TW202317749A - A composition for improving skin condition and a novel sphingomonas olei strain and a lysate, a culture solution, an extract thereof - Google Patents
A composition for improving skin condition and a novel sphingomonas olei strain and a lysate, a culture solution, an extract thereof Download PDFInfo
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- TW202317749A TW202317749A TW111139530A TW111139530A TW202317749A TW 202317749 A TW202317749 A TW 202317749A TW 111139530 A TW111139530 A TW 111139530A TW 111139530 A TW111139530 A TW 111139530A TW 202317749 A TW202317749 A TW 202317749A
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- skin
- oleosphingomonas
- extract
- lysate
- sphingomonas
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Abstract
Description
本發明關於一種油鞘氨醇單胞菌(Sphingomonas olei)用於改善皮膚狀態的用途。更具體地,本發明關於一種包含油鞘氨醇單胞菌或其溶解產物(lysate)、培養液或提取物作為有效成分的組合物藉由加快受損表皮的修復、增加皮膚屏障相關因子的表達、並降低炎症因子的表達,從而改善皮膚狀態的用途。The present invention relates to the use of Sphingomonas olei for improving skin condition. More specifically, the present invention relates to a composition containing Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient, by accelerating the repair of damaged epidermis and increasing skin barrier-related factors. Express and reduce the expression of inflammatory factors to improve skin condition.
由於人體皮膚表面有大量角質、汗液、皮脂等代謝廢物,故各種微生物棲息並形成皮膚微生物群落(skin microbiome),即皮膚常居菌群。在皮膚常居菌群中,有益菌和有害菌共存,皮膚越健康,有益菌的分佈越多,而特應性皮膚或痤瘡性皮膚的情況下,有害菌的分佈越多。因此,眾所周知,維持皮膚中存在的常居菌的平衡有助於維持健康的皮膚狀態。然而,關於這些皮膚常居菌對皮膚的影響的研究仍然較少。Since there is a large amount of metabolic waste such as cutin, sweat, and sebum on the surface of human skin, various microorganisms inhabit and form skin microbiome, which is the resident flora of the skin. In the skin's resident flora, beneficial bacteria and harmful bacteria coexist. The healthier the skin, the more beneficial bacteria are distributed, and in the case of atopic skin or acne-prone skin, the more harmful bacteria are distributed. Therefore, it is known that maintaining the balance of resident bacteria present in the skin helps maintain a healthy skin condition. However, there are still few studies on the effects of these skin-resident bacteria on the skin.
通常,常駐於皮膚上的皮膚常居菌會形成皮膚屏障,以防止其他更有害的微生物(病原體)的入侵,且有助於皮膚的免疫作用。由於皮膚有益菌不會引起疾病,也不刺激免疫系統,故如果可以使用皮膚有益菌製造藥物或化妝品等,則可以以不產生刺激皮膚等的副作用的狀態安心使用。Normally, skin-resident bacteria that live on the skin form a skin barrier to prevent the invasion of other more harmful microorganisms (pathogens) and contribute to the skin's immune function. Since skin-friendly bacteria do not cause diseases and do not stimulate the immune system, if skin-friendly bacteria can be used to produce drugs, cosmetics, etc., they can be used safely without causing side effects such as skin irritation.
當皮膚出現傷口或發生感染時,皮膚組織會受損或發炎,如果其程度不嚴重,皮膚通常會自行治癒。在本發明中,為了開發有助於皮膚再生和修復以及炎症治癒過程的皮膚常居菌而進行研究的結果,鑒定出了可以藉由皮膚再生和炎症治癒來改善皮膚狀態的皮膚有益菌,從而完成了本發明。When a wound or infection occurs on the skin, the skin tissue becomes damaged or inflamed, but the skin usually heals on its own if it is not severe. In the present invention, as a result of research conducted to develop skin-resident bacteria that contribute to skin regeneration and repair and inflammation healing processes, skin-friendly bacteria that can improve skin condition through skin regeneration and inflammation healing have been identified. The present invention was completed.
[發明要解決的問題][Problem to be solved by invention]
基於此,本發明提供一種用於改善皮膚狀態的化妝品材料組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。Based on this, the present invention provides a cosmetic material composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
本發明提供一種用於改善皮膚狀態的醫藥部外品組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。The present invention provides a quasi-drug composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
本發明提供一種用於改善皮膚狀態的皮膚外用劑組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。The present invention provides an external skin preparation composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
本發明提供一種用於改善皮膚狀態的保健功能食品組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。The present invention provides a health functional food composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
本發明提供一種用於預防或治療炎症性皮膚病或治療或改善皮膚傷口的藥物組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物以及藥學上可接受的載體。The present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases or treating or improving skin wounds, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract and a pharmaceutically acceptable carrier .
本發明提供一種以寄存編號KACC 81169BP寄存的油鞘氨醇單胞菌菌株。The present invention provides an Oleosphingomonas strain registered under the registration number KACC 81169BP.
本發明提供一種油鞘氨醇單胞菌菌株的溶解產物、培養液、提取物或它們的混合物。 [用於解決問題的手段] The present invention provides a lysate, culture solution, extract or mixture thereof of a strain of Sphingomonas oleosphingomonas. [Means used to solve problems]
在一實施方式中,本發明提供一種用於改善皮膚狀態的化妝品材料組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。In one embodiment, the present invention provides a cosmetic material composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
在本發明中,油鞘氨醇單胞菌可以是活菌(活著的菌)或死菌(死亡的菌)。更詳細地,死菌可以是藉由熱處理的死菌。In the present invention, Oleosphingomonas bacteria may be live bacteria (living bacteria) or dead bacteria (dead bacteria). In more detail, the dead bacteria may be dead bacteria by heat treatment.
在一實施例中,油鞘氨醇單胞菌可以是以寄存編號KACC 81169BP寄存的油鞘氨醇單胞菌菌株。In one embodiment, the Sphingomonas oleosphingomonas may be the Sphingomonas oleosphingomonas strain registered under accession number KACC 81169BP.
本文所用的術語“溶解產物”是指如裂解的油鞘氨醇單胞菌的微生物的細胞的水性培養基中的溶液或懸浮液。細胞溶解產物包括如DNA、RNA、蛋白質、肽、碳水化合物、脂質等的大分子和/或如氨基酸、糖、脂肪酸等的小分子或其級分。另外,溶解產物還包括細胞碎片,其可以是光滑的或顆粒狀的結構。The term "lysate" as used herein refers to a solution or suspension in an aqueous culture medium of cells of a microorganism such as lysed Sphingomonas oleosphingomonas. Cell lysates include macromolecules such as DNA, RNA, proteins, peptides, carbohydrates, lipids, etc. and/or small molecules such as amino acids, sugars, fatty acids, etc. or fractions thereof. In addition, lysates also include cell debris, which can be in a smooth or granular structure.
作為實現微生物的細胞裂解的方法,可以使用各種公知的方法,可以使用能夠實現微生物的細胞裂解的任意方法。例如,細胞的打開/破壞可藉由酶、化學性或物理性方式進行。酶和酶混合物的非限制性示例為如蛋白酶K的蛋白酶、脂肪酶或糖苷酶;化學物質的非限制性示例為離子載體、如十二烷基硫酸鈉的洗滌劑、酸或鹼;物理手段的非限制性示例為如法式擠壓的高壓、滲透壓、如熱或冷的溫度。另外,也可以適當組合使用除蛋白水解酶之外的酶、酸、鹼等的方法。As a method for achieving cell lysis of microorganisms, various known methods can be used, and any method capable of achieving cell lysis of microorganisms can be used. For example, cell opening/destruction can be carried out enzymatically, chemically, or physically. Non-limiting examples of enzymes and enzyme mixtures are proteases such as proteinase K, lipases or glycosidases; non-limiting examples of chemical substances are ionophores, detergents such as sodium lauryl sulfate, acids or bases; physical means Non-limiting examples of are high pressure such as French extrusion, osmotic pressure, temperature such as hot or cold. In addition, a method in which enzymes other than proteolytic enzymes, acids, bases, etc. are used in appropriate combination is also possible.
本文所用的術語“培養液”可以與“培養上清液”、“條件培養液”或“條件培養基”互換使用,並且可以意指包括在培養基中培養油鞘氨醇單胞菌一段時間而獲得的微生物、其代謝物和殘餘營養物質的整體培養基,其中,培養油鞘氨醇單胞菌的培養基為能夠提供營養物質而使得油鞘氨醇單胞菌可以在體外生長和存活的培養基。此外,該培養液也可以指從培養微生物而獲得的菌體培養液中去除菌體的培養液。另一方面,從該培養基中去除菌體的液體也稱為“上層液”,其可以藉由如下方式獲得:僅取除將培養液靜置一段時間而下沉至下層的部分之外的上層液體;或藉由過濾去除菌體;或藉由離心培養液來去除下層沉澱後僅取上層液體。該“菌體”意指本發明的微生物本體,包括藉由從皮膚試樣等中分離而篩選出的微生物本體、或藉由培養該微生物後從培養液中分離出的微生物。該菌體可以藉由如下方式獲得:即離心後取下沉至下層的部分;或由於其會因重力而下沉至培養液的下層,故可以藉由靜置一段時間後,再去除上層液體而獲得。The term "culture broth" as used herein may be used interchangeably with "culture supernatant", "conditioned culture broth" or "conditioned medium" and may mean a culture medium obtained by culturing Sphingomonas oleois for a period of time. A whole culture medium of microorganisms, their metabolites and residual nutrients, wherein the culture medium for culturing Oleosphingomonas is a culture medium that can provide nutrients so that Oleosphingomonas can grow and survive in vitro. In addition, the culture liquid may refer to a culture liquid in which bacterial cells are removed from a bacterial cell culture liquid obtained by culturing microorganisms. On the other hand, the liquid from which the bacterial cells are removed from the culture medium is also called the "supernatant liquid" and can be obtained by removing only the upper layer except the portion that sinks to the lower layer by letting the culture liquid stand for a period of time. liquid; or remove the bacterial cells by filtration; or remove the lower sediment by centrifuging the culture solution and only take the upper liquid. The "bacterial cell" means the microorganism body of the present invention, including a microorganism body screened by isolation from a skin sample or the like, or a microorganism isolated from a culture solution after culturing the microorganism. The bacterial cells can be obtained by centrifuging and removing the part that sinks to the lower layer; or because they will sink to the lower layer of the culture medium due to gravity, they can be left to stand for a period of time and then the upper liquid is removed. And get.
在一實施方式中,本發明的油鞘氨醇單胞菌的培養物可以使用本領域技術人員根據目的而從用於培養微生物的培養基中輕易進行選擇的培養基,具體地,可以使用用於培養鞘氨醇單胞菌的培養基,例如強化梭菌培養基(Reinforced Clostridium Medium;RCM)、胰蛋白酶大豆肉湯(Tryptic Soy Broth;TSB)或腦心浸液(Brain Heart Infusion;BHI)培養基,但不限於此。根據一實施例,本發明的油鞘氨醇單胞菌培養物藉由在微生物培養基中接種油鞘氨醇單胞菌,並根據本領域公知的微生物培養方法(例如,靜置培養等)製備。In one embodiment, the culture of Sphingomonas oleae of the present invention can use a medium that can be easily selected by those skilled in the art according to the purpose from the culture medium for culturing microorganisms. Specifically, the culture medium for culturing microorganisms can be used. Culture media for Sphingomonas, such as Reinforced Clostridium Medium (RCM), Tryptic Soy Broth (TSB), or Brain Heart Infusion (BHI) media, but not Limited to this. According to one embodiment, the Oleosphingomonas culture of the present invention is prepared by inoculating Oleosphingomonas in a microbial culture medium and according to microbial culture methods known in the art (for example, static culture, etc.) .
上述培養液可以包括藉由培養微生物而獲得的培養液、其濃縮物或凍乾物、或從培養液中去除微生物而獲得的培養上清液、其濃縮物或凍乾物。The above-mentioned culture solution may include a culture solution, a concentrate or a lyophilizate thereof obtained by culturing a microorganism, or a culture supernatant obtained by removing microorganisms from a culture solution, a concentrate thereof or a lyophilizate thereof.
上述培養液可以藉由將油鞘氨醇單胞菌在合適的培養基(例如,R2A培養基或TSB培養基)中,以10℃至40℃中的任何溫度培養一定時間(例如,4至50小時)而獲得。The above culture solution can be cultured by culturing Sphingomonas oleosphingomonas in a suitable medium (for example, R2A medium or TSB medium) at any temperature from 10°C to 40°C for a certain period of time (for example, 4 to 50 hours) And get.
在一實施例中,微生物的培養上清液可以藉由將微生物培養液離心或過濾以去除微生物的步驟而獲得。In one embodiment, the culture supernatant of the microorganism can be obtained by centrifuging or filtering the microorganism culture solution to remove the microorganisms.
在另一實施例中,濃縮物可以藉由對微生物培養液、或將該培養液藉由離心或使用篩檢程式過濾後獲得的上清液進行濃縮的步驟而獲得。In another embodiment, the concentrate can be obtained by concentrating the microbial culture solution or the supernatant obtained by centrifuging or filtering the culture solution using a screening program.
用於培養該油鞘氨醇單胞菌的培養用培養基及培養條件可以藉由本領域技術人員進行適當選擇或變更。The culture medium and culture conditions used for culturing the oleosphingomonas can be appropriately selected or changed by those skilled in the art.
本文所用的術語“提取物”意指從溶解產物、培養液或其濃縮物中提取的物質,且其可以是提取液、提取液的稀釋液或濃縮物、藉由乾燥提取液而獲得的乾燥物、或這些的粗提物或純化物、對其進行分級的級分。The term "extract" as used herein means a substance extracted from a lysate, a culture broth, or a concentrate thereof, and it may be an extract, a dilution or a concentrate of the extract, or a dried extract obtained by drying the extract. substances, or crude extracts or purified substances of these, or fractions thereof.
油鞘氨醇單胞菌或其溶解產物、培養液或提取物具有皮膚狀態改善效果,例如,選自由改善皮膚屏障、皮膚保濕、改善皮膚免疫力、增強皮膚抵抗力、抑制皮膚炎症、鎮靜皮膚和皮膚再生所組成的組中的一種以上的皮膚狀態改善效果。Oleosphingomonas or its lysate, culture solution or extract has a skin condition improving effect, for example, selected from the group consisting of improving skin barrier, skin moisturizing, improving skin immunity, enhancing skin resistance, inhibiting skin inflammation, and calming skin. More than one skin condition improving effect in the group consisting of skin regeneration and skin regeneration.
本文所用的術語“改善皮膚屏障”是指包括強化皮膚屏障或保護功能的含義,其中,皮膚屏障(skin barrier)是表皮的最外層,即角質層(stratum corneum),主要由無核扁平角質細胞(corneocyte)組成。由藉由正常表皮細胞的***和分化過程維持的皮膚屏障的角質細胞所合成的神經醯胺、膽固醇和脂肪酸等細胞間脂質形成的多層脂質膜(multi lamella lipid layer)起著保護屏障的作用,以防止皮膚水分蒸發。該改善皮膚屏障意指緩解由皮膚屏障功能減弱引起的皮膚病(如牛皮癬、接觸性皮炎、濕疹性皮炎、光敏性皮炎、脂漏性皮炎、皰疹樣皮炎、扁平式苔蘚、硬化性苔蘚、壞疽性膿皮病、天皰瘡、大皰性表皮鬆弛症、全身性硬化症或麻風病),或提高受損皮膚屏障功能的所有作用。The term "improving skin barrier" used herein refers to the meaning of strengthening the skin barrier or protective function. The skin barrier is the outermost layer of the epidermis, namely the stratum corneum, and is mainly composed of anucleate flat keratinocytes. (corneocyte) composition. The multi-lamella lipid layer formed by intercellular lipids such as ceramide, cholesterol and fatty acids synthesized by keratinocytes that maintain the skin barrier through normal epidermal cell division and differentiation processes plays a protective role in the barrier. to prevent skin moisture from evaporating. Improving the skin barrier means alleviating skin diseases caused by weakened skin barrier function (such as psoriasis, contact dermatitis, eczematous dermatitis, photosensitivity dermatitis, seborrheic dermatitis, dermatitis herpetiformis, lichen planus, lichen sclerosus , pyoderma gangrenosum, pemphigus, epidermolaxia bullosa, systemic sclerosis, or leprosy), or any effect that increases the barrier function of compromised skin.
本文所用的術語“皮膚保濕”意指保持皮膚水分或防止水分流失的所有作用。The term "skin moisturizing" as used herein means any action that maintains moisture in the skin or prevents moisture loss.
本文所用的術語“皮膚再生”意指皮膚重新再生的所有情況,不僅包括由於老化引起的皮膚的再生,還包括因劃傷等受損的表皮被治癒的情況等。The term "skin regeneration" used herein means all cases of skin regeneration, including not only regeneration of skin due to aging, but also cases where damaged epidermis is healed due to scratches or the like.
上述皮膚炎症選自由皮炎、過敏性皮炎、刺激性皮炎、脂漏性皮炎、特應性皮炎、敏感性皮膚病、瘙癢症、濕疹性皮膚病、乾性濕疹、紅斑、蕁麻疹、牛皮癬、藥疹和痤瘡所組成的組中的任意一種。The above-mentioned skin inflammation is selected from dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin disease, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, Any of the group consisting of drug eruptions and acne.
油鞘氨醇單胞菌或其溶解產物、培養液或提取物促進受損皮膚表面上的角質形成細胞(keratinocyte)的遷移,以加快受損表皮的修復。Oleosphingomonas or its lysate, culture solution or extract promotes the migration of keratinocytes on the damaged skin surface to accelerate the repair of damaged epidermis.
油鞘氨醇單胞菌或其溶解產物、培養液或提取物,可以增加皮膚屏障相關因子(例如兜甲蛋白(Loricrin),其為一種角質形成所需的蛋白質)的表達。Oleosphingomonas or its lysate, culture medium or extract can increase the expression of skin barrier-related factors (such as loricrin, a protein required for keratin formation).
此外,油鞘氨醇單胞菌或其溶解產物、培養液或提取物可以降低炎症因子,例如特應症和瘙癢相關因子(如胸腺基質淋巴細胞生成素(Thymicstromal lymphopoietin;TSLP))及促炎細胞因子(如IL-6、IL-8或IL-1α)的表達。In addition, Sphingomonas oleosphingomonas or its lysate, culture medium or extract can reduce inflammatory factors, such as atopy and pruritus-related factors (such as thymic stromal lymphopoietin (TSLP)) and pro-inflammatory factors Expression of cytokines (such as IL-6, IL-8 or IL-1α).
在一實施例中,油鞘氨醇單胞菌或其溶解產物、培養液或提取物可促進角質形成細胞的遷移,或者增加兜甲蛋白的表達,或者降低選自由TSLP、IL-6、IL-8和IL-1α所組成的組中的一種以上的表達。In one embodiment, Sphingomonas oleosphingomonas or its lysate, culture solution or extract can promote the migration of keratinocytes, or increase the expression of loricrin, or reduce the amount selected from TSLP, IL-6, IL Expression of more than one member of the group consisting of -8 and IL-1α.
相對於總重量,可包含0.00001wt%至80wt%的,例如可包含0.00001wt%至60wt%、0.00001wt%至40wt%、0.00001wt%至30wt%、0.00001wt%至20wt%、0.00001wt%至10wt%、0.00001wt%至5wt%、0.05wt%至60wt%、0.05wt%至40wt%、0.05wt%至30wt%、0.05wt%至20wt%、0.05wt%至10wt%、0.05wt%至5wt%、0.1wt%至60wt%、0.1wt%至40wt%、0.1wt%至30wt%、0.1wt%至20wt%、0.1wt%至10wt%或0.1wt%至5wt%的微生物、或其溶解產物、培養液或提取物。Relative to the total weight, it may include 0.00001wt% to 80wt%, for example, it may include 0.00001wt% to 60wt%, 0.00001wt% to 40wt%, 0.00001wt% to 30wt%, 0.00001wt% to 20wt%, 0.00001wt% to 10wt%, 0.00001wt% to 5wt%, 0.05wt% to 60wt%, 0.05wt% to 40wt%, 0.05wt% to 30wt%, 0.05wt% to 20wt%, 0.05wt% to 10wt%, 0.05wt% to 5wt %, 0.1wt% to 60wt%, 0.1wt% to 40wt%, 0.1wt% to 30wt%, 0.1wt% to 20wt%, 0.1wt% to 10wt% or 0.1wt% to 5wt% microorganisms, or their lysates , culture solution or extract.
本文所用的術語“包含……作為有效成分”意指以可以表現出該效果的程度添加微生物或其溶解產物、培養液或提取物,並且還包括為了藥物遞送和穩定化等,添加各種成分作為副成分而配製(formulation)出的各種形式。The term "comprising... as an active ingredient" as used herein means adding a microorganism or a lysate thereof, a culture fluid or an extract to an extent that can exhibit the effect, and also includes adding various ingredients as an active ingredient for drug delivery, stabilization, etc. Various forms formulated with by-products.
上述化妝品材料組合物可以製備成包括化妝水(潤膚乳液)、潤膚劑(skin)、潤膚柔膚劑(skin softner)、潤膚爽膚水(skin toner)、收斂劑(astringent)、乳液(lotion)、牛奶乳液(milk lotion)、保濕乳液(moisture lotion)、營養乳液、按摩霜、營養霜、保濕霜、護手霜、手部清潔劑、粉底、精華、營養精華、面膜(pack)、香皂、潔面泡沫、潔面乳液、潔面霜、身體乳、沐浴露、懸浮液、啫喱(jelly)、粉末、糊劑、貼片式面膜(mask pack)及片劑的劑型。這種劑型的組合物可以根據本領域常規方法製備。本領域技術人員可以在不損害本發明的目的和效果的範圍內,容易地選擇如上所述的保濕劑等添加成分的混合量。The above-mentioned cosmetic material composition can be prepared to include lotion (moisturizing lotion), emollient (skin), skin softener (skin softner), skin toner (skin toner), astringent (astringent), emulsion ( lotion), milk lotion, moisturizing lotion, nutritional lotion, massage cream, nutritional cream, moisturizing cream, hand cream, hand cleanser, foundation, essence, nutritional essence, facial mask (pack), Dosage forms of soap, cleansing foam, cleansing lotion, cleansing cream, body lotion, shower gel, suspension, jelly, powder, paste, mask pack and tablets. Compositions of this dosage form can be prepared according to conventional methods in the art. Those skilled in the art can easily select the mixing amounts of the above-mentioned moisturizing agents and other additional ingredients within a range that does not impair the purpose and effects of the present invention.
上述化妝品材料組合物中除本發明所公開的有效成分之外,還可以進一步包括功能性添加劑及包含於普通化妝品材料組合物中的成分,還可以包括常用的純淨水、增稠劑、防腐劑、穩定劑、增溶劑、表面活性劑、載體、香料或它們的組合。上述功能性添加劑可以包括選自水溶性維生素、油溶性維生素、高分子肽、高分子多糖、鞘脂和海藻提取物所組成的組中的成分。上述載體的示例包括醇、油、表面活性劑、脂肪酸、矽油、潤濕劑、保濕劑、黏度調節劑、乳劑、穩定劑、紫外線散射劑、紫外線吸收劑、顯色劑、香料等。用於上述醇、油、表面活性劑、脂肪酸、矽油、潤濕劑、保濕劑、黏度調節劑、乳液、穩定劑、紫外線散射劑、紫外線吸收劑、著色劑、香料的化合物/組合物等是本領域公知的,故本領域的技術人員可以選擇並使用適當的相應物質/組合物。此外,在化妝品材料組合物中,根據需要還添加紫外線阻斷劑、抗氧化劑(丁基羥基苯甲醚、沒食子酸丙酯、異抗壞血酸、生育酚乙酸酯、丁基化羥基甲苯等)、防腐劑(對羥基苯甲酸甲酯、對羥基苯甲酸丁酯等、對羥基苯甲酸丙酯、苯氧乙醇、咪唑烷基脲、氯苯甘醚等)、著色劑、pH調節劑(三乙醇胺、檸檬酸(citric acid)、枸櫞酸、檸檬酸鈉、蘋果酸、蘋果酸鈉、富馬酸、富馬酸鈉、琥珀酸、琥珀酸鈉、氫氧化鈉、磷酸氫二鈉等)、保濕劑(甘油、山梨糖醇、丙二醇、丁二醇、己二醇、雙甘油、甜菜鹼、甘油聚醚-26、甲基葡糖醇聚醚-20等)、潤滑劑等。In addition to the active ingredients disclosed in the present invention, the above-mentioned cosmetic material composition may further include functional additives and ingredients contained in ordinary cosmetic material compositions, and may also include commonly used purified water, thickeners, and preservatives. , stabilizers, solubilizers, surfactants, carriers, fragrances or combinations thereof. The above-mentioned functional additives may include components selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high molecular peptides, high molecular polysaccharides, sphingolipids and seaweed extracts. Examples of the above-mentioned carriers include alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity regulators, emulsions, stabilizers, ultraviolet scattering agents, ultraviolet absorbers, color developers, fragrances, and the like. Compounds/compositions used in the above-mentioned alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity regulators, emulsions, stabilizers, UV scattering agents, UV absorbers, colorants, fragrances, etc. It is well known in the art, so those skilled in the art can select and use appropriate corresponding substances/compositions. In addition, in the cosmetic material composition, ultraviolet blockers, antioxidants (butylated hydroxyanisole, propyl gallate, erythorbic acid, tocopherol acetate, butylated hydroxytoluene, etc.) are added as needed. ), preservatives (methylparaben, butylparaben, etc., propylparaben, phenoxyethanol, imidazolidinyl urea, chlorphenesin, etc.), colorants, pH adjusters ( Triethanolamine, citric acid, citric acid, sodium citrate, malic acid, sodium malate, fumaric acid, sodium fumarate, succinic acid, sodium succinate, sodium hydroxide, disodium hydrogen phosphate, etc. ), moisturizers (glycerin, sorbitol, propylene glycol, butylene glycol, hexylene glycol, diglycerin, betaine, glyceryl polyether-26, methyl glucol polyether-20, etc.), lubricants, etc.
此外,在各種劑型的化妝品材料組合物中,可以根據化妝品材料的劑型或使用目的來選擇適當的成分混合。由於混合成分和方法可以遵循常規技術,故本發明中將省略其詳細描述。In addition, in various dosage forms of cosmetic material compositions, appropriate ingredients can be selected and mixed according to the dosage form or purpose of use of the cosmetic material. Since the mixing ingredients and methods can follow conventional techniques, detailed descriptions thereof will be omitted in the present invention.
另一實施方式,本發明提供一種用於改善皮膚狀態的醫藥部外品組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。In another embodiment, the present invention provides a quasi-drug composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
其中,油鞘氨醇單胞菌或其溶解產物、培養液或提取物、及其皮膚狀態改善效果與上述說明相同。Among them, Sphingomonas oleosphingomonas or its lysate, culture solution or extract, and its skin condition improving effect are the same as described above.
本文所用的術語“醫藥部外品”意指用於診斷、治療、改善、緩解、治療或預防人類或動物疾病的物品中的作用比藥物溫和的物品。例如,根據藥事法,醫藥部外品意指除了以醫藥品的用途使用的物品之外的物品,其包括用於治療或預防人類和動物疾病的纖維/橡膠製品、對人體的作用輕微或不直接作用於人體且並非儀器或機器的物品及其類似物、用於預防傳染病的消毒劑和殺蟲劑等。As used herein, the term "quasidrug" means an article intended to diagnose, treat, ameliorate, alleviate, treat, or prevent disease in humans or animals that has a milder effect than a drug. For example, according to the Pharmaceutical Affairs Act, quasi-drugs refer to items other than items used for medicinal purposes, including fiber/rubber products used to treat or prevent human and animal diseases, products that have minor or minor effects on the human body or Items and their analogues that do not directly act on the human body and are not instruments or machines, disinfectants and pesticides used to prevent infectious diseases, etc.
本發明的醫藥部外品組合物的種類或劑型沒有特別限定,但它可以是繃帶、紗布、脫脂棉、創可貼、消毒清潔劑、沐浴泡沫、漱口水、濕紙巾、洗衣皂、洗手液、加濕器填充物、面罩或篩檢程式填充物等。The type or dosage form of the quasi-drug composition of the present invention is not particularly limited, but it can be bandages, gauze, absorbent cotton, band-aids, disinfectant cleaners, bath foam, mouthwash, wet wipes, laundry soap, hand sanitizer, humidification device filling, mask or screening program filling, etc.
為改善皮膚狀態的用途而將本發明的組合物包含於醫藥部外品中時,可以直接將組合物本身包含於醫藥部外品來使用,也可以與其他醫藥部外品成分一同使用,其可根據常規方法適當地使用。有效成分的混合量可以根據使用目的適當地確定,相對於組合物總重量,本發明的醫藥部外品組合物可以包含0.01wt%至20wt%的微生物及其溶解產物、培養液或它們的混合物。When the composition of the present invention is included in a quasi-drug for the purpose of improving skin condition, the composition itself can be directly included in the quasi-drug and used, or it can be used together with other quasi-drug ingredients. It can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined according to the purpose of use. The quasidrug composition of the present invention can contain 0.01 wt% to 20 wt% of microorganisms and their lysates, culture solutions, or mixtures thereof relative to the total weight of the composition. .
又一實施方式,本發明提供一種用於改善皮膚狀態的皮膚外用劑組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。In yet another embodiment, the present invention provides an external skin preparation composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
其中,油鞘氨醇單胞菌或其溶解產物、培養液或提取物及其皮膚狀態改善效果與上述說明相同。Among them, Sphingomonas oleosphingomonas or its lysate, culture solution or extract and its skin condition improving effect are the same as described above.
皮膚外用劑可以是霜劑、凝膠劑、軟膏劑、皮膚乳化劑、皮膚混懸劑、透皮貼劑、乳液或它們的組合。該皮膚外用劑可根據需要適當與普通化妝品或醫藥品等皮膚外用劑中使用的成分(例如水性成分、油性成分、粉末成分、醇類、保濕劑、增稠劑、紫外線吸收劑、美白劑、防腐劑、抗氧化劑、表面活性劑、香料、著色劑、各種皮膚營養劑或它們的組合)進行混合。該皮膚外用劑也可以適當地與金屬封鎖劑(依地酸二鈉、依地酸三鈉、檸檬酸鈉、聚磷酸鈉、偏磷酸鈉、葡萄糖酸等)、藥劑(咖啡因、單寧、維拉帕米、甘草提取物、光甘草定、飛廉草果實的熱水提取物、各種生藥,醋酸生育酚、甘草酸、傳明酸及其衍生物或其鹽等)、糖類(維生素C、抗壞血酸磷酸酯鎂、抗壞血酸葡糖苷、熊果苷、曲酸、葡萄糖、果糖、海藻糖等)等進行混合。The skin topical agent may be a cream, gel, ointment, skin emulsifier, skin suspension, transdermal patch, lotion, or a combination thereof. This external preparation for skin can be appropriately combined with ingredients used in external preparations for skin such as general cosmetics or pharmaceuticals (for example, water-based ingredients, oil-based ingredients, powder ingredients, alcohols, moisturizers, thickeners, ultraviolet absorbers, whitening agents, preservatives, antioxidants, surfactants, fragrances, colorants, various skin nutrients or combinations thereof). This external preparation for skin can also be appropriately combined with metal blocking agents (disodium edetate, trisodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate, gluconic acid, etc.), pharmaceutical agents (caffeine, tannin, Verapamil, licorice extract, glabridin, hot water extract of philanthropy fruit, various crude drugs, tocopherol acetate, glycyrrhizic acid, tranexamic acid and its derivatives or their salts, etc.), sugars (vitamin C , magnesium ascorbyl phosphate, ascorbyl glucoside, arbutin, kojic acid, glucose, fructose, trehalose, etc.) are mixed.
相對於組合物總重量,根據本發明的皮膚外用劑組合物可以包含0.00001wt%至80wt%的微生物、其溶解產物、培養液或它們的混合物。The external skin preparation composition according to the present invention may contain 0.00001 wt% to 80 wt% of microorganisms, lysates thereof, culture solutions, or mixtures thereof relative to the total weight of the composition.
上述皮膚包括面部、手部、手臂、腿部、足部、胸部、腹部、背部、臀部和頭皮的身體的所有皮膚部位。The above-mentioned skin includes all skin parts of the body such as the face, hands, arms, legs, feet, chest, abdomen, back, buttocks and scalp.
又一實施方式,本發明提供一種用於改善皮膚狀態的保健功能食品組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物作為有效成分。In yet another embodiment, the present invention provides a health functional food composition for improving skin condition, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract as an active ingredient.
其中,油鞘氨醇單胞菌或其溶解產物、培養液或提取物及其皮膚狀態改善效果與上述說明相同。Among them, Sphingomonas oleosphingomonas or its lysate, culture solution or extract and its skin condition improving effect are the same as described above.
上述保健功能食品組合物可以單獨使用油鞘氨醇單胞菌或其培養液,也可以與其他食品或食品成分一同使用、還可以根據常規方法適當地使用。有效成分的混合量可以根據使用目的(預防、保健或治療性處理)適當地確定。通常,在製造食品或飲料時,相對於原料,可添加15 重量份以下的本申請說明書的組合物。The above-mentioned health functional food composition can be used alone with Sphingomonas oleosphingomonas or its culture solution, or can be used together with other foods or food ingredients, or can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined depending on the purpose of use (prevention, health care, or therapeutic treatment). Normally, when producing food or beverages, 15 parts by weight or less of the composition described in this application can be added to the raw materials.
對上述保健功能食品的種類沒有特別限制。可以添加上述物質的食品的示例包括選自散劑、顆粒劑、片劑、膠囊劑、丸劑、凝膠劑、果凍劑、懸浮劑、乳劑、糖漿劑、茶包劑、浸出茶和保健飲料組成的組中選擇的劑型等,且包括常規意義上的所有保健食品。如常規飲料,保健功能食品種類中的飲料組合物可以包含各種調味劑或天然碳水化合物等作為附加成分。There are no particular restrictions on the types of the above-mentioned health functional foods. Examples of foods to which the above-mentioned substances may be added include those selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jelly, suspensions, emulsions, syrups, tea bags, infused teas and health drinks. The dosage forms selected from the group, etc., and include all health foods in the conventional sense. Like conventional beverages, beverage compositions in the category of health functional foods can contain various flavoring agents or natural carbohydrates as additional ingredients.
上述天然碳水化合物可以為單糖(如葡萄糖和果糖),二糖(如麥芽糖和蔗糖),多糖(如糊精和環糊精)以及糖醇(如木糖醇、山梨糖醇和赤蘚糖醇等)。作為甜味劑,可以使用天然甜味劑(如索馬甜、甜葉菊提取物)、合成甜味劑(如糖精、阿斯巴甜)等。該保健食品組合物還可以包含:營養劑、維生素、電解質、調味劑、著色劑、果膠酸及其鹽;海藻酸及其鹽;有機酸;保護膠體增稠劑;pH調節劑;穩定劑;防腐劑;甘油;醇;用於碳酸飲料的碳酸化劑或其組合。該保健功能食品組合物還可以包含用於製造天然果汁、果汁飲料、蔬菜飲料的果肉或其組合。The above-mentioned natural carbohydrates can be monosaccharides (such as glucose and fructose), disaccharides (such as maltose and sucrose), polysaccharides (such as dextrin and cyclodextrin) and sugar alcohols (such as xylitol, sorbitol and erythritol). wait). As sweeteners, natural sweeteners (such as thaumatin, stevia extract), synthetic sweeteners (such as saccharin, aspartame), etc. can be used. The health food composition may also include: nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and its salts; alginic acid and its salts; organic acids; protective colloid thickeners; pH regulators; stabilizers ; Preservative; Glycerin; Alcohol; Carbonating agent for carbonated beverages or combinations thereof. The health functional food composition may also include fruit pulp used for manufacturing natural juices, juice drinks, vegetable drinks, or combinations thereof.
又一實施方式,本發明提供一種用於預防或治療炎症性皮膚病或治療或改善皮膚傷口的藥物組合物,其包含油鞘氨醇單胞菌或其溶解產物、培養液或提取物以及藥學上可接受的載體作為有效成分。In yet another embodiment, the present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases or treating or improving skin wounds, which contains Sphingomonas oleosphingomonas or its lysate, culture solution or extract and pharmaceutical composition. an acceptable carrier as the active ingredient.
其中,對於油鞘氨醇單胞菌或其溶解產物、培養基和提取物的描述與上述說明相同。Among them, the description of Sphingomonas oleosphingomonas or its lysate, culture medium and extract is the same as the above description.
上述炎症性皮膚病可以為選自皮炎、過敏性皮炎、刺激性皮炎、脂漏性皮炎、特應性皮炎、敏感性皮膚病、瘙癢症、濕疹性皮膚病、乾性濕疹、紅斑、蕁麻疹、牛皮癬、藥疹和痤瘡所組成的組中的任意一種。The above-mentioned inflammatory skin disease may be selected from the group consisting of dermatitis, allergic dermatitis, irritant dermatitis, seborrheic dermatitis, atopic dermatitis, sensitive skin disease, pruritus, eczematous skin disease, dry eczema, erythema, and urticaria. Any of the group consisting of measles, psoriasis, drug eruptions, and acne.
皮膚傷口也被稱為“創傷”,意指活體的損傷。上述損傷可以為由外部因素(如物理損傷、輻射、因化學物質的刺激、微生物增殖等)或內部因素(如壓力)引起的,但不限於此。該傷口可以包括切創、刺創、砍創、挫傷、裂傷、射傷和咬傷等所有種類的傷口。“傷口治療”可以指修復傷口的一系列生物反應。“改善傷口”可以指減輕面部或身體上的傷口或減少其程度的所有作用。Skin wounds are also called "wounds," meaning damage to a living body. The above-mentioned damage can be caused by external factors (such as physical damage, radiation, stimulation by chemical substances, microbial proliferation, etc.) or internal factors (such as pressure), but is not limited to this. The wound can include all types of wounds including cuts, stab wounds, slashes, contusions, lacerations, shots and bites. "Wound therapy" can refer to a series of biological responses that repair wounds. "Amelioration of wounds" may refer to any action that relieves wounds on the face or body or reduces their extent.
本文所用的術語“藥物組合物”可以指當施用於受試者時賦予某些有益效果的分子或化合物。有益效果可以包括:使診斷決策成為可能;疾病、症狀、障礙或病態的改善;減少或預防疾病、症狀、障礙或病症的發作;以及通常對疾病、症狀、障礙或病態的應對。The term "pharmaceutical composition" as used herein may refer to a molecule or compound that confers certain beneficial effects when administered to a subject. Beneficial effects may include: enabling diagnostic decisions; amelioration of a disease, symptom, disorder, or pathology; reducing or preventing the onset of a disease, symptom, disorder, or pathology; and generally responding to a disease, symptom, disorder, or pathology.
本文所用的術語“預防(prevention)”是指部分或完全延遲或防止病症、障礙或其附屬症狀的發作或復發;防止病症或障礙的獲得或重新獲得;或減少獲得病症或障礙的風險。例如,上述預防是指藉由施用根據本發明的組合物,以抑制或延遲皮膚損傷或症狀的發生的所有行為。The term "prevention" as used herein means to partially or completely delay or prevent the onset or recurrence of a condition, disorder, or associated symptoms; to prevent the acquisition or reacquisition of a condition or disorder; or to reduce the risk of acquiring a condition or disorder. For example, the above-mentioned prevention refers to all actions to inhibit or delay the occurrence of skin damage or symptoms by applying the composition according to the present invention.
本文所用的術語“治療”包括抑制、減輕或消除疾病的發展。The term "treatment" as used herein includes inhibiting, reducing or eliminating the progression of disease.
本文所用的術語“改善”可以意指至少減小與狀態的緩解或治療相關的參數(例如,症狀的程度)的所有行為。The term "improvement" as used herein may mean any act that at least reduces a parameter (eg, the extent of a symptom) associated with the alleviation or treatment of a condition.
上述藥物組合物在臨床給藥時可以以非口服方式給藥,並且可以以常規醫藥品製劑的形式使用。非口服給藥可以意指藉由除口服給藥之外的途徑給藥,例如直腸、靜脈、腹膜、肌肉、動脈、經皮、鼻腔(Nasal)、吸入、眼部和皮下給藥。當本發明的藥物組合物用於醫藥品時,可以進一步包含一種以上的具有相同或相似功能的有效成分。The above pharmaceutical composition can be administered non-orally during clinical administration, and can be used in the form of conventional pharmaceutical preparations. Parenteral administration may mean administration by routes other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal (Nasal), inhalational, ocular and subcutaneous administration. When the pharmaceutical composition of the present invention is used as medicine, it may further contain one or more active ingredients with the same or similar functions.
上述藥物組合物可以進一步包含一種以上藥學上可接受的載體來製備。藥學上可接受的載體可以與鹽水、無菌水、林格氏液、緩衝鹽水、葡萄糖溶液(dextrose solution)、麥芽糖糊精溶液、甘油、乙醇及這些成分中的一種以上混合使用,並且可以根據需要添加其他常規添加劑(如抗氧化劑、緩衝液、抑菌劑等)。此外,還可以額外添加稀釋劑、分散劑、表面活性劑、黏合劑及潤滑劑來製劑化成注射用劑型(如水溶液、懸浮液、乳濁液等)、丸劑、膠囊劑、顆粒劑或片劑。更進一步地,可以藉由本領域適當的方法,根據每種病症或成分較佳地進行製劑化。The above-mentioned pharmaceutical composition can be prepared further including one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier can be mixed with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerin, ethanol, and one or more of these ingredients, and can be used as needed Add other conventional additives (such as antioxidants, buffers, bacteriostatic agents, etc.). In addition, additional diluents, dispersants, surfactants, binders and lubricants can be added to formulate injection dosage forms (such as aqueous solutions, suspensions, emulsions, etc.), pills, capsules, granules or tablets . Furthermore, it can be formulated optimally according to each disease or ingredient by appropriate methods in the art.
當對上述藥物組合物進行製劑化時,可利用通常使用的填充劑、增量劑、黏合劑、潤濕劑、崩解劑、表面活性劑等稀釋劑或賦形劑來進行調製。用於非口服給藥的製劑包括無菌水溶液、非水溶劑、混懸劑、乳劑、凍乾製劑和栓劑。作為非水溶劑和懸浮液劑可使用丙二醇(Propylene glycol)、聚乙二醇(Plyethylene glycol)、植物油(如橄欖油)和可注射酯(如油酸乙酯)等。作為栓劑的基質,可以使用半合成脂肪酸酯(Witepsol)、聚乙二醇(Macrogol)、吐溫(Tween)61、可哥脂、月桂酸甘油酸酯(Laurin oil)、甘油明膠等。When the pharmaceutical composition is formulated, it can be prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterile aqueous solutions, nonaqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspension agents, propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable esters (such as ethyl oleate) can be used. As the base of suppositories, semi-synthetic fatty acid esters (Witepsol), polyethylene glycol (Macrogol), Tween 61, cocoa butter, laurin oil, glycerin gelatin, etc. can be used.
為了提高上述藥物組合物的穩定性和吸收性,可以使用碳水化合物(如葡萄糖、蔗糖或右旋糖)、抗氧化劑(Antioxidants)(如抗壞血酸(Ascorbic acid)或谷胱甘肽(Glutathione))、螯合劑(Chelatingagents)和低分子蛋白質或其他穩定劑(Stabilizers)作為藥劑。In order to improve the stability and absorption of the above pharmaceutical composition, carbohydrates (such as glucose, sucrose or dextrose), antioxidants (Antioxidants) (such as ascorbic acid or glutathione), can be used. Chelating agents and low molecular proteins or other stabilizers are used as pharmaceutical agents.
又一實施方式,本發明提供一種預防、改善或治療個體狀態的方法,其包括將該藥物組合物施用於個體的步驟。In yet another embodiment, the present invention provides a method of preventing, improving or treating an individual's condition, which includes the step of administering the pharmaceutical composition to the individual.
上述個體的狀態可以是與皮膚有關的狀態或與炎症有關的狀態。The condition of the individual may be a skin-related condition or an inflammation-related condition.
可以藉由本領域已知的方法進行給藥。可以藉由任何方式直接施用於個體,例如靜脈內、肌內、口服、經皮(transdermal)、黏膜、鼻內(intranasal)、氣管內(intratracheal)或皮下給藥。上述給藥可以是全身性地或局部性地進行給藥。Administration can be carried out by methods known in the art. Administration may be performed directly to an individual by any means, such as intravenous, intramuscular, oral, transdermal, mucosal, intranasal, intratracheal or subcutaneous administration. The above-mentioned administration may be carried out systemically or locally.
上述個體可以是哺乳動物,例如人、牛、馬、豬、狗、綿羊、山羊或貓。上述個體可以是需要改善皮膚狀態或治療炎症性皮膚病的個體。The individual may be a mammal, such as a human, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat. The above-mentioned individual may be an individual in need of improving the condition of the skin or treating an inflammatory skin disease.
上述給藥可以為向每名個體每天施用0.00001mg至1000mg的根據本發明的組合物,例如,可施用0.00001mg至500mg、0.00001mg至100mg、0.00001mg至50mg、0.00001mg至25mg、1mg至1000mg、1mg至500mg、1mg至100mg、1mg至50mg、1mg至25mg、5mg至1000mg、5mg至500mg、5mg至100mg、5mg至50mg、5mg至25mg、10mg至1000mg、10mg至500mg、10mg至100mg、10mg至50mg或10mg至25mg的根據本發明的組合物。然而,給藥量可以根據製劑化方法、給藥方式、患者年齡、體重、性別、病理狀態、食物、給藥時間、給藥途徑、***速率和反應敏感性等因素以各種方式處方,並且,本領域技術人員可以考慮這些因素來適當地調整給藥量。給藥次數可以是一天一次,或在臨床上可接受副作用的範圍內,給藥次數可以是兩次以上;就給藥部位而言,可以在1處或2處以上進行給藥;就給藥天數而言,可以以每天或間隔2~5天的方式,在1至30 天的範圍內進行給藥。如有必要,在適當時間後,可以重複相同的治療。對於除人之外的動物,可以以與人相同的每kg給藥量進行給藥,或者可以以換算的給藥量進行給藥,如基於以目標動物與人的器官(心臟等)的體積比(如平均值)等進行換算的量。The above-mentioned administration may be 0.00001 mg to 1000 mg of the composition according to the present invention administered to each individual per day. For example, 0.00001 mg to 500 mg, 0.00001 mg to 100 mg, 0.00001 mg to 50 mg, 0.00001 mg to 25 mg, 1 mg to 1000 mg may be administered. , 1mg to 500mg, 1mg to 100mg, 1mg to 50mg, 1mg to 25mg, 5mg to 1000mg, 5mg to 500mg, 5mg to 100mg, 5mg to 50mg, 5mg to 25mg, 10mg to 1000mg, 10mg to 500mg, 10mg to 100mg, 10mg to 50 mg or 10 mg to 25 mg of a composition according to the invention. However, the dosage can be prescribed in various ways depending on factors such as formulation method, administration method, patient's age, weight, gender, pathological state, food, administration time, administration route, excretion rate and reaction sensitivity, and, Those skilled in the art can consider these factors to appropriately adjust the dosage. The number of administrations can be once a day, or within the range of clinically acceptable side effects, the number of administrations can be more than two times; in terms of administration sites, administration can be at 1 or more than 2 places; in terms of administration sites In terms of days, administration can be carried out every day or at intervals of 2 to 5 days, ranging from 1 to 30 days. If necessary, after an appropriate period of time, the same treatment can be repeated. For animals other than humans, administration can be performed at the same dosage per kg as for humans, or administration can be performed at a converted dosage, such as based on the volumes of the organs (heart, etc.) of the target animal and humans. Ratio (such as average value), etc. for conversion.
又一實施方式,本發明提供一種以寄存編號KACC 81169BP寄存的油鞘氨醇單胞菌菌株。In yet another embodiment, the present invention provides an Oleosphingomonas strain registered with registration number KACC 81169BP.
上述油鞘氨醇單胞菌菌株可以是包括SEQ ID NO: 3的16S rRNA的菌株。The above-mentioned Sphingomonas oleosphingomonas strain may be a strain including the 16S rRNA of SEQ ID NO: 3.
上述菌株可以具有皮膚狀態改善效果,例如,具有選自由改善皮膚屏障、皮膚保濕、改善皮膚免疫力、增強皮膚抵抗力、抑制皮膚炎症、鎮靜皮膚和皮膚再生所組成的組中的一種以上的皮膚狀態改善效果。The above-mentioned strain may have a skin condition improving effect, for example, having at least one selected from the group consisting of improving skin barrier, skin moisturizing, improving skin immunity, enhancing skin resistance, inhibiting skin inflammation, calming skin, and regenerating skin. Status improvement effect.
上述菌株可以促進在受損皮膚表面的角質形成細胞的遷移,從而加快受損表皮的修復。上述菌株可以增加皮膚屏障相關因子(如兜甲蛋白)的表達。此外,上述菌株還可降低炎症因子的表達,例如,特應性和瘙癢相關因子(如TSLP)及促炎細胞因子(如IL-6、IL-8或IL-1α)的表達。The above strains can promote the migration of keratinocytes on the surface of damaged skin, thereby accelerating the repair of damaged epidermis. The above strains can increase the expression of skin barrier-related factors (such as loricrin). In addition, the above strains can also reduce the expression of inflammatory factors, such as atopy and pruritus-related factors (such as TSLP) and pro-inflammatory cytokines (such as IL-6, IL-8 or IL-1α).
又一實施方式,本發明提供一種油鞘氨醇單胞菌菌株的溶解產物、培養液、提取物或它們的混合物。In yet another embodiment, the present invention provides a lysate, culture fluid, extract or mixture thereof of a Sphingomonas oleosphingomonas strain.
對於上述油鞘氨醇單胞菌菌株、其溶解產物、培養液以及提取物的描述與上述說明相同。 發明的效果 Descriptions of the above-mentioned Oleosphingomonas strains, their lysates, culture fluids and extracts are the same as those described above. Effect of the invention
根據本發明的一實施方式的包含油鞘氨醇單胞菌、其溶解產物、培養液或提取物作為有效成分的組合物,其具有加快受損表皮的修復、增加皮膚屏障相關因子的表達、降低炎症因子的表達的效果,故可以有效地用於改善皮膚狀態。According to one embodiment of the present invention, a composition containing Sphingomonas oleosphingomonas, its lysate, culture solution or extract as an active ingredient has the functions of accelerating the repair of damaged epidermis and increasing the expression of skin barrier-related factors. It has the effect of reducing the expression of inflammatory factors, so it can be effectively used to improve skin condition.
在下文中,將藉由實施例更詳細地描述本發明。然而,這些實施例僅用於示例性地說明本發明,本發明的範圍不限於這些實施例。In the following, the invention will be described in more detail by means of examples. However, these examples are only used to illustrate the present invention, and the scope of the present invention is not limited to these examples.
實施例1. 菌株的分離和鑒定Example 1. Isolation and identification of bacterial strains
從健康的韓國人的皮膚中採集樣本,將其放入磷酸鹽緩衝液(Phosphate Buffered Saline,PBS)溶液中混合,然後藉由連續稀釋法(Serial dilution)接種到R2A(Reasoner’s 2A agar)培養基中。篩選在30℃的培養箱中培養139小時而形成於平板上的細菌菌落,並在相同的培養基和條件下,使用劃線法(Streaking)進行純化分離。為了確認已完成培養的菌株的分子系統學特徵,進行了16S rRNA基因鹼基序列分析。PCR(Polymerase chain reaction)擴增以如下方式實施:以95℃條件30秒、 55℃條件30秒、72℃條件1分45秒的方式重複32次,最後以72℃條件處理5分鐘。此時,使用了以實現細菌共同基因的特異性反應而提出的引物(SEQ ID NO:1和SEQ ID NO:2)。藉由對PCR擴增產物進行純化,並確認了鹼基序列(Macrogen,韓國),並且使用美國國立生物技術資訊中心(National Center for Biotechnology Information,NCBI)的BLAST程式分析了該序列。與先前登記的其他菌株相比,將具有99%以上鹼基序列同源性的物種確定為近緣種。在本實施例中分離的菌株具有SEQ ID NO: 3(互補DNA(complementary DNA))的16S rRNA序列,其被鑒定為油鞘氨醇單胞菌(Sphingomonas olei)。本發明人將該菌株命名為油鞘氨醇單胞菌CBN003(Sphingomonas olei CBN003),於2021年9月10日寄存於農業遺傳資源保藏中心,獲得的寄存編號為KACC 81169BP。Samples were collected from the skin of healthy Koreans, mixed in Phosphate Buffered Saline (PBS) solution, and then inoculated into R2A (Reasoner's 2A agar) medium by serial dilution. . Screen the bacterial colonies formed on the plate after culturing in an incubator at 30°C for 139 hours, and purify and isolate them using the streaking method under the same culture medium and conditions. In order to confirm the molecular phylogenetic characteristics of the cultured strains, 16S rRNA gene base sequence analysis was performed. PCR (Polymerase chain reaction) amplification was performed as follows: 32 times of 95°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute and 45 seconds, and finally 72°C for 5 minutes. At this time, primers (SEQ ID NO: 1 and SEQ ID NO: 2) proposed to achieve specific reactions of bacterial common genes were used. The PCR amplification product was purified and the base sequence was confirmed (Macrogen, South Korea), and the sequence was analyzed using the BLAST program of the National Center for Biotechnology Information (NCBI). Compared with other previously registered strains, species with more than 99% base sequence homology were identified as closely related species. The strain isolated in this example had the 16S rRNA sequence of SEQ ID NO: 3 (complementary DNA) and was identified as Sphingomonas olei. The inventor named the strain Sphingomonas olei CBN003 (Sphingomonas olei CBN003) and deposited it at the Agricultural Genetic Resources Collection Center on September 10, 2021. The obtained deposit number is KACC 81169BP.
實施例2:菌株的培養Example 2: Cultivation of bacterial strains
對於藉由實施例1分離的菌株,利用胰蛋白酶大豆肉湯(Tryptic Soy Broth;TSB)培養基在34℃的培養箱中進行培養,在生長曲線上的***期(對數期(Log phase))的最後時間點收集菌株培養液和菌體。以6000rpm離心30分鐘,然後使用0.2μm篩檢程式進行過濾和滅菌,從而製備了培養液,當用於評價和活性分析時,在製備後不超過1天的時間內進行了評價和活性分析。The strain isolated in Example 1 was cultured in a 34°C incubator using Tryptic Soy Broth (TSB) medium. During the division phase (Log phase) on the growth curve, The strain culture fluid and bacterial cells were collected at the final time point. Culture broths were prepared by centrifugation at 6000 rpm for 30 minutes, followed by filtration and sterilization using a 0.2 μm screening program. When used for evaluation and activity analysis, evaluation and activity analysis were performed no more than 1 day after preparation.
實驗例1:細胞毒性評價Experimental example 1: Cytotoxicity evaluation
確認了藉由實施例1中分離的菌株是否對細胞具有毒性。It was confirmed whether the strain isolated in Example 1 was toxic to cells.
具體而言,首先,利用包含10%胎牛血清(Fetal bovine serum;FBS)、100μg/mL鏈黴素和100U/mL青黴素的DMEM培養基(Dubelcco’s modified eagle medium),在37℃的5% CO 2培養箱中培養人角質形成細胞系(Human epidermal keratinocyte cellline;HaCaT)。 Specifically, first, DMEM medium (Dubelcco's modified eagle medium) containing 10% fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 U/mL penicillin was used at 37°C in 5% CO 2 Human epidermal keratinocyte cellline (HaCaT) was cultured in an incubator.
藉由MTT分析法對於菌株的培養液進行了細胞毒性評價。將培養的細胞系以8×10 3/孔分裝在96孔板中,然後在相同條件下培養24小時後,分別以0(僅用TSB培養基處理)、1、2、5、10%(v/v)的濃度處理上述菌株的培養液,並且在相同條件下繼續培養24小時。此時,以0%處理組作為陰性對照組,以每孔的總體積為100μL的方式進行。隨後,在每個細胞培養孔中加入20μL 5mg/mL MTT,在相同條件下繼續培養4小時後,加入100μL DMSO,測定590 nm處的吸光度,並將其結果示於圖1中。 The cytotoxicity of the culture medium of the strain was evaluated by MTT assay. The cultured cell lines were distributed in 96-well plates at 8 × 10 3 /well, and then cultured under the same conditions for 24 hours, and then treated with 0 (only treated with TSB medium), 1, 2, 5, and 10% ( v/v) and continue culturing for 24 hours under the same conditions. At this time, the 0% treatment group was used as the negative control group, and the total volume of each well was 100 μL. Subsequently, 20 μL of 5 mg/mL MTT was added to each cell culture well. After continuing to culture for 4 hours under the same conditions, 100 μL of DMSO was added, and the absorbance at 590 nm was measured, and the results are shown in Figure 1.
圖1是顯示藉由MTT分析來評價利用實施例1分離的菌株的培養液處理的人角質形成細胞生存能力的結果的圖。FIG. 1 is a graph showing the results of evaluation of the viability of human keratinocytes treated with the culture medium of the strain isolated in Example 1 by MTT analysis.
如圖1所示,當以1、2、5和10%(v/v)處理菌株培養液時,與未用菌株培養液處理的陰性對照組相比,在人角質形成細胞的存活能力上沒有顯著差異,可見該菌株的培養液無細胞毒性。這樣的結果意味著將該菌株的培養液塗敷於皮膚上是安全的。As shown in Figure 1, when the strain culture medium was treated with 1, 2, 5 and 10% (v/v), compared with the negative control group that was not treated with the strain culture liquid, the viability of human keratinocytes was There was no significant difference, indicating that the culture medium of this strain had no cytotoxicity. Such results mean it is safe to apply cultures of this strain to the skin.
實驗例2:皮膚再生功能活性分析Experimental Example 2: Analysis of skin regeneration function activity
分析了在實施例1中分離的菌株對皮膚再生功能的影響。The effects of the strains isolated in Example 1 on the skin regeneration function were analyzed.
具體而言,首先,利用包含10%胎牛血清(Fetal bovine serum;FBS)、100μg/mL鏈黴素和100U/mL青黴素的DMEM培養基(Dubelcco’s modified eagle medium),在37℃的5% CO 2培養箱中培養人角質形成細胞系(Human epidermal keratinocyte cellline;HaCaT)。將培養的細胞系以5×10 5/孔分裝在6孔板中,之後,為了使細胞貼附而在相同條件下培養24小時。在單層細胞上劃出一個直線劃痕(scratch)後,利用以0(僅處理TSB培養基)、1、2、5、10%(v/v)包含菌株的培養液的無血清DMEM培養基(Dubelcco’s modified eagle medium),在37℃的5% CO 2培養箱中培養24小時。作為陽性對照組,以10ng/mL處理表皮生長因子(Epidermal growth factor,EGF),並以相同方式培養。在顯微鏡下對0小時的劃痕間隔和24小時的間隔進行觀察比較,並將其結果示於圖2中。 Specifically, first, DMEM medium (Dubelcco's modified eagle medium) containing 10% fetal bovine serum (FBS), 100 μg/mL streptomycin and 100 U/mL penicillin was used at 37°C in 5% CO 2 Human epidermal keratinocyte cellline (HaCaT) was cultured in an incubator. The cultured cell lines were distributed in a 6-well plate at 5×10 5 /well, and then cultured under the same conditions for 24 hours to allow cell attachment. After making a straight scratch on the monolayer of cells, use serum-free DMEM medium containing the culture medium of the strain at 0 (only TSB medium), 1, 2, 5, and 10% (v/v). Dubelcco's modified eagle medium), cultured in a 5% CO2 incubator at 37°C for 24 hours. As a positive control group, epidermal growth factor (EGF) was treated with 10ng/mL and cultured in the same way. The scratching interval of 0 hours and the interval of 24 hours were observed and compared under a microscope, and the results are shown in Figure 2.
圖2是顯示評價利用藉由實施例1分離的菌株的培養液處理的人角質形成細胞的遷移的結果(N-CNT,陰性對照組(0% FBS);P-CNT,陽性對照組(EGF 10ng/mL);培養基CNT(Media CNT),TSB培養基)。Figure 2 shows the results of evaluating the migration of human keratinocytes treated with the culture medium of the strain isolated in Example 1 (N-CNT, negative control group (0% FBS); P-CNT, positive control group (EGF) 10ng/mL); medium CNT (Media CNT), TSB medium).
如圖2所示,當利用藉由實施例1分離的菌株的培養液進行處理時,整體上可確認,與0小時的劃痕間隔相比,培養24小時的角質形成細胞的遷移活躍,使得劃痕間隔顯著縮短。這樣的結果表明,該菌株可以有效地用於皮膚再生和傷口治療。As shown in Figure 2, when the culture medium of the strain isolated in Example 1 was used, it was confirmed that the migration of keratinocytes cultured for 24 hours was more active compared with the scratching interval of 0 hours. The scratch interval is significantly shortened. Such results indicate that this strain can be effectively used for skin regeneration and wound treatment.
實驗例3:增強皮膚屏障及保濕活性分析Experimental Example 3: Analysis of skin barrier enhancement and moisturizing activity
分析了藉由實施例1分離的菌株對皮膚屏障的強化和皮膚保濕活性的影響。The effects of the strains isolated in Example 1 on skin barrier strengthening and skin moisturizing activity were analyzed.
具體而言,分析了皮膚屏障功能標誌物兜甲蛋白的表達。首先,對人表皮角質形成細胞系(Human epidermal keratinocyte cellline,HaCaT)進行了分化。將細胞系以5×10 5/孔分裝在6孔板中,並利用包含0.09mM低濃度鈣、表皮生長因子(Epidermal growth factor,EGF)和牛腦垂體提取物(Bevine pituirary extract,BPE)的無血清培養基,在37℃的5% CO 2培養箱中培養2天,以誘導分化。之後,利用包含2.8mM高濃度鈣、表皮生長因子(Epidermal growth factor,EGF)和牛腦垂體提取物(Bevine pituirary extract,BPE)的無血清培養基,在37℃的5% CO 2培養箱中培養7天,並且每2~3天更換一次培養基。向藉由上述過程分化的細胞系添加Poly I:C(10μg/mL)和IL-4(50ng/mL),以誘導炎症反應。在相同條件的培養箱中培養24小時後,以0(僅TSB培養基)、10%(v/v)處理菌株培養液並繼續培養24小時。作為陰性對照組使用了未處理組(僅處理Poly I:C+IL-4),作為陽性對照組使用了1μM***(dexamethasone)處理組。隨後,使用TRIZOL(Invitrogen,美國)從用每個樣品處理的細胞中提取RNA,並使用奈米分光光度計定量RNA,然後分別使用1μg RNA在擴增儀中合成cDNA。以合成的cDNA作為範本,將SYBRV綠(SYBR Green)(Thermo Fisher Scientific,美國)與引物一起加入靶基因進行即時(Real-time)PCR。此時,靶基因使用了作為皮膚屏障功能標誌物兜甲蛋白基因,最後藉由GAPDH(甘油醛3-磷酸脫氫酶)基因的校正來分析了基因的表達量,並將其結果示於圖3中。 Specifically, the expression of loricrin, a marker of skin barrier function, was analyzed. First, the human epidermal keratinocyte cellline (HaCaT) was differentiated. The cell line was distributed in a 6-well plate at 5 × 10 5 /well, and a solution containing 0.09mM low concentration calcium, epidermal growth factor (EGF) and bovine pituitary extract (BPE) was used. Serum-free medium and culture in a 5% CO2 incubator at 37°C for 2 days to induce differentiation. Afterwards, serum-free medium containing 2.8mM high concentration of calcium, epidermal growth factor (EGF) and bovine pituitary extract (BPE) was used to culture in a 5% CO2 incubator at 37°C for 7 days, and the culture medium should be changed every 2 to 3 days. Poly I:C (10 μg/mL) and IL-4 (50 ng/mL) were added to the cell lines differentiated by the above process to induce an inflammatory response. After culturing in an incubator under the same conditions for 24 hours, treat the strain culture medium with 0 (TSB medium only) and 10% (v/v) and continue culturing for 24 hours. An untreated group (poly I:C+IL-4 only) was used as a negative control group, and a 1 μM dexamethasone-treated group was used as a positive control group. Subsequently, RNA was extracted from cells treated with each sample using TRIZOL (Invitrogen, USA), and RNA was quantified using a nanospectrophotometer, and then 1 μg of RNA was used to synthesize cDNA in an amplifier respectively. Using the synthesized cDNA as a template, SYBR Green (Thermo Fisher Scientific, USA) and primers were added to the target gene for real-time PCR. In this case, the loricrin gene, which is a marker of skin barrier function, was used as the target gene. Finally, the expression level of the gene was analyzed by calibration of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene, and the results are shown in the figure. 3 in.
圖3是顯示藉由即時PCR來測定利用本發明一實施例的菌株的培養液處理的人角質形成細胞中兜甲蛋白(LOR)mRNA表達變化的結果的圖(相對於空白組(None)(Poly I:C+IL-4未處理組)時為#;相對於Poly I:C + IL-4處理組時為*;#,* p<0.05;##,** p<0.001;###,*** p<0.0001;紅色,顯著負值(減少);藍色,顯著正值(增加))。Figure 3 is a graph showing the results of measuring loricrin (LOR) mRNA expression changes in human keratinocytes treated with the culture medium of a strain according to an embodiment of the present invention by real-time PCR (relative to the blank group (None) ( Poly I:C + IL-4 untreated group) is #; relative to Poly I:C + IL-4 treated group is *; #, * p<0.05; ##, ** p<0.001; ## #, *** p<0.0001; red, significant negative value (decrease); blue, significant positive value (increase)).
如圖3所示,當利用藉由實施例1分離的菌株進行10%培養液處理時,與陰性對照組相比,兜甲蛋白的表達顯著增加。這樣的結果表明,該菌株可以有效地用於皮膚屏障的強化和皮膚保濕。As shown in Figure 3, when the strain isolated in Example 1 was used for 10% culture solution treatment, the expression of loricrin was significantly increased compared with the negative control group. Such results indicate that this strain can be effectively used for skin barrier strengthening and skin moisturizing.
實驗例4:抗炎症活性分析Experimental Example 4: Analysis of anti-inflammatory activity
為了分析藉由實施例1分離的菌株對抗炎活性的影響,分析了TSLP的表達和促炎細胞因子IL-6、IL-8和IL-1α的表達。In order to analyze the effect of the strains isolated by Example 1 on the anti-inflammatory activity, the expression of TSLP and the expression of pro-inflammatory cytokines IL-6, IL-8 and IL-1α were analyzed.
具體而言,首先,對人表皮角質形成細胞系(Human epidermal keratinocyte cellline;HaCaT)進行了分化。將細胞系以5×10 5/孔分裝在6孔板中,並利用包含0.09mM低濃度鈣、表皮生長因子(Epidermal growth factor,EGF)和牛腦垂體提取物(Bevine pituirary extract,BPE)的無血清培養基,在37℃的5% CO 2培養箱中培養2天,以誘導分化。之後,利用包含2.8mM高濃度鈣、表皮生長因子(Epidermal growth factor,EGF)和牛腦垂體提取物(Bevine pituirary extract;BPE)的無血清培養基,在37℃的5% CO 2培養箱中培養7天,並且每2~3天更換一次培養基。向藉由上述過程分化的細胞系添加Poly I:C(10μg/mL)和IL-4(50ng/mL),以誘導炎症反應。在相同條件的培養箱中培養24小時後,以0(僅TSB培養基)、10%(v/v)處理菌株培養液並繼續培養24小時。作為陰性對照組使用了未處理組(僅處理Poly I:C+IL-4),作為陽性對照組使用了1μM***(dexamethasone)處理組。隨後,使用TRIZOL(Invitrogen,美國)從用每個樣品處理的細胞中提取RNA,並使用奈米分光光度計定量RNA,然後分別使用1μg RNA在擴增儀中合成cDNA。以合成的cDNA為範本,將SYBR Green(Thermo Fisher Scientific,美國)與引物一起加入靶基因進行即時(Real-time)PCR。此時,靶基因使用了TSLP基因和促炎細胞因子IL-6、IL-8和IL-1α基因,最後藉由GAPDH(甘油醛3-磷酸脫氫酶)基因的校正來分析了基因的表達量,並將其結果示於圖4和圖5中。 Specifically, first, the human epidermal keratinocyte cell line (HaCaT) was differentiated. The cell line was distributed in a 6-well plate at 5 × 10 5 /well, and a solution containing 0.09mM low concentration calcium, epidermal growth factor (EGF) and bovine pituitary extract (BPE) was used. Serum-free medium and culture in a 5% CO2 incubator at 37°C for 2 days to induce differentiation. Afterwards, use serum-free medium containing 2.8mM high concentration of calcium, epidermal growth factor (EGF) and bovine pituitary extract (Bevine pituirary extract; BPE), and culture it in a 5% CO2 incubator at 37°C for 7 days, and the culture medium should be changed every 2 to 3 days. Poly I:C (10 μg/mL) and IL-4 (50 ng/mL) were added to the cell lines differentiated by the above process to induce an inflammatory response. After culturing in an incubator under the same conditions for 24 hours, treat the strain culture medium with 0 (TSB medium only) and 10% (v/v) and continue culturing for 24 hours. An untreated group (poly I:C+IL-4 only) was used as a negative control group, and a 1 μM dexamethasone-treated group was used as a positive control group. Subsequently, RNA was extracted from cells treated with each sample using TRIZOL (Invitrogen, USA), and RNA was quantified using a nanospectrophotometer, and then 1 μg of RNA was used to synthesize cDNA in an amplifier respectively. Using the synthesized cDNA as a template, SYBR Green (Thermo Fisher Scientific, USA) and primers were added to the target gene for real-time PCR. At this time, the target genes were the TSLP gene and the pro-inflammatory cytokines IL-6, IL-8 and IL-1α genes, and finally the gene expression was analyzed by calibration of the GAPDH (glyceraldehyde 3-phosphate dehydrogenase) gene. The results are shown in Figures 4 and 5.
圖4是顯示藉由即時PCR來測定利用藉由實施例1分離的菌株的培養基處理的人角質形成細胞中TSLP mRNA表達變化的結果的圖(相對於空白組(None)(Poly I:C+IL-4未處理組)時為#;相對於Poly I:C + IL-4處理組(陰性對照)時為*;#,* p<0.05;##,** p <0.001;###,*** p<0.0001;紅色,顯著負值(減少);藍色,顯著正值(增加))。Figure 4 is a graph showing the results of measuring changes in TSLP mRNA expression in human keratinocytes treated with the culture medium of the strain isolated in Example 1 by real-time PCR (relative to the blank group (None) (Poly I: C+ When compared to the Poly I:C + IL-4 treated group (negative control), it is *; #, * p < 0.05; ##, ** p < 0.001; ### , ***p<0.0001; red, significant negative value (decrease); blue, significant positive value (increase)).
圖5是顯示藉由即時PCR來測定利用藉由實施例1分離的菌株的培養基處理的人角質形成細胞中IL-6、IL-8和IL-1α mRNA表達變化的結果的圖。圖5的a是顯示IL-6 mRNA表達結果的圖,圖5的b是顯示IL-8 mRNA表達結果的圖,圖5的c是顯示IL-1α mRNA表達結果的圖(相對於空白組(None)(Poly I:C+IL-4未處理組)時為#;相對於Poly I:C + IL-4處理組時為*;#,* p<0.05;##,** p<0.001;###,*** p<0.0001;紅色,顯著負值(減少);藍色,顯著正值(增加))。Figure 5 is a graph showing the results of measuring changes in IL-6, IL-8 and IL-1α mRNA expression in human keratinocytes treated with the culture medium of the strain isolated in Example 1 by real-time PCR. Figure 5a is a graph showing the IL-6 mRNA expression results, Figure 5b is a graph showing the IL-8 mRNA expression results, and Figure 5c is a graph showing the IL-1α mRNA expression results (relative to the blank group ( None) (Poly I:C+IL-4 untreated group): #; relative to Poly I:C + IL-4 treated group: *; #, * p<0.05; ##, ** p<0.001 ; ###, ***p<0.0001; red, significant negative value (decrease); blue, significant positive value (increase)).
如圖4所示,當利用藉由實施例1分離的菌株進行10%培養液處理時,與陰性對照相比,TSLP的表達量顯著降低。As shown in Figure 4, when the strain isolated in Example 1 was used for 10% culture solution treatment, the expression level of TSLP was significantly reduced compared with the negative control.
此外,如圖5所示,當利用藉由實施例1分離的菌株進行10%培養液處理時,與陰性對照組相比,IL-6、IL-8和IL-1α的表達量顯著降低,尤其,IL-6和IL-1α顯示出了與陽性對照組相似水準的顯著降低的表達。In addition, as shown in Figure 5, when the strain isolated in Example 1 was used for 10% culture solution treatment, the expression levels of IL-6, IL-8 and IL-1α were significantly reduced compared with the negative control group. In particular, IL-6 and IL-1α showed significantly reduced expression at a similar level to the positive control group.
上述結果表明,上述菌株可以有效地用於預防、改善和治療炎症性皮膚病。The above results indicate that the above strains can be effectively used to prevent, improve and treat inflammatory skin diseases.
無without
圖1是顯示藉由MTT(3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)分析來評價利用本發明一實施例的菌株的培養液處理的人角質形成細胞生存能力的結果的圖。Figure 1 is a diagram showing the use of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide) analysis to evaluate the viability of human keratinocytes treated with the culture medium of the strain according to one embodiment of the present invention.
圖2是評價利用本發明一實施例的菌株的培養液處理的人角質形成細胞的遷移的結果(N-CNT(陰性對照組)-陰性對照組(0% FBS);P-CNT(陽性對照組)-陽性對照組(EGF 10ng/mL);培養基CNT(Media CNT)(對照組),TSB培養基)。Figure 2 is the results of evaluating the migration of human keratinocytes treated with the culture medium of the strain according to one embodiment of the present invention (N-CNT (negative control group) - negative control group (0% FBS); P-CNT (positive control) Group) - Positive control group (EGF 10ng/mL); Media CNT (Control group), TSB medium).
圖3是顯示藉由即時PCR來測定利用本發明一實施例的菌株的培養液處理的人角質形成細胞中兜甲蛋白mRNA表達變化的結果的圖(相對於空白組(None)(Poly I:C+IL-4未處理組)時為#;相對於Poly I:C + IL-4處理組時為*;#,* p<0.05;##,** p<0.001;###,*** p<0.0001;紅色,顯著負值(減少);藍色,顯著正值(增加))。Figure 3 is a graph showing the results of measuring loricrin mRNA expression changes in human keratinocytes treated with the culture medium of a strain of an embodiment of the present invention by real-time PCR (relative to the blank group (None) (Poly I: C+IL-4 untreated group) is #; relative to Poly I:C + IL-4 treated group is *; #, * p<0.05; ##, ** p<0.001; ###, * **p<0.0001; red, significant negative value (decrease); blue, significant positive value (increase)).
圖4是顯示藉由即時PCR來測定利用本發明一實施例的菌株的培養基處理的人角質形成細胞中TSLP mRNA表達變化的結果的圖(相對於空白組(None)(Poly I:C+IL-4未處理組)時為#;相對於Poly I:C + IL-4處理組(陰性對照)時為*;#,* p<0.05;##,** p <0.001;###,*** p<0.0001;紅色,顯著負值(減少);藍色,顯著正值(增加))。Figure 4 is a graph showing the results of measuring changes in TSLP mRNA expression in human keratinocytes treated with the culture medium of a strain according to an embodiment of the present invention by real-time PCR (relative to the blank group (None) (Poly I:C+IL -4 untreated group) is #; relative to the Poly I:C + IL-4 treated group (negative control) is *; #, * p < 0.05; ##, ** p < 0.001; ###, ***p<0.0001; red, significant negative value (decrease); blue, significant positive value (increase)).
圖5是顯示藉由即時PCR來測定利用本發明一實施例的菌株的培養基處理的人角質形成細胞中IL-6、IL-8和IL-1α mRNA表達變化的結果的圖。圖5的a是顯示IL-6 mRNA表達結果的圖,圖5的b是顯示IL-8 mRNA表達結果的圖,圖5的c顯示了IL-1α mRNA表達結果的圖(相對於空白組(None)(Poly I:C+IL-4未處理組)時為#;相對於Poly I:C + IL-4處理組時為*;#,* p<0.05;##,** p<0.001;###,*** p<0.0001;紅色,顯著負值(減少);藍色,顯著正值(增加))。Figure 5 is a graph showing the results of measuring changes in IL-6, IL-8 and IL-1α mRNA expression in human keratinocytes treated with the culture medium of a strain according to an embodiment of the present invention by real-time PCR. Figure 5a is a graph showing the IL-6 mRNA expression results, Figure 5b is a graph showing the IL-8 mRNA expression results, and Figure 5c is a graph showing the IL-1α mRNA expression results (relative to the blank group ( None) (Poly I:C+IL-4 untreated group): #; relative to Poly I:C + IL-4 treated group: *; #, * p<0.05; ##, ** p<0.001 ; ###, ***p<0.0001; red, significant negative value (decrease); blue, significant positive value (increase)).
[登錄號] 寄存機構:韓國農業遺傳資源保藏中心(KACC) 寄存編號:KACC 81169BP 寄存日期:2021年9月10日 [registration number] Depository institution: Korea Agricultural Genetic Resources Collection Center (KACC) Deposit number: KACC 81169BP Deposit date: September 10, 2021
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