TWI772723B - Use of viola mandshurica extract for anti-aging - Google Patents
Use of viola mandshurica extract for anti-aging Download PDFInfo
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- TWI772723B TWI772723B TW108146492A TW108146492A TWI772723B TW I772723 B TWI772723 B TW I772723B TW 108146492 A TW108146492 A TW 108146492A TW 108146492 A TW108146492 A TW 108146492A TW I772723 B TWI772723 B TW I772723B
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
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Abstract
Description
本發明是有關於一種紫花地丁萃取物用於抗老化之用途。 The present invention relates to an anti-aging application of an extract of Viola vulgaris.
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 the human individual, it has the function of resisting water loss, pathogenic bacteria and various environmental damages. Exposure to a large amount of ultraviolet (ultraviolet, UV), ionizing radiation (ionizing radiation), drugs or xenobiotics (xenobiotics) can stimulate the skin to generate reactive oxygen species (ROS) and free radicals (free radicals). When the amount of accumulated reactive oxygen species and free radicals exceeds the antioxidant capacity of cells or tissues, oxidative stress is formed. Next, reactive oxygen species and free radicals react with intracellular constituents (including DNA, proteins, lipids, etc.), thereby having undesired effects on the skin.
近年來，人類對於提升肌膚活性(例如透過促進皮膚纖維母細胞增生)的需求與日俱增，因為一旦提升肌膚活性，就能夠達到抗老化的效用。然而，目前常見用來提升肌膚活性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肌膚活性的健康食品。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, there has been an increasing demand for enhancing skin activity (for example, by promoting dermal fibroblast proliferation), because once skin activity is enhanced, anti-aging effects can be achieved. However, at present, most common ways to enhance skin activity are to use cosmetics, skin care products applied to the skin surface, or oral health foods that claim to enhance skin activity. However, 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 ordinary users.
另一方面，粒線體(mitochondria)亦被稱為細胞的發電站，因為它是細胞內合成三磷酸腺苷(adenosine triphosphate,ATP)(一種傳遞能量的分子)的主要場所，為細胞的各項活動提供了化學能量。粒線體若損壞，對細胞以及生物個體的影響甚鉅。粒線體在合成ATP的過程中會產生很多的自由基，自由基的活性極強，會與體內任何物質發生強烈的氧化反應而破壞其正常功能。自由 基日積月累地傷害粒線體內的酵素與DNA，漸漸地使其功能下降進而使各器官組織的功能衰退。因此，如何提升細胞的粒線體活性，進而達到抗老化之效用，成為本領域的重要課題。 On the other hand, the mitochondria (mitochondria) is also known as the power station of the cell, because it is the main site for the synthesis of adenosine triphosphate (ATP) (a molecule that transmits energy) in the cell, providing various activities of the cell. chemical energy. If mitochondria are damaged, the impact on cells and individual organisms is huge. During the process of synthesizing ATP, mitochondria will generate a lot of free radicals. The activity of free radicals is extremely strong, and they will have strong oxidation reactions with any substances in the body and destroy their normal functions. free Accumulated damage to the enzymes and DNA in mitochondria will gradually reduce the function of each organ and tissue. Therefore, how to enhance the mitochondrial activity of cells to achieve the effect of anti-aging has become an important topic in the field.
為了解決上述問題，本領域的技術人員亟需研發出具有促進皮膚纖維母細胞增生、提升皮膚細胞活性、提升細胞的粒線體活性及抗老化效用的新穎醫藥品、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 problems, those skilled in the art urgently need to develop novel medicines, food products or skin care products with the functions of promoting skin fibroblast proliferation, enhancing skin cell activity, enhancing mitochondrial activity of cells and anti-aging effects for the benefit of A large group of people with this need.
有鑑於此，本發明之目的為提供一種紫花地丁(Viola mandshurica)萃取物用於製備一抗老化之組成物的用途，其中該紫花地丁萃取物係以一溶劑萃取一紫花地丁所獲得，該溶劑為水、醇、或醇水混合物。 In view of this, the purpose of the present invention is to provide the use of a Viola mandshurica extract for preparing an anti-aging composition, wherein the Viola mandshurica extract is obtained by extracting a Viola mandshurica with a solvent , the solvent is water, alcohol, or mixture of alcohol and water.
在本發明的一實施例中，該抗老化包括促進皮膚纖維母細胞增生、提升皮膚細胞活性、及提升細胞的粒線體活性。 In one embodiment of the present invention, the anti-aging comprises promoting skin fibroblast proliferation, enhancing skin cell activity, and enhancing cellular mitochondrial activity.
在本發明的一實施例中，該細胞是一皮膚纖維母細胞。 In one embodiment of the invention, the cell is a dermal fibroblast.
在本發明的一實施例中，該紫花地丁萃取物的有效濃度為至少0.03125mg/mL。 In one embodiment of the present invention, the effective concentration of the extract of Radix Radix et Rhizoma is at least 0.03125 mg/mL.
在本發明的一實施例中，該萃取是於一介於50~100℃的溫度進行。 In an embodiment of the present invention, the extraction is performed at a temperature ranging from 50°C to 100°C.
在本發明的一實施例中，該溶劑與該紫花地丁的體積比介於10~20：1~5。 In an embodiment of the present invention, the volume ratio of the solvent to the violetidine is 10-20:1-5.
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 present invention, the composition is a medicine, a food product or a skin care product.
在本發明的一實施例中，該醫藥品包含一醫藥上可接受的載劑。 In one embodiment of the present invention, the pharmaceutical product comprises a pharmaceutically acceptable carrier.
在本發明的一實施例中，該醫藥品是呈一供局部投藥的劑型。 In one embodiment of the present invention, the medicinal product is in a dosage form for topical administration.
在本發明的一實施例中，該保養品是呈一供局部施用的形式。 In one embodiment of the present invention, the skin care product is in a form for topical application.
綜上所述，本發明紫花地丁萃取物之功效在於：可藉由促進皮膚纖維母細胞增生及提升細胞的粒線體活性，達到維持細胞活力及正常代謝、提升皮膚細胞活性及抗老化的功效。 To sum up, the effect of the extract of the present invention is: by promoting the proliferation of skin fibroblasts and improving the mitochondrial activity of the cells, it can maintain cell vitality and normal metabolism, improve skin cell activity and anti-aging. effect.
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 following examples are used to illustrate the present invention, but not to limit the scope of the present invention. Anyone who is familiar with this technique, without departing from the spirit and scope of the present invention, Some changes and modifications can be made, so the protection scope of the present invention should be determined by the scope of the appended patent application.
圖1是本發明紫花地丁萃取物在作用24小時後促進皮膚纖維母細胞增生上的功效之數據圖，其中“**”表示與對照組比較，p<0.01。 Fig. 1 is a data graph of the efficacy of the extract of the present invention in promoting the proliferation of skin fibroblasts after 24 hours of action, wherein "**" means compared with the control group, p < 0.01.
圖2是本發明紫花地丁萃取物在作用24小時後提升皮膚纖維母細胞的粒線體活性上之效用的數據圖，其中“**”表示與對照組比較，p<0.01。 Figure 2 is a data graph of the effect of the extract of the present invention on the improvement of mitochondrial activity of skin fibroblasts after 24 hours of action, wherein "**" indicates that compared with the control group, p < 0.01.
本文中所使用數值為近似值，所有實驗數據皆表示在20%的範圍內，較佳為在10%的範圍內，最佳為在5%的範圍內。 Numerical values used herein are approximations and all experimental data are expressed within 20%, preferably within 10%, and most preferably within 5%.
使用Excel軟體進行統計分析。數據以平均值±標準差(standard deviation,STDEV)表示，個此之間的差異以學生t檢驗(student's t-test)分析。 Statistical analysis was performed using Excel software. Data were expressed as mean ± standard deviation (STDEV), and differences between them were analyzed by Student's t -test .
依據本發明，紫花地丁(Viola mandshurica)是一種堇菜科(Violaceae)的多年生草本植物。在中醫領域中以全草入藥，性寒、味苦辛，有清熱解毒及涼血消腫功能。 According to the present invention, Viola mandshurica is a perennial herb of the Violaceae family. In the field of traditional Chinese medicine, the whole herb is used as medicine, which is cold in nature, bitter in taste, and has the functions of clearing away heat and detoxifying, cooling blood and reducing swelling.
如本文中所使用的，用語「抗老化(anti-aging)」意指預防、減緩人類皮膚外觀之老化現象，例如：皺紋的產生及失去彈性等。評量實現此目的之程度將根據熟悉此項技藝者已知之諸多因素來決定，諸如消費者的全身狀態、年齡、性別等。 As used herein, the term "anti-aging" means preventing, slowing down the appearance of aging phenomena of human skin, such as the development of wrinkles and loss of elasticity. The extent to which this is assessed will depend upon a number of factors known to those skilled in the art, such as the general condition of the consumer, age, gender, and the like.
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)、口服地(orally)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)、錠劑(tablet)、片劑(troche)、***錠(lozenge)、丸劑(pill)、膠囊(capsule)、分散性粉末(dispersible powder)或細顆粒(granule)、溶液、懸浮液(suspension)、乳劑(emulsion)、糖漿(syrup)、酏劑(elixir)、濃漿(slurry)、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, orally or topically, using techniques well known to those skilled in the art, including, but not limited to: injection [eg, sterile aqueous solution or dispersion], sterile powder, tablet, troche, oral Lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixirs (elixir), slurry, 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 comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may comprise one or more agents 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 agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.
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 comprises a solvent selected from the group consisting of water, normal saline, phosphate buffered saline (PBS), Aqueous solutions 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：腹膜內注射(intraperitoneal injection)、皮下注射(subcutaneous injection)、表皮內注射(intraepidermal injection)、皮內注射(intradermal injection)、肌肉內注射(intramuscular injection)、靜脈內注射(intravenous injection)以及病灶內注射(intralesional injection)。 According to the present invention, the medicinal product may be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intradermal injection Intraepidermal injection, intradermal injection, intramuscular injection, intravenous 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, the medicinal product may be manufactured into an external preparation suitable for topical application to the skin using techniques well known to those skilled in the art, including, but not limited to: emulsions, gels 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 medicinal 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 comprise one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons (such as petroleum, jelly) and white petrolatum], waxes [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers ( absorption enhancers), stabilizing agents, gelling agents [such as Carbopol ® 974P (carbopol ® 974P), microcrystalline cellulose and carboxymethylcellulose], Active agents, humectants, odor absorbers, fragrances, pH adjusting agents, chelating agents, emulsifiers, occlusive agents occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti-irritants, colorants and propellants (propellants) etc. The selection and quantity of these additives are within the professional and routine skills of those skilled in the art.
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 skincare product may further comprise an acceptable adjuvant which is widely used in skincare product manufacturing techniques. 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 agents, thickening agents, fillers, fragrances and odor absorbers. The selection and quantity of these reagents are within the scope of the expertise and routine skills of those skilled in the art.
依據本發明，保養品可利用熟習此技藝者所詳知的技術而被製造成一適合於護膚(skincare)或化妝(makeup)的形式，這包括，但不限於：水性溶液(aqueous solution)、水-醇溶液(aqueous-alcohol solution)或油性溶液(oily solution)、呈水包油型(oil-in-water type)、油包水型(water-in-oil type)或複合型之乳劑、凝膠、軟膏、乳霜、面膜(mask)、貼片、貼布(pack)、擦劑、粉末、氣溶膠、噴霧、乳液、乳漿、糊劑、泡沫、分散液、滴劑、慕斯(mousse)、防曬油(sunblock)、化妝水(tonic water)、粉底(foundation)、卸妝產品(makeup remover products)、肥皂(soap)以及其他身體清潔產品(body cleansing products)等。 According to the present invention, the skin care product can be manufactured into a form suitable for skincare or makeup using techniques well known to those skilled in the art, including, but not limited to: aqueous solution, water -Aqueous-alcohol solution or oily solution, emulsion in oil-in-water type, water-in-oil type or complex type, coagulation glue, ointment, cream, mask, patch, pack, liniment, powder, aerosol, spray, lotion, serum, paste, foam, dispersion, drops, mousse ( mousse), sunblock, tonic water, foundation, makeup remover products, soap and other body cleansing products.
依據本發明，保養品亦可與一或多種選自於下列之已知活性的外用劑(external use agents)一起合併使用：美白劑(whitening agents)[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 may also be used in combination with one or more external use agents with known activity selected from the group consisting of whitening agents (such as tretinoin, catechins) (catechin), koji acid, arbutin and vitamin C], humectants, anti-inflammatory agents, bactericides, ultraviolet absorbers, plant extracts[ such as aloe extract], skin nutrients, anesthetics, anti-acne agents, antipruritics, analgesics, anti-inflammatory agents antidermatitis agents, antihyperkeratolytic 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 topical preparations fall within the scope of the professionalism and routine skills of those skilled in the art.
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, the food product can be regarded as a food additive, which is added during the preparation of raw materials by conventional methods, or added during the production process of the food, and is 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, types of food products include, but are not limited to, beverages, fermented foods, bakery products, health foods, and dietary supplements.
首先，對紫花地丁(Viola mandshurica)，來源為中國陝西產的紫花地丁，購買來源為：台灣的欣大農產行，進行均質處理，然後於50~100℃下，以溶劑對經均質處理的紫花地丁以10~20：1~5的體積比進行萃取0.5~2小時而得到一粗萃取物，其中溶劑是水、醇、或醇水混合物。接著，冷卻至室溫，然後以400網目(mesh)的濾網對粗萃取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紫花地丁萃取物。 First, the Viola mandshurica ( Viola mandshurica ), sourced from Viola mandshurica produced in Shaanxi, China, purchased from: Xinda Agricultural Production Co., Ltd. in Taiwan, was subjected to homogenization treatment, and then homogenized with a solvent at 50~100 ° C. The treated violacean is extracted at a volume ratio of 10-20:1-5 for 0.5-2 hours to obtain a crude extract, wherein the solvent is water, alcohol, or a mixture of alcohol and water. Next, it was cooled to room temperature, and then the crude extract was filtered through a 400 mesh filter to obtain a filtrate, and then the filtrate was concentrated under reduced pressure at 45-70° C. to obtain the extract of Radix Radix et Rhizoma.
首先，以添加有0.1mM非必需胺基酸、1.5g/L碳酸氫鈉(sodium bicarbonate)、1mM丙酮酸鈉(sodium pyruvate)(佔比90%)、及10%胎牛血清(FBS)(Gibco)的最低必需培養基(Minimum essential medium,MEM)(Eagle)(配於厄爾平衡鹽溶液(Earle's Balanced Salt Solution,Earle's BSS))(GIBCO公司，編號41500-034，美國)培養人類皮膚纖維母細胞CCD-966SK(購自台灣生物資源保存及研究中心(Bioresource Collection and Research Center,BCRC)，編號BCRC 60153)於6-孔盤，細胞濃度為1×105細胞/孔。 First, add 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate, 1 mM sodium pyruvate (90%), and 10% fetal bovine serum (FBS) ( Gibco) minimum essential medium (Minimum essential medium, MEM) (Eagle) (mixed with Earle's Balanced Salt Solution, Earle's BSS) (GIBCO Company, No. 41500-034, USA) to culture human skin fibroids Cells CCD-966SK (purchased from Taiwan Bioresource Collection and Research Center, BCRC, No. BCRC 60153) were placed in a 6-well plate at a cell concentration of 1×10 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(即實驗組1及實驗組2)。將0.03125mg/mL紫花地丁萃取物添加至實驗組1的細胞中，及將0.0625mg/mL紫花地丁萃取物添加至實驗組2的細胞中並作用24小時。至於對照組的細胞則不做任何處理。接著，以10μM EdU(組分A，即5-乙炔基-2'-脫氧尿苷(5-ethynyl-2’-deoxyuridine)，其為一種胸苷類似物，可摻入***中的細胞)於培養基中培養細胞1至2小時，然後收取細胞。之後，以1%牛血清白蛋白(Bovine serum albumin,BSA)(配於磷酸鹽緩衝液(phosphate buffered saline，PBS，GIBCO公司，編號14200-075，美國)中)清洗細胞1次，然後移除上清液。接著，添加100μL的Click-iTTM固定劑(Click-iTTM fixative)(組分D，含有4%三聚甲醛(paraformaldehyde)溶於PBS中)並充分混合，於室溫黑暗環境中作用15分鐘。之後，以1% BSA(配於PBS中)清洗細胞1次，然後移除上清液。接著，添加100μL的1X Click-iTTM以皂苷為基礎的透化與洗滌試劑(Click-iTTM saponin-based permeabilization and wash reagent)(組分E)，於室溫黑暗環境中作用15分鐘。之後，以1% BSA(配於PBS中)清洗細胞1次，然後移除上清液。接著，將細胞再懸浮於 100μL的1X Click-iTTM以皂苷為基礎的透化與洗滌試劑中，然後進行Click-iTTM反應。 After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental group 1 and experimental group 2). 0.03125 mg/mL Radix radix extract was added to the cells of experimental group 1, and 0.0625 mg/mL Radix Radix Radix was added to the cells of experimental group 2 and acted for 24 hours. As for the cells of the control group, no treatment was performed. Next, 10 μM EdU (component A, 5-ethynyl-2'-deoxyuridine, a thymidine analog that can be incorporated into dividing cells) was added to Cells are grown in medium for 1 to 2 hours, and then harvested. After that, the cells were washed once with 1% Bovine serum albumin (BSA) (in phosphate buffered saline (PBS, GIBCO, No. 14200-075, USA)), and then removed supernatant. Next, 100 μL of Click-iT TM fixative ( Component D, containing 4% paraformaldehyde in PBS) was added and mixed well, and allowed to act in the dark at room temperature for 15 minutes . Afterwards, cells were washed once with 1% BSA (in PBS) and the supernatant was removed. Next, 100 μL of 1X Click-iT TM saponin-based permeabilization and wash reagent (Component E ) was added and allowed to act in the dark at room temperature for 15 minutes. Afterwards, cells were washed once with 1% BSA (in PBS) and the supernatant was removed. Next, cells were resuspended in 100 μL of 1X Click-iT ™ Saponin-based Permeabilization and Washing Reagent prior to Click-iT ™ reaction.
Click-iTTM反應流程如下：首先製備1X Click-iTTMEdU緩衝添加劑(buffer additive)(組分G)(在-20℃下以2mL的低氘水(deuterium depleted water,DDW)稀釋10X緩衝液)。接著，製備Click-iTTM Plus反應混合物(reaction cocktails)，包括PBS(或杜貝可氏磷酸鹽緩衝液(Dulbecco's phosphate-buffered saline,D-PBS)或TriS-Hcl鹽緩衝液(Tris-Hcl Buffered Saline,TBS))、銅保護劑(copper protectant)(組分F)、光染料疊氮甲基吡啶(fluorescent dye picolyl azide)(組分B)、及反應緩衝添加劑(於15分鐘內製備)。之後，於每個試管添加0.1mL的Click-iTTM Plus反應混合物(總體積200μL)，然後於室溫黑暗環境中作用30分鐘。接著，以1X Click-iTTM以皂苷為基礎的透化與洗滌試劑清洗細胞，然後移除上清液。之後，將細胞再懸浮於500μL的1X Click-iTTM以皂苷為基礎的透化與洗滌試劑中。接著，以流式細胞儀進行分析，激發(excitation)波長為488nm，發射(emission)波長為530/30nm，以對數放大(logarithmic amplification)進行偵測。本實施例的結果顯示於圖1。 The Click-iT ™ reaction scheme is as follows: First prepare 1X Click-iT ™ EdU buffer additive (component G) (dilute 10X buffer with 2 mL of deuterium depleted water (DDW) at -20°C ). Next, click-iT ™ Plus reaction cocktails were prepared, including PBS (or Dulbecco's phosphate-buffered saline (D-PBS) or TriS-Hcl Buffered saline (D-PBS) Saline, TBS)), copper protectant (component F), fluorescent dye picolyl azide (component B), and reaction buffer additives (prepared within 15 minutes). After that, 0.1 mL of Click-iT ™ Plus reaction mixture (total volume 200 μL) was added to each test tube and allowed to react in the dark at room temperature for 30 minutes. Next, cells were washed with 1X Click-iT ™ Saponin-based Permeabilization and Washing Reagent, and the supernatant was removed. Afterwards, cells were resuspended in 500 μL of 1X Click-iT ™ Saponin-based Permeabilization and Washing Reagent. Next, the analysis was performed by flow cytometry, the excitation wavelength was 488 nm, the emission wavelength was 530/30 nm, and the detection was performed by logarithmic amplification. The results of this example are shown in FIG. 1 .
圖1是本發明紫花地丁萃取物在作用24小時後促進皮膚纖維母細胞增生上的功效之數據圖。由圖1可見，與對照組相較之下，實驗組1及實驗組2的相對EdU結合(relative EdU incorporation)倍數有顯著的增加。本實施例的結果顯示，本發明紫花地丁萃取物在24小時後可促進皮膚纖維母細胞增生，提升肌膚細胞活性，藉此達到提升皮膚細胞活性及抗老化的功效。 Fig. 1 is a data graph of the efficacy of the extract of the present invention in promoting the proliferation of skin fibroblasts after 24 hours of action. It can be seen from FIG. 1 that compared with the control group, the relative EdU incorporation multiples of experimental group 1 and experimental group 2 were significantly increased. The results of this example show that the extract of the present invention can promote the proliferation of skin fibroblasts and enhance the activity of skin cells after 24 hours, thereby achieving the effects of enhancing skin cell activity and anti-aging.
本實施例以人類皮膚纖維母細胞進行皮膚細胞粒線體活性分析，並利用流式細胞儀粒線體膜電位偵測套組(Flow cytometry Mitochrondrial membrane potential detection kit,BD)進行實驗。人類皮膚纖維母細胞購自台灣生物資源保存及研究中心(Bioresource Collection and Research Center,BCRC)，編號BCRC 60153。將該細胞培養於添加10%胎牛血清(fetal bovine serum，FBS)(GIBCO公司，編號10438-026，美國)、0.1mM非必需胺基酸、1.5g/L碳酸氫鈉(Sigma公司，編號S5761，美國)、1mM丙酮酸鈉(GIBCO公司，編號 11360-070，美國)的最低必需培養液(Minimum essential medium,MEM)(Eagle)(配於厄爾平衡鹽溶液(Earle's Balanced Salt Solution,Earle's BSS))(GIBCO公司，編號41500-034，美國)。 In this example, human skin fibroblasts were used to analyze the mitochondrial activity of skin cells, and the flow cytometry Mitochrondrial membrane potential detection kit (BD) was used to conduct experiments. Human skin fibroblasts were purchased from Taiwan Bioresource Collection and Research Center (BCRC), number BCRC 60153. The cells were cultured in supplemented with 10% fetal bovine serum (FBS) (GIBCO, No. 10438-026, USA), 0.1 mM non-essential amino acids, 1.5 g/L sodium bicarbonate (Sigma, No. S5761, USA), 1 mM sodium pyruvate (GIBCO, No. 11360-070, USA) minimum essential medium (Minimum essential medium, MEM) (Eagle) (in Earle's Balanced Salt Solution, Earle's BSS) (GIBCO, No. 41500-034, USA) .
在含有2mL上述培養基的6孔培養盤中每個孔洞接種1×105個人類皮膚纖維母細胞(n=3)。之後，將細胞分成3組，其中包括1個對照組及2個實驗組(即實驗組1及實驗組2)。將0.03125mg/mL紫花地丁萃取物添加至實驗組1的細胞中，及將0.0625mg/mL紫花地丁萃取物添加至實驗組2的細胞中。至於對照組的細胞則添加培養基。接著，將各組細胞於37℃下培養24小時，然後於37℃下預熱10X分析緩衝液。之後，以無菌的1X PBS製備1X分析緩衝液，混合均勻並置於37℃，然後添加130μL的二甲基亞碸(Dimethyl sulfoxide,DMSO)至凍乾JC-1粒線體染劑以製備JC-1儲備溶液，儲備溶液可儲存於-20℃歷時6個月。接著，將JC-1粒線體染劑與1X分析緩衝液以1：100的比例製備工作溶液，然後移除培養基並以1XPBS潤洗兩次。之後，加入胰蛋白酶(trypsin)/EDTA處理3分鐘後，吸取懸浮的細胞至1.5mL的微離心管，以400g轉速離心5分鐘收集沈澱的細胞。 1 x 105 human dermal fibroblasts (n=3) were seeded per well in a 6-well culture dish containing 2 mL of the above medium. After that, the cells were divided into 3 groups, including 1 control group and 2 experimental groups (ie, experimental group 1 and experimental group 2). 0.03125 mg/mL Radix radix extract was added to the cells of experimental group 1, and 0.0625 mg/mL Radix Radix radix extract was added to the cells of experimental group 2. As for the cells of the control group, culture medium was added. Next, groups of cells were incubated at 37°C for 24 hours and then pre-warmed with 10X assay buffer at 37°C. Afterwards, prepare 1X assay buffer with sterile 1X PBS, mix well and place at 37°C, then add 130 μL of Dimethyl sulfoxide (DMSO) to lyophilized JC-1 mitochondrial dye to prepare JC- 1 stock solution, the stock solution can be stored at -20°C for 6 months. Next, a working solution of JC-1 mitochondrial stain and 1X assay buffer was prepared at a ratio of 1:100, then the medium was removed and rinsed twice with 1X PBS. Then, after adding trypsin/EDTA for 3 minutes, the suspended cells were pipetted into a 1.5 mL microcentrifuge tube, and the precipitated cells were collected by centrifugation at 400 g for 5 minutes.
在移除上清液後，以1mL的1X PBS再懸浮細胞，然後轉移至1.5mL的離心管，以400g轉速離心5分鐘。在移除上清液後，添加100μL的JC-1工作溶液，混合均勻後在避光下作用15分鐘。之後，以400g轉速離心5分鐘，再以1mL的1X清洗緩衝液清洗並以400g轉速離心5分鐘，然後以1mL的1X清洗緩衝液清洗並以400g轉速離心5分鐘。以含有2%FBS的500μL之1X PBS再懸浮細胞，然後利用流式細胞儀(Beckman)分析觀察細胞凋亡時粒線體膜電位改變，並以Excel進行t-檢驗(student t-test)統計分析樣品群體之間差異的統計學意義。 After removing the supernatant, cells were resuspended in 1 mL of IX PBS, then transferred to a 1.5 mL centrifuge tube and centrifuged at 400 g for 5 min. After removing the supernatant, add 100 μL of the JC-1 working solution, mix well, and act in the dark for 15 minutes. Afterwards, centrifuge at 400g for 5 minutes, wash with 1 mL of 1X wash buffer and centrifuge at 400g for 5 minutes, then wash with 1 mL of 1X wash buffer and centrifuge at 400g for 5 minutes. Cells were resuspended with 500 μL of 1X PBS containing 2% FBS, and then the mitochondrial membrane potential changes during apoptosis were observed by flow cytometry (Beckman) analysis, and t -test (student t -test) statistics were performed with Excel Statistical significance of differences between sample populations was analyzed.
圖2是本發明紫花地丁萃取物在作用24小時後提升皮膚纖維母細胞的粒線體活性上之效用的數據圖。由圖2可見，與對照組相較之下，實驗組1及實驗組2的皮膚纖維母細胞的粒線體活性(即相對的JC-1聚合體(aggregate))有顯著提升。本實施例的結果顯示，本發明紫花地丁萃取物在刺激人類皮膚纖維母細胞24小時後，可以顯著提升細胞粒線體活性，維持細胞活力及正常代謝，達到抗老之潛力。 Fig. 2 is a data graph showing the effect of the extract of the present invention on the mitochondrial activity of dermal fibroblasts after acting for 24 hours. It can be seen from Figure 2 that, compared with the control group, the mitochondrial activity (ie, the relative JC-1 aggregate) of the skin fibroblasts in the experimental group 1 and the experimental group 2 was significantly improved. The results of this example show that, after stimulating human skin fibroblasts for 24 hours, the extract of the present invention can significantly enhance the mitochondrial activity of cells, maintain cell vitality and normal metabolism, and achieve anti-aging potential.
綜上所述，本發明紫花地丁萃取物可藉由促進皮膚纖維母細胞增生及提升細胞的粒線體活性，達到維持細胞活力及正常代謝、提升皮膚細胞活性及抗老化的功效。 To sum up, the extract of the present invention can achieve the effects of maintaining cell vitality and normal metabolism, enhancing skin cell activity and anti-aging by promoting skin fibroblast proliferation and enhancing mitochondrial activity of cells.
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 description is exemplary only, not limiting. Any equivalent modifications or changes that do not depart from the spirit and scope of the present invention shall be included in the appended patent application scope.
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|---|---|
| TWI819235B (en) | 2023-10-21 |
| CN112386643A (en) | 2021-02-23 |
| TW202106330A (en) | 2021-02-16 |
| TW202106324A (en) | 2021-02-16 |
| CN112386665B (en) | 2022-05-06 |
| CN115227774A (en) | 2022-10-25 |
| CN112386627A (en) | 2021-02-23 |
| TWI729618B (en) | 2021-06-01 |
| CN112386655A (en) | 2021-02-23 |
| TW202106328A (en) | 2021-02-16 |
| CN112386534A (en) | 2021-02-23 |
| TWI809299B (en) | 2023-07-21 |
| TW202106323A (en) | 2021-02-16 |
| TW202106329A (en) | 2021-02-16 |
| CN112386665A (en) | 2021-02-23 |
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