TWI729618B - Use of phalaenopsis plant extract for anti-glycation and improving skin appearance - Google Patents
Use of phalaenopsis plant extract for anti-glycation and improving skin appearance Download PDFInfo
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- TWI729618B TWI729618B TW108145367A TW108145367A TWI729618B TW I729618 B TWI729618 B TW I729618B TW 108145367 A TW108145367 A TW 108145367A TW 108145367 A TW108145367 A TW 108145367A TW I729618 B TWI729618 B TW I729618B
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- Prior art keywords
- phalaenopsis
- skin
- extract
- present
- large white
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Abstract
Description
本發明係關於一種蝴蝶蘭屬植物用於抗醣化與改善皮膚外觀的用途，尤其是一種大白蝴蝶蘭萃取物用於提升清除糖化終產物之能力、提升KRT14基因、及SMAD1基因的表現量、提高角質細胞分泌玻尿酸的量、及/或提升個體皮膚之光澤度的用途。 The present invention relates to the use of a Phalaenopsis plant to resist glycation and improve the appearance of the skin, in particular, a large white Phalaenopsis extract is used to improve the ability to remove the end product of glycation, increase the expression level of the KRT14 gene and the SMAD1 gene, and improve Keratinocytes secrete the amount of hyaluronic acid and/or improve the gloss of the individual's skin.
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 the 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 damages. 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 in the epidermis, although the keratin cells are dead cells, its 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 structural 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 UV light is exposed Stimulation will cause the keratinocytes to be unable to maintain the normal metabolic cycle, and the skin's water retention capacity will also decrease, and cause damage to the skin epidermal barrier, making the skin rough, dry and desquamated, fragile, irritated, and sensitive to redness. The health and water retention capacity of the stratum corneum is very important for resisting 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地複雜，且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 complicated and involve countless physiological phenomena, including ultraviolet radiation damage, free radical damage, reduction of collagen, slower cell renewal, The appearance of abnormal cells, 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.
糖化終產物(Advanced Glycation End Products,AGEs)是一群高度氧化的化合物，因此被認為是一種糖毒素(Glycotoxin)，研究顯示糖化終產物會與細胞表面的接受器結合，而改變其結構與功能，並促使細胞的氧化壓力與發炎反應增加，更與糖尿病、動脈硬化及腎臟慢性疾病等的形成關係密不可分。而除了人體正常代謝過程中會產生糖化終產物外，許多加工食品中也會含有糖化終產物，研究也已指出避免從食物攝取糖化終產物，能有助於延緩慢性疾病與老化的進展。 Advanced Glycation End Products (AGEs) are a group of highly oxidized compounds, so they are considered to be a kind of glycotoxin (Glycotoxin). Studies have shown that the glycation end products will bind to receptors on the cell surface to change its structure and function. It also promotes the increase of oxidative stress and inflammation of cells, and is inseparable from the formation of diabetes, arteriosclerosis and chronic kidney diseases. In addition to the glycation end products produced during the normal metabolism of the human body, many processed foods also contain glycation end products. Studies have also pointed out that avoiding the intake of glycation end products from food can help delay the progression of chronic diseases and 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，同時又能有效維持角質細胞的正常生理代謝、維持角質層結構完整之組合物、及增加抗醣化活性之有效成分組成的，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 to maintain the normal physiological metabolism of keratinocytes, maintain the structure of the stratum corneum intact, and increase the composition of active ingredients that increase the anti-glycation activity.
緣此，本發明之一目的在提供一種蝴蝶蘭屬(Phalaenopsis)植物萃取物用於製備提升角蛋白14(Keratin 14,KRT14)基因、及母抗Dpp同源物(Mothers against decapentaplegic homolog,SMAD)基因表現量之組合物的用途， 其中該蝴蝶蘭屬植物萃取物係為一大白蝴蝶蘭(Phalaenopsis Sogo Yukidian ukid)萃取物，該大白蝴蝶蘭萃取物係以一溶劑萃取一大白蝴蝶蘭所獲得，且該溶劑為水、醇、或醇水混合物。 For this reason, one purpose of the present invention is to provide a Phalaenopsis plant extract for preparing Keratin 14 (Keratin 14, KRT14 ) gene and mothers against decapentaplegic homolog (Mothers against decapentaplegic homolog, SMAD ) The use of the gene expression composition, wherein the Phalaenopsis plant extract is a Phalaenopsis Sogo Yukidian ukid (Phalaenopsis Sogo Yukidian ukid) extract, and the Phalaenopsis Sogo Yukidian ukid extract is obtained by extracting a large Phalaenopsis Sogo Yukidian ukid with a solvent, And the solvent is water, alcohol, or a mixture of alcohol and water.
本發明之又一目的在提供一種蝴蝶蘭屬(Phalaenopsis)植物萃取物用於製備抗醣化及改善皮膚外觀之組合物的用途，其中該蝴蝶蘭屬植物萃取物係為一大白蝴蝶蘭(Phalaenopsis Sogo Yukidian ukid或Phalaenopsis Sogo Yukidian V3)萃取物，該大白蝴蝶蘭萃取物係以一溶劑萃取一大白蝴蝶蘭所獲得，且該溶劑為水、醇、或醇水混合物。 Another object of the present invention is to provide a Phalaenopsis ( Phalaenopsis ) plant extract for the preparation of anti-glycation and improve skin appearance composition, wherein the Phalaenopsis plant extract is a large white phalaenopsis ( Phalaenopsis Sogo Yukidian ukid or Phalaenopsis Sogo Yukidian V3) extract, the great white phalaenopsis extract is obtained by extracting a large white phalaenopsis with a solvent, and the solvent is water, alcohol, or a mixture of alcohol and water.
在本發明之一實施例中，該大白蝴蝶蘭與該溶劑之重量比為1-5：5-20；且該萃取之溫度係75-95℃。 In an embodiment of the present invention, the weight ratio of the large white phalaenopsis to the solvent is 1-5:5-20; and the extraction temperature is 75-95°C.
在本發明之又一實施例中，該大白蝴蝶蘭萃取物係維持皮膚角質細胞排列、及使皮膚角質層結構完整。 In another embodiment of the present invention, the large white Phalaenopsis extract maintains the arrangement of skin keratinocytes and completes the structure of the skin stratum corneum.
在本發明之又一實施例中，該大白蝴蝶蘭萃取物係提高角質細胞分泌玻尿酸的量。 In another embodiment of the present invention, the large white phalaenopsis extract increases the amount of hyaluronic acid secreted by keratinocytes.
在本發明之又一實施例中，該大白蝴蝶蘭萃取物係提升皮膚的保水力。 In another embodiment of the present invention, the great white phalaenopsis extract improves the water retention capacity of the skin.
在本發明之又一實施例中，該大白蝴蝶蘭萃取物係提升個體皮膚之光澤度。 In another embodiment of the present invention, the large white Phalaenopsis extract improves the gloss of the individual's skin.
在本發明之另一實施例中，該大白蝴蝶蘭萃取物的濃度為至少0.0625mg/mL。 In another embodiment of the present invention, the concentration of the great white phalaenopsis extract is at least 0.0625 mg/mL.
本發明之大白蝴蝶蘭萃取物具有強效之抑制糖化反應的活性，可有效提升清除糖化終產物之能力，抗醣化能夠抑制非酵素褐變，以避免體內功能性蛋白質之變性，有助於延緩慢性疾病與老化的進展；還能有效提高角質細胞分泌玻尿酸的量、有效提升皮膚細胞中KRT14基因、及SMAD1基因的表現量，能有效維持皮膚角質細胞排列，以使皮膚角質層結構完整，並提升皮膚保 水力，及提升皮膚屏障及防禦功能，以防止皮膚水分散失，使皮膚處於保水的狀態、及維持皮膚年輕光滑有彈性；另外，其亦能有效提升個體皮膚之光澤度，具有改善皮膚暗沉、枯黃的情況使皮膚透亮有光澤之功效。因此，本發明之大白蝴蝶蘭萃取物可用於製備抗醣化與改善皮膚外觀之組合物的用途，其中該組合物是一醫藥品、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 large white phalaenopsis extract of the present invention has a strong activity of inhibiting saccharification reaction, which can effectively improve the ability to remove glycation end products. Anti-glycation can inhibit non-enzymatic browning to avoid the denaturation of functional proteins in the body and help delay The progression of chronic diseases and aging; it can also effectively increase the amount of hyaluronic acid secreted by keratinocytes, effectively increase the expression of KRT14 gene and SMAD1 gene in skin cells, and can effectively maintain the arrangement of skin keratinocytes to make the skin stratum corneum structure intact, and Enhance skin water retention capacity, and enhance skin barrier and defense functions to prevent skin water loss, keep skin in a water-retaining state, and maintain skin youthful, smooth and elastic; in addition, it can also effectively enhance the gloss of individual skin and improve skin The dull and yellow condition makes the skin translucent and shiny. Therefore, the large white phalaenopsis extract of the present invention can be used to prepare a composition for anti-glycation and improving skin appearance, wherein the composition is a medicine, a food or a skin care product, which can be taken orally, applied to the skin, etc. Give a body.
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 following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone who is familiar with the art will not depart from the spirit and spirit of the present invention. Within the scope, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the attached patent scope.
圖1係為本發明之一實施例的大白蝴蝶蘭萃取物抗醣化活性之效果的長條圖。 Fig. 1 is a bar graph showing the anti-glycation activity of the Phalaenopsis phalaenopsis extract in an embodiment of the present invention.
圖2係為本發明之一實施例的大白蝴蝶蘭萃取物促進細胞分泌玻尿酸之效果的長條圖。 Fig. 2 is a bar graph showing the effect of a large white phalaenopsis extract in promoting the secretion of hyaluronic acid from cells according to an embodiment of the present invention.
圖3係為本發明之一實施例的大白蝴蝶蘭萃取物提升KRT14基因、及SMAD1基因表現量之效果的長條圖。* p<0.05；** p<0.01。 Fig. 3 is a bar graph showing the effect of an extract of Great White Phalaenopsis on enhancing the expression of KRT14 gene and SMAD1 gene according to an embodiment of the present invention. * p<0.05; ** p<0.01.
圖4係為本發明之一實施例的大白蝴蝶蘭萃取物提升個體皮膚光澤度之效果的長條圖。 Fig. 4 is a bar graph showing the effect of the great white phalaenopsis extract in enhancing the skin gloss of an individual according to an embodiment of the present invention.
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，各組之間的差異以學生t檢驗(student's t-test)分析。 Use Excel software for statistical analysis. Data mean ± standard deviation (SD), said difference between the groups in a Student's t-test (student's t -test) analysis.
大白蝴蝶蘭(Phalaenopsis Sogo Yukidian ukid或Phalaenopsis Sogo Yukidian V3)為蘭科(Orchidaceae)蝴蝶蘭屬(Phalaenopsis)單軸多年生草本植物，為Phal.Yukimai與Phal.Taisuco Kochdian配種所得之蘭花品系。 Phalaenopsis Sogo Yukidian ukid (Phalaenopsis Sogo Yukidian ukid or Phalaenopsis Sogo Yukidian V3) is a uniaxial perennial herb of the genus Phalaenopsis in the Orchidaceae family. It is an orchid strain derived from Phal. Yukimai and Phal. Taisuco Kochdian.
如本文中所使用的，用語「大白蝴蝶蘭萃取物」意為大白蝴蝶蘭的花與溶劑以1-5：5-20(w/w)比例經一特定時間與溫度萃取而得。 As used in this article, the term "Extract of Great White Phalaenopsis" means that the flower and solvent of Great White Phalaenopsis are extracted at a ratio of 1-5:5-20 (w/w) for a specific time and temperature.
本文所述之「有效濃度」或「有效量」係表示能有效提升清除糖化終產物之能力、有效提高角質細胞分泌玻尿酸的量、有效提升皮膚細胞中KRT14基因、及SMAD1基因的表現量、及/或有效提升個體皮膚之光澤度所需本發明之大白蝴蝶蘭萃取物的濃度。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" or "effective amount" mentioned in this article means that it can effectively improve the ability to clear the end products of glycation, effectively increase the amount of hyaluronic acid secreted by keratinocytes, effectively increase the expression of KRT14 gene and SMAD1 gene in skin cells, and / Or the concentration of the great white phalaenopsis extract of the present invention required to effectively improve the gloss of the individual's skin. The effective concentration may vary depending on the target, but the effective concentration can be determined experimentally by, for example, a dose escalation test.
依據本發明，醫藥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非經腸道地(parenterally)或局部地(topically)投藥的劑型，這包括，但不限於：注射品(injection)[例如，無菌的水性溶液(sterile aqueous solution)或分散液(dispersion)]、無菌的粉末(sterile powder)、外部製劑(external preparation)以及類似之物。 According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally or topically by using techniques well known to those skilled in the art. This includes, but is not limited to: injections (injection) [e.g., sterile aqueous solution or dispersion], sterile powder, external preparation, and the like.
依據本發明，醫藥品可進一步包含有一被廣泛地使用於藥物製造技術之醫藥上可接受的載劑(pharmaceutically acceptable carrier)。例如，該醫藥上可接受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。 According to the present invention, the medicine may further include a pharmaceutically acceptable carrier which is widely used in medicine manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more reagents selected from the group consisting of solvent, buffer, emulsifier, suspending agent, decomposer ), disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent, diluent , 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，該醫藥上可接受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 pharmaceutically acceptable carrier includes a solvent selected from the group consisting of water, normal saline (normal saline), phosphate buffered saline (PBS), Aqueous solution containing alcohol and combinations thereof.
依據本發明，該醫藥品可以一選自於由下列所構成之群組中的非經腸道途徑(parenteral routes)來投藥：皮下注射(subcutaneous injection)、表皮內注射(intraepidermal injection)、皮內注射(intradermal injection)以及病灶內注射(intralesional injection)。 According to the present invention, the drug can be administered by a parenteral route selected from the group consisting of: subcutaneous injection, intraepidermal injection, intradermal injection Injection (intradermal injection) and intralesional injection (intralesional injection).
依據本發明，醫藥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)、貼片(patch)、擦劑(liniment)、粉末(powder)、氣溶膠(aerosol)、噴霧(spray)、乳液(lotion)、乳漿(serum)、糊劑(paste)、泡沫(foam)、滴劑(drop)、懸浮液(suspension)、油膏(salve)以及繃帶(bandage)。 According to the present invention, the medicine can be manufactured into an external preparation suitable for topical application to the skin using techniques well-known to those skilled in the art. This includes, but is not limited to: emulsion, coagulation Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serum, paste, foam, drop, suspension, salve and bandage.
依據本發明，該外部製劑是藉由將本發明的醫藥品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 medicine of 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)[諸如卡波普®974P(carbopol®974P)、微結晶纖維素(microcrystalline cellulose)以及羧基甲基纖維素 (carboxymethylcellulose)]、活性劑(active agents)、保濕劑(humectants)、氣味吸收劑(odor absorbers)、香料(fragrances)、pH調整劑(pH adjusting agents)、螯合劑(chelating agents)、乳化劑(emulsifiers)、閉塞劑(occlusive agents)、軟化劑(emollients)、增稠劑(thickeners)、助溶劑(solubilizing agents)、滲透增強劑(penetration enhancers)、抗刺激劑(anti-irritants)、著色劑(colorants)以及推進劑(propellants)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 agent (gelling agents) [such as Carbopol ® 974P (carbopol ® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)] , Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusion Occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants, and propellants (Propellants) and so on. 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-watertype)、油包水型(water-in-oiltype)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, Ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, cream, paste, foam, dispersion, drops, 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)[諸如維生素A酸(tretinoin)、兒茶素(catechin)、麴酸、熊果苷以及維生素C]、保濕劑、抗發炎劑(anti-inflammatory agents)、殺菌劑(bactericides)、紫外線吸收劑(ultraviolet absorbers)、植物萃取物(plant extracts)[諸如蘆薈萃取物(aloe extract)]、皮膚營養劑(skin nutrients)、麻醉劑(anesthetics)、抗痘劑(anti-acne agents)、止癢劑(antipruritics)、止痛劑(analgesics)、抗皮膚炎劑(antidermatitis agents)、抗過角化劑(antihyperkeratolytic agents)、抗乾皮膚劑(anti-dry skin agents)、抗汗劑(antipsoriatic agents)、抗老化劑(antiaging agents)、抗皺劑(antiwrinkle agents)、抗皮脂溢出劑(antiseborrheic agents)、傷口治療劑(wound-healing agents)、皮質類固醇(corticosteroids)以及激素(hormones)。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 activities selected from the following: whitening agents (such as tretinoin, catechins) (catechin), kojic acid, arbutin and vitamin C), moisturizers, anti-inflammatory agents, bactericides, ultraviolet absorbers absorbers, plant extracts (such as aloe extract), skin nutrients, anesthetics, anti-acne agents, antipruritics , Analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents ( antiaging agents, antiwrinkle agents, antiseborrheic agents, wound-healing agents, corticosteroids and hormones. 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)、烘培產品(bakery products)、健康食品(health foods)以及膳食補充品(dietary supplements)。 According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.
本發明提供一種大白蝴蝶蘭萃取物用於抗醣化與改善皮膚外觀的用途，本發明之大白蝴蝶蘭萃取物係以一溶劑萃取一大白蝴蝶蘭所獲得，該溶劑為水、醇、或醇水混合物，本發明之大白蝴蝶蘭萃取物具有強效之抗醣化活性、亦能有效提高角質細胞分泌玻尿酸的量、有效提升皮膚細胞中KRT14基因、及SMAD1基因的表現量、及/或能有效提升個體皮膚之光澤度。 The present invention provides a use of a large white phalaenopsis extract for anti-glycation and improvement of skin appearance. The large white phalaenopsis extract of the present invention is obtained by extracting a large white phalaenopsis with a solvent, the solvent being water, alcohol, or alcohol water The mixture, the large white phalaenopsis extract of the present invention has strong anti-glycation activity, can also effectively increase the amount of hyaluronic acid secreted by keratinocytes, effectively increase the expression of KRT14 gene and SMAD1 gene in skin cells, and/or can effectively increase The gloss of the individual's skin.
同時，本發明之大白蝴蝶蘭萃取物可用於製備抗醣化與改善皮膚外觀之組合物，其中可包含一有效量之大白蝴蝶蘭萃取物及一醫藥上可接受之載體，該組合物係一醫藥品、一食品或一保養品。 At the same time, the large white phalaenopsis extract of the present invention can be used to prepare a composition for anti-glycation and skin appearance improvement, which may contain an effective amount of large white phalaenopsis extract and a pharmaceutically acceptable carrier. The composition is a medicine. Product, a food or a skin care product.
以下將詳細說明本發明大白蝴蝶蘭萃取物之詳細萃取方法、該大白蝴蝶蘭萃取物抗醣化活性之功效的測試、該大白蝴蝶蘭萃取物促進角質細胞分泌玻尿酸之功效的測試、該大白蝴蝶蘭萃取物提升KRT14基因、及SMAD1基 因表現量之功效的測試、及該大白蝴蝶蘭萃取物於提升個體皮膚光澤度之功效的測試，以證實本發明之大白蝴蝶蘭萃取物能有效提升清除糖化終產物之能力、有效維持皮膚角質細胞排列、及/或有效提升個體皮膚之光澤度，能有助於延緩慢性疾病與老化的進展，並能使皮膚角質層結構完整、提升皮膚保水力，及提升皮膚屏障及防禦功能，亦能改善皮膚暗沉、枯黃的情況使皮膚透亮有光澤。 In the following, the detailed extraction method of the great white phalaenopsis extract of the present invention, the test of the anti-glycation activity of the great white phalaenopsis extract, the test of the effect of the great white phalaenopsis extract in promoting the secretion of hyaluronic acid by keratinocytes, the great white phalaenopsis The test of the effect of the extract in enhancing the expression of KRT14 gene and SMAD1 gene, and the test of the effect of the large white phalaenopsis extract in enhancing the skin gloss of individuals, to confirm that the large white phalaenopsis extract of the present invention can effectively improve the elimination of glycation end The ability of the product to effectively maintain the arrangement of skin keratinocytes, and/or effectively enhance the gloss of individual skin, can help delay the progression of chronic diseases and aging, and can complete the skin stratum corneum structure, enhance skin moisture retention, and enhance The skin barrier and defense function can also improve the dull and yellow skin and make the skin bright and shiny.
在本發明之一實施例中，將大白蝴蝶蘭的花清洗並切成每塊12毫米的大小後，取洗淨後之大白蝴蝶蘭與水、醇、或醇水混合物之萃取溶劑，萃取溶劑較佳為水，以1-5：5-20之體積比混合，均質後在溶劑中以75-95℃進行萃取0.5-3小時後，將粗萃取後冷卻至室溫後進行離心，離心條件為以300g離心10分鐘後取得上清液；再以400mesh之濾網過濾該上清液以獲得濾液。最後，將該濾液於50-60℃進行減壓濃縮後，即得到本發明之大白蝴蝶蘭萃取物。 In an embodiment of the present invention, after the flowers of the large white phalaenopsis are washed and cut into pieces of 12 mm in size, the washed large white phalaenopsis is extracted with water, alcohol, or a mixture of alcohol and water. The extraction solvent Preferably it is water, mixed in a volume ratio of 1-5:5-20, homogenized and extracted in a solvent at 75-95°C for 0.5-3 hours, then the crude extraction is cooled to room temperature and then centrifuged. Centrifugation conditions To obtain the supernatant after centrifugation at 300 g for 10 minutes; filter the supernatant with a 400 mesh filter to obtain the filtrate. Finally, after the filtrate is concentrated under reduced pressure at 50-60° C., the great white phalaenopsis extract of the present invention is obtained.
本實施例係為了進行本發明大白蝴蝶蘭萃取物抗醣化活性之功效的測試，以抑制D-果糖(D-fructose)使膠原蛋白(Collagen)產生糖化的效率，進行糖化活性之定量；抗醣化能夠抑制非酵素褐變，以避免體內功能性蛋白質之變性。首先，分別取0.2mL濃度為2.5%的本發明之大白蝴蝶蘭萃取物溶液，以及0.2mL之水作為控制組溶液，並加入0.2mL的60mg/mL之膠原蛋白溶液(以200mM之磷酸鹽緩衝溶液配置，pH7.4，其中含有0.06%之疊氮化鈉(Sodium azide,NaN3))以及0.2mL的1.5M之D-果糖溶液(以200mM之磷酸鹽緩衝液配置，pH7.4)混合均勻後，再取0.1mL之混合溶液於激發光360nm、放射光460nm下測定螢光值，以此為反應前之零點，並取0.1mL之混合溶液於50℃下培養24 小時後，取出0.1mL測定螢光值，以此為反應之終點；其中該大白蝴蝶蘭萃取物係以水為溶劑進行萃取。並以等量的3mM之氨基胍(Aminoguanidine,AG)(以200mM之磷酸鹽緩衝液配置，pH7.4)回溶溶劑至相等體積以作為正對照組，其中已知Aminoguanidine(AG)具有抑制糖化作用的功效。最後，以下列公式計算清除糖化終產物能力之效率，以此代表各組樣品抗醣化之活性。 This example is to test the anti-glycation activity of the Great White Phalaenopsis extract of the present invention to inhibit the efficiency of D-fructose to produce collagen (Collagen) glycation, and to quantify the glycation activity; anti-glycation It can inhibit non-enzyme browning to avoid the denaturation of functional proteins in the body. First, take 0.2 mL of the 2.5% concentration of the large white phalaenopsis extract solution of the present invention and 0.2 mL of water as the control solution, and add 0.2 mL of 60 mg/mL collagen solution (with 200 mM phosphate buffer Solution configuration, pH 7.4, which contains 0.06% sodium azide (Sodium azide, NaN 3 )) and 0.2 mL of 1.5 M D-fructose solution (configured with 200 mM phosphate buffer, pH 7.4) After homogenization, take 0.1mL of the mixed solution and measure the fluorescence value at 360nm of excitation light and 460nm of emission light. This is the zero point before the reaction, and then take 0.1mL of the mixed solution and incubate at 50℃ for 24 hours, then take out 0.1 Measure the fluorescence value in mL, which is the end of the reaction; the extract of the large white phalaenopsis is extracted with water as a solvent. And with the same amount of 3mM aminoguanidine (Aminoguanidine, AG) (with 200mM phosphate buffer, pH7.4) re-dissolved the solvent to the same volume as a positive control group, in which Aminoguanidine (AG) is known to inhibit glycation The effect of the effect. Finally, use the following formula to calculate the efficiency of the ability to remove the final glycation product, which represents the anti-glycation activity of each group of samples.
本發明之大白蝴蝶蘭萃取物抗醣化活性之功效測試結果如圖1所示。經本發明之大白蝴蝶蘭萃取物作用後，能夠減少約20%之糖化終產物的生成量，此結果顯示本發明之大白蝴蝶蘭萃取物具有強效之抑制糖化反應的活性，可有效提升清除糖化終產物之能力，有助於延緩慢性疾病與老化的進展。 The results of the efficacy test of the anti-glycation activity of the Great White Phalaenopsis extract of the present invention are shown in FIG. 1. After the action of the large white phalaenopsis extract of the present invention, it can reduce about 20% of the amount of saccharification final product. This result shows that the large white phalaenopsis extract of the present invention has a strong activity to inhibit the saccharification reaction and can effectively improve the elimination of saccharification The ability of the final product helps to delay the progression of chronic diseases and aging.
由於已知角質細胞會分泌玻尿酸等物質作為細胞間質，以維持表皮層屏障之完整，及防止皮膚水分散失及形成完整防護，因此本發明之一實施例以人類初代皮膚角質細胞(Human primary epidermal keratinocytes,HPEK)進行本發明之大白蝴蝶蘭萃取物促進角質細胞分泌玻尿酸之功效的測試。該人類初級皮膚角質細胞係購自CELLnTEC公司(瑞士)編號HPEK-50，該細胞係培養於無血清之角質細胞培養液(Keratinocyte-SFM)(購自Thermo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。 Since it is known that keratinocytes secrete hyaluronic acid and other substances as intercellular substance to maintain the integrity of the epidermal barrier, prevent skin water loss and form complete protection, one embodiment of the present invention uses human primary epidermal keratinocytes. keratinocytes, HPEK) to test the effect of the present invention's large white Phalaenopsis extract in promoting the secretion of hyaluronic acid from keratinocytes. This human primary skin keratinocyte cell line was purchased from CELLnTEC (Switzerland) with the number HPEK-50, and the cell line was cultured in serum-free keratinocyte-SFM (purchased from Thermo Company, USA, number #17005042), And cultivated in a 37℃ cell incubator containing 5% CO 2.
首先，於96孔培養盤中，每孔加入200μL之上述培養液並植入1x104個人類初代皮膚角質細胞，並於37℃培養16-18小時，接著將細胞分成三組：(1)僅加入細胞培養液之控制組、(2)加入0.125mg/mL本發明之大白蝴蝶蘭萃取物之實驗組、及(3)加入0.0625mg/mL本發明之大白蝴蝶蘭萃取物之實驗 組，並將各組分別於37℃培養24小時後，在不擾動貼附細胞的情況下，每孔分別收集100μL之培養液；其中該大白蝴蝶蘭萃取物係以水為溶劑進行萃取。 First, in a 96-well culture plate, add 200μL of the above culture medium to each well and implant 1x10 4 human primary skin keratinocytes, and incubate at 37°C for 16-18 hours, then divide the cells into three groups: (1) Only The control group added with cell culture solution, (2) the experimental group added 0.125mg/mL of the great white phalaenopsis extract of the present invention, and (3) the experimental group added 0.0625mg/mL of the great white phalaenopsis extract of the present invention, and After each group was cultured at 37°C for 24 hours, 100 μL of culture solution was collected from each well without disturbing the attached cells; the large white Phalaenopsis extract was extracted with water as a solvent.
接著，利用玻尿酸(Human Hyaluronic Acid,HA又稱人類透明質酸)之ELISA分析檢測試劑套組(購自Cusabio Biotech公司，中國，編號CSB-E04805h)進行分析。首先，於在底部覆蓋一層人類透明質酸捕捉抗體之96孔培養盤中，加入前述100μL之每孔收集之培養液，或溶於含1%牛血清白蛋白之磷酸鹽緩衝溶液的標準品，於37℃下與該捕捉抗體進行結合2小時，時間到後將液體移除，並直接於每孔中加入100μL之生物素(Biotin)偵測抗體，於37℃下偵測捕捉抗體1小時後，移除每孔中的溶液，並以200μL之清洗溶液(Washing solution，其中含0.05%聚氧乙烯無水山梨醇單月桂酸酯(tween 20)之磷酸鹽緩衝溶液)沖洗96孔培養盤中每個孔洞共3次；其中每次加入清洗溶液後須靜置2分鐘，並且須完全地去除所有清洗溶液，特別係最後一次清洗後須以抽吸(Aspirating)或傾析(Decanting)去除任何剩餘的清洗溶液，並將96孔培養盤倒置以乾淨的紙巾擦拭乾剩餘的清洗溶液，以使後續量測之結果較為準確。接著，再於每孔中加入100μL之1倍辣根過氧化物酶標記抗生物素蛋白(1x HRP-avidin)，並於37℃下作用1小時，以使其與偵測抗體進行結合，接著再以前述方法加入200μL之清洗溶液，沖洗96孔培養盤中每個孔洞共5次，接著再於每孔中加入90μL之TMB(3,3',5,5'-Tetramethylbenzidine)呈色溶液，於37℃下作用15-30分鐘並且避光，再於每孔中加入50μL之終止溶液以終止反應，其中以輕拍的方式以使溶液混合均勻，最後以酵素免疫分析儀(ELISA reader,BioTek)測量其5分鐘內於450nm之吸光值。再以Excel軟體進行student t-test以決定各組之變異係數是否在統計上具有顯著差異性(* p值<0.05；** p值<0.01；*** p值<0.001)。 Then, the analysis was performed using the ELISA analysis and detection reagent kit of hyaluronic acid (Human Hyaluronic Acid, HA also known as human hyaluronic acid) (purchased from Cusabio Biotech, China, No. CSB-E04805h). First, in a 96-well culture dish covered with a human hyaluronic acid capture antibody at the bottom, add 100 μL of the culture medium collected in each well, or a standard product dissolved in a phosphate buffer solution containing 1% bovine serum albumin. Binding with the capture antibody at 37°C for 2 hours, remove the liquid when the time is up, and directly add 100μL of Biotin detection antibody to each well. After detecting the capture antibody at 37°C for 1 hour , Remove the solution in each well, and wash each 96-well culture plate with 200μL of washing solution (Washing solution containing 0.05% polyoxyethylene anhydrous sorbitol monolaurate (tween 20) phosphate buffer solution) A total of 3 times for each hole; each time the cleaning solution is added, it must be allowed to stand for 2 minutes, and all the cleaning solution must be completely removed, especially after the last cleaning, aspirating or decanting must be used to remove any remaining The cleaning solution of the 96-well culture plate is turned upside down and the remaining cleaning solution is wiped dry with a clean paper towel to make the subsequent measurement results more accurate. Then, add 100μL of 1x horseradish peroxidase-labeled avidin (1x HRP-avidin) to each well, and react at 37°C for 1 hour to bind to the detection antibody, and then Then add 200μL of washing solution in the above method, rinse each hole in the 96-well culture plate 5 times, and then add 90μL of TMB (3,3',5,5'-Tetramethylbenzidine) coloring solution to each well. Incubate at 37°C for 15-30 minutes and protect from light. Then add 50μL of stop solution to each well to stop the reaction. Tap gently to make the solution evenly mixed. Finally, use an enzyme immunoassay analyzer (ELISA reader, BioTek). ) Measure the absorbance at 450nm within 5 minutes. Then use Excel software to perform student t-test to determine whether the coefficient of variation of each group is statistically significant (* p value <0.05; ** p value <0.01; *** p value <0.001).
本發明之大白蝴蝶蘭萃取物促進角質細胞分泌玻尿酸之功效的測試結果如圖2所示。人類初代皮膚角質細胞經0.125mg/mL本發明之大白蝴蝶蘭萃取物作用後，相較於控制組，能提高11.83%之玻尿酸分泌量；而經0.0625 mg/mL本發明之大白蝴蝶蘭萃取物作用後，相較於控制組，能提高24.75%之玻尿酸分泌量。此結果顯示本發明之大白蝴蝶蘭萃取物能有效提高角質細胞分泌玻尿酸的量，能有效使皮膚保水力提升，以使皮膚角質層結構完整，並提升皮膚屏障功能。 The test result of the effect of the large white phalaenopsis extract of the present invention in promoting the secretion of hyaluronic acid by keratinocytes is shown in FIG. 2. After the first generation of human skin keratinocytes were treated with 0.125mg/mL of the large white phalaenopsis extract of the present invention, compared with the control group, it could increase the secretion of hyaluronic acid by 11.83%; and after 0.0625 After mg/mL of the present invention's large white phalaenopsis extract, it can increase the secretion of hyaluronic acid by 24.75% compared to the control group. This result shows that the large white phalaenopsis extract of the present invention can effectively increase the amount of hyaluronic acid secreted by keratinocytes, can effectively increase the water retention capacity of the skin, so as to complete the skin stratum corneum structure and enhance the skin barrier function.
本發明之一實施例以人類初代皮膚角質細胞HPEK-50進行本發明之大白蝴蝶蘭萃取物提升KRT14基因、及SMAD1基因表現量之功效的測試。首先，將1.5x105個人類初代皮膚角質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二組：(1)僅加入細胞培養液之控制組、及(2)加入0.25mg/mL本發明之大白蝴蝶蘭萃取物的實驗組，並將該二組分別於37℃下作用24小時後，測試各組人類初代皮膚角質細胞中目標基因的表現量；其中該大白蝴蝶蘭萃取物係以水為溶劑進行萃取。首先，將該二組之細胞以細胞裂解液(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)分別收集該二組細胞內之總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，並以表一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)將該二組反轉錄後產物分別以表一之組合引子進行定量即時聚合酶連鎖反應(Quantitative real-time polymerase chainreaction,qPCR)試驗，條件為95℃反應1秒，60℃反應20秒，總共40個循環。用以定量該二組人類初代皮膚角質細胞中KRT14基因、及SMAD1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-ΔΔCt，其 中ΔCT=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)；ΔΔCT=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-ΔΔCt。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)。 In one embodiment of the present invention, human primary skin keratinocytes HPEK-50 were used to test the efficacy of the present invention's large white Phalaenopsis extract to enhance the expression of KRT14 gene and SMAD1 gene. First, culture the 1.5x10 5 human primary skin keratinocytes 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 two groups: (1) Add only The control group of cell culture solution and (2) the experimental group added 0.25mg/mL of the great white phalaenopsis extract of the present invention, and after the two groups were treated at 37°C for 24 hours, the keratin of the primary human skin of each group was tested The expression level of the target gene in the cell; wherein the large white phalaenopsis extract is extracted with water as a solvent. First, the two groups of 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. ) Collect the total RNA in the two 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 primers and reversers in Table 1 The transcriptase performs reverse transcription to produce the cDNA products corresponding to the mRNAs of these genes, and then uses the ABI StepOnePlus TM Real-Time PCR system (Thermo Fisher Scientific Company, USA) and KAPA SYBR FAST (purchased from Sigma Company, USA) , No. 38220000000) The two 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. The conditions were 95℃ for 1 second and 60℃ for 20 Seconds, a total of 40 cycles. For the quantitative expression levels KRT14 gene and mRNA in the two groups of human First generation of skin keratinocytes SMAD1 gene, wherein the quantitative values based taken by the threshold cycle number (Ct), and mRNA of the target gene relative amounts lines derived from equations 2 - ΔΔCt , where ΔC T =C T comparison group or experimental group target gene/control group target gene- C T TBP (TATA-binding protein); ΔΔC T =C T comparison group or experimental group target gene- C T The target gene of the control group ; the fold change of each gene in each group is 2 -ΔΔCt . Then, 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).
本發明之大白蝴蝶蘭萃取物提升KRT14基因、及SMAD1基因表現量之功效的測試結果如圖3所示。其中，KRT會形成角蛋微絲，與維持角質細胞結構相關；而SMAD是一種訊號傳遞因子與調控膠原蛋白表現相關。人類初代皮膚角質細胞經0.25mg/mL本發明之大白蝴蝶蘭萃取物作用後，相較於控制組，能分別顯著提升KRT14基因、及SMAD1基因的表現量達控制組的1.24倍、及1.28倍。此結果顯示本發明之大白蝴蝶蘭萃取物能有效提升皮膚細胞中KRT14基因、及SMAD1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，以強化皮膚屏障及防禦功能、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 test results of the efficacy of the large white phalaenopsis extract of the present invention in enhancing the expression of KRT14 gene and SMAD1 gene are shown in FIG. 3. Among them, KRT forms keratin filaments, which are related to maintaining the structure of keratinocytes; while SMAD is a signaling factor and is related to regulating the expression of collagen. Compared with the control group, human primary skin keratinocytes treated with 0.25 mg/mL of the present invention's large white phalaenopsis extract can significantly increase the expression levels of KRT14 gene and SMAD1 gene by 1.24 times and 1.28 times that of the control group. . This result shows that the large white phalaenopsis extract of the present invention can effectively enhance the expression of KRT14 gene and SMAD1 gene in skin cells, can effectively maintain the arrangement of skin keratinocytes, make the skin stratum corneum structure complete, and strengthen the skin barrier and defense functions, Prevent skin moisture loss, keep the skin in a water-retaining state, and keep the skin young, smooth and elastic.
本發明之一實施例為進行本發明之大白蝴蝶蘭萃取物於提升個體皮膚光澤度之功效的測試。首先，製備添加1%(w/w)本發明之大白蝴蝶蘭萃取物之精華液的大白蝴蝶蘭萃取物面膜；以及不含本發明大白蝴蝶蘭萃取物之 精華液的面膜作為控制組；其中，該精華液成分皆為水、馨鲜酮、己二醇、1,3-丁二醇、三仙膠、增稠劑以及三乙醇胺；其中該大白蝴蝶蘭萃取物係以水為溶劑進行萃取。接著，進行瞬效實驗檢測，募集8位受試者，每位受試者於每日早晚清洗完臉部後，將控制組面膜、及含本發明之大白蝴蝶蘭萃取物的面膜分別貼敷於左右半臉之皮膚15分鐘後取下，並以指腹稍加按摩促進吸收，並於敷面膜前與敷面膜後以C+K Cutometer® MPA 580多探頭膚質分析儀(C+K electronic，德國)中的Glossymeter_GL200光澤探頭進行全臉皮膚光澤度的檢測。藉由儀器打到皮膚表面的反射光和散射光，可以精確且便利的測試肌膚的光澤度，其平行白光係由探頭頂部的LED所產生；其中，以敷面膜前的肌膚光澤度為100%，相對於敷面膜前所得出的百分比，可得到敷面膜後相對的肌膚光澤度改善百分比。 An embodiment of the present invention is to test the effect of the present invention's large white phalaenopsis extract in improving the gloss of the individual's skin. First, prepare a large white phalaenopsis extract mask with the essence of 1% (w/w) of the invention’s large white phalaenopsis extract; and a mask that does not contain the large white phalaenopsis extract of the present invention The facial mask of the essence is used as the control group; among them, the essence of the essence is water, saccharin, hexylene glycol, 1,3-butanediol, trixian gum, thickener and triethanolamine; among them, the great white phalaenopsis The extract is extracted with water as a solvent. Then, a transient effect test was conducted and 8 subjects were recruited. After each subject cleans his face every morning and evening, he applied the mask of the control group and the mask containing the large white phalaenopsis extract of the present invention. Take off the skin of the left and right sides of the face after 15 minutes, massage it with your fingertips to promote absorption, and apply the C+K Cutometer® MPA 580 multi-probe skin analyzer (C+K electronic) before and after applying the mask. , Germany) Glossymeter_GL200 gloss probe for the detection of the skin gloss of the whole face. The reflected light and scattered light hit the skin surface by the instrument can accurately and conveniently test the gloss of the skin. The parallel white light is generated by the LED on the top of the probe; among them, the skin gloss before applying the mask is 100% , Relative to the percentage obtained before applying the mask, the relative skin gloss improvement percentage after applying the mask can be obtained.
本發明之大白蝴蝶蘭萃取物於提升個體皮膚光澤度之結果如圖4所示。使用含本發明之大白蝴蝶蘭萃取物之面膜後，比使用前提高了皮膚光澤度10.4%，未含本發明之大白蝴蝶蘭萃取物之控制組僅提高3.6%。此結果顯示本發明之大白蝴蝶蘭萃取物能有效提升個體皮膚之光澤度，具有改善皮膚暗沉、枯黃的情況使皮膚透亮有光澤之功效。 The results of the present invention of the great white phalaenopsis extract in improving the gloss of the individual's skin are shown in Figure 4. After using the mask containing the great white phalaenopsis extract of the present invention, the skin gloss increased by 10.4% compared to before use, and the control group without the great white phalaenopsis extract of the present invention only increased by 3.6%. This result shows that the large white phalaenopsis extract of the present invention can effectively enhance the gloss of the individual's skin, and has the effect of improving the dull and yellow skin and making the skin bright and shiny.
綜上所述，本發明之大白蝴蝶蘭萃取物具有強效之抑制糖化反應的活性，可有效提升清除糖化終產物之能力，有助於延緩慢性疾病與老化的進展；還能有效提高角質細胞分泌玻尿酸的量、有效提升皮膚細胞中KRT14基因、及SMAD1基因的表現量，能有效維持皮膚角質細胞排列，以使皮膚角質層結構完整，並提升皮膚保水力，及提升皮膚屏障及防禦功能，以防止皮膚水分散失，使皮膚處於保水的狀態、及維持皮膚年輕光滑有彈性；另外，其亦能有效提升個體皮膚之光澤度，具有改善皮膚暗沉、枯黃的情況使皮膚透亮有光澤之功效。因此，本發明之大白蝴蝶蘭萃取物可用於製備抗醣化與改善皮膚外觀之組 合物的用途，其中該組合物是一醫藥品、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 great white phalaenopsis extract of the present invention has potent activity in inhibiting glycation reaction, can effectively improve the ability to clear the end products of glycation, and help delay the progression of chronic diseases and aging; it can also effectively improve keratinocytes Secreting the amount of hyaluronic acid, effectively enhancing the expression of KRT14 gene and SMAD1 gene in skin cells, can effectively maintain the arrangement of skin keratinocytes, so that the skin stratum corneum structure is intact, and skin water retention capacity is improved, and skin barrier and defense functions are improved. To prevent skin water loss, keep the skin in a water-retaining state, and keep the skin young, smooth and elastic; in addition, it can also effectively improve the gloss of the individual's skin, improve the dull and yellow skin, and make the skin bright and shiny. . Therefore, the large white phalaenopsis extract of the present invention can be used to prepare a composition for anti-glycation and improving skin appearance, wherein the composition is a medicine, a food or a skin care product, which can be taken orally, applied to the skin, etc. Give a body.
<110> 大江生醫股份有限公司 <110> Dajiang Biomedical Co., Ltd.
<120> 大白蝴蝶蘭萃取物用於抗醣化與改善皮膚外觀的用途 <120> The use of large white Phalaenopsis extract for anti-glycation and improving skin appearance
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<210> 6 <210> 6
<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> 6 <400> 6
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| TW108146492A TWI772723B (en) | 2019-08-14 | 2019-12-18 | Use of viola mandshurica extract for anti-aging |
| TW109127832A TWI819235B (en) | 2019-08-14 | 2020-08-14 | Fermentation broth of natural raw materials and uses thereof for inhibiting allergic reaction or for enhancing immunity |
| TW109127837A TW202106323A (en) | 2019-08-14 | 2020-08-14 | Use ofrosmarinus officinaliscallus extract for manufacturing composition inhibiting skin aging and a culture medium for inducingrosmarinus officinaliscallus |
| TW109127833A TWI809299B (en) | 2019-08-14 | 2020-08-14 | Preparation method of viola mandshurica extract |
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| TW109127832A TWI819235B (en) | 2019-08-14 | 2020-08-14 | Fermentation broth of natural raw materials and uses thereof for inhibiting allergic reaction or for enhancing immunity |
| TW109127837A TW202106323A (en) | 2019-08-14 | 2020-08-14 | Use ofrosmarinus officinaliscallus extract for manufacturing composition inhibiting skin aging and a culture medium for inducingrosmarinus officinaliscallus |
| TW109127833A TWI809299B (en) | 2019-08-14 | 2020-08-14 | Preparation method of viola mandshurica extract |
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| CN115607582A (en) * | 2021-07-16 | 2023-01-17 | 大江生医股份有限公司 | Chinese medicinal fermentation liquor and its application in refreshing, relieving fatigue, protecting liver, enhancing immunity, and reducing blood lipid |
| CN113398186B (en) * | 2021-08-20 | 2021-11-02 | 山东中医药大学附属医院 | Viola yedoensis makino extract and preparation method and application thereof |
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| TW201642833A (en) * | 2015-06-02 | 2016-12-16 | 青山蘭花生物科技有限公司 | The active extract of phalaenopsis, preparation method and use thereof |
| TW201700108A (en) * | 2015-06-29 | 2017-01-01 | Guang-Ding Biotech Co | Phalaenopsis extract for whitening skin and resisting skin aging capable of reducing the activity of tyrosinase and being anti-oxidative , capable of whitening the skin and slowing down the phenomenon of skin aging |
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| JP4880816B2 (en) * | 2000-12-15 | 2012-02-22 | 株式会社ヤクルト本社 | Skin anti-aging agent |
| CN100471514C (en) * | 2005-12-30 | 2009-03-25 | 王志民 | External use preparation for treating vulnus slow agglutination and its preparation method |
| CN101601780A (en) * | 2008-06-10 | 2009-12-16 | 中国医学科学院药物研究所 | The application of Chinese violet in anti-diabetic and control correlated metabolism diseases |
| CN102274142B (en) * | 2011-07-19 | 2013-01-23 | 北京联合大学生物化学工程学院 | Preparation method of compound Chinese medicine washing liquid |
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| CN102669666A (en) * | 2011-11-30 | 2012-09-19 | 宁夏天瑞产业集团现代农业有限公司 | Chinese date slices and production method thereof |
| TW201420107A (en) * | 2012-11-28 | 2014-06-01 | Taiwan Orchid Professionals Co Ltd | Extract of Phalaenopsis amabilis petals, and the preparation process and uses thereof |
| TW201420127A (en) * | 2012-11-28 | 2014-06-01 | Taiwan Orchid Professionals Co Ltd | Extract of Phalaenopsis amabilis meristem, and the preparation process and uses thereof |
| CN103417749B (en) * | 2013-09-11 | 2015-08-12 | 河南中医学院 | A kind of preparation method of Herba Violae extract and the application in hypoglycemic medicine thereof |
| KR101672274B1 (en) * | 2013-10-28 | 2016-11-04 | 한국 한의학 연구원 | Compositions comprising a Viola Herba extract, or an extract of Viola Herba, Persicae Semen, Cinnamomi Ramulus, and Glycyrrhiza spp. for the prevention or treatment of lipid-related cardiovascular diseases and obesity |
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| TWI624226B (en) * | 2015-08-25 | 2018-05-21 | a method for preparing a fermentation culture and a method thereof Fermentation culture and its use | |
| KR101793098B1 (en) * | 2016-02-26 | 2017-11-03 | 정의수 | Natural plant mixed extracts that regulate heat of the skin and a cosmetic composition for skin heat anti-aging that containing the same |
| TWI727159B (en) * | 2017-04-10 | 2021-05-11 | 大江生醫股份有限公司 | Uses of orchid leaf callus extract |
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| CN108309855A (en) * | 2018-03-31 | 2018-07-24 | 佛山娇芙生物科技有限公司 | A kind of Cranberry beauty care composition and its application |
| CN109939022A (en) * | 2019-05-12 | 2019-06-28 | 辽宁石油化工大学 | A kind of plant bacteriostatic skin-care composition |
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| TW201642833A (en) * | 2015-06-02 | 2016-12-16 | 青山蘭花生物科技有限公司 | The active extract of phalaenopsis, preparation method and use thereof |
| TW201700108A (en) * | 2015-06-29 | 2017-01-01 | Guang-Ding Biotech Co | Phalaenopsis extract for whitening skin and resisting skin aging capable of reducing the activity of tyrosinase and being anti-oxidative , capable of whitening the skin and slowing down the phenomenon of skin aging |
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| TWI772723B (en) | 2022-08-01 |
| CN112386643A (en) | 2021-02-23 |
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| CN112386665A (en) | 2021-02-23 |
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