TWI687237B - Use of extract of victoria cruziana for inducing expression of keratin gene and hyaluronan synthase 2 gene, and enhancing moisture-retaining capacity of skin - Google Patents

Use of extract of victoria cruziana for inducing expression of keratin gene and hyaluronan synthase 2 gene, and enhancing moisture-retaining capacity of skin Download PDF

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TWI687237B
TWI687237B TW108102976A TW108102976A TWI687237B TW I687237 B TWI687237 B TW I687237B TW 108102976 A TW108102976 A TW 108102976A TW 108102976 A TW108102976 A TW 108102976A TW I687237 B TWI687237 B TW I687237B
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TW202027724A (en
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林詠翔
李姿儀
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大江生醫股份有限公司
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    • AHUMAN NECESSITIES
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P17/16Emollients or protectives, e.g. against radiation
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin

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Abstract

The present disclosure provides a use of an extract of Victoria cruziana for inducing expression of keratin gene and hyaluronan synthase 2 gene, and enhancing the moisture-retaining capacity of the skin.

Description

克魯茲王蓮的萃取物用於提升角蛋白基因及透明質酸合 成酶2基因的表現量及提升皮膚的保濕能力之用途 The extract of King Cruz is used to enhance keratin gene and hyaluronic acid synthesis Gene expression level of genase 2 gene and use for enhancing skin's moisturizing ability

本發明是有關於一種魯茲王蓮的萃取物用於提升角蛋白基因及透明質酸合成酶2基因的表現量,及提升皮膚的保濕能力的用途。 The present invention relates to a use of the extract of King Lutzia to enhance the expression of keratin gene and hyaluronic acid synthase 2 gene and enhance the skin's moisturizing ability.

皮膚是保護人類個體的最大屏障,它具有對抗水分散失、病原菌以及各種環境損害之功能。暴露於大量的紫外線(ultraviolet,UV)、游離輻射(ionizing radiation)、藥物或異生物質(xenobiotics)會促使皮膚生成活性氧族(reactive oxygen species,ROS)以及自由基(free radicals)。當所累積的活性氧族以及自由基的數量超過細胞或組織本身的抗氧化能力時,便會形成氧化性壓力(oxidative stress)。接著,活性氧族以及自由基會與細胞內的組成物(包括DNA、蛋白質以及脂質等)相反應,進而對皮膚產生非所欲的影響。 The skin is the largest barrier to protect human individuals, it has the function to resist water loss, pathogenic bacteria and various environmental damage. Exposure to a large amount of ultraviolet (UV), ionizing radiation, drugs or xenobiotics will cause the skin to generate reactive oxygen species (ROS) and free radicals. When the accumulated amount of reactive oxygen species and free radicals exceeds the antioxidant capacity of the cell or tissue itself, oxidative stress will be formed. Next, the reactive oxygen species and free radicals will react with intracellular components (including DNA, proteins, lipids, etc.), which will undesirably affect the skin.

近年來,人類對於皮膚保濕(moisturizing)的需求與日俱增,然而,目前常見用來提升皮膚保濕能力的方式大多為利用塗抹於皮膚表面的化妝品、保養品,或口服宣稱具有提升皮膚保濕效用的健康食品。然而,習知的化妝品、保養品及健康食品大多由化學成分所製成,長期使用不但對人體健康有害無益,且這些產品往往價格昂貴,並非為一般使用者所能負擔。 In recent years, human demand for skin moisturizing has been increasing day by day. However, the most common ways to improve skin moisturizing ability at present are to use cosmetics, skin care products applied to the skin surface, or oral health foods that claim to enhance skin moisturizing effect. . However, the conventional cosmetics, skin care products and health foods are mostly made of chemical ingredients. Long-term use is not only harmful to human health, but also these products are often expensive and not affordable for general users.

為了解決上述問題,本領域的技術人員亟需研發出具有提升皮膚的保濕能力的新穎醫藥品、食品產品或保養品以造福有此需求的廣大族群。 In order to solve the above-mentioned problems, those skilled in the art urgently need to develop novel medicines, food products, or skin care products that enhance the moisturizing ability of the skin, so as to benefit the broad ethnic groups in need.

有鑑於此,本發明之目的為提供一種克魯茲王蓮(Victoria cruziana)的萃取物用於製備一提升角蛋白(keratin,KRT)基因及透明質酸合成酶2(hyaluronan synthase 2,HAS2)基因的表現量之組成物的用途,其中該克魯茲王蓮的萃取物是以水、醇類、含水醇類或其組合作為一萃取溶劑對該克魯茲王蓮進行萃取而製得。 In view of this, the object of the present invention is to provide an extract of Victoria cruziana for preparing a keratin ( KRT ) gene and hyaluronan synthase 2 ( HAS2 ) gene The use of the expressive composition, in which the extract of King Cruz is made by extracting King Lotus from water, alcohol, water-containing alcohol or a combination thereof as an extraction solvent.

本發明之另一目的為提供一種克魯茲王蓮的萃取物用於製備一提升皮膚的保濕能力之組成物的用途,其中該克魯茲王蓮的萃取物是以水、醇類、含水醇類或其組合作為一萃取溶劑對該克魯茲王蓮進行萃取而製得。 Another object of the present invention is to provide a use of the extract of Cruz Royale for the preparation of a composition for enhancing the moisturizing capacity of the skin, wherein the extract of Royal Lotus is water, alcohols, hydrous alcohols or The combination is used as an extraction solvent to extract the Cruz royal lotus.

在本發明的一實施例中,該克魯茲王蓮與該萃取溶劑的體積比例介於1~5:10~20。 In an embodiment of the invention, the volume ratio of the Cruz King Lotus to the extraction solvent is between 1 and 5: 10 and 20.

在本發明的一實施例中,該KRT基因是KRT1基因、KRT10基因或KRT14基因。 In an embodiment of the invention, the KRT gene is KRT1 gene, KRT10 gene or KRT14 gene.

在本發明的一實施例中,該克魯茲王蓮的萃取物的有效濃度為至少0.03125mg/mL。 In an embodiment of the present invention, the effective concentration of the extract of King Cruz Cruz is at least 0.03125 mg/mL.

在本發明的一實施例中,該克魯茲王蓮的萃取物為一克魯茲王蓮第1天開花的花萃取物。 In an embodiment of the invention, the extract of King Cruz is a flower extract of King Lotus blooming on the first day.

在本發明的一實施例中,該組成物是一醫藥品、一食品產品或一保養品。 In an embodiment of the invention, the composition is a pharmaceutical product, a food product or a maintenance product.

綜上所述,本發明克魯茲王蓮的萃取物之功效在於:可藉由提升角蛋白基因及透明質酸合成酶2基因的表現量,達到提升皮膚的保濕能力的功效。 To sum up, the effect of the extract of the Royal Lotus of Cruz of the present invention is that the effect of enhancing the moisturizing ability of the skin can be achieved by enhancing the expression of the keratin gene and hyaluronic acid synthase 2 gene.

以下將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. 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 scope of the present invention, Some changes and retouching can be done, so the scope of protection of the present invention shall be deemed as defined by the scope of the attached patent application.

圖1是本發明克魯茲王蓮的萃取物的總多酚含量檢測之數據圖,其中“***”表示與比較組比較,p<0.001。 FIG. 1 is a data graph of the detection of total polyphenol content of the extract of the Royal Lotus of Cruz of the present invention, where “***” indicates comparison with the comparison group, p <0.001.

圖2是本發明克魯茲王蓮的萃取物在作用6小時之提升與皮膚細胞保濕相關的KRT1基因、KRT10基因、KRT14基因及HAS2基因表現上的功效之數據圖,其中“*”表示與對照組比較,p<0.05;“**”表示與對照組比較,p<0.01。 FIG. 2 is a data graph showing the efficacy of the extract of Kreuz king lotus of the present invention in enhancing the KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization after 6 hours of action, where “*” represents the control group For comparison, p <0.05;"**" indicates comparison with the control group, p <0.01.

圖3是本發明克魯茲王蓮的萃取物在作用24小時之提升與皮膚細胞保濕相關的KRT1基因、KRT10基因、KRT14基因及HAS2基因表現上的功效之數據圖,其中“*”表示與對照組比較,p<0.05;“**”表示與對照組比較,p<0.01;“***”表示與對照組比較,p<0.001。 FIG. 3 is a data graph showing the efficacy of the extract of the King of Lotus of the present invention in enhancing the KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization for 24 hours, where “*” represents the control group For comparison, p <0.05;"**" means comparison with the control group, p <0.01;"***" means comparison with the control group, p <0.001.

定義definition

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%.

依據本發明,克魯茲王蓮(Victoria cruziana)別名為巴拉圭王蓮或阿根廷王蓮,為睡蓮科(Nymphaeaceae)王蓮屬(Victoria)的水生植物,原產於南美熱帶地區。克魯茲王蓮具地下莖,根狀莖直立塊狀,肥厚,具刺,具粗壯發達之側根,質地軟而脆,單葉互生。 According to the present invention, Victoria cruziana ( Victoria cruziana ) is aliased as Paraguay king lotus or Argentine king lotus, which is an aquatic plant of Nymphaeaceae (Nymphaeaceae) King lotus ( Victoria ), native to tropical South America. King Cruz has underground stems, rhizomes erect and massive, thick, thorny, with thick and strong lateral roots, soft and brittle texture, single leaf alternate.

依據本發明,克魯茲王蓮的花可具有不同的開花期(flowering stage)。較佳地,克魯茲王蓮的花是第1、2天開的花。更佳地,克魯茲王蓮的花是第1天開的花。 According to the present invention, the flowers of King Lotus can have different flowering stages. Preferably, the flowers of King Cruz are blooming on days 1 and 2. More preferably, the flowers of King Cruz Cruz bloom on the first day.

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, pharmaceutical products can be manufactured into a dosage form suitable for parenterally or topically administration using techniques well known to those skilled in the art, including, but not limited to: injections (injection) [for example, 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 pharmaceutical product may further include a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, and decomposers ), 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 professional qualities and routine techniques of those who are familiar with this technology.

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 contains a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), Aqueous solution containing alcohol and their combination.

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 medicinal product can be administered via parenteral routes selected from the group consisting of subcutaneous injection, intraepidermal injection, and intradermal injection Intradermal injection and 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, pharmaceutical products can be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, which include, but are not limited to: emulsion, gel Gel, ointment, cream, patch, liniment, powder, aerosol, spray, lotion, milk Serums, pastes, foams, drops, suspensions, salves and bandages.

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 pharmaceutical product 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 include one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons [such as petroleum, jelly, and 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, occlusive agents (occlusive agents), softeners (emollients), thickeners (thickeners), solubilizing agents (solubilizing agents), penetration enhancers (penetration enhancers), anti-irritants (anti-irritants), colorants (colorants) and propellants (propellants) etc. The selection and quantity of these additives fall within the scope of professional qualities and routine skills 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 maintenance product may further include an acceptable adjuvant widely used in the maintenance 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 professional qualities and routine techniques of those who are familiar with this technology.

依據本發明,保養品可利用熟習此技藝者所詳知的技術而被製造成一適合於護膚(skincare)或化妝(makeup)的形式,這包括,但不限於:水性溶液(aqueous solution)、水-醇溶液(aqueous-alcohol solution)或油性溶液(oily solution)、呈水包油型(oil-in-water type)、油包水型(water-in-oil type)或複合型之乳劑、凝膠、軟膏、乳霜、面膜(mask)、貼片、貼布(pack)、擦劑、粉末、氣溶膠、噴霧、乳液、乳漿、糊劑、泡沫、分散液、滴劑、慕斯(mousse)、防曬油(sunblock)、化妝水(tonic water)、粉底(foundation)、卸妝產品(makeup remover products)、肥皂(soap)以及其他身體清潔產品(body cleansing products)等。 According to the present invention, skin care products can be manufactured into a form suitable for skincare or makeup using techniques well known to those skilled in the art. This includes, but is not limited to: aqueous solution (aqueous solution), water -Aqueous-alcohol solution or oily solution, oil-in-water type, water-in-oil type or compound emulsion, gel Glue, ointment, cream, mask, patch, pack, wipe, powder, aerosol, spray, emulsion, emulsion, paste, foam, dispersion, drops, mousse ( mousse, sunblock, tonic water, foundation, foundation remover products, 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, the skin care product can also be used in combination with one or more external use agents of known activity selected from the following: whitening agents [such as tretinoin, catechins (catechin), kojic acid, arbutin and vitamin C], moisturizer, anti-hair Anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts [such as aloe extract], skin nutrients, anesthetics (anesthetics), anti-acne agents, antipruritics, analgesics, antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents (anti-dry skin agents), antipsoriatic agents, antiaging agents, antiwrinkle agents, antiseborrheic agents, wound-healing agents, Corticosteroids and hormones. The selection and quantity of these external agents fall within the professional qualities and routine technologies of those who are familiar with this technology.

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 is added during the preparation of raw materials by conventional methods, or added during the production of food, and formulated with any edible material for 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.

實施例1. 克魯茲王蓮的萃取物之製備Example 1. Preparation of the extract of King Cruz

首先,將未開花、第1天開花或第2天開花的克魯茲王蓮(來自於台南市白河區蓮合國休閒蓮園)的花進行均質,接而以水、醇類、含水醇類或其組合作為萃取溶劑對均質後的克魯茲王蓮進行萃取0.5~3小時,其中萃取的溫度介於50℃至100℃,萃取溶劑與克魯茲王蓮的體積比介於10~20:1~5。接著,將所得到的產物冷卻至室溫,以400網目(mesh)的濾網對產物過濾而得到一濾液,然後於45~70℃下對濾液進行減壓濃縮而得到一濃縮產物。之後,以5μm孔徑的濾心對濃縮產物進行過濾,得到本發明克魯茲王蓮的萃取物。 First, homogenize the flowers of King Cruz Cruz (from Lianhe United Leisure Garden in Baihe District, Tainan City), which is not blooming, blooming on the first day or blooming on the second day, and then use water, alcohol, water-containing alcohol or The combination is used as an extraction solvent to extract the homogenized Cruz king lotus for 0.5~3 hours, wherein the extraction temperature is between 50°C and 100°C, and the volume ratio of the extraction solvent to the Cruz king lotus is between 10-20: 1~5. Next, the obtained product was cooled to room temperature, and the product was filtered with a 400-mesh filter to obtain a filtrate, and then the filtrate was concentrated under reduced pressure at 45 to 70°C to obtain a concentrated product. After that, the concentrated product was filtered with a filter core with a pore size of 5 μm to obtain an extract of the King Cruz of the present invention.

實施例2. 克魯茲王蓮的萃取物的總多酚含量檢測Example 2. Determination of total polyphenol content in the extract of King Lotus of Cruz

首先,製備標準溶液及繪製標準曲線,將10mg的沒食子酸(gallic acid)(Sigma G7384)溶於水中並添加10mL的體積至量瓶中,然後儲存於-20℃作為儲備溶液。之後,將儲備溶液稀釋10倍至100μg/mL的濃度,未使用完之液體可儲存於-20℃。接著,於玻璃試管中分別配製0μg/mL、20μg/mL、40 μg/mL、60μg/mL、80μg/mL及100μg/mL的沒食子酸標準溶液,配製方式顯示於表1。 First, prepare a standard solution and draw a standard curve, dissolve 10 mg of gallic acid (Sigma G7384) in water and add a volume of 10 mL to a measuring flask, and then store at -20°C as a stock solution. After that, dilute the stock solution 10 times to a concentration of 100 μg/mL, and the unused liquid can be stored at -20°C. Next, prepare 0 μg/mL, 20 μg/mL, and 40 in glass test tubes, respectively The standard solutions of gallic acid for μg/mL, 60μg/mL, 80μg/mL and 100μg/mL are shown in Table 1.

Figure 108102976-A0101-12-0007-1
Figure 108102976-A0101-12-0007-1

之後,添加500μL的佛蕭酚試劑(Folin-Ciocalteu’s phenol reagent)(Merck 1.09001.0100)、混合均勻並靜置3分鐘,接而添加400μL的7.5%碳酸鈉(sodium carbonate)(Sigma 31432,溶於水中)、混合均勻並反應30分鐘。接著,經由渦旋(vortex)確保無氣泡後取200μL的標準溶液於750nm下測量吸光值,並繪製標準曲線。 After that, add 500 μL of Folin-Ciocalteu's phenol reagent (Merck 1.09001.0100), mix well and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate (Sigma 31432, dissolved in (In water), mix well and react for 30 minutes. Next, after ensuring no bubbles through vortex, 200 μL of the standard solution was taken and the absorbance was measured at 750 nm, and a standard curve was drawn.

另外,將實施例1所得到的第1天開花的克魯茲王蓮的萃取物作為實驗組1,第2天開花的克魯茲王蓮的萃取物作為實驗組2,未開花的克魯茲王蓮的萃取物作為比較組。將實驗組1、實驗組2及比較組分別以水予以稀釋並取100μL的體積至玻璃試管中,每組樣品需三重複。之後,添加500μL的佛蕭酚試劑、混合均勻並靜置3分鐘,接而添加400μL的7.5%碳酸鈉、混合均勻並反應30分鐘~1小時。接著,經由渦旋確保無氣泡後取200μL的各組反應溶液於750nm下測量吸光值,然後以內差法算出濃度後再回乘稀釋倍數以取得原萃取物濃度。本實施例的結果顯示於圖1。 In addition, the extract of the Royal Lotus of Cruzia that bloomed on the first day obtained in Example 1 was taken as the experimental group 1, and the extract of the Royal Lotus of Cruzia that bloomed on the second day was the experimental group 2, and the extract of the Royal Lotus of Cruzia unpublished As a comparison group. The experimental group 1, the experimental group 2 and the comparative group were respectively diluted with water and a volume of 100 μL was taken into a glass test tube. The samples of each group were repeated three times. Afterwards, add 500 μL of flavanol reagent, mix well and let stand for 3 minutes, then add 400 μL of 7.5% sodium carbonate, mix well and react for 30 minutes to 1 hour. Next, after vortexing to ensure that there are no bubbles, 200 μL of each group of reaction solution was taken to measure the absorbance at 750 nm, and then the concentration was calculated by the internal difference method, and then the dilution factor was multiplied to obtain the original extract concentration. The results of this example are shown in Figure 1.

圖1是本發明克魯茲王蓮的萃取物的總多酚含量檢測之數據圖。由圖1可見,與比較組相較之下,實驗組1及實驗組2的總多酚含量有顯著的提升,其中與比較組相較之下,實驗組1的總多酚含量提升7.1倍。本實施例的結果顯示,本發明克魯茲王蓮的萃取物會大量釋出總多酚。由於第1天開花的克魯茲王蓮的萃取物之總多酚含量最高,因此以第1天開花的克魯茲王蓮的萃取物拿來進行後續實驗。 FIG. 1 is a data graph of the detection of the total polyphenol content of the extract of the Royal Lotus of Cruz. As can be seen from Figure 1, compared with the comparison group, the total polyphenol content of the experimental group 1 and the experimental group 2 has a significant increase, and compared with the comparison group, the total polyphenol content of the experimental group 1 increases by 7.1 times . The results of this example show that the extract of Kreuz Wanglian of the present invention will release a large amount of total polyphenols. Since the extract of the flowering Kr. Cruze on the first day had the highest total polyphenol content, the extract of the flowering Kr. Cruze on the first day was used for subsequent experiments.

實施例3. 克魯茲王蓮的萃取物在提升皮膚的保濕能力上的效用評估Example 3. Evaluation of the effectiveness of the extract of Cruz Royale in enhancing the moisturizing ability of the skin

本實施例藉由探討克魯茲王蓮的萃取物可否藉由調控與皮膚細胞保濕相關的基因表現來達到提升皮膚的保濕能力之功效。 In this embodiment, by exploring whether the extract of King Cruz Cruz can regulate the gene expression related to skin cell moisturization, the effect of enhancing the moisturizing capacity of the skin can be achieved.

於角質細胞專用之無血清培養基(Keratinocyte-SFM;購自Thermo,產品編號:17005042)中培養人類表皮角質細胞(HPEK-50;購自CELLnTEC)於6-孔盤,2mL培養基的細胞濃度為1.5×105細胞/孔。 Culture human epidermal keratinocytes (HPEK-50; purchased from CELLnTEC) in a serum-free medium for keratinocytes (Keratinocyte-SFM; purchased from Thermo, product number: 17005042) in a 6-well dish, with a cell concentration of 1.5 in 2 mL of medium ×10 5 cells/well.

之後,將細胞分成3組,其中包括1個對照組及2個實驗組(亦即實驗組1與2)。將第1天開花的克魯茲王蓮的萃取物以培養基稀釋為具有0.0625mg/mL及0.03125mg/mL濃度的稀釋液,繼而分別將0.0625mg/mL稀釋液添加至實驗組1的細胞中,及將0.03125mg/mL稀釋液添加至實驗組2的細胞中,至於對照組的細胞則添加培養基。接著,於培養箱中培養各組細胞6小時,接而收取各組細胞培養物並拿來進行基因表現分析。 After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental groups 1 and 2). Dilute the extract of the King Cruz lotus flower blooming on Day 1 with the medium to a dilution with a concentration of 0.0625 mg/mL and 0.03125 mg/mL, and then add the dilution of 0.0625 mg/mL to the cells of Experimental Group 1, respectively, and A dilution of 0.03125 mg/mL was added to the cells of experimental group 2, and the cells of the control group were added with culture medium. Next, cells of each group were cultured in an incubator for 6 hours, and then cell cultures of each group were collected and used for gene expression analysis.

在本實施例中,用來分析與皮膚細胞保濕相關的基因包括角蛋白1(keratin 1,KRT1)基因、角蛋白10(keratin 10,KRT10)基因、角蛋白14(keratin 14,KRT14)基因及透明質酸合成酶2(hyaluronan synthase 2,HAS2)基因。 In this embodiment, the genes used to analyze skin cell moisturization include keratin 1 (keratin 1, KRT1 ) gene, keratin 10 (keratin 10, KRT10 ) gene, keratin 14 (keratin 14, KRT14 ) gene and Hyaluronan synthase 2, HAS2 gene.

以RNA萃取套組(Geneaid)對上面所得到的各組細胞培養物進行RNA的萃取。對由此所得到的各組RNA取2,000ng並以SuperScript® III反轉錄酶(Invitrogen)將萃取出的RNA反轉錄為cDNA。接著,以cDNA作為模版,並且使用用來擴增標的基因的引子對,包括KRT1KRT10KRT14HAS2TBP(作為內部對照組),它們的核苷酸序列顯示於下表2,在StepOne Plus即時PCR系統(ABI)中利用KAPA CYBR FAST qPCR套組(2x)(KAPA Biosystems)來進行定量即時PCR,俾以對標的基因進行擴增及定量。PCR產物的熔化曲線是在定量即時PCR反應期間進行確認。 RNA extraction was performed on the cell cultures of each group obtained above with an RNA extraction kit (Geneaid). Take 2,000 ng of each set of RNA thus obtained and reverse transcribe the extracted RNA into cDNA with SuperScript ® III reverse transcriptase (Invitrogen). Next, using cDNA as a template and using primer pairs to amplify the target gene, including KRT1 , KRT10 , KRT14 , HAS2, and TBP (as internal control groups), their nucleotide sequences are shown in Table 2 below, in StepOne Plus real-time PCR system (ABI) uses KAPA CYBR FAST qPCR kit (2x) (KAPA Biosystems) to perform quantitative real-time PCR to amplify and quantify the target gene. The melting curve of the PCR product is confirmed during the quantitative real-time PCR reaction.

Figure 108102976-A0101-12-0009-2
Figure 108102976-A0101-12-0009-2

標的基因的相對表現量是推導自方程式2-△△Ct,並利用TBP基因(作為內部對照組)及基準基因的循環閾值及藉由標準差來計算相對倍數變化,其中△Ct=Ct目標基因/基準基因-CtTBP,△△Ct=△Ct目標基因-△Ct基準基因,倍數變化=2-△△Ct 平均值。以對照組的標的基因表現量作為1的比較基準。各組之間的統計學顯著差異是藉由單尾史徒登氏t-檢定來決定。本實施例的結果顯示於圖2。 The relative expression of the target gene is derived from Equation 2 -△△Ct , and the cycle threshold of the TBP gene (as an internal control group) and the reference gene and the relative fold change are calculated by standard deviation, where △Ct=Ct target gene / Reference gene- Ct TBP , △△Ct = △Ct target gene-△Ct reference gene , fold change = 2- △△Ct average . The target gene expression amount of the control group was used as the comparison criterion of 1. The statistically significant differences between the groups were determined by the one-tailed Stein's t-test. The results of this example are shown in FIG. 2.

圖2是本發明克魯茲王蓮的萃取物在作用6小時之時提升與皮膚細胞保濕相關的KRT1基因、KRT10基因、KRT14基因及HAS2基因表現上的功效之數據圖。由圖2可見,無論是KRT1基因、KRT10基因、KRT14基因或HAS2基因, 與對照組相較之下,實驗組1的基因相對表現量有顯著的提升。就KRT14基因及HAS2基因而言,與對照組相較之下,實驗組2的基因相對表現量有顯著的提升。本實施例的結果顯示,本發明克魯茲王蓮的萃取物可藉由提升KRT1基因、KRT10基因及KRT14基因的表現量來維持角質細胞排列,使皮膚角質組織完整,防止肌膚水分散失。另一方面,本發明克魯茲王蓮的萃取物可藉由提升HAS2基因的表現量來促進內生性玻尿酸生成,透過吸附水分支撐肌膚,維持肌膚彈性及光澤。 FIG. 2 is a data graph of the effect of the extract of the king lotus of Cruz of the present invention on the performance of KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization at 6 hours of action. As can be seen from Figure 2, whether it is the KRT1 gene, KRT10 gene, KRT14 gene or HAS2 gene, compared with the control group, the relative performance of the gene in the experimental group 1 has been significantly improved. In terms of KRT14 gene and HAS2 gene, compared with the control group, the relative performance of the gene in the experimental group 2 has been significantly improved. The results of this example show that the extract of Kreuz king lotus of the present invention can maintain the arrangement of keratinocytes by enhancing the expression levels of KRT1 gene, KRT10 gene and KRT14 gene to complete the skin keratinous tissue and prevent the skin from losing water. On the other hand, the extract of King Cruz of the present invention can promote the production of endogenous hyaluronic acid by enhancing the expression level of the HAS2 gene, support the skin by adsorbing moisture, and maintain skin elasticity and gloss.

另外,本發明利用濃度為0.03125mg/mL的克魯茲王蓮的萃取物來探討其可否提升KRT1基因、KRT10基因及KRT14基因的表現量,實驗流程同上所述,不同之處在於,萃取物添加至實驗組細胞之後的作用時間是以24小時來取代6小時。本實施例的結果顯示於圖3。 In addition, the present invention utilizes the extract of King Cruz Cruz with a concentration of 0.03125 mg/mL to explore whether it can enhance the performance of the KRT1 gene, KRT10 gene and KRT14 gene. The experimental procedure is the same as above, the difference is that the extract is added to After the cells in the experimental group, the action time was replaced by 24 hours for 6 hours. The results of this example are shown in Figure 3.

圖3是本發明克魯茲王蓮的萃取物在作用24小時之時提升與皮膚細胞保濕相關的KRT1基因、KRT10基因、KRT14基因及HAS2基因表現上的功效之數據圖。由圖3可見,無論是KRT1基因、KRT10基因或KRT14基因,與對照組相較之下,實驗組的基因相對表現量皆有顯著的提升。本實施例的結果顯示,本發明克魯茲王蓮的萃取物在24小時均可藉由提升KRT1基因、KRT10基因及KRT14基因的表現量來維持角質細胞排列,使皮膚角質組織完整,防止肌膚水分散失。 FIG. 3 is a data graph of the effect of the extract of the Royal Lotus of Cruz of the present invention on enhancing the expression of the KRT1 gene, KRT10 gene, KRT14 gene and HAS2 gene related to skin cell moisturization when acting for 24 hours. As can be seen from Figure 3, whether it is the KRT1 gene, KRT10 gene or KRT14 gene, compared with the control group, the relative expression of the gene in the experimental group has significantly improved. The results of this example show that the extract of Kreuz King Lotus of the present invention can maintain the arrangement of keratinocytes by increasing the expression levels of KRT1 gene, KRT10 gene and KRT14 gene in 24 hours, so that the skin keratinous tissue is intact and prevents skin water loss .

綜上所述,本發明克魯茲王蓮的萃取物可藉由提升角蛋白基因(包括KRT1基因、KRT10基因及KRT14基因)及透明質酸合成酶2(HAS2)基因的表現量,透過維持肌膚細胞排列及維持肌膚水分恆定,提升玻尿酸生成,整體提升角質細胞保濕基因表現,達到提升皮膚的保濕能力的功效。 To sum up, the extract of Kreuz King Lotus of the present invention can enhance the expression of keratin genes (including KRT1 gene, KRT10 gene and KRT14 gene) and hyaluronic acid synthase 2 ( HAS2 ) gene by maintaining skin cells Arrange and maintain constant moisture in the skin, increase the production of hyaluronic acid, enhance the overall performance of keratinocyte moisturizing genes, and achieve the effect of enhancing the moisturizing ability of the skin.

以上所述僅為舉例性,而非為限制性者。任何未脫離本發明之精神與範疇,而對其進行之等效修改或變更,均應包含於後附之申請專利範圍中。 The above is only exemplary, and not restrictive. Any equivalent modifications or changes made without departing from the spirit and scope of the present invention shall be included in the scope of the attached patent application.

<110> 大江生醫股份有限公司 <110> Dajiang Biomedical Co., Ltd.

<120> 克魯茲王蓮的萃取物用於提升角蛋白基因及透明質酸合成酶2基因的表現量及提升皮膚的保濕能力之用途 <120> The use of the extract of Cruz Royale to enhance the expression of keratin gene and hyaluronic acid synthase 2 gene and enhance the moisturizing ability of the skin

<130> 107B0592-I1 <130> 107B0592-I1

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Figure 108102976-A0101-12-0011-3
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Figure 108102976-A0101-12-0011-3

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Figure 108102976-A0101-12-0012-4
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Figure 108102976-A0101-12-0012-4

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Figure 108102976-A0101-12-0012-5
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Figure 108102976-A0101-12-0012-5

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Figure 108102976-A0101-12-0012-6
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Figure 108102976-A0101-12-0012-6

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Figure 108102976-A0101-12-0013-7
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Figure 108102976-A0101-12-0013-7

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Figure 108102976-A0101-12-0013-8
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Figure 108102976-A0101-12-0015-12
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Figure 108102976-A0101-12-0015-12

Claims (9)

一種克魯茲王蓮(Victoria cruziana)的花萃取物用於製備一提升角蛋白(keratin,KRT)基因及透明質酸合成酶2(hyaluronan synthase 2,HAS2)基因的表現量之醫藥組成物的用途,其中該克魯茲王蓮的萃取物是以水、醇類、含水醇類或其組合作為一萃取溶劑對該克魯茲王蓮進行萃取而製得。 The use of a flower extract of Victoria cruziana to prepare a pharmaceutical composition that enhances the expression of keratin ( KRT ) gene and hyaluronan synthase 2 ( HAS2 ) gene, Wherein the extract of King Cruz Cruz is prepared by extracting King Lotus Cruz with water, alcohol, water-containing alcohol or a combination thereof as an extraction solvent. 如申請專利範圍第1項所述的用途,其中該克魯茲王蓮與該萃取溶劑的體積比例介於1~5:10~20。 The use as described in Item 1 of the patent application scope, wherein the volume ratio of the Cruz King Lotus to the extraction solvent is between 1 and 5: 10 and 20. 如申請專利範圍第1項所述的用途,其中該KRT基因是KRT1基因、KRT10基因或KRT14基因。 The use as described in item 1 of the patent application range, wherein the KRT gene is a KRT1 gene, a KRT10 gene or a KRT14 gene. 如申請專利範圍第1項所述的用途,其中該克魯茲王蓮的萃取物的有效濃度為至少0.03125mg/mL。 The use as described in item 1 of the scope of the patent application, wherein the effective concentration of the extract of King Cruz Cruz is at least 0.03125 mg/mL. 如申請專利範圍第1項所述的用途,其中該克魯茲王蓮的萃取物為一克魯茲王蓮第1天開花的花萃取物。 The use as described in item 1 of the scope of the patent application, wherein the extract of King Cruz is a flower extract of King Lotus blooming on the first day. 一種克魯茲王蓮的花萃取物用於製備一提升皮膚的保濕能力之醫藥組成物的用途,其中該克魯茲王蓮的萃取物是以水、醇類、含水醇類或其組合作為一萃取溶劑對該克魯茲王蓮進行萃取而製得。 The use of a flower extract of Cruzia crassifolia to prepare a medicinal composition that enhances the skin's moisturizing ability, wherein the extract of Cruzia crassifolia is based on water, alcohols, water-containing alcohols or a combination thereof as an extraction solvent The Cruz King Lotus is prepared by extraction. 如申請專利範圍第6項所述的用途,其中該克魯茲王蓮與該萃取溶劑的體積比例介於1~5:10~20。 The use as described in Item 6 of the patent application scope, wherein the volume ratio of the Cruz King Lotus to the extraction solvent is between 1 and 5: 10 and 20. 如申請專利範圍第6項所述的用途,其中該克魯茲王蓮的萃取物的有效濃度為至少0.03125mg/mL。 The use as described in item 6 of the patent application range, wherein the effective concentration of the extract of Kreuz king lotus is at least 0.03125 mg/mL. 如申請專利範圍第6項所述的用途,其中該克魯茲王蓮的萃取物為一克魯茲王蓮第1天開花的花萃取物。 The use as described in Item 6 of the patent application range, wherein the extract of King Cruz is a flower extract of King Lotus blooming on the first day.
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CN111707836A (en) * 2020-07-08 2020-09-25 无限极(中国)有限公司 Protein chip for skin moisture detection, preparation method, application and detection method thereof

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* Cited by examiner, † Cited by third party
Title
李鵬程,"王蓮類黃酮成分分析",南京農業大學全日制專業學位碩士學位論文,2017年5月28日 *
李鵬程,"王蓮類黃酮成分分析",南京農業大學全日制專業學位碩士學位論文,2017年5月28日。

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111707836A (en) * 2020-07-08 2020-09-25 无限极(中国)有限公司 Protein chip for skin moisture detection, preparation method, application and detection method thereof
CN111707836B (en) * 2020-07-08 2022-08-30 无限极(中国)有限公司 Protein chip for skin moisture detection, preparation method, application and detection method thereof

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