萃取物之重量百分比為1~2。具體來說，荷葉萃取物、水、丙二醇及凝膠得為1：4：10：75或1：6.5：5：37.5。 Another embodiment of the present invention discloses a composition comprising a lotus leaf extract, water, propylene glycol, and a gel, wherein the weight percentage of the lotus leaf extract is 1 to 2. Specifically, the lotus leaf extract, water, propylene glycol, and gel were 1:4:10:75 or 1:6.5:5:37.5.
第一圖係為細胞存活率試驗之結果。 The first panel is the result of a cell viability assay.
第二圖係為合成黑色素之標準曲線。 The second figure is the standard curve for the synthesis of melanin.
第三圖為經不同處理後之黑色素瘤細胞B16-F1，其黑色素累積之結果。 The third panel shows the results of melanin accumulation in melanoma cells B16-F1 after different treatments.
第四圖為分析經不同處理之黑色素瘤細胞B16-F1之黑色素含量之結果。 The fourth panel is the result of analyzing the melanin content of the differently treated melanoma cells B16-F1.
第五圖為分析經不同處理之黑色素瘤細胞B16-F1之黑色素分泌量之結果。 The fifth panel is the result of analyzing the melanin secretion of the differently treated melanoma cells B16-F1.
第六圖係顯示各該組細胞內酪氨酸酶、MITF、TRP1之表現。 The sixth panel shows the expression of tyrosinase, MITF, and TRP1 in each group of cells.
第七圖係為分析各該組細胞內酪氨酸酶、MITF、TRP1表現量之結果。 The seventh graph is the result of analyzing the expression levels of tyrosinase, MITF, and TRP1 in each of the cells.
第八圖係顯示各該組細胞內CREB、PKA之表現。 The eighth panel shows the expression of CREB and PKA in each group of cells.
第九圖係為分析各該組細胞內CREB、PKA表現量之結果。 The ninth figure is the result of analyzing the expression of CREB and PKA in each group of cells.
第十圖係顯示各該組小鼠皮膚組織內酪氨酸酶、MITF、TRP1之表現。 The tenth graph shows the expression of tyrosinase, MITF, and TRP1 in the skin tissues of each group of mice.
第十一圖係為分析各該組小鼠皮膚組織內酪氨酸酶、MITF、TRP1表現量之結果。 The eleventh figure is the result of analyzing the expression levels of tyrosinase, MITF, and TRP1 in the skin tissues of each group of mice.
第十二圖係顯示各該組小鼠皮膚組織內CREB、PKA、ERK之表現。 The twelfth graph shows the expression of CREB, PKA, and ERK in the skin tissues of each group of mice.
第十三圖係為分析各該組小鼠皮膚組織內CREB、PKA、ERK表現量之結果。 The thirteenth figure is the result of analyzing the expression of CREB, PKA and ERK in the skin tissue of each group of mice.
第十四圖係為各該組小鼠皮膚組織切片經H&E染色之結果，其中A~D依序為第一組至第四組放大倍率為40x。 The fourteenth figure is the result of H&E staining of the skin tissue sections of each group of mice, wherein A~D sequentially has a magnification of 40x from the first group to the fourth group.
第十五圖係為各該組小鼠皮膚組織切片經Fontana-Masson染色之結果，其中A~D依序為第一組至第四組，放大倍率為40x。 The fifteenth figure is the result of staining the skin tissue sections of each group of mice by Fontana-Masson, wherein A~D are sequentially from the first group to the fourth group, and the magnification is 40x.
第十六圖係為各該組小鼠皮膚組織內黑色素含量。 The sixteenth figure is the melanin content in the skin tissue of each group of mice.
為能驗證本發明之功效，以下將藉由實例並搭配圖式做更進一步說明如后。 In order to be able to verify the efficacy of the present invention, the following will be further explained by way of examples and with the drawings.
以下實例中細胞實驗之數據以student's t-test進行分析，而動物實驗之數據係以one way ANOVA試算，再以student's t-test進行分析，結果皆以means±SD表示，當p<0.05時，表示為統計學上有意義的差異。 The data of the cell experiments in the following examples were analyzed by student's t-test, and the data of the animal experiments were calculated by one way ANOVA, and then analyzed by student's t-test. The results were expressed as mean±SD, when p<0.05, Expressed as a statistically significant difference.
實例中所揭動物試驗係經中山醫學大學實驗動物照護及使用委員會審查同意，同意書編號為1606。 The animal test revealed in the example was approved by the Laboratory Animal Care and Use Committee of Zhongshan Medical University. The consent number is 1606.
實例一：製備荷葉萃取物 Example 1: Preparation of lotus leaf extract
將荷葉乾燥後製成粉末後，取200公克荷葉粉末，加入二次水至5公升，於室溫下攪拌一小時後，於冷房靜置12小時，過濾去除雜質後進行收集，直至15公升，再經冷凍乾燥處理，得到粉末狀之荷葉萃取物，其產率約為1.5%。該荷葉萃取物係保存於室溫下，供後續實例使用，其中該荷葉萃取物製備為各濃度荷葉萃取物於各實例使用前，應先進行過濾除菌之程序，舉例來說，以0.22μm之過濾器進行過濾。 After the lotus leaf is dried and made into a powder, 200 g of lotus leaf powder is taken, secondary water is added to 5 liters, stirred at room temperature for one hour, and then allowed to stand in a cold room for 12 hours, filtered to remove impurities, and collected until 15 liters. Further, it was subjected to freeze-drying to obtain a powdery lotus leaf extract having a yield of about 1.5%. The lotus leaf extract is stored at room temperature for use in subsequent examples, wherein the lotus leaf extract is prepared for each concentration of the lotus leaf extract before use in each instance, and the procedure of filtration sterilization should be performed, for example, at 0.22 μm. The filter is filtered.
實例二：細胞培養 Example 2: Cell culture
自食品工業發展研究所生物資源保存暨應用中心購買之黑色素瘤細胞株B16F1(ATCC CRL-6323)。 Melanoma cell line B16F1 (ATCC CRL-6323) purchased from the Center for Bioresource Conservation and Application of the Food Industry Development Institute.
培養黑色素瘤細胞株B16F1之培養基為包含1.5g/L碳酸氫鈉之 90% DMEM培養基，並額外添加5mL、200mM之麩醯胺酸、5mL青黴素/鏈黴素及10%胎牛血清，其中培養基及添加物均購買於GBICO-life technologies。所有細胞培養於5%二氧化碳、37℃之恆溫環境。每2-3天更新一次培養基，繼代培養之稀釋比例為1：10。 The medium for culturing melanoma cell line B16F1 was 1.5 g/L sodium bicarbonate. 90% DMEM medium with 5 mL, 200 mM branic acid, 5 mL penicillin/streptomycin and 10% fetal bovine serum, both of which were purchased from GBICO-life technologies. All cells were cultured in a constant temperature environment of 5% carbon dioxide and 37 °C. The medium was updated every 2-3 days and the dilution ratio of the subculture was 1:10.
實例三：細胞毒性試驗 Example 3: Cytotoxicity test
本實例係參考根據Mosmann等人之研究所設計者。將黑色素瘤細胞B16-F1(3×104 Cell)培養於24孔培養盤中。於培養基中分別加入不同濃度之該荷葉萃取物溶液：0.1、0.2、0.3、0.4、0.5mg/mL，並且，不同濃度之該荷葉萃取物溶液係分別處理細胞24、48、72小時，其中每24小時更換一次，再加入新的培養基和荷葉萃取物溶液。完成試驗後，以磷酸鹽緩衝液清洗培養盤，再加入含MTT試劑(0.5mg/mL)之培養基，混合反應3小時，移除培養基後，以異丙醇將結晶溶出，在OD 563nm下測吸光值的變化，結果如第一圖所示。 This example is based on the design of the Institute according to Mosmann et al. Melanoma cells B16-F1 (3 x 104 Cell) were cultured in 24-well plates. Different concentrations of the lotus leaf extract solution were added to the culture medium: 0.1, 0.2, 0.3, 0.4, 0.5 mg/mL, and the lotus leaf extract solution was treated at different concentrations for 24, 48, and 72 hours, respectively. Change it 24 hours, then add new medium and lotus leaf extract solution. After the test was completed, the culture plate was washed with phosphate buffer, and the medium containing MTT reagent (0.5 mg/mL) was added, and the reaction was mixed for 3 hours. After the medium was removed, the crystal was dissolved in isopropanol and measured at OD 563 nm. The change in absorbance value is shown in the first figure.
由第一圖之結果可知，經本發明所揭之荷葉萃取物處理過之細胞存活率相近於空白組，顯示本發明所揭荷葉萃取物係具有安全性。 As can be seen from the results of the first graph, the cell survival rate of the lotus leaf extract treated by the present invention is similar to that of the blank group, indicating that the lotus leaf extract of the present invention is safe.
實例四：黑色素含量試驗 Example 4: Melanin content test
本實例係參考根據Bellei等人之研究所設計者。 This example is based on the design of the research institute according to Bellei et al.
將黑色素瘤細胞B16-F1接種於6孔培養盤(1×105 Cell)24小時後，分別添加10μMα-MSH及/或0.5mg/mL之該荷葉萃取物溶液。培養72小時後，移除培養基，以磷酸鹽緩衝液漂洗，再用胰蛋白酶將細胞拆下，離心留下細胞，用含1% Triton X-100之磷酸鹽緩衝液破壞細胞後，將其蛋白質及細胞碎片以12000rpm速度離心10分鐘，將上清液測吸光確定蛋白質含量；另取200μL 1M之NaOH加入細胞碎片置於70℃下2小時，將黑色素溶出來後，以分光光度計 405nm之波長測吸光值，比對合成黑色素(0~500μg/ml)之標準曲線(如第二圖所示)，測出黑色素總量再比上蛋白質總量，即可得知結果(μg的黑色素/mg的蛋白質)。本實例之結果係如第三至五圖所示。 After inoculation of melanoma cells B16-F1 in a 6-well culture plate (1×105 Cell) for 24 hours, 10 μM α-MSH and/or 0.5 mg/mL of the lotus leaf extract solution were added, respectively. After 72 hours of culture, the medium was removed, rinsed with phosphate buffer, the cells were removed by trypsin, the cells were centrifuged, and the cells were disrupted with phosphate buffer containing 1% Triton X-100. And the cell debris was centrifuged at 12000 rpm for 10 minutes, and the supernatant was measured to absorb the light to determine the protein content; another 200 μL of 1 M NaOH was added to the cell debris and placed at 70 ° C for 2 hours to dissolve the melanin, and then spectrophotometer The absorbance at 405nm wavelength is compared with the standard curve of synthetic melanin (0~500μg/ml) (as shown in the second figure). The total amount of melanin is measured and the total amount of protein is measured. The result (μg Melanin/mg protein). The results of this example are shown in Figures 3 through 5.
請參閱第三至五圖，由於α-MSH會促使B16-F1細胞內進行黑色素合成之反應，因此，經α-MSH單獨處理後之細胞，黑色素含量最高；而將B16-F1細胞僅以本發明所揭荷葉萃取物溶液處理後，發現細胞內黑色素之含量明顯下降，顯示本發明所揭荷葉萃取物係能抑制黑色素生成，使黑色素含量與空白組無差異。又，同時以α-MSH與本發明所揭荷葉萃取物處理細胞，結果顯示細胞內黑色素之含量仍然不會增加。由此可知，本發明所揭荷葉萃取物確實能夠抑制細胞內黑色素之生成及累積。 Please refer to the third to fifth figures. Since α-MSH will promote the reaction of melanin synthesis in B16-F1 cells, the cells treated with α-MSH alone have the highest melanin content; while B16-F1 cells are only used for this. After the treatment of the lotus leaf extract solution of the invention, it was found that the content of melanin in the cells was significantly decreased, indicating that the lotus leaf extract system of the present invention can inhibit melanin production, so that the melanin content is not different from the blank group. Further, while the cells were treated with α-MSH and the lotus leaf extract of the present invention, the results showed that the content of melanin in the cells did not increase. From this, it is understood that the lotus leaf extract of the present invention can indeed inhibit the production and accumulation of melanin in cells.
實例五：黑色素生成酶(melanogenic enzyme)之表現 Example 5: Performance of melanogenic enzyme
將B16-F1細胞分為六組，第一組為空白組，第二到六組分別以下列條件處理之：10μMα-MSH、0.5mg/mL之該荷葉萃取物溶液、10μMα-MSH及0.3mg/mL之該荷葉萃取物溶液、10μMα-MSH及0.4mg/mL之該荷葉萃取物溶液、10μMα-MSH及0.5mg/mL之該荷葉萃取物。各該組細胞培養24小時後，以西方墨點法分析各該組細胞內酪氨酸酶、MITF(Microphthalmia transcription factor)、TRP1(Tyrosinase related protein-1)、磷酸化CREB(cAMP responseelement-binding protein)、磷酸化PKA(Protein kinase A)等與黑色素生成相關酵素之表現，其中西方墨點法為本發明所屬技術領域者之周知技術，因此於此不加以冗言。結果如第六至九圖所示。 B16-F1 cells were divided into six groups, the first group was a blank group, and the second to sixth groups were treated under the following conditions: 10 μM α-MSH, 0.5 mg/mL of the lotus leaf extract solution, 10 μM α-MSH and 0.3 mg. /mL of the lotus leaf extract solution, 10 μM α-MSH and 0.4 mg/mL of the lotus leaf extract solution, 10 μM α-MSH and 0.5 mg/mL of the lotus leaf extract. After 24 hours of cell culture in each group, tyrosinase, MITF (Microphthalmia transcription factor), TRP1 (Tyrosinase related protein-1), and phosphorylated CREB (cAMP responseelement-binding protein) were analyzed by Western blotting method. ), phosphorylated PKA (Protein kinase A) and the like, and the expression of melanin-related enzymes, and the Western blotting method is a well-known technique of those skilled in the art, and therefore will not be redundant. The results are shown in Figures 6 to 9.
由第六至九圖之結果可知，以α-MSH處理後之細胞，其內與黑色素生成路徑相關之酵素表現量確實會增加，顯示α-MSH會使細胞合成黑色素； 而以本發明所揭荷葉萃取物處理之細胞，其內與黑色素生成路徑之酵素表現係明顯降低，並且酪氨酸酶、MITF、磷酸化CREB及磷酸化PKA之表現量皆低於第一組，可知本發明所揭荷葉萃取物能夠黑色素生成路徑之酵素表現而降低黑色素之生成及累積。又，將第四至六組之結果分別對比第二組之結果，顯示於處理α-MSH之條件下，若額外給予本發明所揭荷葉萃取物，係能夠有效地抑制與黑色素生成路徑相關之酵素表現，達到抑制黑色素生成及累積之功效，並且，隨著本發明所揭荷葉萃取物之劑量越高，其抑制黑色素生成路徑相關之酵素表現之效果越佳。 From the results of the sixth to the ninth graph, it is known that the amount of the enzyme involved in the melanin production pathway is indeed increased in the cells treated with α-MSH, indicating that α-MSH causes the cells to synthesize melanin; In the cells treated with the lotus leaf extract of the present invention, the enzyme expression in the melanin production pathway was significantly decreased, and the expression levels of tyrosinase, MITF, phosphorylated CREB and phosphorylated PKA were lower than those in the first group. It can be seen that the lotus leaf extract of the present invention can exhibit the enzyme expression of the melanin production pathway and reduce the production and accumulation of melanin. Further, comparing the results of the fourth to sixth groups with the results of the second group, respectively, under the condition of treating α-MSH, if the lotus leaf extract of the present invention is additionally administered, the melanin production path can be effectively suppressed. The enzyme exhibits an effect of inhibiting the production and accumulation of melanin, and the higher the dose of the lotus leaf extract of the present invention, the better the effect of inhibiting the enzyme expression associated with the melanin production pathway.
實例六：動物試驗 Example 6: Animal test
自國家實驗動物中心購入若干隻體重約為300~350克之雌性天竺鼠N：HARTLEY，飼養於中山醫學大學之動物中心，環境條件設有自動空氣調節及自動光照控制(12小時白晝、12小時黑夜)、溫度平均為22±2℃、相對溼度為50-55%；飼料為LABDIET公司之Guinea pig diet 5025。 A number of female guinea pigs N:HARTLEY weighing approximately 300-350 grams were purchased from the National Experimental Animal Center, and were raised at the Animal Center of Zhongshan Medical University. The environment was equipped with automatic air conditioning and automatic light control (12 hours of daylight, 12 hours of darkness). The average temperature is 22±2° C. and the relative humidity is 50-55%; the feed is Guinea pig diet 5025 of LABDIET Company.
將實例一之荷葉萃取物粉末與丙二醇、凝膠、水製備為荷葉凝膠，其內分別含有重量百分比1%或2%之荷葉萃取物，其中凝膠為水及2%高分子凝膠所組成者。舉例來說，取2g丙二醇及15g凝膠，添加0.2g荷葉萃取物粉末及2.8g水可製備出含有1%wt荷葉萃取物之荷葉凝膠，而添加0.4g荷葉萃取物粉末及2.6g水時，則可完成含有2%wt荷葉萃取物之荷葉凝膠。 The lotus leaf extract powder of Example 1 was prepared as a lotus leaf gel with propylene glycol, gel and water, respectively containing 1% or 2% by weight of lotus leaf extract, wherein the gel was water and 2% polymer gel. Component. For example, 2 g of propylene glycol and 15 g of gel, 0.2 g of lotus leaf extract powder and 2.8 g of water can be added to prepare a lotus leaf gel containing 1% by weight of lotus leaf extract, and 0.4 g of lotus leaf extract powder and 2.6 g of water are added. At the time, a lotus leaf gel containing 2% by weight of lotus leaf extract can be completed.
待各該天竺鼠體重成長至500克後，參照Li-Hua Penga等人的UV照射皮膚誘導色素沉積方式進行實驗。將該天竺鼠隨積分為4組，其中第一組為空白組；第二組為UV組；第三組為低劑量實驗組，其係照射UV及塗抹含有1%wt荷葉萃取物之荷葉凝膠；第四組為高劑量實驗組，其係照射UV及塗抹含有2%wt荷葉萃取物之荷葉凝膠。第二至四組之照射部位為背部除毛後分成三個區 域，每個區域(1.5公分x1.5公分)，UVB劑量每次500mj/cm2照射15分鐘(Spectroline SelectTM Series,Philips TL/12 lamp emitting 280-305nm)，連續照射兩周，每周三次，而第三及四組分別於照射完後每天塗上不同濃度之荷葉凝膠。兩周後犧牲取皮膚，進行後續實例。 After each day of the squirrel weight growth to 500 g, the experiment was carried out by referring to the UV-induced skin-induced pigmentation method of Li-Hua Penga et al. The guinea pigs were integrated into 4 groups, the first group was a blank group; the second group was a UV group; the third group was a low-dose experimental group, which was irradiated with UV and applied with a lotus leaf gel containing 1% wt lotus leaf extract. The fourth group is a high-dose experimental group that irradiates UV and applies a lotus leaf gel containing 2% by weight of lotus leaf extract. The second to fourth groups were irradiated into three areas, each area (1.5 cm x 1.5 cm), and the UVB dose was irradiated for 15 minutes at 500 mj/cm2 (Spectroline Select TM Series, Philips TL/12). Lamp emitting 280-305 nm), continuous irradiation for two weeks, three times a week, and the third and fourth groups were coated with different concentrations of lotus leaf gel every day after irradiation. After two weeks, the skin was sacrificed for subsequent examples.
實例七：皮膚組織之蛋白質表現 Example 7: Protein Expression of Skin Tissue
自實例六各組天竺鼠所取下之皮膚組織萃取出皮膚蛋白萃取物，以西方墨點法分析各該組天竺鼠皮膚組織內如酪氨酸酶、MITF、TRP1、磷酸化CREB、磷酸化PKA等與黑色素生成相關酵素之表現，結果如第十至十三圖所示。 The skin protein extracts were extracted from the skin tissues taken from the guinea pigs of the sixth group, and the skin tissues of the guinea pigs such as tyrosinase, MITF, TRP1, phosphorylated CREB, phosphorylated PKA, etc. were analyzed by Western blotting method. The performance of enzymes related to melanin production is shown in the tenth to thirteenth figures.
由第十至十三圖之結果可知，照射UV光確實會使皮膚組織內與與黑色素生成相關酵素表現量明顯增加，如第二組所示，顯示皮膚組織受到UV之刺激會促使生成黑色素。而相較於第二組，第三組及第四組天竺鼠之皮膚組織內與與黑色素生成相關酵素表現量係分別明顯降低，並且又以濃度高者之抑制效果更佳，顯示本發明所揭荷葉萃取物係能夠抑制皮膚組織內與與黑色素生成相關酵素表現，而能有效抑制黑色素形成且降低黑色素累積之情形，以達到治療色素沈積症之功效。 From the results of the tenth to thirteenth graphs, it is known that the irradiation of UV light does cause a significant increase in the amount of enzymes associated with melanin production in the skin tissue, as shown in the second group, indicating that skin tissue is stimulated by UV to promote the production of melanin. Compared with the second group, the expression levels of the enzymes related to melanin production in the skin tissues of the third group and the fourth group of guinea pigs were significantly reduced, respectively, and the inhibition effect was higher in the higher concentration, indicating that the present invention was revealed. The lotus leaf extract system can inhibit the expression of melanin-related enzymes in skin tissues, and can effectively inhibit melanin formation and reduce melanin accumulation, so as to achieve the effect of treating pigmentation.
實例八：皮膚組織之染色結果 Example 8: Staining results of skin tissue
將實例六各組天竺鼠所取下之皮膚組織先以石蠟包埋後，進行切片，再分別以H&E及Fontana-Masson法進行染色，其中組織切片及染色係為本發明所屬技術領域之通常知識，故於此不加以贅述。各該組組織染色切片之結果係如第十四及十五圖所示。 The skin tissues removed from the guinea pigs of the sixth group were firstly paraffin-embedded, sliced, and then stained by H&E and Fontana-Masson methods respectively, wherein the tissue sections and staining were the general knowledge in the technical field of the present invention. Therefore, it will not be repeated here. The results of staining sections of each of the group tissues are shown in Figures 14 and 15.
由第十四及十五圖之結果可知，經UV照射之第二組天竺鼠之表 皮層係明顯增厚、黑色素沈積於表皮層，顯示經UV照射後不僅會使皮膚生成黑色素，更會造成皮膚代謝不佳而使黑色素累積於表皮層，而形成色素沈積症。而相較於第二組，第三組及第四組之皮膚組織厚度皆明顯變薄，顯示皮膚組織之代謝係較佳，並且，黑色素於表皮層堆積之情形係明顯改善。換言之，本發明所揭荷葉萃取物不僅能夠抑制黑色素之生成，並且，能夠有效地改善黑色素沈積於表皮層之情形。因此，本發明所揭荷葉萃取物應具有改善或治療色素沈積症之用途。 From the results of the fourteenth and fifteenth figures, the second group of guinea pigs irradiated by UV The cortical system is obviously thickened and melanin is deposited on the epidermis layer. It shows that not only the skin will produce melanin after UV irradiation, but also the skin metabolism is poor and melanin accumulates in the epidermis layer to form pigmentation. Compared with the second group, the skin tissue thicknesses of the third group and the fourth group were significantly thinner, indicating that the metabolic system of the skin tissue was better, and the accumulation of melanin in the epidermal layer was significantly improved. In other words, the lotus leaf extract of the present invention can not only inhibit the formation of melanin, but also effectively improve the deposition of melanin on the skin layer. Therefore, the lotus leaf extract of the present invention should have the use of improving or treating pigmentation.
實例九：皮膚組織之黑色素含量 Example 9: Melanin content of skin tissue
取實例六中各組天竺鼠皮膚組織3克，RIPA緩衝液與蛋白水解酶抑制劑(protease inhibitor)，於冰上研磨，而後以4℃/20,000×g/20min之方式離心，抽取上清液，以分光光度計405nm之波長測吸光值，比對合成黑色素之標準曲線，分析出各該組天竺鼠皮膚組織內黑色素之含量，結果如第十六圖所示。 Take 3 g of skin tissue of each group of guinea pigs in Example 6, RIPA buffer and protease inhibitor, grind on ice, then centrifuge at 4 ° C / 20,000 × g / 20 min, and extract the supernatant. The absorbance was measured by a spectrophotometer at a wavelength of 405 nm, and the standard curve of synthetic melanin was compared to analyze the content of melanin in the skin tissue of each group of guinea pigs. The results are shown in Fig. 16.
由第十六圖之結果可知，第二組天竺鼠皮膚組織內之黑色素含量係明顯高於第一組小鼠，而相較於第二組，第三組及第四組天竺鼠皮膚組織內之黑色素含量係明顯降低，並且，相近於第一組天竺鼠。由上述結果再次顯示，本發明所揭荷葉萃取物確實具有降低黑色素之生成及累積之功效。 From the results of the sixteenth figure, the melanin content in the skin tissue of the second group of guinea pigs was significantly higher than that of the first group of mice, and the melanin in the skin tissue of the third group and the fourth group of guinea pigs compared to the second group. The content was significantly reduced and was similar to the first group of guinea pigs. It is again shown by the above results that the lotus leaf extract of the present invention does have the effect of reducing the production and accumulation of melanin.
根據上述實例之說明係證實本發明所揭荷葉萃取物係能夠透過調控細胞內與黑色素生成相關之酵素，使酪氨酸酶不被活化，抑制黑色素生成，並且，使皮膚組織之代謝反應不受外界刺激影響，以維持正常之代謝反應，達到改善或治療黑色素累積、色斑形成之功效。據此，本發明所揭荷葉萃取物確實具有治療色素沉積綜合症之功效，能夠有效避免或改善皮膚發生如黃斑、雀斑、老人斑等色斑或是色斑顏色變深之情形。此外，本發明所揭荷葉萃取物係為非化學合成之成份，因此能夠安心地被使用於人體，具有極高供產業利用之經濟價值。 According to the description of the above examples, it was confirmed that the lotus leaf extract of the present invention is capable of modulating intracellular melanin-related enzymes, inhibiting tyrosinase activation, inhibiting melanin production, and preventing metabolic reactions of skin tissues. External stimuli affect the normal metabolic response to improve or treat melanin accumulation and pigmentation. Accordingly, the lotus leaf extract of the present invention has the effect of treating pigmentation syndrome, and can effectively avoid or improve skin spots such as yellow spots, freckles, age spots, and dark spots. In addition, the lotus leaf extract of the present invention is a non-chemically synthesized component, and therefore can be safely used in the human body, and has an economic value that is extremely high for industrial use.
Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1-2): p. 55-63. Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65(1-2): p. 55-63.
Bellei, B., et al., p38 regulates pigmentation via proteasomal degradation of tyrosinase. J Biol Chem, 2010. 285(10): p. 7288-99. Bellei, B., et al., p38 regulates pigmentation via proteasomal degradation of tyrosinase. J Biol Chem, 2010. 285(10): p. 7288-99.
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