TW202014176A - Pachyrhizus erosus fermented extracts and the use thereof for enhancing the gene expression of col, timp, lox, eln, has, sod, tcp1 and ung, and for reducing the skin melanin content - Google Patents
Pachyrhizus erosus fermented extracts and the use thereof for enhancing the gene expression of col, timp, lox, eln, has, sod, tcp1 and ung, and for reducing the skin melanin content Download PDFInfo
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Abstract
Description
本發明係關於一涼薯發酵物及其用途，尤其是一種涼薯發酵物用於製備提升COL基因、TIMP基因、LOX基因、ELN基因、HAS基因、SOD基因、TCP1基因與UNG基因、以及降低皮膚黑色素含量之組合物的用途。 The present invention relates to a cold potato ferment and its use, especially a cold potato ferment for the preparation of COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and reduction Use of skin melanin content composition.
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, which is the stratum corneum, the granular layer, the spinous layer and the basal layer in sequence from the outer to the inner. The epidermal layer is mainly formed by the continuous differentiation of the undifferentiated cylindrical keratinocytes in the basal layer. This process Called keratinization. Keratinocytes have a high water content. As the cells metabolize and differentiate, the shape of the keratinocytes will gradually become flat, and the nuclei and organelles will begin to degenerate and shrink, and dead cells without nucleus and organelles will form in the stratum corneum. The main function of the epidermal layer is to keep the skin water-retaining and form a skin barrier to resist various external damages. The outermost layer of the epidermal layer is composed of a weakly acidic sebum film and the stratum corneum like a brick wall structure. This barrier can lock the skin. Moisture and oil, resist the invasion of bacteria on the skin surface, and resist the damage of foreign bodies and ultraviolet light, etc., have a very important protective effect on the human body.
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 skin grows older, the skin will gradually age (Ageing). The causes and processes of skin aging are very complex and involve countless physiological phenomena, including ultraviolet damage, free radical damage, Decreased collagen, slower cell renewal, the appearance of abnormal cells, decreased skin fat, interstitial cell deficiencies, cell growth arrest, hormonal decline and other common factors. But as of now, most of the skin anti-aging products on the market can only start to increase the antioxidant activity, and cannot directly or effectively improve or delay the occurrence of skin aging.
另外近年來，為達到人們努力追求皮膚白皙及斑點漸少的需求，市面上不斷開發出各種抑制黑色素形成或淡化斑點之美白產品，然而，該些產品為訴求達到特殊效果，而加入過多致癌的化學物質或環境荷爾蒙，使消費者在不知情的狀況下使用會傷害皮膚影響健康的成分。 In addition, in recent years, in order to meet people's demand for fair skin and less spots, various whitening products on the market have been developed to suppress the formation of melanin or lighten spots. However, these products have added too many carcinogens in order to achieve special effects. Chemical substances or environmental hormones cause consumers to use ingredients that can harm skin and affect health without knowing it.
因此，綜合上面所述，因應講求天然健康的現代，開發一種能直接且有效地改善或延緩皮膚老化發生，又能同時兼顧皮膚美白訴求之綜合皮膚保健組合物，著實有其必要性。 Therefore, it is necessary to develop a comprehensive skin health care composition that can directly and effectively improve or delay the occurrence of skin aging, and at the same time take into account the skin whitening demands, in light of the above-mentioned natural health.
緣此，本發明之一目的在提供一種涼薯發酵物，其係涼薯的水萃取物經由酵母菌(Saccharomyces cerevisiae)、乳酸桿菌(Lactobacillus plantarum)、及醋酸桿菌(Acetobacter aceti)依序進行發酵而獲得。 Therefore, an object of the present invention is to provide a cold potato fermented product, in which the water extract of cold potato is fermented in order via yeast ( Saccharomyces cerevisiae ), Lactobacillus plantarum , and Acetobacter aceti And get.
本發明之又一目的在提供一種如前所述之涼薯發酵物用於製備一提升膠原蛋白(Collagens,COL)基因、基質金屬蛋白酶(Tissue inhibitor of metallopeptidases,TIMP)基因、離胺酸氧化酶(Lysyl oxidase,LOX)基因、彈性蛋白(elastin,ELN)基因、及玻尿酸合成酶(Hyaluronan synthase,HAS)基因的表現量之組合物的用途。 Another object of the present invention is to provide a cold potato fermented product as described above for the preparation of an elevated collagen (Collagens, COL) gene, matrix metalloproteinase (Tissue inhibitor of metallopeptidases, TIMP) gene, lysine oxidase (Lysyl oxidase (LOX) gene, elastin (ELN) gene, and hyaluronan synthase (HAS) gene).
本發明之又一目的在提供一種如前所述之涼薯發酵物用於製備一提升超氧化物歧化酶(Superoxide dismutase,SOD)基因、TCP1的折疊蛋白(Chaperonin containing TCP1,CCT)基因、及尿嘧啶DNA糖基化酶(Uracil DNA glycosylase,UNG)基因的表現量之組合物的用途。 Another object of the present invention is to provide a cold potato fermented product as described above for preparing a superoxide dismutase (SOD) gene, a TCP1 folding protein (Chaperonin containing TCP1, CCT) gene, and Uracil DNA glycosylase (Uracil DNA glycosylase, UNG) gene expression level of the use of the composition.
本發明之另一目的在提供一種如申請專利範圍第1項所述之涼薯發酵物用於製備降低皮膚黑色素含量之組合物的用途。 Another object of the present invention is to provide a use of the cold potato fermented product as described in item 1 of the patent application scope for the preparation of a composition for reducing skin melanin content.
在本發明之一實施例中，其中該酵母菌之添加量為0.01-0.5%(v/v)、該乳酸桿菌之添加量0.01-0.25%(v/v)、及該醋酸桿菌之添加量為3-10%(v/v)；且該酵母菌之發酵時間為1至2.5天、該乳酸桿菌之發酵時間為1至3天、及該醋酸桿菌之發酵時間為3至10天。 In one embodiment of the present invention, the addition amount of the yeast is 0.01-0.5% (v/v), the addition amount of the Lactobacillus is 0.01-0.25% (v/v), and the addition amount of the Acetobacter 3-10% (v/v); and the fermentation time of the yeast is 1 to 2.5 days, the fermentation time of the Lactobacillus is 1 to 3 days, and the fermentation time of the Acetobacter is 3 to 10 days.
在本發明之又一實施例中，其中該膠原蛋白基因係為膠原蛋白23型A1(Collagen,type XXIII,alpha 1；COL23A1)基因、膠原蛋白25型A1(Collagen,type XXV,alpha 1；COL25A1)基因、膠原蛋白6型A2(Collagen,type VI,alpha 2；COL6A2)基因、及膠原蛋白4型A4(Collagen,type IV,alpha 4；COL4A4)基因；該基質金屬蛋白酶基因係為基質金屬蛋白酶1(Tissue inhibitor of metallopeptidases 1,TIMP1)基因、及基質金屬蛋白酶3(Tissue inhibitor of metallopeptidases 3,TIMP3)基因；該玻尿酸合成酶基因係為玻尿酸合成酶2(Hyaluronan synthase 2,HAS2)基因及玻尿酸合成酶3(Hyaluronan synthase 3,HAS3)基因。
In yet another embodiment of the present invention, wherein the collagen gene line is a collagen 23 type A1 (Collagen, type XXIII, alpha 1; COL23A1) gene, a collagen type 25 A1 (Collagen, type XXV, alpha 1; COL25A1 ) Gene, collagen 6 type A2 (Collagen, type VI, alpha 2; COL6A2) gene, and
在本發明之又一實施例中，其中該涼薯發酵物係提升彈性蛋白之生成。 In yet another embodiment of the present invention, wherein the cold potato fermentation system enhances the production of elastin.
在本發明之又一實施例中，其中該涼薯發酵物係提升皮膚緊緻度及玻尿酸之生成。 In yet another embodiment of the present invention, wherein the cold potato fermented material enhances skin firmness and hyaluronic acid production.
在本發明之又一實施例中，其中該超氧化物歧化酶基因係為超氧化物歧化酶2(Superoxide dismutase 2,SOD2)基因、及超氧化物歧化酶3(Superoxide dismutase 3,SOD3)基因；該TCP1的折疊蛋白基因係為TCP1的折疊蛋白2(Chaperonin containing TCP1 2,CCT2)基因、TCP1的折疊蛋白4(Chaperonin containing TCP1 4,CCT4)基因、TCP1的折疊蛋白5(Chaperonin
containing TCP1 5,CCT5)基因、及TCP1的折疊蛋白7(Chaperonin containing TCP1 7,CCT 7)基因。
In yet another embodiment of the present invention, wherein the superoxide dismutase gene line is a superoxide dismutase 2 (Superoxide dismutase 2, SOD2) gene, and a superoxide dismutase 3 (Superoxide
在本發明之又一實施例中，其中該涼薯發酵物係促進DNA修復能力。 In yet another embodiment of the present invention, wherein the cold potato fermentation strain promotes DNA repair ability.
本發明將涼薯以酵母菌、乳酸桿菌及醋酸桿菌進行三段式發酵所得之涼薯發酵物，能有效提升其中的總多酚含量、提升皮膚細胞抗紫外光能力、且也能有效提升皮膚細胞中COL4A4基因、TIMP1基因、LOX基因、HAS2基因、HAS3基因、CCT2基因、CCT5基因、CCT7基因、及SOD3基因之表現量，且較涼薯水萃取物有更佳之效果，因此能更加有效的用於製備增加皮膚抗氧化活性、增加皮膚抗老化活性、增加皮膚緊緻度、及增加皮膚美白能力之醫藥組合物，並達到綜合地皮膚保健功效。 According to the invention, the cold potato fermented product obtained by three-stage fermentation of cold potato with yeast, lactobacilli and acetobacter can effectively increase the total polyphenol content therein, improve the skin cell's ability to resist ultraviolet light, and can also effectively improve the skin The expression level of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, HAS3 gene, CCT2 gene, CCT5 gene, CCT7 gene, and SOD3 gene in cells, and has better effect than cold potato water extract, so it can be more effective It is used to prepare pharmaceutical compositions that increase skin antioxidant activity, increase skin anti-aging activity, increase skin firmness, and increase skin whitening ability, and achieve comprehensive skin health benefits.
以下將配合圖式進一步說明本發明的實施方式，下述所列舉的實施例係用以闡明本發明，並非用以限定本發明之範圍，任何熟習此技藝者，在不脫離本發明之精神和範圍內，當可做些許更動與潤飾，因此本發明之保護範圍當視後附之申請專利範圍所界定者為準。 The embodiments of the present invention will be further described below with reference to the drawings. The examples listed below are used to clarify the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with this art without departing from the spirit and spirit of the present invention Within the scope, some changes and retouching can be done, so the protection scope of the present invention shall be subject to the scope defined in the appended patent application.
圖1係為本發明之涼薯發酵物提升總多酚含量之長條圖。 FIG. 1 is a bar graph of the cold potato fermented product of the present invention to increase the total polyphenol content.
圖2係為本發明之涼薯發酵物降低黑色素生成量之長條圖。 Fig. 2 is a bar graph showing that the fermentation product of cold potato of the present invention reduces melanin production.
圖3係為本發明之涼薯發酵物於DNA微陣列分析中提升COL基因、TIMP基因、ELN基因、及HAS基因表現量之長條圖。 FIG. 3 is a bar graph of the expression of the COL gene, TIMP gene, ELN gene, and HAS gene of the cold potato fermented product of the present invention in DNA microarray analysis.
圖4係為本發明之涼薯發酵物於DNA微陣列分析中提升SOD基因、CCT基因、及UNG基因表現量之長條圖。 FIG. 4 is a bar graph of the improved expression of SOD gene, CCT gene, and UNG gene of the cold potato fermented product of the present invention in DNA microarray analysis.
圖5係為本發明之涼薯發酵物提升COL4A4基因表現量之長條圖。 FIG. 5 is a bar graph of the cold potato fermented product of the present invention enhancing the expression level of the COL4A4 gene.
圖6係為本發明之涼薯發酵物提升TIMP1基因表現量之長條圖。 FIG. 6 is a bar graph of the cold potato fermented product of the present invention enhancing TIMP1 gene expression.
圖7係為本發明之涼薯發酵物提升LOX基因表現量之長條圖。 FIG. 7 is a bar graph of the cold potato fermented product of the present invention enhancing the expression level of the LOX gene.
圖8係為本發明之涼薯發酵物提升HASs基因表現量之長條圖。 FIG. 8 is a bar graph of the cold potato fermented product of the present invention enhancing the expression level of HASs genes.
圖9係為本發明之涼薯發酵物提升CCTs基因表現量之長條圖。 FIG. 9 is a bar graph of the cold potato fermented product of the present invention enhancing the CCTs gene expression.
圖10係為本發明之涼薯發酵物提升SOD3基因表現量之長條圖。 FIG. 10 is a bar graph of the cold potato fermented product of the present invention enhancing the expression level of the SOD3 gene.
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 in this article are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.
本發明提供一種涼薯發酵物及其用於製備提升COL基因、TIMP基因、LOX基因、ELN基因、HAS基因、SOD基因、TCP1基因與UNG基因、以及降低皮膚黑色素含量之組合物的用途，本發明之涼薯發酵物係將涼薯以酵母菌、乳酸桿菌及醋酸桿菌進行三段式發酵所得，其可用於提升其中的總多酚含量、提升皮膚細胞抗紫外光能力、且也能有效提升皮膚細胞中COL4A4基因、TIMP1基因、LOX基因、HAS2基因、HAS3基因、CCT2基因、CCT5基因、CCT7基因、及SOD3基因之表現量。 The invention provides a cold potato fermented substance and its use for preparing a composition for enhancing COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and reducing skin melanin content. The invented cold potato fermentation system is obtained by three-stage fermentation of cold potato with yeast, Lactobacillus and Acetobacter, which can be used to increase the total polyphenol content, improve the skin cell's ability to resist ultraviolet light, and can also effectively improve The expression levels of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, HAS3 gene, CCT2 gene, CCT5 gene, CCT7 gene, and SOD3 gene in skin cells.
同時，本發明用於製備提升COL基因、TIMP基因、LOX基因、ELN基因、HAS基因、SOD基因、TCP1基因與UNG基因、以及降低皮膚黑色素含量之組合物，亦可包含一有效量之涼薯發酵物及一醫藥上可接受之載體，該組合物係以粉末狀、顆粒狀、液狀、膠狀或膏狀之劑型存在，且其劑型係以食品、飲品、藥品、試劑或營養補充劑之形式提供。 At the same time, the present invention is used to prepare a composition for enhancing the COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and reducing the skin melanin content, and may also contain an effective amount of cold potatoes Fermented product and a pharmaceutically acceptable carrier, the composition is in the form of powder, granule, liquid, gel or paste, and the dosage form is food, drink, medicine, reagent or nutritional supplement Provided.
以下將詳細說明本發明涼薯發酵物之詳細製備方法，與該涼薯發酵物之總多酚含量測試、抗UVA能力測試、調控基因之初步測試、調控彈性蛋白基因能力之測試、及調控抗老化基因能力之測試，以證實該涼薯發酵物對於 提升COL基因、TIMP基因、LOX基因、ELN基因、HAS基因、SOD基因、TCP1基因與UNG基因表現量、以及提升皮膚抗老化能力之效果。 The detailed preparation method of the cold potato fermented product of the present invention, the total polyphenol content test of the cold potato fermented product, the anti-UVA capability test, the preliminary test of the regulatory gene, the test of the regulatory elastin gene capability, and the regulatory resistance will be described in detail below Aging gene ability test to confirm that the cold potato fermentation product is Improve the expression of COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and enhance the effect of skin anti-aging ability.
本發明以人類皮膚纖維母細胞(CCD-966sk)進行本發明之涼薯發酵物之細胞實驗。該人類皮膚纖維母細胞係購自美國典型培養物保藏中心(美國)，編號CRL-1881。該細胞係培養於含有10%之胎牛血清(Fetal Bovine Serum)之MEM(Minimum essential medium，購自Gibco，美國，12100-046)培養液，其中加入1mM之丙酮酸鈉(sodium pyruvate)以及1%之青黴素/鏈黴素。 In the present invention, human skin fibroblasts (CCD-966sk) are used for the cell experiment of the cold potato fermented product of the present invention. The human skin fibroblast cell line was purchased from the American Type Culture Collection (USA), number CRL-1881. The cell line was cultured in MEM (Minimum essential medium, purchased from Gibco, USA, 12100-046) medium containing 10% fetal bovine serum (Fetal Bovine Serum), to which 1 mM sodium pyruvate and 1 were added % Of penicillin/streptomycin.
涼薯(Pachyrhizus erosus)為豆科(Legumen)豆薯屬(Pachyrhizus)多年生藤本狀草本植物，又稱豆薯、葛薯、番葛，涼薯為豆類因根如薯塊而得名，涼薯塊根可生食或熟食，直徑約20-30公分，富含糖類、蛋白質，並可加工製成沙葛粉，有清涼去熱的功效。 Cold potato ( Pachyrhizus erosus ) is a legumen ( Pachyrhizus ) perennial vine-like herb. It is also known as bean potato, kudzu potato, and pueraria lobata . The cold potato is named after beans like roots. Roots can be eaten raw or cooked, and are about 20-30 cm in diameter. They are rich in sugar and protein, and can be processed into Shage powder, which has the effect of cooling and removing heat.
在本發明一實施例中，將涼薯徹底清洗，取洗淨後之涼薯以1：5-10之重量比例與水混合，在50-100℃下滅菌萃取0.5-1.5小時，以獲得涼薯水萃取物，該涼薯水萃取物冷卻至室溫供後續三段式發酵使用，首先殖入0.01-0.5%酵母菌(Saccharomyces cerevisiae，購買於生物資源保存與研究中心，台灣，編號為BCRC20271)於該涼薯水萃取物內進行發酵1-2.5天後，接著直接加入0.01-0.25%乳酸桿菌(Lactobacillus plantarum TCI028，專利寄存於生物資源保存與研究中心，台灣，編號為BCRC910805)發酵1-3天，再加入3-10%醋酸桿菌(Acetobacter aceti，購買於生物資源保存與研究中心，台灣，編號為BCRC11688)發酵3-10天；其中，乳酸桿菌TCI028係已於中華民國專利申請號106145146完成專利寄存，此三種菌之發酵依序為：酵母菌、乳酸桿菌、醋酸 桿菌，且無法前後對調，最後在不移除此三種菌之情況下，使用設定的糖度範圍2-10°、pH2-4、無酒精、多酚等規格，如檢驗符合該規格，則判定發酵完成並得到發酵液，其總多酚含量至少佔20%。接著，將該發酵液於45-70℃進行減壓濃縮，並以200-400mesh網篩過濾，再添加1-3%檸檬酸及40-70%異麥芽寡糖調整規格後滅菌，即得到本發明之涼薯發酵物，其中藉由微生物發酵製成，使涼薯之效性物質大量釋出。 In an embodiment of the present invention, the cold potatoes are thoroughly washed, the washed cold potatoes are mixed with water at a weight ratio of 1:5-10, and sterilized and extracted at 50-100°C for 0.5-1.5 hours to obtain cold Potato water extract. The cold potato water extract is cooled to room temperature for the subsequent three-stage fermentation. It is first planted with 0.01-0.5% yeast ( Saccharomyces cerevisiae) , purchased from the Biological Resources Conservation and Research Center, Taiwan, number BCRC20271 ) After fermentation in the cold potato water extract for 1-2.5 days, then directly add 0.01-0.25% Lactobacillus plantarum TCI028, the patent is deposited in the Biological Resources Conservation and Research Center, Taiwan, number BCRC910805) Fermentation 1- 3 days, then add 3-10% Acetobacter aceti (purchased from the Biological Resources Conservation and Research Center, Taiwan, number BCRC11688) and ferment for 3-10 days; of which, Lactobacillus TCI028 has been registered in the Republic of China Patent Application No. 106145146 After completing the patent deposit, the fermentation of these three bacteria is in order: yeast, lactobacilli, and acetobacter, and cannot be adjusted back and forth. Finally, without removing these three bacteria, use the set sugar degree range 2-10°, pH2 -4. Non-alcohol, polyphenol and other specifications, if the test meets the specifications, it is judged that the fermentation is completed and the fermentation broth is obtained, and its total polyphenol content accounts for at least 20%. Next, the fermentation broth is concentrated under reduced pressure at 45-70°C, filtered through a 200-400 mesh screen, then 1-3% citric acid and 40-70% isomalt oligosaccharide are added to adjust the specifications, and then sterilized to obtain The cold potato fermented substance of the present invention is made by microbial fermentation to release a large amount of the effective substance of cold potato.
本實施例係為了比較本發明之涼薯發酵物中總多酚含量是否較涼薯水萃取物高，使用Folin-Ciocalteu比色法測定其中總多酚含量，該測定法係利用試劑中的鎢鉬酸定量總多酚含量，其自身被還原(由Mo6+變為Mo5+)後會生成藍色化合物，而該藍色化合物的深淺與總多酚含量呈正相關。首先，分別將本發明之涼薯發酵物及涼薯水萃取物以水稀釋1倍後取100mL到離心管中，接著加入500μL之Folin-Ciocalteu酚試劑混合並靜置3分鐘後，加入400μL之7.5%碳酸鈉混勻靜置30分鐘後，取200μL反應溶液96孔板中，並測量750nm之吸光度。其中以沒食子酸(Gallic acid)作為標準品以製作標準曲線，將10g之沒食子酸溶於水中，並配置標準0、20、40、60、80、及100μL/mL之沒食子酸，並分別取100μL之各濃度的標準溶液至10mL離心管中，並加入500μL之Folin-Ciocalteu酚試劑混合靜置3分鐘後，加入400μL之7.5%碳酸鈉混勻靜置30分鐘後，取200μL反應溶液96孔板中，並測量750nm之吸光度，以獲得標準曲線，再經由標準曲線換算本發明之涼薯發酵物及涼薯水萃取物中總多酚含量。 In this example, in order to compare whether the total polyphenol content in the cold potato fermentation of the present invention is higher than that of the cold potato water extract, the total polyphenol content in the Folin-Ciocalteu colorimetric method is used to determine the total polyphenol content, which uses tungsten in the reagent Molybdenum acid quantifies the total polyphenol content. When it is reduced by itself (from Mo 6+ to Mo 5+ ), a blue compound is formed, and the depth of the blue compound is positively correlated with the total polyphenol content. First, dilute the cold potato fermented product and cold potato water extract of the present invention twice with water, take 100 mL into a centrifuge tube, then add 500 μL of Folin-Ciocalteu phenol reagent and mix for 3 minutes, then add 400 μL of After 7.5% sodium carbonate was mixed and allowed to stand for 30 minutes, 200 μL of the reaction solution was taken in a 96-well plate, and the absorbance at 750 nm was measured. Among them, using gallic acid as the standard product to prepare a standard curve, dissolve 10g of gallic acid in water, and configure standard 0, 20, 40, 60, 80, and 100μL/mL gallic acid Acid, and take 100μL of each standard solution of each concentration into a 10mL centrifuge tube, add 500μL of Folin-Ciocalteu phenol reagent and mix for 3 minutes, add 400μL of 7.5% sodium carbonate and mix and let stand for 30 minutes. 200 μL of the reaction solution was measured in 96-well plates, and the absorbance at 750 nm was measured to obtain a standard curve, and then the total polyphenol content in the cold potato fermented product and cold potato water extract of the present invention was converted through the standard curve.
本發明之涼薯發酵物提升總多酚含量之結果如圖1所示。多酚為一種抗氧化劑，先前研究指出多酚可以預防紫外光造成之氧化損傷，能夠提高細胞之抗氧化壓力、協助去除自由基、並防止DNA損傷。涼薯水萃取物經本發 明之三段式發酵作用後，其中總多酚為單純涼薯水萃取物之13倍之高，此結果顯示本發明之涼薯發酵物在經過特定之微生物發酵步驟後，可有效提升其中的總多酚含量，該特定發酵步驟能提升涼薯的抗氧化能力，並可用於製備提高皮膚抗氧化能力的組合物，使皮膚抗老化及抗紫外線傷害能力提升。 The result of raising the total polyphenol content of the cold potato fermentation product of the present invention is shown in FIG. 1. Polyphenols are an antioxidant. Previous studies have pointed out that polyphenols can prevent oxidative damage caused by ultraviolet light, can increase the antioxidant pressure of cells, help remove free radicals, and prevent DNA damage. Cold potato water extract by this hair After the three-stage fermentation of Ming Dynasty, the total polyphenols are 13 times as high as the pure cold potato water extract. This result shows that the cold potato fermentation product of the present invention can effectively increase the total polyphenols after a specific microbial fermentation step. The content of polyphenols, this specific fermentation step can enhance the antioxidant capacity of cold potatoes, and can be used to prepare a composition that enhances the skin's antioxidant capacity, so that the skin's ability to resist aging and UV damage.
本實施例係為了比較本發明之涼薯發酵物降低黑色素含量之能力是否較涼薯水萃取物高，因此利用黑色素瘤細胞(B16F10，編號ATCC® CRL-6475TM)測試，其中使用含有10%之胎牛血清(Fetal Bovine Serum)、1%青黴素-鏈黴素(購自Gibco，美國)、以及90%之DMEM(Dulbecco's Modified Eagle Medium，購自Gibco，美國，12100-046)培養液進行細胞培養。首先，於6孔培養盤之每孔中加入3mL培養液，並植入1.5×105個細胞，將細胞於37℃培養24小時後，在不擾動貼附細胞的情況下去除培養液，接著將細胞分成以下四組(各組n=4)：(1)空白控制組、(2)0.25%麴酸為正控制組、(3)0.25%涼薯水萃取物、及(4)0.25%本發明之涼薯發酵物，並在3mL新鮮培養液中於37℃作用48小時後，移除培養液並使用1倍磷酸鹽緩衝溶液(1x Phosphate buffered saline,1xPBS)清洗細胞兩次，再加入200μL胰蛋白酶(Trypsin)反應3分鐘，使細胞由培養盤上脫落，加入200μL的培養液終止反應，並將細胞連同培養液收集至15mL離心管，以400g離心5分鐘後移除上清液，再以1mL之1xPBS沖洗細胞二次後，以200μL之1xPBS重新懸浮細胞，再將細胞溶液置於液態氮中急速冷凍10分鐘，接著將細胞在室溫下靜置30分鐘以解凍，待完全解凍後，以12,000g離心30分鐘並去除上清液，接著加入並混合120μL之1N的NaOH(以ddH2O配置)於60℃中乾浴1小時後，取100μL待測溶液至96孔培養盤中測量OD450nm，其中以下列公式測定黑色素含量： 黑色素含量(%)=(%)=(實驗組OD值/控制組OD值)×100%再利用Excel軟體進行student t-test以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)。 This example is to compare whether the ability of the cold potato ferment of the present invention to reduce the melanin content is higher than that of the cold potato water extract, so the melanoma cells (B16F10, number ATCC ® CRL-6475 TM ) were used for testing, which contained 10% Fetal Bovine Serum (Fetal Bovine Serum), 1% penicillin-streptomycin (available from Gibco, USA), and 90% DMEM (Dulbecco's Modified Eagle Medium, purchased from Gibco, USA, 12100-046) to cultivate. First, add 3mL of culture medium to each well of the 6-well culture plate and implant 1.5×10 5 cells. After culturing the cells at 37°C for 24 hours, remove the culture medium without disturbing the attached cells, then Divide the cells into the following four groups (each group n=4): (1) blank control group, (2) 0.25% aspartic acid as positive control group, (3) 0.25% cold potato water extract, and (4) 0.25% The cold potato fermented product of the present invention, after being used in 3 mL fresh culture medium at 37°C for 48 hours, remove the culture medium and wash the cells twice with 1x Phosphate buffered saline (1xPBS), and then add 200μL of trypsin was reacted for 3 minutes to make the cells detach from the culture plate. 200μL of culture medium was added to stop the reaction, and the cells and culture medium were collected into a 15mL centrifuge tube. After centrifugation at 400g for 5 minutes, the supernatant was removed. After rinsing the cells twice with 1 mL of 1xPBS again, resuspend the cells with 200 μL of 1xPBS, and then freeze the cell solution in liquid nitrogen for 10 minutes, then let the cells stand at room temperature for 30 minutes to thaw, to be completely thawed After that, centrifuge at 12,000g for 30 minutes and remove the supernatant, then add and mix 120 μL of 1N NaOH (configured with ddH 2 O) to dry bath at 60°C for 1 hour, then take 100 μL of the test solution to a 96-well culture dish Measure OD 450nm in the middle, and determine the melanin content with the following formula: Melanin content (%)=(%)=(Experimental group OD value/Control group OD value)×100% Then use Excel software to conduct student t-test to determine the coefficient of variation There is a statistically significant difference (*p value <0.05; **p value <0.01; ***p value <0.001).
本發明之涼薯發酵物降低黑色素生成量之結果如圖2所示。人類皮膚纖維母細胞經本發明之涼薯發酵物處理後，降低了14%之細胞黑色素含量，與麴酸之正控制組數值相近，而未經發酵之涼薯水萃取物僅降低約8%之細胞黑色素含量，此結果顯示本發明之涼薯發酵物在經過特定之微生物發酵步驟後，可有效降低細胞之黑色素含量，該特定發酵步驟能提升涼薯減少細胞黑色素含量的功效，並可用於製備提高皮膚美白的組合物使皮膚淨透亮白。 The result of the cold potato fermented product of the present invention reducing the amount of melanin production is shown in FIG. 2. The human skin fibroblasts treated with the cold potato fermentation product of the present invention reduced the cell melanin content by 14%, which was similar to the value of the positive control group of kojic acid, while the unfermented cold potato water extract only reduced by about 8%. Cellular melanin content. This result shows that the cold potato fermented product of the present invention can effectively reduce the melanin content of cells after a specific microbial fermentation step. This specific fermentation step can enhance the efficiency of cold potato to reduce the cell melanin content, and can be used for preparation The skin whitening enhancing composition makes the skin clear and bright.
為了初步檢測本發明之涼薯發酵物會影響細胞中哪些基因的表現，藉由DNA微陣列分析(DNA microarray)經本發明之涼薯發酵物處理過後之細胞中表現量上升之基因。首先，收集經2%涼薯發酵物刺激24小時之人類皮膚纖維母細胞後，將細胞以細胞裂解液(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)萃取人類皮膚纖維母細胞內之RNA，接著利用SuperScript® III反轉錄酶(購自Invitrogene公司，美國，編號18080-044)以1μg之萃取RNA為模板並以T7 Oligo(dT)引子進行反轉錄反應(42℃,2小時)產生具有T7啟動子(promoter)之相應cDNA產物，接著同時加入1μL之DNA合成酶(Polymerase)及0.5μL之RNA分解酶(RNase H)將該cDNA產物之原始RNA模板分解，並以該單股cDNA為模板進行DNA合成反應(16℃,2小時)以形成雙股cDNA產物，將該雙股cDNA產物進行純化以移除可能抑制體外轉錄反應(in vitro transcription)的RNA、引子、酶和鹽類。其中純化係將50μL的cDNA產物加入50μL的 Nuclease-free water混合均勻，再加入250μL的cDNA binding buffer混合均勻後，放入cDNA spin column中，以10,000g離心1分鐘，接著將離心管下方液體移除，再將cDNA spin column放入離心機內，以離心10,000g離心3分鐘，使spin column內殘餘酒精完全去除，再將spin column移到新的1.5mL離心管，加入已於55℃預熱之12μL Nuclease-free water，於室溫靜置5分鐘後，以離心10,000g離心2分鐘，即得到純化之雙股cDNA。接著使用Amino Allyl MessageAmpTM II aRNA Amplification Kit(購自Ambion，美國，編號AM1753)，將Amino Allyl UTP(aaUTP)以該雙股cDNA為模板進行體外轉錄反應，以合成具有胺基烯丙基修飾的aRNA(amino allyl-modified antisensesRNA)，使待測RNA之量擴增，接著將該aRNA產物使用Amino Allyl MessageAmpTM II aRNA Amplification Kit進行純化(購自美國，編號AM1753)，以去除未嵌入產物之NTP、鹽類、酶和無機磷酸鹽，以改善aRNA的穩定性，再透過Labeling Reagent and Hybridization Kit(購自Phalanx Biotech Group，美國，編號HOA v7)將Cy5 NHS ester染料(購自AAT Bioquest，美國，編號Cat.151)與aRNA上具有胺基烯丙基修飾的UTP殘基進行染料偶聯反應(Dye coupling reaction)，接著將剩餘的染料去除後，將緩衝溶液置換成不含核酸分解酶的水(Nuclease-free Water，購自Ambion，美國，編號AM1753)，並建立Whole Genome Experssion微陣列雜交系統(購自Phalanx Biotech Group，美國，編號HOA v7)，以Agilent Microarray Scanner進行掃描和信號分割，於標準化原始數據後，通過使用log2之比率方法測定不同基因的相對表現量。再利用Excel軟體進行非成對單尾student t-test，以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)。 In order to preliminarily detect which genes of the cold potato fermented product of the present invention will affect the performance of cells, DNA microarray analysis (DNA microarray) is used to analyze the genes in the cells that have been treated by the cold potato fermented product of the present invention. First, after collecting human skin fibroblasts stimulated with 2% cold potato ferment for 24 hours, the cells were recovered in cell lysate (RB buffer, purchased from Geanaid Corporation, Taiwan, Cat. No. RBD300), and RNA was used. Extraction reagent kit (purchased from Geneaid, Taiwan, Cat. No. RBD300) extracts RNA from human skin fibroblasts, and then uses SuperScript ® III reverse transcriptase (purchased from Invitrogene, USA, No. 18080-044) to 1μg of extracted RNA was used as a template and a reverse transcription reaction (42°C, 2 hours) was performed with T7 Oligo (dT) primers to produce the corresponding cDNA product with T7 promoter (promoter), followed by the simultaneous addition of 1 μL of DNA synthetase (Polymerase) and 0.5 μL of RNA degrading enzyme (RNase H) decomposes the original RNA template of the cDNA product, and performs DNA synthesis reaction (16° C., 2 hours) using the single-stranded cDNA as a template to form a double-stranded cDNA product. The cDNA product is purified to remove RNA, primers, enzymes, and salts that may inhibit in vitro transcription. In the purification system, 50 μL of cDNA product was added to 50 μL of Nuclease-free water to mix well, then 250 μL of cDNA binding buffer was added to mix well, put into cDNA spin column, centrifuged at 10,000 g for 1 minute, then the liquid under the centrifuge tube was transferred In addition, put the cDNA spin column in the centrifuge again, centrifuge at 10,000g for 3 minutes to completely remove the residual alcohol in the spin column, then move the spin column to a new 1.5mL centrifuge tube, add it and preheat at 55℃ 12μL Nuclease-free water, after standing at room temperature for 5 minutes, centrifuged at 10,000g for 2 minutes to obtain purified double-stranded cDNA. Then use Amino Allyl MessageAmp TM II aRNA Amplification Kit (purchased from Ambion, USA, number AM1753), Amino Allyl UTP (aaUTP) using the double-stranded cDNA as a template for in vitro transcription reaction to synthesize amino allyl modified aRNA (amino allyl-modified antisensesRNA) to amplify the amount of RNA to be tested, and then purify the aRNA product using Amino Allyl MessageAmp TM II aRNA Amplification Kit (purchased from the United States, number AM1753) to remove NTPs that are not embedded in the product , Salts, enzymes and inorganic phosphate to improve the stability of aRNA, and then through the Labeling Reagent and Hybridization Kit (purchased from Phalanx Biotech Group, United States, number HOA v7) Cy5 NHS ester dye (purchased from AAT Bioquest, United States, Cat. 151) Dye coupling reaction with UTP residues modified with amino allyl groups on aRNA, and then after removing the remaining dye, the buffer solution was replaced with water without nucleolytic enzyme (Nuclease-free Water, purchased from Ambion, United States, number AM1753), and established the Whole Genome Experssion microarray hybridization system (purchased from Phalanx Biotech Group, United States, number HOA v7), using Agilent Microarray Scanner for scanning and signal segmentation, in After normalizing the original data, the relative performance of different genes was determined by using the log2 ratio method. Then use Excel software to perform unpaired one-tailed student t-test to determine whether the coefficient of variation is statistically significant (*p value<0.05; **p value<0.01; ***p value<0.001).
本發明之涼薯發酵物於DNA微陣列分析之結果如圖3及圖4所示。圖3中可看出經本發明之涼薯發酵物處理過後，皮膚纖維母細胞中COL23A1基因、COL25A1基因、COL6A2基因、TIMP3基因、ELN基因、及HAS2基因表 現量顯著上升20.56-21.37倍不等，其中COL23A1、COL25A1、及COL6A2為膠原蛋白之結構蛋白；TIMP3已知可抑制與癌症轉移相關之MMP(Matrix metalloproteinases)酵素活性；ELN為彈性蛋白(Elastin)；HAS則可表現玻尿酸合成酶，能促進角質細胞分泌玻尿酸的能力。 The results of DNA microarray analysis of the cold potato fermentation product of the present invention are shown in FIGS. 3 and 4. It can be seen in FIG. 3 that after treatment with the cold potato fermented material of the present invention, the expression levels of COL23A1 gene, COL25A1 gene, COL6A2 gene, TIMP3 gene, ELN gene, and HAS2 gene in skin fibroblasts significantly increased by 2 0.56 -2 1.37 times. Among them, COL23A1, COL25A1, and COL6A2 are structural proteins of collagen; TIMP3 is known to inhibit MMP (Matrix metalloproteinases) enzyme activity associated with cancer metastasis; ELN is elastin; HAS can express hyaluronic acid synthase, Can promote the ability of keratinocytes to secrete hyaluronic acid.
圖4中則可看出經本發明之涼薯發酵物處理過後，皮膚纖維母細胞中SOD2基因、CCT2基因、CCT4基因、及UNG基因表現量也顯著上升20.37-21.46倍不等，其中SOD是一種能夠催化超氧化物通過歧化反應，並使其轉化為氧氣和過氧化氫的酵素，是生物體內一種重要的抗氧化劑，保護暴露於氧氣中的細胞；CCT基因為折疊蛋白(Chaperonin)的一種，主要功能為矯正錯誤摺疊的蛋白質，其被認為與個體之老化調節相關；UNG則為DNA修復機制中一重要蛋白質，亦被認為與老化調節相關。 4 can be seen in FIG. Pachyrhizus treated after fermentation product of the present invention, skin fibroblasts SOD2 gene, gene CCT2, CCT4 gene, and the gene expression amount UNG increased significantly 0.37 -2 1.46 times ranging from 2 wherein SOD It is an enzyme that can catalyze superoxide through disproportionation and convert it into oxygen and hydrogen peroxide. It is an important antioxidant in the body and protects cells exposed to oxygen; CCT gene is folded protein (Chaperonin) One type of protein whose main function is to correct misfolding is considered to be related to the individual's aging regulation; UNG is an important protein in the DNA repair mechanism and is also considered to be related to aging regulation.
此些結果可初步地觀察出本發明之涼薯發酵物可能與膠原蛋白、彈性蛋白及玻尿酸生成有關，也可能與抗氧化、抗老及DNA修復之作用有關，為了更詳盡了解本發明之涼薯發酵物，對細胞之影響，將於後續實施例中更進一步的探討本發明之涼薯發酵物於各種調節彈性蛋白相關基因及各種抗老化相關基因表現之功效。 From these results, it can be preliminary observed that the cold potato fermented product of the present invention may be related to the production of collagen, elastin and hyaluronic acid, and may also be related to the effects of antioxidant, anti-aging and DNA repair. In order to understand the cooling of the present invention in more detail The effect of the fermented potato on the cells will be further explored in the subsequent examples on the efficacy of the cold potato fermented material of the present invention in regulating the expression of various elastin-related genes and various anti-aging related genes.
以人類皮膚纖維母細胞進行本發明之涼薯發酵物對彈性蛋白基因表現之測試。將1.5x105個人類皮膚纖維母細胞培養於含有2mL上述培養液之六孔培養盤中，並分成以下三組：(1)加入2%(v/v)前述之涼薯水萃取物之對照組、(2)加入2%(v/v)本發明之涼薯發酵物之實驗組，以及(3)僅含培養液之空白控制組於37℃下培養6小時，接著將纖維母細胞以細胞裂解液(RB buffer，購自Geanaid公司，臺灣，編號RBD300)回收細胞後，使用RNA萃取試劑套組(購自Geneaid公司，台灣，Lot No.FC24015-G)分別收集三組纖維母細胞內之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為模板並以引子產生mRNA反轉錄之相應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)將三組反轉錄後產物分別以表一之組合引子進行定量即時反轉錄聚合酶連鎖反應(quantitative real-time reverse transcription polymerase chain reaction)試驗，條件為95℃反應1秒，60℃反應20秒，總共40個迴圈。用以定量COL1A1基因、COL1A2基因、COL4A1基因、COL4A3基因、COL4A4基因、COL4A5基因、MMP1基因、MMP9基因、MMP2基因、TGM1基因、TIMP1基因、ELN基因、FBN1基因、LOX基因、HAS2基因、及HAS3基因之mRNA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目標基因-CtGAPDH(甘油醛-3-磷酸脫氫酶，Glyceraldehyde-3-phosphate dehydrogenase)，再利用Excel軟體進行非成對單尾student t-test，以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)。 Human skin fibroblasts were used to test the expression of elastin gene of the cold potato fermentation product of the present invention. To 1.5x10 5 based personal skin fibroblasts were cultured in six-well culture plates containing 2mL of the culture solution and divided into three groups: (1) was added 2% (v / v) control of the water extract of pachyrhizus Group, (2) the experimental group added with 2% (v/v) of the cold potato fermentation product of the present invention, and (3) the blank control group containing only the culture solution was cultured at 37°C for 6 hours, and then the fibroblasts were After recovering the cells with cell lysate (RB buffer, purchased from Geanaid, Taiwan, number RBD300), use RNA extraction reagent kit (purchased from Geneaid, Taiwan, Lot No. FC24015-G) to collect three groups of fibroblasts respectively RNA, and then use SuperScript ® III reverse transcriptase (purchased from Invitrogene, USA, 18080-051) using 2000ng of extracted RNA as a template and using primers to generate the corresponding cDNA product of mRNA reverse transcription, and then use ABI StepOnePlus TM Real- Time PCR system (Thermo Fisher Scientific, USA) and KAPA SYBR FAST (purchased from Sigma, USA, No. 38220000000) The three sets of reverse transcription products were quantified by the combination primers in Table 1 for real-time reverse transcription polymerase chain reaction (quantitative real-time reverse transcription polymerase chain reaction) test, the conditions are 95 ℃ reaction for 1 second, 60 ℃ reaction for 20 seconds, a total of 40 cycles. Used to quantify COL1A1 gene, COL1A2 gene, COL4A1 gene, COL4A3 gene, COL4A4 gene, COL4A5 gene, MMP1 gene, MMP9 gene, MMP2 gene, TGM1 gene, TIMP1 gene, ELN gene, FBN1 gene, LOX gene, HAS2 gene, and HAS3 The mRNA expression level of the gene, where the quantitative value is taken from the threshold cycle number (Ct), and the relative mRNA level of the target gene is derived from Equation 2 -△Ct , where △Ct=Ct target gene- Ct GAPDH (glyceraldehyde-3 -Phosphate dehydrogenase, Glyceraldehyde-3-phosphate dehydrogenase), and then use Excel software for unpaired single-tailed student t-test to determine whether the coefficient of variation is statistically significant (*p value<0.05; ** p value <0.01; ***p value <0.001).
本發明之涼薯發酵物於提升COL4A4基因表現量之結果如圖5所示；於提升TIMP1基因表現量之結果如圖6所示；提升LOX基因表現量之結果如圖7所示，其中LOX主要功能為使膠原蛋白和彈性蛋白產生交聯，以穩定膠 原纖維和成熟之彈性蛋白間的完整性和彈性；提升HAS基因表現量之結果如圖8所示。其中，單純以涼薯水萃取物處理之組別皆與空白控制組之數據相近；然而，以本發明之涼薯發酵物處理後，COL4A4基因之表現量為空白控制組之1.78倍，TIMP1基因之表現量為空白控制組之1.37倍，LOX基因之表現量為空白控制組之1.24倍，HAS2基因之表現量為空白控制組之2.24倍，HAS3基因之表現量則為空白控制組之2.74倍。此結果顯示本發明之涼薯發酵物在經過特定之微生物發酵步驟後，可有效提升COL4A4基因、TIMP1基因、LOX基因、HAS2基因、及HAS3基因之表現量，使膠原蛋白、彈性蛋白、及玻尿酸之生成提升，並可用於製備提高皮膚緊緻度之組合物，使皮膚光滑有彈性。 The results of the cold potato fermented product of the present invention in enhancing the expression level of the COL4A4 gene are shown in FIG. 5; the results in increasing the expression level of the TIMP1 gene are shown in FIG. 6; the results in increasing the expression level of the LOX gene are shown in FIG. 7, wherein LOX The main function is to cross-link collagen and elastin to stabilize the glue The integrity and elasticity between fibrils and mature elastin; the results of enhancing the expression of the HAS gene are shown in Figure 8. Among them, the group treated only with cold potato water extract is similar to the data of the blank control group; however, after treatment with the cold potato fermented material of the present invention, the expression amount of the COL4A4 gene is 1.78 times that of the blank control group, TIMP1 gene The expression level is 1.37 times that of the blank control group, the LOX gene performance is 1.24 times that of the blank control group, the HAS2 gene performance is 2.24 times that of the blank control group, and the HAS3 gene performance is 2.74 times that of the blank control group. . This result shows that the cold potato fermented product of the present invention can effectively enhance the expression of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, and HAS3 gene after a specific microbial fermentation step, so that collagen, elastin, and hyaluronic acid The generation is improved, and it can be used to prepare a composition for improving skin firmness, making the skin smooth and elastic.
以人類皮膚纖維母細胞進行本發明之涼薯發酵物對抗老基因表現之測試。將1.5x105個人類皮膚纖維母細胞培養於含有2mL上述培養液之六孔培養盤，並分成以下三組：(1)加入2%前述之涼薯水萃取物之對照組、(2)加入2%本發明之涼薯發酵物之實驗組，以及(3)僅含培養液之空白控制組於37℃下培養24小時，並以實施例5之方法分析經本發明之涼薯發酵物處理過之皮膚纖維母細胞中CCT2基因、CCT5基因、CCT6A基因、CCT7基因、CCT8基因、Pink1基因、Parkin基因、Atg1基因、Atg8基因、SIRT1基因、FOXO基因、PARP1基因、PARP2基因、NADSYN基因、MRPS5基因、Ubl-5基因、及SOD3基因之表現情形。 Human skin fibroblasts were used to test the anti-aging gene expression of the cold potato fermentation product of the present invention. To 1.5x10 5 based personal skin fibroblasts were cultured in six-well culture plates containing 2mL of the culture solution and divided into three groups: (1) 2% of the control group of cold potato extract water, (2) was added 2% of the experimental group of the cold potato fermentation product of the present invention, and (3) the blank control group containing only the culture solution were incubated at 37°C for 24 hours, and analyzed by the method of Example 5 after being processed by the cold potato fermentation product of the present invention CCT2 gene, CCT5 gene, CCT6A gene, CCT7 gene, CCT8 gene, Pink1 gene, Parkin gene, Atg1 gene, Atg8 gene, SIRT1 gene, FOXO gene, PARP1 gene, PARP2 gene, NADSYN gene, MRPS5 gene in skin fibroblasts , Ubl-5 gene, and SOD3 gene performance.
本發明之涼薯發酵物於提升CCT2基因、CCT5基因、及CCT7基因表現量之結果如圖9所示；於提升SOD3基因表現量之結果如圖10所示。其中，單純以涼薯水萃取物處理之組別皆與空白控制組之數據相近或較低；然而，以本發明之涼薯發酵物處理後，CCT2基因之表現量為空白控制組之1.1倍，CCT5基因之表現量為空白控制組之1.3倍，CCT7基因之表現量為空白控制組之2.5倍，SOD3基因之表現量則為空白控制組之1.5倍。此結果顯示本發明之涼薯發酵物在經過特定之微生物發酵步驟後，可有效提升CCT2基因、CCT5基因、CCT7基因、及SOD3基因之表現量，而能有效提升細胞代謝之能力，以及有效提高清除細胞內自由基的效率，並可用於製備提高皮膚抗老化以及抗氧化力能力之組合物，使皮膚回復年輕狀態。 The results of the cold potato fermented product of the present invention in enhancing the expression levels of the CCT2 gene, CCT5 gene, and CCT7 gene are shown in FIG. 9; the results in increasing the expression level of the SOD3 gene are shown in FIG. Among them, the group treated only with cold potato water extract is similar to or lower than the data of the blank control group; however, after treatment with the cold potato fermented material of the present invention, the expression amount of the CCT2 gene is 1.1 times that of the blank control group The expression level of the CCT5 gene is 1.3 times that of the blank control group, the expression level of the CCT7 gene is 2.5 times that of the blank control group, and the expression amount of the SOD3 gene is 1.5 times that of the blank control group. This result shows that the cold potato fermented product of the present invention can effectively enhance the expression of the CCT2 gene, CCT5 gene, CCT7 gene, and SOD3 gene after a specific microbial fermentation step, and can effectively improve the ability of cell metabolism and effectively improve The efficiency of removing free radicals in cells, and can be used to prepare a composition that improves the skin's anti-aging and antioxidant capacity to restore the skin to a youthful state.
綜上所述，本發明將涼薯以酵母菌、乳酸桿菌及醋酸桿菌進行三段式發酵所得之涼薯發酵物，能有效提升其中的總多酚含量、提升皮膚細胞抗紫外光能力、且也能有效提升皮膚細胞中COL4A4基因、TIMP1基因、LOX基因、HAS2基因、HAS3基因、CCT2基因、CCT5基因、CCT7基因、及SOD3基因之表現量，且較涼薯水萃取物有更佳之效果，因此能更加有效的用於製備增加皮膚抗氧化活性、增加皮膚抗老化活性、增加皮膚緊緻度、及增加皮膚美白能力之醫藥組合物，並達到綜合地皮膚保健功效。 In summary, in the present invention, the cold potato fermented product obtained by three-stage fermentation of cold potato with yeast, lactobacilli and acetobacter can effectively increase the total polyphenol content in it and enhance the ability of skin cells to resist ultraviolet light, and It can also effectively enhance the expression of COL4A4 gene, TIMP1 gene, LOX gene, HAS2 gene, HAS3 gene, CCT2 gene, CCT5 gene, CCT7 gene, and SOD3 gene in skin cells, and has better effect than cold potato water extract. Therefore, it can be more effectively used to prepare pharmaceutical compositions that increase skin antioxidant activity, increase skin anti-aging activity, increase skin firmness, and increase skin whitening ability, and achieve comprehensive skin health benefits.
<110> 大江生醫股份有限公司 <110> Dajiang Biomedical Co., Ltd.
<120> 涼薯發酵物及其用於製備提升COL基因、TIMP基因、LOX基因、ELN基因、HAS基因、SOD基因、TCP1基因與UNG基因、以及提升皮膚抗老化能力之組合物的用途 <120> Cold potato ferment and its use for preparing COL gene, TIMP gene, LOX gene, ELN gene, HAS gene, SOD gene, TCP1 gene and UNG gene, and a composition for enhancing skin anti-aging ability
<130> 107B0099-I1 <130> 107B0099-I1
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<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 33
<210> 34 <210> 34
<211> 21 <211> 21
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 34
<210> 35 <210> 35
<211> 18 <211> 18
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 35
<210> 36 <210> 36
<211> 22 <211> 22
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 36
<210> 37 <210> 37
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 37
<210> 38 <210> 38
<211> 17 <211> 17
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 38
<210> 39 <210> 39
<211> 23 <211> 23
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 39
<210> 40 <210> 40
<211> 25 <211> 25
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 40
<210> 41 <210> 41
<211> 24 <211> 24
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 41
<210> 42 <210> 42
<211> 19 <211> 19
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 42
<210> 43 <210> 43
<211> 17 <211> 17
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 43
<210> 44 <210> 44
<211> 25 <211> 25
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 44
<210> 45 <210> 45
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 45
<210> 46 <210> 46
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 46
<210> 47 <210> 47
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 47
<210> 48 <210> 48
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 48
<210> 49 <210> 49
<211> 23 <211> 23
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 49
<210> 50 <210> 50
<211> 23 <211> 23
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 50
<210> 51 <210> 51
<211> 21 <211> 21
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 51
<210> 52 <210> 52
<211> 22 <211> 22
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 52
<210> 53 <210> 53
<211> 23 <211> 23
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 53
<210> 54 <210> 54
<211> 22 <211> 22
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 54
<210> 55 <210> 55
<211> 19 <211> 19
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 55
<210> 56 <210> 56
<211> 21 <211> 21
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 56
<210> 57 <210> 57
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 57
<210> 58 <210> 58
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 58
<210> 59 <210> 59
<211> 22 <211> 22
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 59
<210> 60 <210> 60
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 60
<210> 61 <210> 61
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 61
<210> 62 <210> 62
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 62
<210> 63 <210> 63
<211> 18 <211> 18
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 63
<210> 64 <210> 64
<211> 21 <211> 21
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 64
<210> 65 <210> 65
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 65
<210> 66 <210> 66
<211> 20 <211> 20
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 66
<210> 67 <210> 67
<211> 18 <211> 18
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 67
<210> 68 <210> 68
<211> 21 <211> 21
<212> DNA <212> DNA
<213> 人工序列 <213> Artificial sequence
<220> <220>
<223> 合成引子 <223> Synthetic primer
<400> 68
Claims (11)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| TW107135620A TWI702055B (en) | 2018-10-09 | 2018-10-09 | Pachyrhizus erosus fermented extracts and the use thereof for enhancing the gene expression of col, timp, lox, eln, has, sod, tcp1 and ung, and for reducing the skin melanin content |
| CN201811449321.4A CN111012700B (en) | 2018-10-09 | 2018-11-28 | Yam bean fermentation product and application thereof in preparing composition for improving gene expression amount and reducing skin melanin content |
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| TW107135620A TWI702055B (en) | 2018-10-09 | 2018-10-09 | Pachyrhizus erosus fermented extracts and the use thereof for enhancing the gene expression of col, timp, lox, eln, has, sod, tcp1 and ung, and for reducing the skin melanin content |
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| TWI749734B (en) * | 2019-08-27 | 2021-12-11 | 大江生醫股份有限公司 | Bacillus coagulans or it’s metabolite and use of bacillus coagulans in preparing compositions for stomach health |
| CN112708574B (en) * | 2019-10-24 | 2022-03-08 | 大江生医股份有限公司 | Lactobacillus bulgaricus TCI904, compositions thereof and use thereof for reducing body weight |
| TWI743754B (en) * | 2020-04-23 | 2021-10-21 | 大江生醫股份有限公司 | Manufacturing method of increasing geniposide content applied to cardiovascular health care |
| CN113876833B (en) * | 2020-07-03 | 2023-09-01 | 百岳特生物技术（上海）有限公司 | White mulberry fermented product and preparation method and application thereof |
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| JP2004352658A (en) * | 2003-05-29 | 2004-12-16 | Shiseido Co Ltd | External preparation for skin |
| JP6177595B2 (en) * | 2012-06-29 | 2017-08-09 | ライオン株式会社 | Skin cosmetics |
| KR101566320B1 (en) * | 2015-08-27 | 2015-11-05 | 정용문 | Cosmetic composition comprising an Impatiens balsamina L, Maca, Rosa multiflora Thunberg, Eryobotrya japonica and Pachyrrhizus erosus Complex-fermented extract |
| KR101791549B1 (en) * | 2015-12-16 | 2017-10-30 | 경상북도(농업기술원생물자원연구소장) | An antibacterial composition comprising the pachyrhizus erosus root bark extract as an effective component |
| KR20170072055A (en) * | 2015-12-16 | 2017-06-26 | 경상북도(농업기술원생물자원연구소장) | An antioxidant composition comprising the pachyrhizus erosus root bark extract |
| NL2016115B1 (en) * | 2016-01-18 | 2017-07-25 | Innoso B V | Food composition with fermented starch. |
| KR101916953B1 (en) * | 2016-10-31 | 2018-11-08 | 상주시 | Extract of Pachyrhizus erosus having functuon of Whitening |
| CN107581596A (en) * | 2017-09-14 | 2018-01-16 | 深圳市淳睿贸易有限公司 | A kind of enzyme composition |
| CN107518389A (en) * | 2017-09-14 | 2017-12-29 | 深圳市淳睿贸易有限公司 | A kind of preparation method of ferment and enzyme liquid |
| CN107581597A (en) * | 2017-09-14 | 2018-01-16 | 深圳市淳睿贸易有限公司 | A kind of enzyme liquid |
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