TW202010507A - Use of extract of rhinacanthus communis callus tissue for inducing expression of collagen type i gene, eln gene, has3 gene, xrcc1 gene, msh gene, sod1 gene, gpx1 gene, cat gene, tgm gene and aqp gene, promoting collagen production, and anti-oxidation, method for inducing and proliferating rhinacanthus communis callus tissue, and medium for culturing rhinacanthus communis callus tissue - Google Patents
Use of extract of rhinacanthus communis callus tissue for inducing expression of collagen type i gene, eln gene, has3 gene, xrcc1 gene, msh gene, sod1 gene, gpx1 gene, cat gene, tgm gene and aqp gene, promoting collagen production, and anti-oxidation, method for inducing and proliferating rhinacanthus communis callus tissue, and medium for culturing rhinacanthus communis callus tissue Download PDFInfo
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- TW202010507A TW202010507A TW107131942A TW107131942A TW202010507A TW 202010507 A TW202010507 A TW 202010507A TW 107131942 A TW107131942 A TW 107131942A TW 107131942 A TW107131942 A TW 107131942A TW 202010507 A TW202010507 A TW 202010507A
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- callus
- ganoderma lucidum
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Abstract
Description
本發明是有關於一種白鶴靈芝(Rhinacanthus communis)癒傷組織的萃取物用於提升膠原蛋白第I型基因、ELN基因、HAS3基因、XRCC1基因、MSH基因、SOD1基因、GPX1基因、CAT基因、TGM基因、AQP基因及GBA基因的表現量、促進膠原蛋白產生及抗氧化之用途、用於誘導及增殖白鶴靈芝癒傷組織的方法、及用於培養白鶴靈芝癒傷組織的培養基。 The present invention relates to an extract of Rhinacanthus communis callus for raising collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene, TGM The expression level of genes, AQP gene and GBA gene, the use of promoting collagen production and anti-oxidation, the method for inducing and propagating the callus of Ganoderma lucidum, and the medium for cultivating the callus of Ganoderma lucidum.
皮膚組織是由表皮、真皮及皮下組織所構成,其中真皮含有大量膠原蛋白及玻尿酸,而與皮膚之保水性及彈力息息相關。人類皮膚會隨著年紀、生理因素或環境因素,而有老化、膚質粗糙或產生皺紋等現象,如正常年 輕人的皮膚都具一定的彈性和張力,當表情肌鬆弛後,皮膚會很快復原,使皺紋消失;但進入中年後,皮膚開始明顯老化,皮膚變薄、變硬、乾燥、張力降低;真皮膠原蛋白減少、彈力纖維變性、斷裂,使皮膚的張力和彈性降低,因此,當表情肌鬆弛後,皮膚不能很快復原,久之則使皺紋成形;且隨年齡的增大,皮膚和皮下組織更加鬆弛,加上面部支持組織的萎縮或缺失,以及肌肉的鬆軟,皮膚會在重力的作用下發生滑墜,形成更深的皺紋。而肌膚粗糙係由於乾燥、紫外線、清潔劑或化學物質等刺激性物質等外在重要因素,或激素平衡之紊亂等內在重要因素而產生之肌膚困擾,並伴隨角質層屏障功能之下降、角質層水分量之下降、表皮代謝回轉之亢進、鱗屑之產生而角質粗糙化等現象。因此皮膚上的細胞若失去彈性及保濕功能,會引起皮膚褶皺、乾燥及失去光澤等現象。 Skin tissue is composed of epidermis, dermis and subcutaneous tissue. The dermis contains a lot of collagen and hyaluronic acid, which is closely related to the skin's water retention and elasticity. Human skin will have aging, rough skin or wrinkles according to age, physiological factors or environmental factors. For example, the skin of normal young people has certain elasticity and tension. When the expression muscles relax, the skin will quickly Recovery, making wrinkles disappear; but after entering middle age, the skin begins to age significantly, the skin becomes thinner, harder, dry, and tension is reduced; dermal collagen is reduced, elastic fibers are degenerated, broken, and the skin's tension and elasticity are reduced, therefore, When the expression muscles are relaxed, the skin cannot recover quickly, and over time it will form wrinkles; and as the age increases, the skin and subcutaneous tissues become more relaxed, plus the atrophy or loss of facial support tissues, and the softness of the muscles, the skin will be in Under the effect of gravity, a slip occurs, forming deeper wrinkles. Rough skin is caused by externally important factors such as dryness, ultraviolet rays, detergents, chemical substances and other irritating substances, or internally important factors such as hormonal balance disorder, and is accompanied by a decrease in the barrier function of the stratum corneum Phenomenon such as the decrease of water content, the increase of epidermal metabolic rotation, the generation of scales and the roughening of keratin. Therefore, if the cells on the skin lose their elasticity and moisturizing function, they will cause skin wrinkles, dryness and loss of luster.
目前常見用來解決皮膚問題的方式大多為利用塗抹於皮膚表面的化妝品、保養品,或口服具有抗氧化效用的健康食品。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。 At present, most of the common methods used to solve skin problems are to use cosmetics, skin care products applied to the skin surface, or oral health foods with antioxidant effects. 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促進膠原蛋白產生、抗氧化、保濕、及抗老化等功效的新穎醫藥品、化妝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, cosmetics, or maintenance products with the effects of promoting collagen production, antioxidant, moisturizing, and anti-aging effects, 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白鶴靈芝(Rhinacanthus communis)癒傷組織的萃取物用於製備一提升膠原蛋白第I型(collagen type I)基因、彈力蛋白(elastin,ELN)基因、玻尿酸合成酶3(hyaluronan synthase 3,HAS3)基因、X射線修復交叉互補蛋白1(X-ray repair cross complementary protein 1,XRCC1)基因、MSH基因、超氧化物歧化酶1(superoxide dismutase 1,SOD1)基因、榖胱甘肽過氧化酶1(glutathione peroxidase 1,GPX1)基因、氯黴素乙醯基轉移酶(chloramphenicol acetyltransferase,CAT)基因、轉穀胺醯胺酶(transglutaminase,TGM)基因、水通道蛋白(aquaporin,AOP)基因,及GBA基因的表現量之醫藥品、化妝品或保養品的用途,其中白鶴靈芝癒傷組織的萃取物是以水或醇類作為萃 取溶劑對白鶴靈芝癒傷組織進行萃取而製得,白鶴靈芝癒傷組織與萃取溶劑的體積比例介於1:5至1:10,且萃取的溫度介於60℃至80℃。 In view of this, the object of the present invention is to provide an extract of the callus of Rhinacanthus communis for the preparation of a collagen type I gene, elastin (ELN) gene, hyaluronic acid Synthase 3 (hyaluronan synthase 3, HAS3) gene, X-ray repair cross complementary protein 1 (XRCC1) gene, MSH gene, superoxide dismutase 1 (superoxide dismutase 1, SOD1) gene , Glutathione peroxidase 1, GPX1 gene, chloramphenicol acetyltransferase (CAT) gene, transglutaminase (TGM) gene, aquaporin (aquaporin, AOP) gene, and the use of medicinal products, cosmetics or maintenance products of the expression level of GBA gene, in which the extract of Baihe Ganoderma lucidum callus extracts the callus of Baihe Ganoderma lucidum with water or alcohol as the extraction solvent The volume ratio of the callus of Ganoderma lucidum to the extraction solvent is between 1:5 and 1:10, and the extraction temperature is between 60°C and 80°C.
本發明之另一目的為提供一種白鶴靈芝癒傷組織的萃取物用於製備一促進膠原蛋白產生及抗氧化之醫藥品、化妝品或保養品的用途,其中白鶴靈芝癒傷組織的萃取物是以水或醇類作為萃取溶劑對白鶴靈芝癒傷組織進行萃取而製得,白鶴靈芝癒傷組織與萃取溶劑的體積比例介於1:5至1:10,且萃取的溫度介於60℃至80℃。 Another object of the present invention is to provide an extract of Baihe Ganoderma lucidum callus for the preparation of a medicinal product, cosmetic or maintenance product that promotes collagen production and anti-oxidation, wherein the extract of Baihe Ganoderma lucidum callus is Water or alcohol is used as the extraction solvent to extract Baihe Ganoderma lucidum callus, the volume ratio of Baihe Ganoderma lucidum callus to extraction solvent is between 1:5 and 1:10, and the extraction temperature is between 60℃ and 80 ℃.
本發明之另一目的為提供一種用於誘導及增殖一白鶴靈芝癒傷組織的方法,包含使用一添加2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid)及6-苯胺嘌呤(6-benzyladenine)的MS培養基(Murashige and Skoog medium)來培養白鶴靈芝癒傷組織。 Another object of the present invention is to provide a method for inducing and proliferating a callus of Ganoderma lucidum, which includes the use of an addition of 2,4-dichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid) and 6-aniline purine ( 6-benzyladenine) MS medium (Murashige and Skoog medium) to cultivate the white crane Ganoderma lucidum callus.
本發明之另一目的為提供一種用於培養一白鶴靈芝癒傷組織的培養基,包含2,4-二氯苯氧乙酸及6-苯胺嘌呤。 Another object of the present invention is to provide a culture medium for culturing a callus of Ganoderma lucidum, which contains 2,4-dichlorophenoxyacetic acid and 6-aniline purine.
在本發明的一實施例中,白鶴靈芝癒傷組織是經由添加2,4-二氯苯氧乙酸及6-苯胺嘌呤的MS培養基培養而得。 In one embodiment of the present invention, the callus of Ganoderma lucidum is obtained by culturing with MS medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-aniline purine.
在本發明的一實施例中,MS培養基包含3%蔗糖及0.8%瓊脂。 In an embodiment of the invention, the MS medium contains 3% sucrose and 0.8% agar.
在本發明的一實施例中,膠原蛋白第I型基因為膠原蛋白第I型,α 1(collagen type I,alpha 1,COL1A1)基因或膠原蛋白第I型,α 2(collagen type I,alpha 2,COL1A2)基因。 In an embodiment of the present invention, the collagen type I gene is collagen type I, α 1 (collagen type I, alpha 1, COL1A1) gene or collagen type I, α 2 (collagen type I,
在本發明的一實施例中,MSH基因為MSH2基因或MSH6基因。 In an embodiment of the invention, the MSH gene is the MSH2 gene or the MSH6 gene.
在本發明的一實施例中,TGM基因為TGM1基因。 In an embodiment of the invention, the TGM gene is the TGM1 gene.
在本發明的一實施例中,AQP基因為AQP3基因。 In an embodiment of the invention, the AQP gene is the AQP3 gene.
在本發明的一實施例中,2,4-二氯苯氧乙酸的濃度為0.5mg/L至5mg/L,6-苯胺嘌呤的濃度為0.1mg/L至1mg/L。 In an embodiment of the present invention, the concentration of 2,4-dichlorophenoxyacetic acid is 0.5 mg/L to 5 mg/L, and the concentration of 6-aniline purine is 0.1 mg/L to 1 mg/L.
在本發明的一實施例中,白鶴靈芝癒傷組織具有一至少4倍的生長量。 In an embodiment of the present invention, the white crane Ganoderma lucidum callus has a growth amount of at least 4 times.
在本發明的一實施例中,培養基為一MS培養基。 In an embodiment of the invention, the medium is an MS medium.
綜上所述,本發明白鶴靈芝癒傷組織的萃取物之功效在於:可藉由提升膠原蛋白第I型基因、ELN基因、HAS3基因、XRCC1基因、MSH基因、SOD1基因、GPX1基因、CAT基因、TGM基因、AQP基因及GBA基因的表現量來達到保濕及抗老化的功效;促進皮膚細胞膠原蛋白產生;及藉由降低皮膚受到氧化的傷害來達到抗氧化之功效。另一方面,本發明利用變化的MS培養基配方,以達到大量培養白鶴靈芝癒傷組織的效果,使本發明之白鶴靈芝癒傷組織的萃取物更有利於產業利用。 In summary, the efficacy of the extract of the callus of Ganoderma lucidum of the present invention is that it can be improved by collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene , TGM gene, AQP gene and GBA gene expression to achieve moisturizing and anti-aging effects; promote skin cell collagen production; and by reducing skin damage caused by oxidation to achieve antioxidant effect. On the other hand, the present invention utilizes the changed MS medium formulation to achieve the effect of cultivating a large amount of Baihe Ganoderma lucidum callus tissue, so that the extract of Baihe Ganoderma lucidum callus tissue of the present invention is more conducive to industrial utilization.
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是本發明白鶴靈芝癒傷組織的萃取物在促進膠原蛋白產生上的效用之數據圖,其中“*”表示與對照組比較,p<0.05。 FIG. 1 is a data graph of the effect of the extract of the callus extract of Baihe Ganoderma lucidum of the present invention on promoting collagen production, where “*” indicates comparison with the control group, p <0.05.
圖2是本發明白鶴靈芝癒傷組織的萃取物在皮膚細胞抗氧化上的效用之數據圖,其中“***”表示與過氧化氫組比較,p<0.001。 FIG. 2 is a data graph of the effect of the extract of Baihe Ganoderma lucidum callus of the present invention on the anti-oxidation of skin cells, where “***” indicates comparison with the hydrogen peroxide group, p <0.001.
圖3是本發明白鶴靈芝癒傷組織的萃取物在調控與皮膚細胞彈力相關的基因(包括COL1A1基因、COL1A2基因、ELN基因及HAS3基因)表現上的功效之數據圖。 FIG. 3 is a data graph showing the efficacy of the extract of the white crane Ganoderma lucidum callus of the present invention in regulating the expression of genes related to skin cell elasticity (including COL1A1 gene, COL1A2 gene, ELN gene and HAS3 gene).
圖4是本發明白鶴靈芝癒傷組織的萃取物在調控與皮膚細胞抗光老化相關的基因(包括XRCC1基因、MSH2基因、MSH6基因、SOD1基因、GPX1基因及CAT基因)表現上的功效之數據圖。 Fig. 4 is data on the efficacy of the extract of the callus of Ganoderma lucidum of the present invention in regulating the genes related to the anti-aging of skin cells (including XRCC1 gene, MSH2 gene, MSH6 gene, SOD1 gene, GPX1 gene and CAT gene) Figure.
圖5是本發明白鶴靈芝癒傷組織的萃取物在調控TGM1基因表現上的功效之數據圖。 FIG. 5 is a data graph of the efficacy of the extract of the callus of Ganoderma lucidum of the present invention in regulating the expression of TGM1 gene.
圖6是本發明白鶴靈芝癒傷組織的萃取物在調控AQP3基因表現上的功效之數據圖。 FIG. 6 is a data graph of the efficacy of the extract of Baihe Ganoderma lucidum callus of the present invention in regulating the expression of AQP3 gene.
圖7是本發明白鶴靈芝癒傷組織的萃取物在調控GBA基因及HAS3基因表現上的功效之數據圖。 7 is a data graph of the efficacy of the extract of the callus of Ganoderma lucidum of the present invention in regulating the expression of GBA gene and HAS3 gene.
本文中所使用數值為近似值,所有實驗數據皆表示在20%的範圍內,較佳為在10%的範圍內,最佳為在5%的範圍內。 The numerical values used in this article are approximate values, and all experimental data are expressed within a range of 20%, preferably within a range of 10%, and most preferably within a range of 5%.
依據本發明,白鶴靈芝(Rhinacanthus communis)別名為仙鶴草或靈芝草,為爵床科(Acanthaceae)、白鶴靈芝屬(Rhinacanthus)的灌木植物,多年生,亦為中國珍貴的中藥材,原產於中國雲貴山區,多分布於平原荒地,適合溫暖、潮濕之環境。野生的白鶴靈芝含有許多活性成分,包括羽扇豆醇(lupeol)、β-榖甾醇(β-sitosterol)、豆甾醇(stigmasterol)及白鶴靈芝醌A(rhinacanthin A)。白鶴靈芝已知可用來治療高血壓、糖尿病、肝病、及肺結核。 According to the present invention, Rhinacanthus communis is also known as Agrimonia or Ganoderma lucidum. It is a shrub plant of the family Acanthaceae and Rhinacanthus . It is a perennial and also a precious Chinese medicinal material that is native to China. Mountain areas in Yunnan and Guizhou are mostly distributed in plains and wastelands, suitable for warm and humid environments. Wild Siberian Ganoderma lucidum contains many active ingredients, including lupeol, β-sitosterol, stigmasterol and rhinacanthin A. Baihe Ganoderma is known to treat hypertension, diabetes, liver disease, and tuberculosis.
依據本發明,癒傷組織(callus)是植物原始尚未分化的狀態,來自於莖與根的頂端分生組織(apical meristem)或是體細胞(somatic cell)。癒傷組織具有超基因(epigentic)作用與全能分化能力(totipotent)能夠分化成植物胚胎細胞進而形成新的植株,並有幫助細胞代謝新生、延緩老化與賦予活力等功效。近代利用植物組織培養方法,已經成功發展出一套屬於植物癒傷組織增殖的技術,然而對於不同品種的植株尚須經由詳盡實驗找出適合的培養配方,方能產出有利用價值的植物癒傷組織,提供產業利用。 According to the present invention, callus is the original undifferentiated state of the plant, which comes from the apical meristem or somatic cell of the stem and root. The callus has epigenetic effect and totipotent differentiation ability (totipotent), which can differentiate into plant embryo cells and form new plants, and has the functions of helping cell metabolism to regenerate, delaying aging and energizing. In modern times, a method of plant tissue culture has been used to successfully develop a set of plant callus proliferation techniques. However, for different varieties of plants, it is necessary to find a suitable cultivation formula through detailed experiments before they can produce valuable plant healing. Injury tissue, provide industrial use.
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 and slowing down the aging phenomenon of the appearance of human skin, such as the generation of wrinkles and loss of elasticity. The degree to which this objective is achieved will be determined based on many factors known to those skilled in the art, such as the consumer’s overall state, age, and gender.
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 invention, the substrate may contain one or more additives selected from the group consisting of water, alcohols, glycols, hydrocarbons [such as petroleum jelly and white White petrolatum], wax [such as paraffin and yellow wax], preserving agents, antioxidants, surfactants, absorption enhancers enhancers), stabilizers (stabilizing agents), gelling agent (gelling agents) [such as Carbopol ® 974P (carbopol ® 974P), microcrystalline cellulose (microcrystalline cellulose) and carboxymethyl cellulose (carboxymethylcellulose)], the activity of 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 fall within the scope of professional qualities and routine skills of those who are familiar with this technology.
依據本發明,化妝品可進一步包含有一被廣泛地使用於化妝品製造技術之化妝品上可接受的佐劑(cosmetically acceptable adjuvant)。例如,該化妝品上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, cosmetics may further include a cosmetically acceptable adjuvant widely used in cosmetic manufacturing technology. For example, the cosmetically 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 , 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,化妝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, cosmetics 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 solutions (aqueous solution), water- Aqueous-alcohol solution or oily solution, oil-in-water type, water-in-oil type or compound emulsion or gel , 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,化妝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, cosmetics can also be used in combination with one or more external use agents selected from the following known actives: whitening agents [such as tretinoin, catechins ( catechin), kojic 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-dermatitis agents ( antidermatitis agents, antihyperkeratolytic agents, anti-dry skin agents, antipsoriatic agents, antiaging agents, antiwrinkle agents, anti-wrinkle 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.
首先,取白鶴靈芝作為培植體並將白鶴靈芝進行滅菌處理以除去植物表面的微生物,可以任意方式使白鶴靈芝產生傷口,舉例而言,可以割劃、撕裂或剪切之方式,獲得帶有傷口的白鶴靈芝。 First, take Baihe Ganoderma lucidum as the inoculum and sterilize the Baihe Ganoderma lucidum to remove the microorganisms on the surface of the plant. The Baihe Ganoderma lucidum can be wounded in any way. Ganoderma lucidum on the wound.
接著,使用MS培養基,加入0.5~5mg/L的2,4-二氯苯氧乙酸(2,4-dichlorophenoxyacetic acid)、0.1~1mg/L的6-苯胺嘌呤(6-benzyladenine)、3%蔗糖及0.8%瓊脂至MS培養基,並均勻混合。之後,以0.1N氫氧化鈉溶液來調整pH值至5.7~5.8,接而進行白鶴靈芝癒傷組織增殖培養,經由30天培養周期後可以得到4倍的癒傷組織生長量。 Next, using MS medium, add 0.5~5mg/L of 2,4-dichlorophenoxyacetic acid (2,4-dichlorophenoxyacetic acid), 0.1~1mg/L of 6-aniline purine (6-benzyladenine), 3% sucrose And 0.8% agar to MS medium, and mix evenly. Afterwards, the pH value was adjusted to 5.7~5.8 with 0.1N sodium hydroxide solution, and then the white crane Ganoderma lucidum callus proliferation culture was carried out. After 30 days of culture period, the growth amount of callus can be obtained 4 times.
接著,將經培養的白鶴靈芝癒傷組織以冷凍乾燥機於-40℃進行乾燥16小時,接而收取產物確保水分低於5%,得到乾燥的白鶴靈芝癒傷組織。之後,以75%乙醇於70℃下對乾燥的白鶴靈芝癒傷組織(白鶴靈芝癒傷組織與乙醇的重量比例介於1:5至1:10)進行萃取20~40分鐘(較佳為30分鐘),得到本發明白鶴靈芝癒傷組織的萃取物。 Next, the cultured Baihe Ganoderma lucidum callus was dried in a freeze dryer at -40°C for 16 hours, and then the product was collected to ensure that the water content was less than 5% to obtain dried Baihe Ganoderma lucidum callus. Afterwards, extract the dried white crane Ganoderma lucidum callus (the weight ratio of white crane Ganoderma lucidum callus to ethanol between 1:5 and 1:10) with 75% ethanol at 70°C for 20 to 40 minutes (preferably 30 Minutes) to obtain the extract of the callus of Ganoderma lucidum of the present invention.
首先,以添加有10%胎牛血清(FBS)、1%青黴素(penicillin)/鏈黴素(streptomycin)及1mM丙酮酸鈉(sodium pyruvate)的最低必需培養基(MEM)(Gibco)培養人類皮膚纖維母細胞(human skin fibroblast)CCD-966SK(ATCC CRL-1881)於24-孔盤,500μL培養基的細胞濃度為2×104細胞/孔,接而 於37℃進行培養24小時,並以PBS清洗。接著,將經培養的細胞分成2組,其中包括1個對照組及1個實驗組。將實施例1得到的白鶴靈芝癒傷組織的萃取物以無血清培養基稀釋為具有2%濃度的稀釋液,繼而添加500μL的稀釋液至實驗組的細胞中,至於對照組的細胞則添加500μL的無血清培養基。之後,收取1,000μL的培養基並轉移至1.5mL微量離心管中,接而添加200μL的膠原蛋白分離&濃縮試劑(collagen isolation & concentration reagent),並翻轉離心管6~8次。接著,於4℃進行培養隔夜。之後,取出離心管並以12,000rpm離心10分鐘,接而移除1,000μL的上清液。接著,以SircolTM可溶性膠原蛋白分析套組(SircolTM Soluble Collagen Assay Kit)(Biocolor Life Science Assays,Northern Ireland,UK)來分析各組的膠原蛋白相對百分比。添加1,000μL的Sircol®染劑至離心管中,並輕微振盪30分鐘,接而以12,000rpm離心10分鐘,並移除上清液。接著,添加750μL的酸-鹽類清洗試劑(acid-salt washing reagent),並以12,000rpm離心10分鐘。之後,移除上清液並翻轉離心管,接而以棉花棒移除離心管中的液體。接著,添加250μL的鹼性試劑並充分振盪混合,繼而以光譜訊號感測器測量波長555nm的吸光值,藉此計算出膠原蛋白分泌率,其中以對照組的膠原蛋白分泌率作為100%的基準。本實施例的結果顯示於圖1。 First, human skin fibers were cultured with the minimum necessary medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1 mM sodium pyruvate. Human skin fibroblast CCD-966SK (ATCC CRL-1881) in a 24-well dish, 500 μL of medium with a cell concentration of 2×10 4 cells/well, followed by incubation at 37°C for 24 hours, and washed with PBS . Next, the cultured cells were divided into two groups, including a control group and an experimental group. The extract of Baihe Ganoderma lucidum callus obtained in Example 1 was diluted with serum-free medium to a dilution with a concentration of 2%, and then 500 μL of the dilution was added to the cells of the experimental group, and 500 μL of the cells of the control group were added Serum-free medium. Then, collect 1,000 μL of culture medium and transfer to a 1.5 mL microcentrifuge tube, then add 200 μL of collagen isolation & concentration reagent, and invert the centrifuge tube 6-8 times. Next, the culture was carried out at 4°C overnight. After that, the centrifuge tube was taken out and centrifuged at 12,000 rpm for 10 minutes, and then 1,000 μL of supernatant was removed. Next, soluble collagen Sircol TM Analysis kit (Sircol TM Soluble Collagen Assay Kit) (Biocolor Life Science Assays, Northern Ireland, UK) to analyze the collagen relative percentage of each group. The Sircol ® dye added 1,000μL centrifuge tube, and gently shaken for 30 minutes and then centrifuged for 10 minutes at 12,000rpm, and the supernatant removed. Next, 750 μL of acid-salt washing reagent was added and centrifuged at 12,000 rpm for 10 minutes. After that, the supernatant was removed and the centrifuge tube was turned over, and then the liquid in the centrifuge tube was removed with a cotton swab. Next, add 250 μL of alkaline reagent and mix thoroughly, and then use the spectral signal sensor to measure the absorbance at a wavelength of 555 nm to calculate the collagen secretion rate. The collagen secretion rate of the control group is taken as a benchmark of 100% . The results of this example are shown in Figure 1.
圖1是本發明白鶴靈芝癒傷組織的萃取物在促進膠原蛋白產生上的效用之數據圖。由圖1可見,與對照組相較之下,實驗組所測得的膠原蛋白相對百分比有提升的現象。本實施例的結果顯示,本發明白鶴靈芝癒傷組織的萃取物確實具有促進膠原蛋白產生的功效。 FIG. 1 is a data graph of the effect of the extract of Baihe Ganoderma lucidum callus of the present invention on promoting collagen production. It can be seen from Figure 1 that compared with the control group, the relative percentage of collagen measured by the experimental group has increased. The results of this example show that the extract of the callus of Ganoderma lucidum of the present invention does have the effect of promoting collagen production.
首先,以添加有10%胎牛血清(FBS)、1%青黴素(penicillin)/鏈黴素(streptomycin)及1mM丙酮酸鈉(sodium pyruvate)的最低必需培養基(MEM)(Gibco)培養人類皮膚纖維母細胞CCD-966SK(BCRC 60153)於6-孔盤,2mL培養基的細胞濃度為2×105細胞/孔,接而於37℃進行培養24小時,並移除培養基。接著,將經培養的細胞分成4組,其中包括1個對照組、1個過氧化氫組,及2個實驗組(亦即實驗組1與2)。將實施例1得到的白鶴靈芝癒傷組織的萃取物以培養基稀釋為具有2%及1%濃度的稀釋液,繼而分別將2%稀釋液及1mM過氧化氫(Sigma)添加至實驗組1的細胞中,以及將1%稀釋液及1mM過氧化氫添加至實 驗組2的細胞中。過氧化氫組的細胞被添加以1mM過氧化氫,至於對照組的細胞則添加培養基。之後,添加5μg/mL二氯二氫螢光黃二乙酸酯(Dichloro-dihydro-fluorescein diacetate,DCFH-DA)(Sigma/SI-D6883-50MG)(儲備溶液為5mg/mL溶於DMSO中)並於37℃反應1小時,接而以1mL的1X PBS(Gibco)清洗每孔兩次。接著,添加200μL胰蛋白酶(trypsin)並於避光環境反應5分鐘,接而將細胞培養物收集至1.5mL體積的離心管,並以400×g進行離心10分鐘。之後,移除上清液並以1X PBS清洗一次,接而以400×g進行離心10分鐘。接著,移除上清液並以1mL的1X PBS再懸浮細胞沉澱物。之後,以流式細胞儀(BD Accuri)藉由激發波長(excitation wavelength)450~490nm及放射波長(emission wavelength)510~550nm來偵測DCFH-DA的螢光訊號,藉此計算出受到活性含氧物(reactive oxygen species,ROS)傷害的細胞百分比。各組之間的統計學顯著差異是藉由史徒登氏t-檢定(Student’s t-test)來決定。本實施例的結果顯示於圖2。 First, human skin fibers were cultured with the minimum necessary medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1 mM sodium pyruvate. The mother cell CCD-966SK (BCRC 60153) was placed in a 6-well dish with a cell concentration of 2×10 5 cells/well in 2 mL of culture medium, and then cultured at 37° C. for 24 hours, and the culture medium was removed. Next, the cultured cells were divided into 4 groups, including 1 control group, 1 hydrogen peroxide group, and 2 experimental groups (ie, experimental groups 1 and 2). The extract of Baihe Ganoderma lucidum callus obtained in Example 1 was diluted with a medium to a dilution with a concentration of 2% and 1%, and then 2% dilution and 1 mM hydrogen peroxide (Sigma) were added to the experimental group 1 Cells, and 1% dilution and 1 mM hydrogen peroxide were added to the cells of
圖2是本發明白鶴靈芝癒傷組織的萃取物在皮膚細胞抗氧化上的效用之數據圖。由圖2可見,與對照組相較之下,過氧化氫組測得的受到ROS傷害的細胞百分比有顯著提升,這表示過氧化氫會對細胞產生大量ROS傷害;而與過氧化氫組相較之下,實驗組1及實驗組2測得的受到ROS傷害的細胞百分比有顯著降低。本實施例的結果顯示,本發明白鶴靈芝癒傷組織的萃取物確實具有抗氧化的功效。 Fig. 2 is a data graph of the effect of the extract of the callus of Ganoderma lucidum of the present invention on the anti-oxidation of skin cells. As can be seen from Figure 2, compared with the control group, the percentage of cells injured by ROS measured in the hydrogen peroxide group has increased significantly, which means that hydrogen peroxide will cause a lot of ROS damage to the cells; compared with the hydrogen peroxide group In contrast, the percentage of cells injured by ROS measured in experimental group 1 and
首先,以添加有10%胎牛血清(FBS)、1%青黴素(penicillin)/鏈黴素(streptomycin)及1mM丙酮酸鈉(sodium pyruvate)的最低必需培養基(MEM)(Gibco)培養人類皮膚纖維母細胞CCD-966SK(BCRC 60153)於6-孔盤,2mL培養基的細胞濃度為2×105細胞/孔,接而於37℃進行培養24小時,並移除培養基。 First, human skin fibers were cultured with the minimum necessary medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1 mM sodium pyruvate. The mother cell CCD-966SK (BCRC 60153) was placed in a 6-well dish with a cell concentration of 2×10 5 cells/well in 2 mL of culture medium, and then cultured at 37° C. for 24 hours, and the culture medium was removed.
之後,將經培養的細胞分成2組,其中包括1個對照組及1個實驗組。將實施例1得到的白鶴靈芝癒傷組織的萃取物以培養基稀釋為具有2%濃度的 稀釋液,繼而將之添加至實驗組1的細胞中並靜置24小時,至於對照組的細胞則添加培養基。接著,收取各組細胞培養物並拿來進行基因表現分析。 After that, the cultured cells were divided into two groups, including a control group and an experimental group. The extract of Baihe Ganoderma lucidum callus obtained in Example 1 was diluted with a medium to a dilution with a concentration of 2%, and then added to the cells of experimental group 1 and allowed to stand for 24 hours, while the cells of the control group were added Culture medium. Next, the 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彈力相關的基因包括膠原蛋白第I型,α 1(collagen type I,alpha 1,COL1A1)基因、膠原蛋白第I型,α 2(collagen type I,alpha 2,COL1A2)基因、彈力蛋白(elastin,ELN)基因,及玻尿酸合成酶3(hyaluronan synthase 3,HAS3)基因。 In this embodiment, the genes used to analyze the elasticity of skin cells include collagen type I, α 1 (collagen type I, alpha 1, COL1A1) gene, collagen type I, α 2 (collagen type I,
以RNA萃取套組(RNA extraction kit)(Geneaid)對上面所得到的各組細胞培養物進行RNA的萃取。之後,以SuperScript® III反轉錄酶(SuperScript® III Reverse Transcriptase)(Invitrogen)將萃取出的RNA反轉錄為cDNA。接著,以cDNA作為模版(template),並且使用用來擴增標的基因的引子對[包括COL1A1、COLIA2、ELN、HAS3,及GAPDH(作為內部對照組),它們的核苷酸序列顯示於下面表1],利用KAPA CYBR FAST qPCR套組(2x)(KAPA Biosystems)來進行定量即時聚合酶鏈反應(quantitative real-time polymerase chain reaction,以下簡稱定量即時PCR),俾以對標的基因進行擴增及定量。PCR產物的熔化曲線(melting curve)是在定量即時PCR反應期間進行確認。 The RNA extraction kit (Geneaid) was used to extract RNA from each group of cell cultures obtained above. Thereafter, SuperScript ® III reverse transcriptase (SuperScript ® III Reverse Transcriptase) ( Invitrogen) The extracted RNA was reverse transcribed to cDNA. Next, using cDNA as a template, and using primer pairs [including COL1A1, COLIA2, ELN, HAS3, and GAPDH (as an internal control group) for amplifying the target gene, their nucleotide sequences are shown in the table below 1], use KAPA CYBR FAST qPCR kit (2x) (KAPA Biosystems) to perform quantitative real-time polymerase chain reaction (hereinafter referred to as quantitative real-time PCR), so as to amplify the target gene and Quantitative. The melting curve of the PCR product is confirmed during the quantitative real-time PCR reaction.
標的基因的相對表現量是推導自方程式,並利用GAPDH基因(作為內部對照組)及基準基因(reference gene)的循環閾值[cycle threshold(C t)value]及藉由標準差(standard deviation,STDEV)來計算相對倍數變化,其中△Ct=Ct目標基因/基準基因-CtGAPDH,△△Ct=△Ct目標基因-△Ct基準基因,倍數變化=。以對照組的標的基因表現量作為1.00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(single tailed Student’s t-test)來決定。本實施例的結果顯示於圖3。 The relative expression of the target gene is derived from the equation , And use GAPDH gene (as internal control group) and reference gene (reference gene) cycle threshold [cycle threshold ( C t ) value] and the standard deviation (standard deviation, STDEV) to calculate the relative fold change, where △Ct =Ct target gene/reference gene- Ct GAPDH , △△Ct=△Ct target gene-△Ct reference gene , fold change= . The target gene expression amount of the control group was used as the comparison standard of 1.00. The statistically significant differences between the groups were determined by single-tailed Student's t-test. The results of this example are shown in Figure 3.
圖3是本發明白鶴靈芝癒傷組織的萃取物在調控與皮膚細胞彈力相關的基因(包括COL1A1基因、COL1A2基因、ELN基因及HAS3基因)表現上的功效之數據圖。由圖3可見,無論是COL1A1基因、COL1A2基因、ELN基因,或是HAS3基因,與對照組相較之下,實驗組測得與皮膚細胞彈力相關的基因相對表現量有顯著提升。這個實驗結果顯示,本發明白鶴靈芝癒傷組織的萃取物可藉由調控與皮膚細胞彈力相關的基因表現來達到抗老化的功效。 FIG. 3 is a data graph showing the efficacy of the extract of the white crane Ganoderma lucidum callus of the present invention in regulating the expression of genes related to skin cell elasticity (including COL1A1 gene, COL1A2 gene, ELN gene and HAS3 gene). As can be seen from Fig. 3, whether it is COL1A1 gene, COL1A2 gene, ELN gene, or HAS3 gene, compared with the control group, the relative performance of genes related to skin cell elasticity measured by the experimental group has significantly improved. This experimental result shows that the extract of the callus of Ganoderma lucidum of the present invention can achieve the anti-aging effect by regulating the gene expression related to the elasticity of skin cells.
首先,以添加有10%胎牛血清(FBS)、1%青黴素(penicillin)/鏈黴素(streptomycin)及1mM丙酮酸鈉(sodium pyruvate)的最低必需培養基(MEM)(Gibco)培養人類皮膚纖維母細胞CCD-966SK(BCRC 60153)於6-孔盤,2mL培養基的細胞濃度為2×105細胞/孔,接而於37℃進行培養24小時,並移除培養基。 First, human skin fibers were cultured with the minimum necessary medium (MEM) (Gibco) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 1 mM sodium pyruvate. The mother cell CCD-966SK (BCRC 60153) was placed in a 6-well dish with a cell concentration of 2×10 5 cells/well in 2 mL of culture medium, and then cultured at 37° C. for 24 hours, and the culture medium was removed.
之後,將經培養的細胞分成3組,其中包括1個對照組、1個UVA組及1個實驗組。將實施例1得到的白鶴靈芝癒傷組織的萃取物以培養基稀釋為 具有2%濃度的稀釋液,繼而將之添加至實驗組的細胞中並靜置24小時,UVA組的細胞被處理以5J/cm2 UVA,及對照組的細胞則不做任何處理。接著,收取各組細胞培養物並拿來進行基因表現分析。 After that, the cultured cells were divided into three groups, including a control group, a UVA group, and an experimental group. The extract of Callus Ganoderma lucidum callus obtained in Example 1 was diluted with a medium to a dilution with a concentration of 2%, and then added to the cells of the experimental group and allowed to stand for 24 hours. The cells of the UVA group were treated with 5J /cm 2 UVA, and the cells of the control group did not do any treatment. Next, the 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抗光老化相關的基因包括X射線修復交叉互補蛋白1(X-ray repair cross complementary protein 1,XRCC1)基因、MSH2基因、MSH6基因、超氧化物歧化酶1(superoxide dismutase 1,SOD1)基因、榖胱甘肽過氧化酶1(glutathione peroxidase 1,GPX1)基因,及氯黴素乙醯基轉移酶(chloramphenicol acetyltransferase,CAT)基因。 In this example, the genes used to analyze the anti-aging of skin cells include X-ray repair cross complementary protein 1, XRC1 gene, MSH2 gene, MSH6 gene, superoxide disproportionation Enzyme 1 (superoxide dismutase 1, SOD1) gene, glutathione peroxidase 1 (glutathione peroxidase 1, GPX1) gene, and chloramphenicol acetyltransferase (CAT) gene.
以RNA萃取套組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XRCC1、MSH2、MSH6、SOD1、GPX1、CAT,及GAPDH(作為內部對照組),它們的核苷酸序列顯示於下面表2],在StepOne Plus即時PCR系統(StepOne Plus Real Time PCR system)(ABI)中利用KAPA SYBR FAST qPCR套組(2x)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。 Each group of cell cultures obtained above was subjected to RNA extraction with an RNA extraction kit. Take 2,000 ng of each group of RNA thus obtained and reverse transcribe the extracted RNA into cDNA with SuperScript ® III reverse transcriptase. Next, using cDNA as a template and using primer pairs [including XRCC1, MSH2, MSH6, SOD1, GPX1, CAT, and GAPDH (as an internal control group) for amplifying the target gene, their nucleotide sequences are shown below Table 2], using the KAPA SYBR FAST qPCR kit (2x) in the StepOne Plus Real Time PCR system (ABI) 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.
標的基因的相對表現量是推導自方程式,並利用GAPDH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-CtGAPDH,△△Ct=△Ct目標基因-△Ct基準基因,倍數變化= 平均值。以對照組的標的基因表現量作為1.00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4。 The relative expression of the target gene is derived from the equation , And use GAPDH gene (as an internal control group) and the reference gene cycle threshold and calculate the relative fold change by standard deviation, where △Ct=Ct target gene/reference gene- Ct GAPDH , △△Ct=△Ct target gene -△Ct reference gene , fold change = Average . The target gene expression amount of the control group was used as the comparison standard of 1.00. 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 Figure 4.
圖4是本發明白鶴靈芝癒傷組織的萃取物在調控與皮膚細胞抗光老化相關的基因(包括XRCC1基因、MSH2基因、MSH6基因、SOD1基因、GPX1基因及CAT基因)表現上的功效之數據圖。由圖4可見,無論是XRCC1基因、MSH2基因、MSH6基因、SOD1基因、GPX1基因,或是CAT基因,與對照組及UVA組相較之下,實驗組測得與皮膚細胞抗光老化相關的基因相對表現量有顯著提升。這個實驗結果顯示,本發明白鶴靈芝癒傷組織的萃取物可藉由調控與皮膚細胞抗光老化相關的基因表現來達到抗老化的功效。 Fig. 4 is data on the efficacy of the extract of the callus of Ganoderma lucidum of the present invention in regulating the genes related to the anti-aging of skin cells (including XRCC1 gene, MSH2 gene, MSH6 gene, SOD1 gene, GPX1 gene and CAT gene) Figure. As can be seen from Figure 4, whether it is XRCC1 gene, MSH2 gene, MSH6 gene, SOD1 gene, GPX1 gene, or CAT gene, compared with the control group and UVA group, the experimental group measured the skin cell anti-aging related The relative expression of genes has improved significantly. The experimental results show that the extract of the callus of Ganoderma lucidum of the present invention can achieve the anti-aging effect by regulating the gene expression related to the anti-aging of skin cells.
在本實施例中,申請人探討白鶴靈芝癒傷組織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保濕的功效。 In this embodiment, the applicant explored whether the extract of the callus extract of Ganoderma lucidum can achieve moisturizing effect by regulating the gene expression related to moisturizing of skin cells.
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1×105細胞/孔,接而於37℃進行培養24小時,並移除培養基。 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 2 mL of medium of 1 ×10 5 cells/well, followed by cultivation at 37°C for 24 hours, and the medium was removed.
之後,將經培養的細胞分成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)。將實施例1得到的白鶴靈芝癒傷組織的萃取物以培養基稀釋為具有0.5mg/mL及1mg/mL濃度的稀釋液,繼而分別將0.5mg/mL稀釋液添加至實驗組1的細胞中,及將1mg/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,收取各組細胞培養物並拿來進行基因表現分析。 After that, the cultured cells were divided into three groups, including a control group and two experimental groups (ie, experimental groups 1 and 2). The extract of the callus of Baihe Ganoderma lucidum obtained in Example 1 was diluted with a medium to a dilution with a concentration of 0.5 mg/mL and 1 mg/mL, and then 0.5 mg/mL of the dilution was added to the cells of Experimental Group 1, respectively. And the 1 mg/mL dilution was added to the cells of the
在本實施例中,用來分析與皮膚細胞保濕相關的基因包括轉穀胺醯胺酶1(transglutaminase 1,TGM1)基因、水通道蛋白3(aquaporin 3,AQP3)基因、GBA基因,及HAS3基因。 In this embodiment, the genes used to analyze skin cell moisturization include transglutaminase 1 (transglutaminase 1, TGM1) gene, aquaporin 3 (Aquaporin 3, AQP3) gene, GBA gene, and HAS3 gene .
以RNA萃取套組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TGM1、AQP3、GBA、HAS3,及TBP(作為內部對照組),它們的核苷酸序列顯示於下面表3],在StepOne Plus即時PCR系統中利用KAPA SYBR FAST qPCR套組(2x)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。 Each group of cell cultures obtained above was subjected to RNA extraction with an RNA extraction kit. Take 2,000 ng of each group of RNA thus obtained and reverse transcribe the extracted RNA into cDNA with SuperScript ® III reverse transcriptase. Next, using cDNA as a template and using primer pairs [including TGM1, AQP3, GBA, HAS3, and TBP (as an internal control group) for amplifying the target gene, their nucleotide sequences are shown in Table 3 below], Use the KAPA SYBR FAST qPCR kit (2x) in the StepOne Plus real-time PCR system 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.
標的基因的相對表現量是推導自方程式,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-CtGAPDH,△△Ct=△Ct目標基因-△Ct基準基因,倍數變化=。以對照組的標的基因表現量作為1.00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5至圖7。 The relative expression of the target gene is derived from the equation , And use the TBP gene (as an internal control group) and the reference gene cycle threshold and calculate the relative fold change by standard deviation, where △Ct=Ct target gene/reference gene- Ct GAPDH , △△Ct=△Ct target gene -△Ct reference gene , fold change = . The target gene expression amount of the control group was used as the comparison standard of 1.00. 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 Figures 5-7.
圖5是本發明白鶴靈芝癒傷組織的萃取物在調控TGM1基因表現上的功效之數據圖;圖6是本發明白鶴靈芝癒傷組織的萃取物在調控AQP3基因表現上的功效之數據圖;圖7是本發明白鶴靈芝癒傷組織的萃取物在調控GBA基因及HAS3基因表現上的功效之數據圖。由圖5至圖7可見,無論是TGM1基因、AQP3基因、GBA基因,或是HAS3基因,與對照組相較之下,實驗組1與實驗組2測得與皮膚細胞保濕相關的基因相對表現量有顯著提升。本實施例的結果顯示,本發明白鶴靈芝癒傷組織的萃取物可藉由調控與皮膚細胞保濕相關的基因表現來達到保濕的功效。 FIG. 5 is a data diagram of the efficacy of the extract of Baihe Ganoderma lucidum callus of the present invention in regulating the expression of TGM1 gene; FIG. 6 is a data diagram of the efficacy of the extract of Baihe Ganoderma lucidum callus of the present invention in regulating the expression of AQP3 gene; 7 is a data graph of the efficacy of the extract of the callus of Ganoderma lucidum of the present invention in regulating the expression of GBA gene and HAS3 gene. As can be seen from Figure 5 to Figure 7, whether it is TGM1 gene, AQP3 gene, GBA gene, or HAS3 gene, compared with the control group, the relative performance of genes related to skin cell moisturization measured in experimental group 1 and
綜上所述,本發明白鶴靈芝癒傷組織的萃取物可藉由提升膠原蛋白第I型基因、ELN基因、HAS3基因、XRCC1基因、MSH基因、SOD1基因、GPX1基因、CAT基因、TGM基因、AQP基因及GBA基因的表現量來達到保濕及抗老化的功效;促進皮膚細胞膠原蛋白產生;及藉由降低皮膚受到氧化的傷害來達到抗氧化之功效。另一方面,本發明利用變化的MS培養基配方,以達到大 量培養白鶴靈芝癒傷組織的效果,使本發明之白鶴靈芝癒傷組織的萃取物更有利於產業利用。 In summary, the extract of the callus of Ganoderma lucidum of the present invention can be improved by enhancing collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene, TGM gene, The expression levels of AQP gene and GBA gene can achieve moisturizing and anti-aging effects; promote the production of collagen in skin cells; and achieve the anti-oxidant effect by reducing the skin's oxidative damage. On the other hand, the present invention utilizes the changed MS medium formulation to achieve the effect of cultivating the callus of Baihe Ganoderma lucidum in large quantities, so that the extract of Baihe Ganoderma lucidum callus of the present invention is more conducive to industrial utilization.
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> 白鶴靈芝癒傷組織的萃取物用於提升膠原蛋白第I型基因、ELN基因、HAS3基因、XRCC1基因、MSH基因、SOD1基因、GPX1基因、CAT基因、TGM基因、AQP基因及GBA基因的表現量、促進膠原蛋白產生及抗氧化之用途、用於誘導及增殖白鶴靈芝癒傷組織的方法、及用於培養白鶴靈芝癒傷組織的培養基 <120> Extracts from Callus of Ganoderma lucidum are used to enhance collagen type I gene, ELN gene, HAS3 gene, XRCC1 gene, MSH gene, SOD1 gene, GPX1 gene, CAT gene, TGM gene, AQP gene and GBA gene The amount of expression, the use of promoting collagen production and anti-oxidation, the method for inducing and proliferating the callus of Ganoderma lucidum, and the medium used to cultivate the callus of Ganoderma lucidum
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US8221766B2 (en) * | 2004-12-22 | 2012-07-17 | Avon Products, Inc. | Use of plant extracts to prevent and/or reduce the signs of subjective discomfort and/or irritation in the topical application of cosmetic products |
US7618662B2 (en) * | 2004-12-22 | 2009-11-17 | Avon Products, Inc | Use of natural plant extracts in cosmetic compositions |
CN108379162A (en) * | 2018-04-25 | 2018-08-10 | 佛山娇芙生物科技有限公司 | A kind of bignose rhinacanthus branchlet and leaf moisture-keeping composition and its application |
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2018
- 2018-09-11 TW TW107131942A patent/TWI743393B/en active
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2019
- 2019-01-14 CN CN201910033751.6A patent/CN110882290A/en active Pending
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CN110882290A (en) | 2020-03-17 |
TWI743393B (en) | 2021-10-21 |
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