TWI743445B - Use of physalis peruviana fruits extract for enhancing the gene expression level of tgm, and/or krt - Google Patents
Use of physalis peruviana fruits extract for enhancing the gene expression level of tgm, and/or krt Download PDFInfo
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Abstract
Description
本發明係關於一黃金莓萃取物之用途，尤其是一種黃金莓萃取物用於製備提升TGM基因及/或KRT基因表現量、以及提升粒線體活性之組合物的用途。 The present invention relates to the use of a golden raspberry extract, in particular to the use of a golden raspberry extract to prepare a composition for enhancing TGM gene and/or KRT gene expression and enhancing mitochondrial activity.
表皮層為皮膚的最外層，由外往內依序為角質層、顆粒層、有棘層及基底層，表皮層主要由基底層中未分化之圓柱型角質細胞持續向上進行分化形成，此過程稱為角質化。角質細胞內含水量高，隨著細胞向上代謝分化，角質細胞形狀會逐漸變成扁平狀，且細胞核及胞器開始退化萎縮，並在角質層形成不具細胞核與胞器之死細胞。表皮層的主要功能為使皮膚保水，並形成皮膚屏障以抵禦各種外來傷害，其中表皮層最外層由一弱酸性的皮脂膜以及如磚牆結構的角質層所構成，此屏障能鎖住皮膚的水分和油脂、抵抗皮膚表面病菌入侵，及對抗外界異物及紫外光等傷害，對人體有非常重要的保護作用。 The epidermal layer is the outermost layer of the skin. From the outside to the inside, it is the stratum corneum, the granular layer, the spinous layer and the basal layer. The epidermal layer is mainly formed by undifferentiated cylindrical keratinocytes in the basal layer. This process It is called keratinization. The water content in keratinocytes is high. As the cells are metabolized and differentiated, the shape of keratinocytes will gradually become flat, and the nucleus and organelles will begin to degenerate and shrink, and dead cells without nuclei and organelles will be formed in the stratum corneum. The main function of the epidermis is to keep the skin water and form a skin barrier to resist various external injuries. The outermost layer of the epidermis is composed of a weakly acidic sebum membrane and a stratum corneum like a brick wall. This barrier can lock the skin. Moisture and grease, resistance to the invasion of bacteria on the skin surface, and resistance to damage from foreign bodies and ultraviolet light, have a very important protective effect on the human body.
表皮層中的角質層，其角質細胞雖然為死細胞，但其主要成分為角蛋白(keratin)，角蛋白能吸收水分使皮膚保持濕潤，角質細胞也會分泌如玻尿酸等物質作為細胞間質，以維持表皮層皮膚屏障之結構完整，以防止皮膚水分散失及形成完整防護。當皮膚接觸過冷或過熱之環境以及照射紫外光等刺激，會導致角質細胞無法維持正常的代謝循環，且皮膚保水能力也會下降，並 導致皮膚表皮層屏障受損，讓皮膚變得粗糙、乾燥脫屑、脆弱易受刺激、敏感泛紅，因此角質層的健康與保水能力對於抵禦外來傷害著實非常重要。 In the stratum corneum of the epidermis, although the keratinocytes are dead cells, the main component is keratin. Keratin can absorb water to keep the skin moist, and keratinocytes also secrete substances such as hyaluronic acid as intercellular substance. To maintain the integrity of the skin barrier of the epidermal layer to prevent the skin from losing water and form a complete protection. When the skin is exposed to a cold or hot environment and irritation such as ultraviolet light, the keratinocytes will not be able to maintain the normal metabolic cycle, and the skin's water retention capacity will also decrease. The skin's epidermal barrier is damaged, and the skin becomes rough, dry and desquamated, fragile and easily irritated, and sensitive to redness. Therefore, the health and water retention capacity of the stratum corneum is very important to resist external damage.
然而，隨著年紀漸長皮膚會逐漸地老化(Ageing)，皮膚老化形成原因與過程非常複雜牽涉了無數的生理現象，其中紫外線傷害、自由基傷害、膠原蛋白減少、細胞更新減緩、異常細胞的出現、皮膚脂肪減少、細胞間質缺乏、細胞生長休止、荷爾蒙下降等係較常見的因素。但截至目前為止，市售的皮膚抗老化產品大多只能著手在增加抗氧化活性，並無法直接且有效地改善或延緩皮膚老化的發生。 However, as the age gets older, the skin will gradually age (Ageing). The causes and processes of skin aging are very complex and involve countless physiological phenomena, including UV damage, free radical damage, reduction of collagen, slower cell renewal, and abnormal cells. Appearance, loss of skin fat, lack of intercellular substance, cessation of cell growth, and decline in hormones are more common factors. But up to now, most of the skin anti-aging products on the market can only start to increase the antioxidant activity, and cannot directly and effectively improve or delay the occurrence of skin aging.
綜合上面所述，為了改善因角質細胞受損、保水能力下降、以及皮膚老化所導致皮膚變得脆弱、易敏等問題，開發一種能直接且有效地改善或延緩皮膚老化發生，同時又能有效使角質細胞分泌更多保濕因子，以維持角質細胞排列、維持角質層結構完整之組合物，著實有其必要性。 Based on the above, in order to improve the skin's fragility and susceptibility caused by damaged keratinocytes, decreased water retention capacity, and skin aging, we have developed a method that can directly and effectively improve or delay the occurrence of skin aging, while being effective It is indeed necessary for the keratinocytes to secrete more moisturizing factors to maintain the arrangement of keratinocytes and maintain the complete structure of the stratum corneum.
緣此，本發明之一目的在提供一種黃金莓萃取物用於製備提升轉谷氨醯胺酶(Transglutaminase,TGM)基因、及角蛋白(Keratin,KRT)基因表現量之組合物的用途，其中該黃金莓萃取物係以一溶劑萃取一黃金莓所獲得，該溶劑為水、醇、或醇水混合物。 For this reason, one purpose of the present invention is to provide a use of golden berry extract for preparing a composition for enhancing expression of transglutaminase (TGM ) gene and keratin ( KRT ) gene, wherein The golden raspberry extract is obtained by extracting a golden raspberry with a solvent, and the solvent is water, alcohol, or a mixture of alcohol and water.
本發明之另一目的在提供一種黃金莓萃取物用於製備提升粒線體活性之組合物的用途，其中該黃金莓萃取物係以一溶劑萃取一黃金莓所獲得，該溶劑為水、醇、或醇水混合物。 Another object of the present invention is to provide a use of golden berry extract for preparing a composition for enhancing mitochondrial activity, wherein the golden berry extract is obtained by extracting a golden berry with a solvent, and the solvent is water and alcohol. , Or a mixture of alcohol and water.
在本發明之一實施例中，該溶劑與該黃金莓之重量比為10-20：1-5；且該萃取之溫度係50-100℃。 In an embodiment of the present invention, the weight ratio of the solvent to the golden berry is 10-20:1-5; and the extraction temperature is 50-100°C.
在本發明之又一實施例中，該轉谷氨醯胺酶基因係轉谷氨酰胺酶1(Transglutaminase 1,TGM1)基因；且該角蛋白基因係角蛋白10(Keratin10,KRT10)基因、角蛋白14(Keratin14,KRT14)基因或其組合。 In another embodiment of the present invention, the transglutaminase gene is a transglutaminase 1 (Transglutaminase 1, TGM1 ) gene; and the keratin gene is a keratin 10 (Keratin 10, KRT10 ) gene, Protein 14 (Keratin14, KRT14 ) gene or a combination thereof.
在本發明之又一實施例中，該黃金莓萃取物係維持皮膚角質層結構完整、及/或提升皮膚保濕能力；且該黃金莓萃取物的濃度為至少0.05mg/mL。 In another embodiment of the present invention, the golden berry extract maintains the integrity of the skin stratum corneum structure and/or improves the skin's moisturizing ability; and the concentration of the golden berry extract is at least 0.05 mg/mL.
在本發明之另一實施例中，該黃金莓萃取物係維持皮膚細胞的正常代謝、及/或提升皮膚細胞的抗老化活性；且該黃金莓萃取物的濃度為至少0.03125mg/mL。 In another embodiment of the present invention, the golden berry extract maintains the normal metabolism of skin cells and/or enhances the anti-aging activity of the skin cells; and the concentration of the golden berry extract is at least 0.03125 mg/mL.
本發明之黃金莓萃取物能有效在6-24小時內即有效提升皮膚角質細胞中與保濕相關的TGM1基因、KRT10基因、及KRT14基因的表現量，能有效維持皮膚角質細胞排列，使皮膚角質層結構完整，以提升皮膚屏障功能、以及防止皮膚水分散失，使皮膚處於保水的狀態、及維持皮膚年輕光滑有彈性；本發明之黃金莓萃取物亦能有效提升皮膚細胞的粒線體活性，以維持皮膚細胞的活力及正常代謝，以提升皮膚細胞抗老化的活性。因此，本發明之黃金莓萃取物具有提升皮膚抗老化活性及保濕能力之功效，可用於製備皮膚保健之組合物的用途，其中該組合物該組合物是一食品或一保養品，可藉由口服、皮膚塗抹等方式給予一個體。 The golden berry extract of the present invention can effectively increase the expression of TGM1 , KRT10 , and KRT14 genes related to moisturizing in skin keratinocytes within 6-24 hours, and can effectively maintain the arrangement of skin keratinocytes and make skin keratinous The layer structure is complete to enhance the skin barrier function, prevent skin water loss, keep the skin in a water-retaining state, and maintain the skin young, smooth and elastic; the golden berry extract of the present invention can also effectively enhance the mitochondrial activity of skin cells, To maintain the vitality and normal metabolism of skin cells to enhance the anti-aging activity of skin cells. Therefore, the golden berry extract of the present invention has the effect of enhancing the anti-aging activity and moisturizing ability of the skin, and can be used to prepare skin health care composition, wherein the composition is a food or a skin care product. Give to a body by oral, skin application, etc.
圖1為本發明之一實施例的黃金莓萃取物後提升角質細胞中TGM1基因、KRT10基因、及KRT14基因表現量之效果的長條圖。* p值<0.05；** p<0.01；*** p<0.001。 Fig. 1 is a bar graph showing the effect of the golden berry extract in enhancing the expression levels of TGM1 , KRT10 , and KRT14 genes in keratinocytes according to an embodiment of the present invention. * p value<0.05; ** p<0.01; *** p<0.001.
圖2係為本發明之一實施例的黃金莓萃取物提升皮膚細胞粒線體活性之效果的長條圖。** p<0.01。 Fig. 2 is a bar graph showing the effect of the golden berry extract in enhancing the mitochondrial activity of skin cells according to an embodiment of the present invention. ** p<0.01.
本文中所使用數值為近似值，所有實驗數據皆表示在20%的範圍內，較佳為在10%的範圍內，最佳為在5%的範圍內。 The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.
使用Excel軟體進行統計分析。數據以平均值±標準差(SD)表示，個此之間的差異以學生t檢驗(student's t-test)分析。 Use Excel software for statistical analysis. Data mean ± standard deviation (SD) represented by the difference between the two herein by Student t test (student's t -test) analysis.
黃金莓為祕魯苦蘵(Physalis peruviana)之果實。祕魯苦蘵係為茄科(Solanaceae)酸漿屬(Physalis)多年生草本植物，原產於秘魯與智利。祕魯苦蘵又稱燈籠果，其果實直徑約1公分且呈球形，被膨大的宿存萼片所包覆，成熟時為金黃色，因味甜而被廣泛食用，果實被稱為黃金莓、燈籠果，其具有清熱解毒及化痰之功效。 Golden berry is the fruit of Peruvian bitter rice ( Physalis peruviana). The Peruvian bitter family is a perennial herb of the Solanaceae ( Solanaceae) physalis (Physalis ), which is native to Peru and Chile. Peruvian bitter is also known as cape gooseberry. Its fruit is about 1 cm in diameter and spherical in shape. It is covered by enlarged persistent sepals. It is golden yellow when mature. It is widely eaten because of its sweet taste. The fruit is called golden raspberry, lantern Fruit, it has the effects of clearing away heat, detoxifying and resolving phlegm.
如本文中所使用的，用語「黃金莓萃取物」意為黃金莓果實與溶劑以1-5：10-20(w/w)比例經一特定時間與溫度萃取而得。 As used herein, the term "golden berry extract" means that the golden berry fruit and the solvent are extracted at a ratio of 1-5:10-20 (w/w) for a specific time and temperature.
本文所述之「有效濃度」係表示能有效提升皮膚細胞粒線體活性、及/或有效提升皮膚細胞中TGM1基因、KRT10基因、及KRT14基因表現量所需本發明之黃金莓萃取物的濃度。有效濃度依所作用的對象而可能不同，但可藉由例如劑量遞增試驗(dose escalation)以實驗決定其有效濃度。 The "effective concentration" mentioned herein refers to the concentration of the golden berry extract of the present invention required to effectively increase the mitochondrial activity of skin cells and/or effectively increase the expression of TGM1 , KRT10 , and KRT14 genes in skin cells . The effective concentration may vary depending on the target, but the effective concentration can be determined experimentally by, for example, a dose escalation test.
本發明可進一步包含有一被廣泛地使用於各式的載劑(pharmaceutically acceptable carrier)。例如，該各式的載劑可包含一或多種選自於下列的試劑：溶劑(solvent)、緩衝液(buffer)、乳化劑(emulsifier)、懸浮劑(suspending agent)、分解劑(decomposer)、崩解劑(disintegrating agent)、分散劑(dispersing agent)、黏結劑(binding agent)、賦形劑(excipient)、安定劑(stabilizing agent)、螯合劑(chelating agent)、稀釋劑(diluent)、膠凝劑(gelling agent)、防腐劑(preservative)、潤濕劑(wetting agent)、潤滑劑(lubricant)、吸收延遲劑(absorption delaying agent)、脂質體(liposome)以及類似之物。有關這些試劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。 The present invention may further include a pharmaceutically acceptable carrier that is widely used in various types. For example, the various carriers may include one or more reagents selected from the following: solvent, buffer, emulsifier, suspending agent, decomposer, Disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent, glue Gelling agent, preservative, wetting agent, lubricant, absorption delaying agent, liposome and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.
依據本發明，該各式的載劑包含有一選自於由下列所構成之群組中的溶劑：水、生理鹽水(normal saline)、磷酸鹽緩衝生理鹽水(phosphate buffered saline,PBS)、含有醇的水性溶液(aqueous solution containing alcohol)以及它們的組合。 According to the present invention, the various carriers include a solvent selected from the group consisting of water, normal saline (normal saline), phosphate buffered saline (PBS), alcohol-containing The aqueous solution containing alcohol (aqueous solution containing alcohol) and their combinations.
本發明可利用熟習此技藝者所詳知的技術而被製造成一適合於局部地施用於皮膚上的外部製劑(external preparation)，這包括，但不限於：乳劑(emulsion)、凝膠(gel)、軟膏(ointment)、乳霜(cream)、擦劑(liniment)、粉末(powder)、噴霧(spray)、乳液(lotion)、泡沫(foam)、以及懸浮液(suspension)等。 The present invention can be manufactured into an external preparation suitable for topical application to the skin by using techniques well known to those skilled in the art. This includes, but is not limited to: emulsion, gel , Ointment, cream, liniment, powder, spray, lotion, foam, suspension, etc.
依據本發明，該外部製劑是藉由將本發明與一為熟習此項技藝者所詳知的基底(base)相混合而被製備。 According to the present invention, the external preparation is prepared by mixing the present invention with a base well known to those skilled in the art.
依據本發明，該基底可包含有一或多種選自於下列的添加劑(additives)：水、醇(alcohols)、甘醇(glycol)、碳氫化合物(hydrocarbons)[諸如石油膠(petroleum,jelly)以及白凡士林(white petrolatum,)]、蠟(wax)[諸如石蠟(paraffin)以及黃蠟(yellow wax)]、保存劑(preserving agents)、抗氧化劑(antioxidants)、界面活性劑(surfactants)、吸收增強劑(absorption enhancers)、安定劑(stabilizing agents)、膠凝劑(gelling agents)等。有關這些添加劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。 According to the present invention, the substrate may contain one or more additives selected from the following: water, alcohols, glycols, hydrocarbons (such as petroleum jelly) and White petrolatum (white petrolatum,)], wax (such as paraffin and yellow wax), preserving agents, antioxidants, surfactants, absorption enhancers (absorption enhancers), stabilizers (stabilizing agents), gelling agents, etc. The selection and quantity of these additives fall within the scope of professionalism and routine techniques of those who are familiar with this technology.
依據本發明，保養品可進一步包含有一被廣泛地使用於保養品製造技術之可接受的佐劑(acceptable adjuvant)。例如，該可接受的佐劑可包含有一或多種選自於下列的試劑：溶劑、膠凝劑、活性劑、防腐劑、抗氧化劑、遮蔽劑(screening agent)、螯合劑、界面活性劑、染色試劑(coloring agent)、增稠劑(thickening agent)、填料(filler)、香料以及氣味吸收劑。有關這些試劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。 According to the present invention, the skin care product can further include an acceptable adjuvant that is widely used in skin care product manufacturing technology. For example, the acceptable adjuvant may contain one or more agents selected from the group consisting of solvents, gelling agents, active agents, preservatives, antioxidants, screening agents, chelating agents, surfactants, dyes Coloring agent, thickening agent, filler, fragrance and odor absorber. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.
依據本發明，保養品可利用熟習此技藝者所詳知的技術而被製造成一適合於護膚(skincare)或化妝(makeup)的形式，這包括，但不限於：水性溶液(aqueous solution)、水-醇溶液(aqueous-alcohol solution)或油性溶液(oily solution)、呈水包油型(oil-in-water type)、油包水型(water-in-oil type)或複合型之乳劑、凝膠、軟膏、乳霜、面膜(mask)、貼片、貼布(pack)、擦劑、粉末、氣溶膠、噴霧、乳液、乳漿、糊劑、泡沫、分散液、滴劑、慕斯(mousse)、防曬油(sunblock)、化妝水(tonic water)、粉底(foundation)、卸妝產品(makeup remover products)、肥皂(soap)以及其他身體清潔產品(body cleansing products)等。 According to the present invention, the skin care product can be manufactured into a form suitable for skincare or makeup by using techniques well known to those skilled in the art. This includes, but is not limited to: aqueous solution, water -Aqueous-alcohol solution or oily solution, oil-in-water type, water-in-oil type or compound emulsion, gel Glue, ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, makeup remover products, soap, and other body cleansing products.
依據本發明，保養品亦可與一或多種選自於下列之已知活性的外用劑(external use agents)一起合併使用：美白劑(whitening agents)、保濕劑、抗發炎劑(anti-inflammatory agents)、殺菌劑(bactericides)、及紫外線吸收劑(ultraviolet absorbers)等。有關這些外用劑的選用與數量是落在熟習此項技術之人士的專業素養與例行技術範疇內。 According to the present invention, skin care products can also be used in combination with one or more external use agents with known activity selected from the following: whitening agents, moisturizers, anti-inflammatory agents ), bactericides, and ultraviolet absorbers, etc. The selection and quantity of these topical agents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.
依據本發明，食品產品可被當作食品添加物(food additive)，藉由習知方法於原料製備時添加，或是於食品的製作過程中添加，而與任一種可食性材料配製成供人類與非人類動物攝食的食品產品。 According to the present invention, a food product can be used as a food additive, which can be added during the preparation of raw materials by a conventional method, or added during the production process of the food, and formulated with any edible material for supply Food products consumed by humans and non-human animals.
依據本發明，食品產品的種類包括但不限於：飲料(beverages)、發酵食品(fermented foods)、健康食品(health foods)以及膳食補充品(dietary supplements)等。 According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, health foods, and dietary supplements.
本發明提供一種黃金莓萃取物用於製備皮膚保健之組合物的用途，本發明之黃金莓萃取物係以一溶劑萃取一黃金莓所獲得，該溶劑為水、醇、或醇水混合物，其可用於維持角質細胞排列，使皮膚角質組織的結構完整，以及提升皮膚細胞的粒線體活性，以維持皮膚細胞的活力及正常代謝。 The present invention provides a use of golden raspberry extract for preparing skin health care composition. The golden raspberry extract of the present invention is obtained by extracting a golden raspberry with a solvent. The solvent is water, alcohol, or a mixture of alcohol and water. It can be used to maintain the arrangement of keratinocytes, complete the structure of skin keratinous tissue, and enhance the mitochondrial activity of skin cells to maintain the vitality and normal metabolism of skin cells.
同時，本發明用於製備皮膚保健之組合物，亦可包含一有效量之黃金莓萃取物及各式可接受之載體，該組合物係一食品或一保養品。 At the same time, the composition for preparing skin health care of the present invention can also include an effective amount of golden berry extract and various acceptable carriers. The composition is a food or a skin care product.
以下將詳細說明本發明黃金莓萃取物之詳細萃取方法；本發明黃金莓萃取物於短時間與較長作用下，提升皮膚角質細胞中TGM1基因、KRT10基因、及/或KRT14基因表現量之功效測試；以及本發明黃金莓萃取物提升皮膚細胞粒線體活性之功效測試。以證實本發明之黃金莓萃取物能有效的維持皮膚角質細胞排列，使皮膚角質層結構完整，以及提升皮膚細胞的粒線體活性，以維持皮膚細胞的活力及正常代謝。以使皮膚處於保水的狀態、及維持皮膚年輕光滑有彈性，且同時能有效提升皮膚細胞抗老化的活性。 The detailed extraction method of the golden raspberry extract of the present invention will be described in detail below; the golden raspberry extract of the present invention can enhance the expression of TGM1 gene, KRT10 gene, and/or KRT14 gene in skin keratinocytes in a short time and a long time. Test; and the efficacy test of the golden berry extract of the present invention to enhance the mitochondrial activity of skin cells. It is confirmed that the golden berry extract of the present invention can effectively maintain the arrangement of skin keratinocytes, complete the structure of the skin stratum corneum, and enhance the mitochondrial activity of skin cells to maintain the vitality and normal metabolism of skin cells. In order to keep the skin in a water-retaining state, and maintain the skin young, smooth and elastic, and at the same time can effectively enhance the anti-aging activity of skin cells.
在本發明之一實施例中，首先將黃金莓果實清洗乾淨後，將該黃金莓果實與水、醇、或醇水混合物之萃取溶劑，萃取溶劑較佳為水，以1-5：10-20之重量比混合，均質後在溶劑中以50-100℃進行萃取0.5-2小時後，將粗萃取物冷卻至室溫，並將該粗萃取物以400mesh之濾網過濾以獲得濾液。最後，將該濾液於45-70℃進行減壓濃縮後，即得到本發明之黃金莓萃取物。 In an embodiment of the present invention, after the golden berry fruit is cleaned first, the extraction solvent of the golden berry fruit and water, alcohol, or a mixture of alcohol and water, the extraction solvent is preferably water, with a ratio of 1-5:10- Mix at a weight ratio of 20, homogenize and extract in a solvent at 50-100°C for 0.5-2 hours, then cool the crude extract to room temperature, and filter the crude extract with a 400 mesh filter to obtain a filtrate. Finally, after the filtrate is concentrated under reduced pressure at 45-70°C, the golden raspberry extract of the present invention is obtained.
本發明之一實施例以人類初代皮膚角質細胞HPEK-50(Human primary epidermal keratinocytes-50,HPEK-50)進行本發明之黃金莓萃取物在短時間作用下提升TGM1基因、KRT10基因、及KRT14基因表現量之功效的測試。該HPEK-50細胞係購自CELLnTEC公司(瑞士)，且培養於無血清之角質細胞培養液(keratinocyte-SFM)，該細胞培養液係購自Gibco公司(美國)，編號為#17005042，並於含有5% CO2之37℃細胞培養箱中進行培養。 An embodiment of the present invention uses human primary epidermal keratinocytes-50 (HPEK-50) to carry out the golden berry extract of the present invention to enhance TGM1 gene, KRT10 gene, and KRT14 gene under a short-term action A test of the efficacy of the manifestation. The HPEK-50 cell line was purchased from CELLnTEC (Switzerland) and was cultured in serum-free keratinocyte-SFM. The cell culture medium was purchased from Gibco (USA), numbered #17005042, and was used in Cultivation is carried out in a 37°C cell incubator containing 5% CO 2.
首先，將1.5x105個HPEK-50細胞培養於每孔含有2mL上述培養液之6孔培養盤中，於37℃培養16-18小時，接著將細胞分成以下三組：(1)僅加
入細胞培養液之控制組、(2)加入0.0625mg/mL本發明之黃金莓萃取物的實驗組、及(3)加入0.03125mg/mL本發明之黃金莓萃取物的實驗組，並將該三組分別作用24小時後，測試各組HPEK-50細胞中目標基因的表現量；其中該黃金莓萃取物係以水為溶劑進行萃取。首先，將該三組之HPEK-50細胞以細胞裂解液(RB buffer，購自Geanaid公司，臺灣，Cat No.RBD300)回收細胞後，使用RNA萃取試劑套組(購自Geneaid公司，臺灣，Cat No.RBD300)分別收集該三組細胞內之總RNA，接著利用SuperScript® III反轉錄酶(購自Invitrogene公司，美國，編號18080-051)以2000ng之萃取RNA為模板，並以表1之組合引子及反轉錄酶進行反轉錄作用，以產生該些基因之mRNA所相應之cDNA產物，接著利用ABI StepOnePlusTM Real-Time PCR system(Thermo Fisher Scientific公司，美國)，以及KAPA SYBR FAST(購自Sigma公司，美國，編號38220000000)將該三組反轉錄後產物分別以表1之組合引子進行定量即時聚合酶連鎖反應(Quantitative real-time polymerase chain reaction,qPCR)試驗，條件為95℃反應1秒，60℃反應20秒，總共40個循環。用以定量該三組HPEK-50細胞中TGM1基因、KRT10基因、及KRT14基因之mRNA的表現量，其中定量數值係取由閾值循環數(Ct)，而目標基因的mRNA相對量係推導自方程式2-△△Ct，其中△CT=CT比較組或實驗組目標基因/控制組目標基因-CT TBP(TATA結合蛋白，TATA-binding protein)；△△CT=CT比較組或實驗組目標基因-CT控制組目標基因；各組中各基因的fold change則為2-△△Ct。接著，再利用Excel軟體決定變異係數與是否在統計上具有顯著差異(*p值<0.05；**p值<0.01；***p值<0.001)。
First, culture 1.5x10 5 HPEK-50 cells in a 6-well culture dish containing 2 mL of the above culture medium per well, and incubate at 37°C for 16-18 hours, and then divide the cells into the following three groups: (1) Add cells only The control group of the culture solution, (2) the experimental group with 0.0625 mg/mL golden berry extract of the present invention, and (3) the experimental group with 0.03125 mg/mL golden berry extract of the present invention, and combine the three groups After acting for 24 hours, the expression level of the target gene in each group of HPEK-50 cells was tested; the golden berry extract was extracted with water as a solvent. First, the three groups of HPEK-50 cells were recovered with cell lysate (RB buffer, purchased from Geanaid Company, Taiwan, Cat No.RBD300), and then RNA extraction reagent kit (purchased from Geneaid Company, Taiwan, Cat No. RBD300) was used to recover the cells. No.RBD300) Collect the total RNA in the three groups of cells, and then use SuperScript ® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-051) with 2000ng of extracted RNA as a template, and use the combination in Table 1 The primers and reverse transcriptase perform reverse transcription to produce the cDNA products corresponding to the mRNAs of these genes, and then use the ABI StepOnePlus TM Real-Time PCR system (Thermo Fisher Scientific Company, USA), and KAPA SYBR FAST (purchased from Sigma Company, U.S., No. 38220000000) The three sets of reverse transcription products were respectively subjected to the quantitative real-time polymerase chain reaction (qPCR) test with the combination primers in Table 1, and the conditions were 95°C for 1 second. Reaction at 60°C for 20 seconds, a total of 40 cycles. It is used to quantify the mRNA expression levels of TGM1 , KRT10 , and KRT14 genes in the three groups of HPEK-50 cells, where the quantitative value is taken from the threshold cycle number (Ct), and the relative amount of mRNA of the target gene is derived from the
先前研究指出TGM會使角質化細胞之細胞膜與結構蛋白間形成強力鍵結，並能增加表皮層的強度與穩定性；KRT會形成角蛋白微絲，為角質細胞的結構蛋白，可維持角質細胞結構的強度和彈性。因此，角質細胞的結構基因-TGM1基因、KRT10基因、及KRT14基因的表現量上升，能夠維持角質細胞排列，使皮膚角質組織的結構完整，並防止皮膚水分散失。 Previous studies have pointed out that TGM will form a strong bond between the cell membrane of keratinocytes and structural proteins, and increase the strength and stability of the epidermal layer; KRT will form keratin filaments, which are structural proteins of keratinocytes, which can maintain keratinocytes. Strength and elasticity of the structure. Therefore, the expression levels of keratinocyte structural genes- TGM1 gene, KRT10 gene, and KRT14 gene are increased, which can maintain the arrangement of keratinocytes, make the structure of skin keratinous tissue intact, and prevent skin water loss.
本發明之黃金莓萃取物提升角質細胞內TGM1基因、KRT10基因、及KRT14表現量之結果如圖1所示。人類初代角質細胞經0.0625mg/mL本發明之黃金莓萃取物處理後，TGM1基因的表現量為控制組之1.15倍、KRT10基因的表現量為控制組之1.72倍、KRT14基因的表現量則為控制組之2.17倍；經0.03125mg/mL本發明之黃金莓萃取物處理後，TGM1基因的表現量為控制組之1.20倍、KRT10基因的表現量為控制組之1.72倍、KRT14基因的表現量則為控制組之2.35倍。此些結果顯示本發明之黃金莓萃取物能在24小時的作用下，有效提高TGM1基因、KRT10基因、及KRT14基因的表現量，能有效維持皮膚角質細胞排列，使皮膚角質層結構完整，以提升皮膚屏障功能、防止皮膚水分散失，使皮膚處於保水的狀態、及維持皮膚年輕光滑有彈性。 The results of the golden berry extract of the present invention enhancing the expression levels of TGM1 gene, KRT10 gene, and KRT14 in keratinocytes are shown in FIG. 1. After human primary keratinocytes were treated with 0.0625mg/mL golden berry extract of the present invention, the expression level of TGM1 gene was 1.15 times that of the control group, the expression level of KRT10 gene was 1.72 times that of the control group, and the expression level of KRT14 gene was 2.17 times of the control group; after gold berry extract 0.03125mg / mL of the process of the present invention, gene expression levels TGM1 is 1.20 times as the control group, the expression levels of the gene KRT10 1.72 times the control group, the expression levels of the gene KRT14 It is 2.35 times that of the control group. These results show that the golden berry extract of the present invention can effectively increase the expression of TGM1 gene, KRT10 gene, and KRT14 gene under the action of 24 hours, can effectively maintain the arrangement of skin keratinocytes, and make the skin stratum corneum structure complete. Improve skin barrier function, prevent skin water loss, keep skin in a state of water retention, and maintain skin youthful, smooth and elastic.
本發明之一實施例以人類皮膚纖維母細胞(Human skin fibroblast)CCD-966SK細胞、以及BDTM MitoScreen(JC-1)試劑套組(Flow cytometry Mitochondrial membrane potential detection kit)，以進行本發明之黃金莓萃取物提升皮膚細胞粒線體活性之功效的測試。其中，該CCD-966SK細胞係購自美國典型培養物保藏中心(ATCC，美國)，編號BCRC No.60153，且該細胞係培養於含有10%之胎牛血清(Fetal Bovine Serum)以及90%之Eagle’s Minimum Essential Medium(EMEM)的培養液(購自Gibco，美國)，其中含有0.1mM之非必需氨基酸(Non-essential amino acids)、1.5g/L之碳酸氫鈉(Sodium bicarbonate)、及1mM之丙酮酸鈉(Sodium pyruvate)。 An embodiment of the present invention uses Human skin fibroblast CCD-966SK cells and BD TM MitoScreen (JC-1) reagent kit (Flow cytometry Mitochondrial membrane potential detection kit) to perform the gold of the present invention. A test of the effect of raspberry extract in enhancing mitochondrial activity of skin cells. Among them, the CCD-966SK cell line was purchased from the American Type Culture Collection (ATCC, USA), numbered BCRC No. 60153, and the cell line was cultured with 10% Fetal Bovine Serum and 90% Eagle's Minimum Essential Medium (EMEM) culture medium (purchased from Gibco, USA), which contains 0.1 mM of non-essential amino acids, 1.5 g/L of sodium bicarbonate (Sodium bicarbonate), and 1 mM of Sodium pyruvate.
其中，在使用該BDTM MitoScreen(JC-1)試劑套組前，需先將10x JC-1染色測定緩衝溶液(JC-1 10x assay buffer)置於37℃下預熱，並以無菌之1x磷酸鹽緩衝溶液(1x Phosphate buffered saline,1xPBS)配置1x JC-1染色測定緩衝溶液，且維持溫度為37℃，接著加入130μL之二甲基亞碸(Dimethyl sulfoxide,DMSO)於該1x JC-1染色測定緩衝溶液中進行冷凍乾燥，即為JC-1染色測定緩衝溶液的儲存溶液，其能夠在-20℃下保存6個月；待使用時，將JC-1染劑與該1x JC-1測定緩衝溶液以1：100的比例均勻，即為JC-1作用染劑。 Among them, before using the BD TM MitoScreen (JC-1) reagent kit, the 10x JC-1 staining assay buffer (JC-1 10x assay buffer) needs to be preheated at 37°C and used aseptic 1x Phosphate buffer solution (1x Phosphate buffered saline, 1xPBS) is equipped with 1x JC-1 staining assay buffer solution, and the temperature is maintained at 37℃, and then 130μL of dimethyl sulfoxide (DMSO) is added to the 1x JC-1 Freeze-drying in the staining assay buffer solution is the storage solution of the JC-1 staining assay buffer solution, which can be stored at -20°C for 6 months; when it is to be used, mix the JC-1 stain with the 1x JC-1 The measuring buffer solution is uniform in the ratio of 1:100, which is the JC-1 dye.
首先，將1.5x105個CCD-966SK細胞培養於每孔含有2mL上述培養液之6孔培養盤中，於37℃培養16-18小時，接著在不影響吸附盤底細胞的情狀下，更換新的細胞培養液，並將細胞分成以下三組(n=3)：(1)僅加入細胞培養液之控制組、(2)加入1mg/mL本發明之黃金莓萃取物的實驗組、及(3)加入0.5mg/mL本發明之黃金莓萃取物的實驗組，並將該三組分別作用24小時；其中該黃金莓萃取物係以水為溶劑進行萃取，接著在不影響吸附盤底細胞的情狀下，移除培養液並以1xPBS沖洗細胞兩次，再加入200μL之胰蛋白酶(Trypsin)反應5分鐘，使細胞由培養盤上脫落，加入適當的培養液終止反應，並將細胞連同培養液收集至1.5mL離心管中，以400g離心5分鐘後移除上清液，再以1mL之1xPBS沖洗細胞一次後，於400g離心5分鐘後除上清液，再加入100μL之前述JC-1作用染劑，以vortex混合均勻後於黑暗中避光反應15分鐘，接著於400g離 心5分鐘後除上清液，再以前述1x JC-1染色測定緩衝溶液沖洗細胞兩次後，將細胞重新懸浮於含有2%胎牛血清之500μL之1xPBS中，以流式細胞儀(Beckman)偵測CCD-966SK細胞中粒線體的膜電位，其中粒線體膜電位越高表示粒線體活性越高。最後再利用Excel軟體決定變異係數與是否在統計上具有顯著差異(*p值<0.05；**p值<0.01；***p值<0.001)。 First, culture 1.5x10 5 CCD-966SK cells in a 6-well culture dish containing 2 mL of the above culture medium per well, and incubate at 37°C for 16-18 hours, and then replace them with new ones without affecting the adsorption of cells at the bottom of the dish. Cell culture medium, and the cells are divided into the following three groups (n=3): (1) control group with only cell culture medium added, (2) experimental group with 1mg/mL golden berry extract of the present invention, and ( 3) Add 0.5 mg/mL golden berry extract of the present invention to the experimental group, and act on the three groups for 24 hours respectively; wherein the golden berry extract is extracted with water as a solvent, and then it does not affect the adsorption of the cells at the bottom of the plate. In the case of circumstance, remove the culture medium and wash the cells twice with 1xPBS, then add 200μL of Trypsin to react for 5 minutes to make the cells fall off the culture plate, add appropriate culture medium to stop the reaction, and culture the cells together Collect the liquid into a 1.5mL centrifuge tube, centrifuge at 400g for 5 minutes, remove the supernatant, then wash the cells with 1mL of 1xPBS once, centrifuge at 400g for 5 minutes, remove the supernatant, and add 100μL of the aforementioned JC-1 Action stain, mix well with vortex and react for 15 minutes in the dark in the dark, then centrifuge at 400g for 5 minutes, remove the supernatant, and rinse the cells twice with the aforementioned 1x JC-1 staining assay buffer solution, then renew the cells Suspended in 500μL of 1xPBS containing 2% fetal bovine serum, and measured the membrane potential of mitochondria in CCD-966SK cells with a flow cytometer (Beckman). The higher the mitochondrial membrane potential, the higher the mitochondrial activity. high. Finally, use Excel software to determine whether the coefficient of variation is statistically significant (*p value<0.05; **p value<0.01; ***p value<0.001).
本發明之黃金莓萃取物提升皮膚細胞粒線體活性之結果如圖2所示。人類纖維母細胞1mg/mL本發明之黃金莓萃取物處理後，相較於控制組，能顯著提升36.5%之粒線體膜電位；經0.5mg/mL本發明之黃金莓萃取物處理後，相較於控制組，則能顯著提升32.3%之粒線體膜電位。此結果顯示本發明之黃金莓萃取物有效提升皮膚細胞的粒線體活性，以維持皮膚細胞的活力及正常代謝，並能有效提升皮膚細胞抗老化的活性。 The results of the golden berry extract of the present invention in enhancing the activity of mitochondria of skin cells are shown in FIG. 2. Human fibroblasts treated with 1 mg/mL golden berry extract of the present invention can significantly increase the mitochondrial membrane potential by 36.5% compared to the control group; after 0.5 mg/mL golden berry extract of the present invention, Compared with the control group, it can significantly increase the mitochondrial membrane potential by 32.3%. This result shows that the golden berry extract of the present invention effectively enhances the mitochondrial activity of skin cells to maintain the vitality and normal metabolism of skin cells, and can effectively enhance the anti-aging activity of skin cells.
綜上所述，本發明之黃金莓萃取物能有效在6-24小時內即有效提升皮膚角質細胞中與保濕相關的TGM1基因、KRT10基因、及KRT14基因的表現量，能有效維持皮膚角質細胞排列，使皮膚角質層結構完整，以提升皮膚屏障功能、以及防止皮膚水分散失，使皮膚處於保水的狀態、及維持皮膚年輕光滑有彈性；本發明之黃金莓萃取物亦能有效提升皮膚細胞的粒線體活性，以維持皮膚細胞的活力及正常代謝，以提升皮膚細胞抗老化的活性。因此，本發明之黃金莓萃取物具有提升皮膚抗老化活性及保濕能力之功效，可用於製備皮膚保健之組合物的用途，其中該組合物該組合物是一食品或一保養品，可藉由口服、皮膚塗抹等方式給予一個體。 In summary, the golden berry extract of the present invention can effectively increase the expression of TGM1 , KRT10 , and KRT14 genes related to moisturizing in skin keratinocytes within 6-24 hours, and can effectively maintain skin keratinocytes Arrangement to complete the skin stratum corneum structure to enhance the skin barrier function and prevent skin water loss, keep the skin in a water-retaining state, and maintain the skin young, smooth and elastic; the golden berry extract of the present invention can also effectively enhance the skin cell Mitochondrial activity to maintain the vitality and normal metabolism of skin cells to enhance the anti-aging activity of skin cells. Therefore, the golden berry extract of the present invention has the effect of enhancing the anti-aging activity and moisturizing ability of the skin, and can be used to prepare skin health care composition, wherein the composition is a food or a skin care product. Give to a body by oral, skin application, etc.
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| JP4560156B2 (en) * | 1999-10-26 | 2010-10-13 | 一丸ファルコス株式会社 | Cosmetic composition containing moisturizing plant extract |
| EP1992322A1 (en) * | 2007-05-11 | 2008-11-19 | Dr. Scheller Cosmetics AG | Composition for percutaneous application |
| CN103445180B (en) * | 2013-09-17 | 2015-05-06 | 王喜军 | Application of physalis pubescens in preparing anti-oxidization health-care food |
| KR101926783B1 (en) * | 2016-11-29 | 2018-12-07 | 주식회사 코리아나화장품 | Cosmetic composition for moisturizing skin containing mixture of amorphophallus konjac root extract, physalis peruviana fruit extract and epiphyllum oxpetalum flower extract |
| CN108685097B (en) * | 2018-03-16 | 2021-06-04 | 通化师范学院 | Application of iris angulatus in anti-aging |
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| Title |
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| 2018/07/20「粒線體在人體健康、老化和幹細胞治療疾病的重要角色」 |
| Journal of Pharmacognosy and Phytochemistry 2018; 7(6): 1751-1755 |
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