KR20230171329A - Cosmetic composition for anti-inflammation, anti-oxidation and skin protection against UV comprising biorenovation extract of black tea as effective component - Google Patents
Cosmetic composition for anti-inflammation, anti-oxidation and skin protection against UV comprising biorenovation extract of black tea as effective component Download PDFInfo
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- KR20230171329A KR20230171329A KR1020220071783A KR20220071783A KR20230171329A KR 20230171329 A KR20230171329 A KR 20230171329A KR 1020220071783 A KR1020220071783 A KR 1020220071783A KR 20220071783 A KR20220071783 A KR 20220071783A KR 20230171329 A KR20230171329 A KR 20230171329A
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- black tea
- extract
- bioconversion
- skin
- ultraviolet rays
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- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/04—Topical preparations for affording protection against sunlight or other radiation; Topical sun tanning preparations
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/522—Antioxidants; Radical scavengers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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- Microbiology (AREA)
- Epidemiology (AREA)
- Birds (AREA)
- Mycology (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Engineering & Computer Science (AREA)
- Medicines Containing Plant Substances (AREA)
- Cosmetics (AREA)
Abstract
본 발명은 홍차 생물전환 추출물을 유효성분으로 함유하는 항염증, 항산화 및 자외선에 대한 피부 보호용 화장료 조성물에 관한 것으로, 상세하게는 생물전환법을 통해 제조된 홍차 생물전환 추출물이 세포독성이 없으며, 홍차 추출물에 대비하여 자외선에 대한 피부보호 활성 및 항산화 활성이 우수하고, LPS에 의해 유도된 NO(nitric oxide), PGE2(prostaglandin E2) 및 전염증성 사이토카인의 생성을 현저하게 억제하는 효과가 있으므로, 본 발명의 피부 보호용, 항산화용 및 항염증용 조성물은 화장품에 적용하여 유용하게 사용할 수 있다.The present invention relates to a cosmetic composition for anti-inflammatory, antioxidant and skin protection against ultraviolet rays containing black tea bioconversion extract as an active ingredient. Specifically, the black tea bioconversion extract prepared through the bioconversion method is non-cytotoxic, and the black tea bioconversion extract is non-cytotoxic. Compared to extracts, it has superior skin protection and antioxidant activity against ultraviolet rays, and has the effect of significantly suppressing the production of NO (nitric oxide), PGE 2 (prostaglandin E 2 ), and pro-inflammatory cytokines induced by LPS. , the skin protection, antioxidant and anti-inflammatory composition of the present invention can be usefully applied to cosmetics.
Description
본 발명은 홍차 생물전환 추출물을 유효성분으로 함유하는 항염증, 항산화 및 자외선에 대한 피부 보호용 화장료 조성물에 관한 것이다.The present invention relates to a cosmetic composition for anti-inflammatory, antioxidant and skin protection against ultraviolet rays containing black tea bioconversion extract as an active ingredient.
염증(inflammation)은 물리적인 상처나 미생물에 감염되었을 때 일어나는 정상적인 생체의 방어기전(defense mechanism)의 일종이며, 이 염증작용을 통하여 발병요인(pathogen)을 중화시키거나 제거하고, 손상된 조직을 복구시켜 정상적인 구조와 기능을 하게 한다. 염증을 동반하는 대부분의 질환은 조직의 손상, 통증 및 가려움증과 같은 삶의 질을 떨어뜨리는 결과를 초래하고, 만성적인 염증상태는 관절염, 천식, 뇌와 척수의 다발성 경화증, 염증성 장 질환 및 동맥 경화증을 일으킨다. 대식세포는 선천면역을 담당하는 주요 세포로, 사이토카인 및 박테리아 지질다당류 내독소(lipopolysaccharide, LPS) 같은 수많은 인자에 의해 활성화되고, 활성화된 대식세포는 산화질소(nitric oxide, NO) 및 프로스타글란딘 E2(prostaglandin E2, PGE2) 같은 염증 인자는 물론 TNF-α(tumor necrosis factor-α), IL-6(interleukin-6), IL-1(interleukin-1) 같은 전염증성 사이토카인을 생산한다. NO는 자유 라디칼(free radical)의 일종으로 산화질소 합성효소(nitric oxide synthase, NOS)에 의해 합성되며, eNOS(endothelial NOS), nNOS(neuronal NOS) 및 iNOS(inducible NOS)의 3개의 동형 단백질(isoform)이 존재한다. IL-1, TNF-α, 인터페론-감마(interferon-gamma) 같은 전염증성 사이토카인들은 iNOS 발현을 유발하고 NO 생산을 일으킬 수 있다. 뇌암, 유방암, 폐암, 전립선암, 췌장암 및 흑색종 같은 많은 악성 암들이 iNOS 발현과 연관되어 있고, 과도한 NO의 생산은 상피세포암, 돌연변이 및 DNA 구조 손상을 일으킬 수 있다.Inflammation is a type of normal body's defense mechanism that occurs when a physical wound or infection occurs with microorganisms. Through this inflammation, pathogens are neutralized or removed and damaged tissues are repaired. It ensures normal structure and function. Most diseases accompanied by inflammation result in a decrease in quality of life such as tissue damage, pain, and itching, and chronic inflammatory conditions include arthritis, asthma, multiple sclerosis of the brain and spinal cord, inflammatory bowel disease, and arteriosclerosis. causes Macrophages are the main cells responsible for innate immunity. They are activated by numerous factors such as cytokines and bacterial lipopolysaccharide (LPS), and activated macrophages produce nitric oxide (NO) and prostaglandin E2 ( It produces inflammatory factors such as prostaglandin E2 and PGE2) as well as pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1 (IL-1). NO is a type of free radical and is synthesized by nitric oxide synthase (NOS), and is composed of three isoforms: endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). isoform) exists. Pro-inflammatory cytokines such as IL-1, TNF-α, and interferon-gamma can induce iNOS expression and cause NO production. Many malignant cancers, such as brain cancer, breast cancer, lung cancer, prostate cancer, pancreatic cancer, and melanoma, are associated with iNOS expression, and excessive production of NO can cause epithelial cell carcinoma, mutations, and DNA structural damage.
생물전환법(biorenovation)이란 미생물이 가지고 있는 효소적 기능을 이용하여 전구물질로부터 원하는 산물을 생산 또는 제조하는 방법을 말하며, 미생물 발효 또는 효소처리 등의 생물학적 방법을 통해 물질의 구조적 변화를 유도시키는 방법을 통칭한다. 상기 변화로 인하여 유효성분의 함량이 증가하거나 흡수율이 개선되고, 새로운 기능성분이 생성된다는 사실이 새롭게 알려지고 있으며 이러한 기법을 이용한 소재 개발이 전 세계적으로 주목을 받고 있다. 이러한 생물전환법은 항생제 및 스테로이드를 포함한 의약품 원료물질의 생산뿐만 아니라 아미노산, 비타민 등의 유용 물질의 생산에 널리 이용되고 있다. 선진국의 경우 생물공학 기법으로 인한 제품의 출시로 고부가치를 목표로 한 제품들이 다수 판매되고 있으며, 이러한 신 바이오 기술 기반 소재는 소비자로부터 안전성이 인정되어 최근에 다양한 생물공학기술을 체계적으로 융합한 다양한 기능성 소재가 등장하고 있다.Biorenovation refers to a method of producing or manufacturing a desired product from precursor materials using the enzymatic function of microorganisms, and is a method of inducing structural changes in materials through biological methods such as microbial fermentation or enzyme treatment. collectively referred to as It is newly known that due to the above changes, the content of active ingredients is increased, absorption rate is improved, and new functional ingredients are created, and the development of materials using these techniques is attracting attention around the world. This bioconversion method is widely used in the production of pharmaceutical raw materials including antibiotics and steroids, as well as useful substances such as amino acids and vitamins. In developed countries, many products targeting high added value are being sold due to the launch of products using biotechnology techniques, and these new biotechnology-based materials have been recognized as safe by consumers, and have recently become a variety of products that systematically combine various biotechnology technologies. Functional materials are emerging.
한편, 홍차는 백차, 황차 및 녹차와 달리 산화 과정을 거친 차의 일종이다. 따라서 향이 더 강하며, 카페인도 더 많이 함유하고 있다. 동양에서는 찻물의 빛이 붉기 때문에 홍차(紅茶)라고 부르지만, 서양에서는 찻잎의 검은 색깔 때문에 '블랙티(black tea)'라고 부른다. 홍차는 차나무(Camellia sinensis)라고 하는 쌍떡잎 식물의 일종으로 주로 열대지방 또는 온대지방에서 자라는 나무로부터 얻어지는 잎의 가공 과정에 따라 얻어진다. 가공 과정에서 홍차는 산화가 많이 일어난다. 산화효소는 단백질 성분으로서 차잎을 막 수확해서 가마솥에 넣어 열을 가해 굴리는 덖음과정을 거치면 산화과정 촉매작용을 못하게 되어 잎이 녹색을 유지한다. 홍차는 잎을 수확해서 말린 다음, 바삭한 잎을 비비거나 굴리면서 잎에 상처를 내어 세포를 파괴시킨다. 그러면 잎 속에 폴리페놀 효소들이 밖으로 흘러나와 공기중의 산소와 산화반응이 일어나서 갈색을 띄는데 이것을 홍차라 한다. 홍차는 산화반응으로 테아플라민이라는 성분과 폴리페놀 성분을 다량 함유하고 있다.Meanwhile, black tea is a type of tea that has gone through an oxidation process, unlike white tea, yellow tea, and green tea. Therefore, it has a stronger flavor and contains more caffeine. In the East, the color of the tea is red, so it is called black tea, but in the West, it is called 'black tea' because of the black color of the tea leaves. Black tea is a type of dicotyledonous plant called Camellia sinensis , and is obtained through the processing of leaves obtained from trees that grow mainly in tropical or temperate regions. During the processing process, black tea undergoes a lot of oxidation. Oxidase is a protein component, and when tea leaves are just harvested and put in a cauldron and then heated and rolled, they are unable to catalyze the oxidation process and the leaves remain green. Black tea leaves are harvested and dried, then the crunchy leaves are rubbed or rolled to damage the leaves and destroy the cells. Then, the polyphenol enzymes in the leaves flow out and undergo an oxidation reaction with oxygen in the air, turning brown. This is called black tea. Black tea contains a large amount of theaflamin and polyphenols due to oxidation reactions.
항염증 관련 선행기술로는 한국등록특허 제1485896호에 '홍차 발효액을 유효성분으로 하는 항산화, 항염증 및 아토피 개선용 화장료 조성물 및 그의 제조방법'이 개시되어 있으며, 한국등록특허 제1412446호에 '녹차 캘러스 추출물을 함유하는 항염증 화장료 조성물의 제조방법'이 개시되어 있다. 하지만, 본 발명의 '홍차 생물전환 추출물을 유효성분으로 함유하는 항염증, 항산화 및 자외선에 대한 피부 보호용 화장료 조성물'에 대해서는 아직까지 개시된 바가 없다.As anti-inflammatory related prior art, Korean Patent No. 1485896 discloses ‘Cosmetic composition for improving antioxidant, anti-inflammatory and atopic dermatitis and manufacturing method thereof using fermented black tea extract as an active ingredient,’ and Korean Patent No. 1412446 discloses ‘Cosmetic composition for improving antioxidant, anti-inflammatory and atopic dermatitis and its manufacturing method. A method for producing an anti-inflammatory cosmetic composition containing green tea callus extract is disclosed. However, the 'cosmetic composition for anti-inflammatory, antioxidant and skin protection against ultraviolet rays containing black tea bioconversion extract as an active ingredient' of the present invention has not yet been disclosed.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 홍차 생물전환 추출물을 유효성분으로 함유하는 항염증, 항산화 및 자외선에 대한 피부 보호용 화장료 조성물을 제공하고, 생물전환법을 통해 제조된 홍차 생물전환 추출물이 세포독성이 없으며, 홍차 추출물에 대비하여 자외선에 대한 피부보호 활성 및 항산화 활성이 우수하고, LPS에 의해 유도된 NO(nitric oxide), PGE2(prostaglandin E2) 및 전염증성 사이토카인의 생성을 현저하게 억제하는 것을 확인함으로써, 본 발명을 완성하였다.The present invention was developed in response to the above-mentioned needs, and provides a cosmetic composition for anti-inflammatory, antioxidant and skin protection against ultraviolet rays containing black tea bioconversion extract as an active ingredient, and provides a black tea bioconversion extract produced through a bioconversion method. It has no cytotoxicity, has superior skin protection activity against ultraviolet rays and antioxidant activity compared to black tea extract, and inhibits the production of NO (nitric oxide), PGE 2 (prostaglandin E 2 ), and pro-inflammatory cytokines induced by LPS. The present invention was completed by confirming that it was significantly suppressed.
상기 과제를 해결하기 위하여, 본 발명은 홍차 생물전환 추출물을 유효성분으로 함유하는 피부 염증의 예방 또는 개선; 자외선에 대한 피부 보호; 및 항산화용 화장료 조성물을 제공한다.In order to solve the above problems, the present invention provides a method for preventing or improving skin inflammation containing black tea bioconversion extract as an active ingredient; Skin protection against ultraviolet rays; and a cosmetic composition for antioxidant use.
또한, 본 발명은 (1) 홍차에 추출용매를 가하여 추출하는 단계; (2) 단계 (1)에서 획득한 홍차 추출물과 바실러스 속(Baillus sp.) 균주를 혼합하여 생물전환 반응을 유도하는 단계; 및 (3) 단계 (2)에서 획득한 생물전환 반응물을 원심분리한 후, 상등액을 수득하는 단계;를 포함하여 제조되는 것을 특징으로 하는 피부 염증의 예방 또는 개선, 자외선에 대한 피부 보호 및 항산화 효과를 갖는 홍차 생물전환 추출물의 제조 방법을 제공한다.In addition, the present invention includes the steps of (1) extracting black tea by adding an extraction solvent; (2) mixing the black tea extract obtained in step (1) with a Baillus sp. strain to induce a bioconversion reaction; And (3) centrifuging the bioconversion reactant obtained in step (2) and then obtaining a supernatant; preventing or improving skin inflammation, skin protection against ultraviolet rays, and antioxidant effect. It provides a method for producing a black tea bioconversion extract having a.
본 발명은 홍차 생물전환 추출물을 유효성분으로 함유하는 항염증, 항산화 및 자외선에 대한 피부 보호용 화장료 조성물에 관한 것으로, 상세하게는 생물전환법을 통해 제조된 홍차 생물전환 추출물이 세포독성이 없으며, 홍차 추출물에 대비하여 자외선에 대한 피부보호 활성 및 항산화 활성이 우수하고, LPS에 의해 유도된 NO(nitric oxide), PGE2(prostaglandin E2) 및 전염증성 사이토카인의 생성을 현저하게 억제하는 효과가 있다.The present invention relates to a cosmetic composition for anti-inflammatory, antioxidant and skin protection against ultraviolet rays containing a black tea bioconversion extract as an active ingredient. Specifically, the black tea bioconversion extract prepared through the bioconversion method is non-cytotoxic, and the black tea bioconversion extract is non-cytotoxic. Compared to extracts, it has excellent skin protection and antioxidant activity against ultraviolet rays, and has the effect of significantly suppressing the production of NO (nitric oxide), PGE 2 (prostaglandin E 2 ), and pro-inflammatory cytokines induced by LPS. .
도 1은 본 발명의 일 실시예에 따른 홍차 추출물 및 홍차 생물전환 추출물의 HPLC 분석 결과를 나타낸 것이다. NC는 바실러스 속(Bacillus sp.) JD3-7 균주 현탁액이다.
도 2는 본 발명의 일 실시예에 따른 자외선에 대한 홍차 추출물 및 홍차 생물전환 추출물의 피부세포 보호 효과를 나타낸 것이다. *는 홍차 추출물 대비 홍차 생물전환 추출물의 피부세포 보호 효과가 통계적으로 유의미하게 증가하였다는 것으로, p<0.05이다.
도 3은 본 발명의 일 실시예에 따른 홍차 추출물 및 홍차 생물전환 추출물의 DPPH 라디칼 소거 활성을 나타낸 것이다. P.C.는 양성대조군인 1% 아스코르브산(ascorbic acid)이며, **는 홍차 추출물 대비 홍차 생물전환 추출물의 DPPH 라디칼 소거 활성이 통계적으로 유의미하게 증가하였다는 것으로, p<0.01이다.
도 4는 본 발명의 일 실시예에 따른 홍차 추출물 및 홍차 생물전환 추출물의 수퍼옥사이드 음이온 라디칼 소거 활성을 나타낸 것이다. P.C.는 양성대조군인 1% 아스코르브산(ascorbic acid)이며, ** 및 ***는 홍차 추출물 대비 홍차 생물전환 추출물의 수퍼옥사이드 음이온 라디칼 소거 활성이 통계적으로 유의미하게 증가하였다는 것으로, **는 p<0.01이고, ***는 p<0.001이다.
도 5는 본 발명의 일 실시예에 따른 홍차 추출물 및 홍차 생물전환 추출물 처리에 따른 대식세포의 세포 생존율을 나타낸 것이다.
도 6은 본 발명의 일 실시예에 따른 홍차 추출물 및 홍차 생물전환 추출물의 NO(nitric oxide) 생성 억제 효과를 나타낸 것이다. ** 및 ***는 홍차 추출물 대비 홍차 생물전환 추출물의 NO 생성 억제 효과가 통계적으로 유의미하게 증가하였다는 것으로, **는 p<0.01이고, ***는 p<0.001이다.
도 7은 본 발명의 일 실시예에 따른 홍차 추출물 및 홍차 생물전환 추출물의 PGE2(prostaglandin E2) 생성 억제 효과를 나타낸 것이다. *는 홍차 추출물 대비 홍차 생물전환 추출물의 PGE2 생성 억제 효과가 통계적으로 유의미하게 증가하였다는 것으로, p<0.05이다.
도 8은 본 발명의 일 실시예에 따른 홍차 추출물 및 홍차 생물전환 추출물의 IL-6 생성 억제 효과를 나타낸 것이다. *는 0.25mg/㎖의 처리농도에서, 홍차 추출물 대비 홍차 생물전환 추출물의 IL-6 생성 억제 효과가 통계적으로 유의미하게 증가하였다는 것으로, p<0.05이다.Figure 1 shows the results of HPLC analysis of black tea extract and black tea bioconversion extract according to an embodiment of the present invention. NC is a suspension of Bacillus sp. strain JD3-7.
Figure 2 shows the skin cell protection effect of black tea extract and black tea bioconversion extract against ultraviolet rays according to an embodiment of the present invention. * indicates a statistically significant increase in the skin cell protection effect of black tea bioconversion extract compared to black tea extract, p<0.05.
Figure 3 shows the DPPH radical scavenging activity of black tea extract and black tea bioconversion extract according to an embodiment of the present invention. PC is 1% ascorbic acid, a positive control, and ** indicates a statistically significant increase in the DPPH radical scavenging activity of the black tea bioconversion extract compared to the black tea extract, p<0.01.
Figure 4 shows the superoxide anion radical scavenging activity of black tea extract and black tea bioconversion extract according to an embodiment of the present invention. PC is 1% ascorbic acid, a positive control, ** and *** indicate a statistically significant increase in superoxide anion radical scavenging activity of black tea bioconversion extract compared to black tea extract, ** is p <0.01, and *** is p<0.001.
Figure 5 shows the cell survival rate of macrophages according to treatment with black tea extract and black tea bioconversion extract according to an embodiment of the present invention.
Figure 6 shows the effect of inhibiting NO (nitric oxide) production of black tea extract and black tea bioconversion extract according to an embodiment of the present invention. ** and *** indicate a statistically significant increase in the NO production inhibition effect of the black tea bioconversion extract compared to the black tea extract, ** is p < 0.01, and *** is p < 0.001.
Figure 7 shows the inhibitory effect of black tea extract and black tea bioconversion extract on PGE 2 (prostaglandin E 2 ) production according to an embodiment of the present invention. * indicates a statistically significant increase in the inhibition effect of PGE 2 production of black tea bioconversion extract compared to black tea extract, p<0.05.
Figure 8 shows the inhibitory effect of black tea extract and black tea biotransformation extract on IL-6 production according to an embodiment of the present invention. * indicates a statistically significant increase in the IL-6 production inhibition effect of black tea bioconversion extract compared to black tea extract at a treatment concentration of 0.25 mg/ml, p<0.05.
본 발명의 목적을 달성하기 위하여, 본 발명은 홍차 생물전환 추출물을 유효성분으로 함유하는 피부 염증의 예방 또는 개선; 자외선에 대한 피부 보호; 및 항산화용 화장료 조성물을 제공한다.In order to achieve the purpose of the present invention, the present invention provides a method for preventing or improving skin inflammation containing black tea bioconversion extract as an active ingredient; Skin protection against ultraviolet rays; and a cosmetic composition for antioxidant use.
본 발명에서 사용되는 용어 "생물전환" 또는 "생물전환법"은 미생물을 이용하여 기질 또는 전구물질로부터 인산화(phosphorylation), 글리코실화(glycosylation) 등의 구조적 변화를 유도한 화합물을 생산, 제조하는 방법을 의미한다. 상기 인산화는 인산화효소(kinase)의 작용에 의하여 인산기(phosphoric acid group, HOPO(OH)O-)가 기질의 수소 원자와 치환됨으로써 기질에 결합하는 것을 의미하며, 상기 글리코실화는 기질에 단당체, 다당체 등의 당류(glucoside)가 첨가되는 것을 의미한다. 상기 홍차 생물전환 추출물은 홍차 추출물을 균주와 함께 배양함으로써 생물전환에 의해 홍차에 존재하는 유효성분의 함량이 증가할 수 있고, 홍차에 함유된 유용한 화합물들의 작용기가 치환되어 기존에 존재하지 않던 신규 화합물의 생성을 유도할 수 있다. The term "bioconversion" or "bioconversion method" used in the present invention refers to a method of producing and manufacturing a compound that induces structural changes such as phosphorylation and glycosylation from a substrate or precursor using microorganisms. means. The phosphorylation means binding to the substrate by replacing a phosphoric acid group (HOPO(OH)O-) with the hydrogen atom of the substrate by the action of a phosphorylation enzyme, and the glycosylation involves binding to the substrate as a monosaccharide, This means that glucosides such as polysaccharides are added. The black tea bioconversion extract can increase the content of active ingredients present in black tea by bioconversion by culturing the black tea extract with the strain, and the functional groups of useful compounds contained in black tea are replaced to create new compounds that did not previously exist. can induce the creation of .
상기 홍차 생물전환 추출물은 하기의 단계를 포함하는 방법에 의해 제조할 수 있으나, 이에 한정하지 않는다:The black tea bioconversion extract can be prepared by a method comprising the following steps, but is not limited to this:
(1) 홍차에 추출용매를 가하여 추출하는 단계;(1) Extracting black tea by adding an extraction solvent;
(2) 단계 (1)에서 획득한 홍차 추출물과 미생물을 혼합하여 생물전환 반응을 유도하는 단계; 및(2) mixing the black tea extract obtained in step (1) with microorganisms to induce a bioconversion reaction; and
(3) 단계 (2)에서 획득한 생물전환 반응물을 원심분리한 후, 상등액을 수득하는 단계.(3) Centrifuging the bioconversion reactant obtained in step (2) and obtaining a supernatant.
상기 단계 (1)에서 추출용매는 물, C1~C4의 저급 알코올 또는 이들의 혼합물 중에서 선택하는 것이 바람직하며, 더 바람직하게는 에탄올이지만 이에 한정하지 않는다. 상기 단계 (2)에서 미생물은 바람직하게는 바실러스 속(Bacillus sp.) 균주일 수 있으며, 더 바람직하게는 바실러스 속(Bacillus sp.) JD3-7 균주일 수 있으나, 이에 제한되지 않는다.In step (1), the extraction solvent is preferably selected from water, lower alcohols of C 1 to C 4 , or mixtures thereof, and more preferably ethanol, but is not limited thereto. In step (2), the microorganism may preferably be a Bacillus sp. strain, more preferably a Bacillus sp. JD3-7 strain, but is not limited thereto.
또한, 본 발명의 홍차 생물전환 추출물은 홍차 추출물에 비해 항산화 활성, 자외선에 대한 피부보호 활성 및 항염증 활성이 증진된 것이 특징이다.In addition, the black tea bioconversion extract of the present invention is characterized by improved antioxidant activity, skin protection activity against ultraviolet rays, and anti-inflammatory activity compared to black tea extract.
본 발명의 일 구현 예에 따른 화장료 조성물에서, 상기 홍차 생물전환 추출물은 화장료 조성물의 총 중량에 대하여 0.01 내지 20 중량%로 포함될 수 있으며, 바람직하며, 0.5 내지 10 중량%로 포함될 수 있으며, 가장 바람직하게는 1~5 중량%로 포함될 수 있으나, 이에 제한되지 않는다.In the cosmetic composition according to one embodiment of the present invention, the black tea bioconversion extract may be included in an amount of 0.01 to 20% by weight, preferably, and 0.5 to 10% by weight, based on the total weight of the cosmetic composition, and is most preferably included. It may be included at 1 to 5% by weight, but is not limited thereto.
본 발명의 화장료 조성물은 피부외용 연고, 크림, 유연화장수, 영양화장수, 팩, 에센스, 헤어토닉, 샴푸, 린스, 헤어 컨디셔너, 헤어트리트먼트, 젤, 스킨 로션, 스킨소프너, 스킨토너, 아스트린젠트, 밀크 로션, 모이스처 로션, 영양 로션, 마사지 크림, 영양크림, 아이크림, 모이스처 크림, 핸드크림, 파운데이션, 영양에센스, 선스크린, 비누, 클렌징폼, 클렌징로션, 클렌징크림, 바디로션 및 바디클렌저로 이루어지는 군으로부터 선택된 어느 하나의 제형으로 제조되는 것이 바람직하다. 이들 각 제형으로 이루어진 화장료 조성물은 그 제형의 제제화에 필요하고 적절한 각종의 기제와 첨가물을 함유할 수 있으며, 이들 성분의 종류와 양은 당업자에 의해 용이하게 선정될 수 있다.The cosmetic composition of the present invention includes ointment for external use, cream, softening lotion, nourishing lotion, pack, essence, hair tonic, shampoo, rinse, hair conditioner, hair treatment, gel, skin lotion, skin softener, skin toner, astringent, milk. A group consisting of lotion, moisture lotion, nutritional lotion, massage cream, nutritional cream, eye cream, moisture cream, hand cream, foundation, nutritional essence, sunscreen, soap, cleansing foam, cleansing lotion, cleansing cream, body lotion and body cleanser. It is preferably prepared in any one formulation selected from. A cosmetic composition composed of each of these formulations may contain various bases and additives necessary and appropriate for the formulation of the formulation, and the types and amounts of these components can be easily selected by a person skilled in the art.
본 발명의 화장료 조성물의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물섬유, 식물섬유, 왁스, 파라핀, 전분, 트라가칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a paste, cream, or gel, animal fiber, plant fiber, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide are used as carrier ingredients. etc. can be used.
본 발명의 화장료 조성물의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가로 클로로플루오로히드로카본, 프로판-부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the cosmetic composition of the present invention is powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder can be used as the carrier ingredient, and especially in the case of spray, chlorofluoro It may contain propellants such as hydrocarbon, propane-butane or dimethyl ether.
본 발명의 화장료 조성물의 제형이 용액 또는 유탁액의 경우에는 담체 성분으로서 용매, 용매화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the cosmetic composition of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene. These include fatty acid esters of glycol, 1,3-butylglycol oil, glycerol aliphatic esters, polyethylene glycol or sorbitan.
본 발명의 화장료 조성물의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a suspension, the carrier component includes water, a liquid diluent such as ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant may be used.
본 발명의 화장료 조성물의 제형이 계면-활성제 함유 클렌징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르설페이트, 설포숙신산 모노에스테르, 아세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the cosmetic composition of the present invention is a surfactant-containing cleansing agent, the carrier ingredients include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, acethionate, imidazolinium derivative, methyl taurate, and sarcosinate. , fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester can be used.
본 발명의 화장료 조성물은 형광물질, 살진균제, 굴수성 유발물질, 방향제, 방향제 담체, 단백질, 용해화제, 당유도체, 일광차단제, 비타민, 식물 추출물 등을 포함하는 부형제를 추가로 함유할 수 있다.The cosmetic composition of the present invention may further contain excipients including fluorescent substances, fungicides, hydrotropes-inducing substances, fragrances, fragrance carriers, proteins, solubilizers, sugar derivatives, sunscreens, vitamins, plant extracts, etc.
또한, 본 발명은 In addition, the present invention
(1) 홍차에 추출용매를 가하여 추출하는 단계;(1) Extracting black tea by adding an extraction solvent;
(2) 단계 (1)에서 획득한 홍차 추출물과 바실러스 속(Baillus sp.) 균주를 혼합하여 생물전환 반응을 유도하는 단계; 및(2) mixing the black tea extract obtained in step (1) with a Baillus sp. strain to induce a bioconversion reaction; and
(3) 단계 (2)에서 획득한 생물전환 반응물을 원심분리한 후, 상등액을 수득하는 단계;를 포함하여 제조되는 것을 특징으로 하는 피부 염증의 예방 또는 개선, 자외선에 대한 피부 보호 및 항산화 효과를 갖는 홍차 생물전환 추출물의 제조 방법을 제공한다.(3) centrifuging the bioconversion reactant obtained in step (2) and then obtaining a supernatant; preventing or improving skin inflammation, protecting the skin against ultraviolet rays, and providing antioxidant effects. A method for producing a black tea bioconversion extract is provided.
본 발명의 홍차 생물전환 추출물의 제조 방법에서, 상기 추출용매는 물, C1~C4의 저급 알코올 또는 이들의 혼합물 중에서 선택하는 것이 바람직하며, 더 바람직하게는 에탄올이지만 이에 한정하지 않는다. 상기 단계 (2)에서 바실러스 속(Bacillus sp.) 균주는 바람직하게는 바실러스 속(Bacillus sp.) JD3-7 균주일 수 있으나, 이에 제한되지 않는다.In the method for producing a black tea bioconversion extract of the present invention, the extraction solvent is preferably selected from water, C 1 -C 4 lower alcohol, or a mixture thereof, more preferably ethanol, but is not limited thereto. In step (2), the Bacillus sp. strain may preferably be a Bacillus sp. JD3-7 strain, but is not limited thereto.
이하, 본 발명의 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
제조예 1. 홍차 추출물 제조Preparation Example 1. Preparation of black tea extract
건조된 홍차 150g에 70%(v/v) 에탄올 1ℓ를 첨가하고 실온에서 24시간 동안 추출하여 1차 추출물을 회수하고, 70%(v/v) 에탄올 1ℓ를 추가로 첨가하여 실온에서 24시간 동안 추출하여 2차 추출물을 회수한 후, 회수한 1차 및 2차 추출물을 혼합하여 5.0㎛의 공극 크기를 갖는 필터(TOYO, Japan)로 여과하여 홍차 추출물을 수득하였다. 여과된 추출물을 5 브릭스(brix) 이상으로 진공농축하여 준비하였다.Add 1 liter of 70% (v/v) ethanol to 150 g of dried black tea and extract at room temperature for 24 hours to recover the primary extract. Add 1 liter of 70% (v/v) ethanol and extract at room temperature for 24 hours. After extracting and recovering the secondary extract, the recovered primary and secondary extracts were mixed and filtered through a filter (TOYO, Japan) with a pore size of 5.0㎛ to obtain a black tea extract. The filtered extract was prepared by vacuum concentration to 5 Brix or more.
제조예 2. 홍차 생물전환 추출물 제조Preparation Example 2. Preparation of black tea bioconversion extract
생물전환(Biorenovation)을 위한 미생물은 제주도 콩 재래 간장에서 분리한 바실러스 속 JD3-7(Bacillus sp. JD3-7, KACC 92346P) 균주를 분양받아 사용하였으며, NB(Nutrient broth) 배지를 이용하여 30℃에서 20시간 동안 배양하였다. 이후, 5,000rpm에서 10분간 원심분리하여 회수한 균체를 PG 버퍼(50mM Phosphate buffer pH 7.2, 2% Glycerin)로 2회 세척한 뒤 5㎖의 PG 버퍼에 현탁하였으며, 플라스크에 상기 제조예 1의 홍차 추출물과 바실러스 속 JD3-7 균주 현탁액을 첨가하여 30℃에서 72시간 동안 동일한 조건하에 생물전환 반응을 수행하였다. 반응 종료 후, 원심분리하여 상등액을 수거하고 감압농축하여 물에 용해시켜 홍차 생물전환 추출물을 준비하였다.The microorganism for biorenovation was the Bacillus JD3-7 ( Bacillus sp. JD3-7, KACC 92346P) strain isolated from traditional soybean sauce from Jeju Island, and cultured at 30°C using NB (Nutrient broth) medium. was cultured for 20 hours. Afterwards, the cells recovered by centrifugation at 5,000 rpm for 10 minutes were washed twice with PG buffer (50mM Phosphate buffer pH 7.2, 2% Glycerin) and suspended in 5 ml of PG buffer. The black tea of Preparation Example 1 was added to the flask. The bioconversion reaction was performed under the same conditions at 30°C for 72 hours by adding the extract and suspension of the Bacillus JD3-7 strain. After completion of the reaction, the supernatant was collected by centrifugation, concentrated under reduced pressure, and dissolved in water to prepare a black tea bioconversion extract.
실시예 1. HPLC 분석Example 1. HPLC analysis
상기 제조예 1 및 2에서 제조된 홍차 추출물 및 홍차 생물전환 추출물에 대해 HPLC(high performance liquid chromatography) 분석을 통해 생물전환 화합물의 생성여부를 확인하였다. The black tea extract and black tea bioconversion extract prepared in Preparation Examples 1 and 2 were tested for production of bioconversion compounds through high performance liquid chromatography (HPLC) analysis.
구체적으로, Prominence HPLC System(Shimadzu, Japan) 장치를 사용하였으며, 검출기로 LC-2030/2040 PDA Detector를, 칼럼으로 Shim-pack GIS를 사용하였다. 용매로 0.1% TFA 및 아세토나이트릴(acetonitrile)을 사용하였으며, 분석 조건은 30분까지 아세토나이트릴 10%에서 100%로 하였다. 파장을 254nm로, 유속을 1 mL/min으로 설정하였고, 주입량을 10㎕로 하였다. Specifically, a Prominence HPLC System (Shimadzu, Japan) device was used, with LC-2030/2040 PDA Detector as a detector and Shim-pack GIS as a column. 0.1% TFA and acetonitrile were used as solvents, and analysis conditions were 10% to 100% acetonitrile for 30 minutes. The wavelength was set to 254 nm, the flow rate was set to 1 mL/min, and the injection volume was 10 μl.
그 결과, 도 1에 개시한 바와 같이, 홍차 생물전환 추출물은 홍차 추출물에 존재하지 않는 다수의 피크(peak)를 포함하였고, 일부 피크의 세기가 강해진 것으로 확인되었다.As a result, as shown in Figure 1, it was confirmed that the black tea bioconversion extract contained a number of peaks that did not exist in the black tea extract, and that the intensity of some peaks became stronger.
실시예 2. 피부세포 보호 효과Example 2. Skin cell protection effect
상기 제조예 1 및 2에서 제조된 홍차 추출물 및 홍차 생물전환 추출물은 0.25 및 0.5㎎/㎖의 농도로 사용하였고, 세포는 ATCC사로부터 구입한 CCD-1064sk 인간 섬유아세포(human fibroblast cell line) 및 RAW 264.7 세포를 사용하였다. CD-1064sk 인간 섬유아세포의 배양 배지로는 10% FBS(fetal bovine serum) 및 1% 페니실린-스트렙토마이신(penicillin-streptomycin)이 첨가된 IMDM(Iscove's modified Dulbeco's medium)을 사용하였으며, RAW 264.7 세포의 배양 배지로는 10% FBS(fetal bovine serum) 및 1% 페니실린-스트렙토마이신(penicillin-streptomycin)이 첨가된 DMEM(Dulbecco's Modified Eagle Medium)을 사용하였다. 상기 각 세포는 10㎝ 세포 배양접시에 10㎖의 배지로 37℃, 5% CO2 조건의 배양기에서 세포가 70~80% 합류(confluency)가 되도록 배양하였다. 배지는 주 2회 교환하고, 합류에 도달한 세포는 트립신(trypsin)-EDTA를 사용하여 트립신 처리(trypsinization)한 후 계대배양하여 유지하였다.The black tea extract and black tea bioconversion extract prepared in Preparation Examples 1 and 2 were used at a concentration of 0.25 and 0.5 mg/ml, and the cells were CCD-1064sk human fibroblast cell line and RAW purchased from ATCC. 264.7 cells were used. IMDM (Iscove's modified Dulbeco's medium) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin was used as a culture medium for CD-1064sk human fibroblasts, and RAW 264.7 cells were cultured. As a medium, DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum) and 1% penicillin-streptomycin was used. Each of the cells was cultured in a 10-cm cell culture dish with 10 ml of medium in an incubator at 37°C and 5% CO 2 conditions until the cells reached 70-80% confluency. The medium was changed twice a week, and cells that reached confluence were trypsinized using trypsin-EDTA and maintained by subculture.
피부세포 보호 효과를 측정하기 위해, 상기 세포 배양액에서 배지를 제거하고 PBS 버퍼로 2번 세척한 후, UVA/UVB 측정기 850009(Sper scientific, Scottsdale, AZ, USA)로 자외선 조사 동안 세포가 건조되지 않도록 1㎖의 PBS를 배양 용기에 분주한 다음, UVB를 선량별로 50, 100, 200, 300mJ/㎠를 조사하였다. 이후 최적화된 UVB 200mJ/㎠의 조건에서 홍차 추출물 및 홍차 생물전환 추출물을 0.25 및 0.5㎎/㎖의 농도로 1시간 처리한 후 UVB 200mJ/㎠를 조사하였다. 1㎖의 PBS 버퍼로 1회 세척한 다음, 홍차 추출물 및 홍차 생물전환 추출물을 0.25 및 0.5㎎/㎖의 농도로 첨가된 배양액으로 교환하였다. 세포에 UVB 200mJ/㎠를 조사하고 4시간이 지난 후에 MTT 분석을 통해 세포생존율을 측정하였다. PBS 버퍼에 용해한 5㎎/㎖의 MTT 용액을 새로운 배지에서 10배 희석하고, 이 용액을 세포 각각의 웰에 100㎕씩 첨가하여 37℃에서 4시간 반응시켰다. 배양 상등액을 제거하고 각각의 웰에 200㎕의 DMSO(dimethyl sulfoxide)를 첨가하여 세포에서 생성된 MTT-포르마잔 결정체를 용해시킨 후 흡수분광광도계(UVmax, Molecular Device, USA)를 이용하여 540㎚에서 흡광도를 측정하였다. 각 시료군에 대한 평균 흡광도를 측정하였으며, 대조군의 흡광도 측정값과 비교하여 세포독성을 평가하였다.To measure the skin cell protection effect, the medium was removed from the cell culture, washed twice with PBS buffer, and then measured with a UVA/UVB meter 850009 (Sper scientific, Scottsdale, AZ, USA) to prevent the cells from drying out during ultraviolet irradiation. 1 ml of PBS was dispensed into the culture vessel, and then UVB was irradiated at doses of 50, 100, 200, and 300 mJ/cm2. Afterwards, black tea extract and black tea bioconversion extract were treated for 1 hour at concentrations of 0.25 and 0.5 mg/ml under the optimized conditions of UVB 200mJ/cm2 and then irradiated with UVB 200mJ/cm2. After washing once with 1 ml of PBS buffer, the black tea extract and black tea biotransformation extract were exchanged for culture medium added at concentrations of 0.25 and 0.5 mg/ml. Cells were irradiated with 200 mJ/cm2 of UVB and 4 hours later, cell viability was measured through MTT analysis. A 5 mg/ml MTT solution dissolved in PBS buffer was diluted 10-fold in new medium, and 100 μl of this solution was added to each cell well and incubated at 37°C for 4 hours. The culture supernatant was removed, 200㎕ of DMSO (dimethyl sulfoxide) was added to each well to dissolve the MTT-formazan crystals generated in the cells, and then measured at 540㎚ using an absorption spectrophotometer (UVmax, Molecular Device, USA). Absorbance was measured. The average absorbance for each sample group was measured, and cytotoxicity was evaluated by comparing it with the absorbance measurement of the control group.
그 결과, 도 2에 개시한 바와 같이 UVB(200mJ/㎠) 조사하고 홍차 추출물 및 홍차 생물전환 추출물을 1시간 처리한 후 세포 생존율을 측정하였을 때, 아무것도 처리하지 않은 대조군 대비 UVB 단독 처리군에서 세포 생존율이 현저하게 감소되었다. 반면, 홍차 추출물 및 홍차 생물전환 추출물 처리군에서 농도 의존적으로 세포 생존율이 모두 증가되었으며, 홍차 추출물 대비 홍차 생물전환 추출물 처리군의 세포 생존율이 통계적으로 유의미하게 더 증가되었다.As a result, when cell viability was measured after irradiation with UVB (200 mJ/㎠) and treatment with black tea extract and black tea bioconversion extract for 1 hour as shown in Figure 2, cells in the group treated with UVB alone compared to the untreated control group were Survival rate was significantly reduced. On the other hand, the cell survival rate increased in a concentration-dependent manner in both the black tea extract and black tea bioconversion extract treatment groups, and the cell survival rate in the black tea bioconversion extract treatment group was statistically significantly increased compared to the black tea extract.
실시예 3. 항산화 활성Example 3. Antioxidant activity
(1) DPPH(1,1-diphenyl-2 picrylhydrazyl) 라디칼 소거활성(1) DPPH (1,1-diphenyl-2 picrylhydrazyl) radical scavenging activity
DPPH(D9132, SIGMA)를 메탄올 및 증류수 6:4(v/v)의 혼합용매에 용해하여 DPPH 용액을 제조한 후, 513nm에서 흡광도 값을 약 0.6으로 조정하여 실험하였다. 제조예 1 및 2에서 제조된 홍차 추출물 및 홍차 생물전환 추출물 각각에 2㎖의 DPPH 용액을 혼합하여 실온에서 1시간 동안 반응시킨 후, 분광광도계(UVmax, Molecular Device, USA)를 이용하여 513nm에서 흡광도를 측정하였다. 양성대조군으로 1% 아스코르브산(ascorbic acid)을 사용하였다.DPPH (D9132, SIGMA) was dissolved in a mixed solvent of methanol and distilled water 6:4 (v/v) to prepare a DPPH solution, and then tested by adjusting the absorbance value to about 0.6 at 513 nm. 2 ml of DPPH solution was mixed with each of the black tea extract and black tea bioconversion extract prepared in Preparation Examples 1 and 2, reacted at room temperature for 1 hour, and then measured for absorbance at 513 nm using a spectrophotometer (UVmax, Molecular Device, USA). was measured. 1% ascorbic acid was used as a positive control.
그 결과, 도 3에 개시한 바와 같이 홍차 추출물 및 홍차 생물전환 추출물은 농도 의존적으로 DPPH 라디칼 소거활성이 증가하였으며, 홍차 추출물 대비 홍차 생물전환 추출물의 DPPH 라디칼 소거활성이 통계적으로 유의미하게 더 증가하였다.As a result, as shown in Figure 3, the DPPH radical scavenging activity of the black tea extract and the black tea bioconversion extract increased in a concentration-dependent manner, and the DPPH radical scavenging activity of the black tea bioconversion extract increased statistically significantly compared to the black tea extract.
(2) 수퍼옥사이드 음이온 라디칼(superoxide anion radical) 소거활성(2) Superoxide anion radical scavenging activity
수퍼옥사이드 음이온 라디칼 소거 활성은 NBT(nitro blue tetrazolium) 환원방법에 의해 측정하였다. 300mM PBS 버퍼(pH 7.8)에 1.6㎖의 트리톤 X-100(triton X-100), 2.9mg의 EDTA, 1mg의 NBT(Sigma) 및 5.4mg의 하이포크산틴(hypoxanthine)을 넣어 기질/반응 용액을 제조하였다. 12㎕의 크산틴 옥시다제를 300mM PBS 버퍼(pH 7.8) 10㎖에 용해시켜 효소 용액을 제조하였다. 분광광도계의 셀 내에 1㎖의 버퍼를 넣은 다음, 상기 제조한 기질/반응 용액 0.5㎖을 첨가하였다. 그 후, 제조예 1 및 2에서 제조된 홍차 추출물 및 홍차 생물전환 추출물을 다양한 농도로 제조하여 각각 100㎕씩 처리하였으며, 상기 제조된 0.5㎖의 효소 용액을 첨가하고 37℃에서 30분간 반응시킨 후 540nm에서 흡광도를 측정하였다.Superoxide anion radical scavenging activity was measured by the NBT (nitro blue tetrazolium) reduction method. Prepare a substrate/reaction solution by adding 1.6 ml of Triton did. An enzyme solution was prepared by dissolving 12㎕ of xanthine oxidase in 10㎖ of 300mM PBS buffer (pH 7.8). 1 ml of buffer was placed in the cell of the spectrophotometer, and then 0.5 ml of the substrate/reaction solution prepared above was added. Afterwards, the black tea extract and the black tea bioconversion extract prepared in Preparation Examples 1 and 2 were prepared at various concentrations and treated at 100 ㎕ each. 0.5 mL of the enzyme solution prepared above was added and reacted at 37°C for 30 minutes. Absorbance was measured at 540 nm.
그 결과, 도 4에 개시한 바와 같이 홍차 추출물 및 홍차 생물전환 추출물은 농도 의존적으로 수퍼옥사이드 음이온 라디칼 소거활성이 증가하였으며, 홍차 추출물 대비 홍차 생물전환 추출물의 수퍼옥사이드 음이온 라디칼 소거활성이 통계적으로 유의미하게 더 증가하였다.As a result, as shown in Figure 4, the superoxide anion radical scavenging activity of the black tea extract and the black tea bioconversion extract increased in a concentration-dependent manner, and the superoxide anion radical scavenging activity of the black tea bioconversion extract was statistically significant compared to the black tea extract. It increased further.
실시예 4. 세포 독성Example 4. Cytotoxicity
RAW264.7 세포에 대한 홍차 추출물 및 홍차 생물전환 추출물의 세포 독성은 MTT 분석을 이용한 세포 생존율 분석을 통해 측정하였다. 10% FBS(fetal bovine serum)가 첨가된 DMEM(Dulbecco's Modified Eagle Medium)을 이용하여 24-웰 플레이트에 8.0×104 cells/㎖의 농도로 분주하여 18시간 동안 전 배양한 후, 홍차 추출물 또는 홍차 생물전환 추출물과 LPS(1㎍/㎖)를 동시에 처리하여 24시간 동안 반응시켰다. 이후, 각 웰에 MTT(Thiazolyl Blue Tetrazolium Bromide) 시약을 첨가하여 37℃에서 4시간 동안 반응시킨 다음, 상층액을 제거한 후 형성된 포르마잔 블루(Formazan blue)에 DMSO를 첨가하여 용해한 뒤, ELISA 리더를 이용하여 570nm에서 흡광도를 측정하였다. The cytotoxicity of black tea extract and black tea biotransformation extract against RAW264.7 cells was measured through cell viability analysis using the MTT assay. Using DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum), the cells were distributed at a concentration of 8.0 The bioconversion extract and LPS (1㎍/㎖) were treated simultaneously and reacted for 24 hours. Afterwards, MTT (Thiazolyl Blue Tetrazolium Bromide) reagent was added to each well and reacted at 37°C for 4 hours. After removing the supernatant, DMSO was added to dissolve the formed Formazan blue, and then ELISA reader was used. Absorbance was measured at 570 nm using
그 결과, 도 5에 개시한 바와 같이 홍차 추출물 및 홍차 생물전환 추출물은 모든 처리농도에서 95% 이상의 세포생존율을 나타내었다.As a result, as shown in Figure 5, black tea extract and black tea bioconversion extract showed a cell viability of more than 95% at all treatment concentrations.
실시예 5. 항염증 효과Example 5. Anti-inflammatory effect
(1) NO(Nitric oxide) 생성 억제(1) Inhibition of NO (Nitric oxide) production
10% FBS(fetal bovine serum)가 첨가된 DMEM(Dulbecco's Modified Eagle Medium)을 이용하여 24-웰 플레이트에 RAW 264.7 세포를 8.0×104 cells/well의 농도로 분주한 뒤, 37℃, 5% CO2 조건의 인큐베이터에서 18시간 동안 전 배양하였다. 이후 홍차 추출물 또는 홍차 생물전환 추출물과 LPS(1㎍/㎖)를 동시 처리하여 24시간 배양한 다음, 이후 배양액을 10,000rpm에서 3분 동안 원심분리하여 침전물을 제거한 뒤, 상등액을 회수하였다. 회수한 상층액 100㎕ 및 그리스 시약[1% (w/v) sulfanilamide, 0.1%(w/v) naphylethylenediamine in 2.5%(v/v) phosphoric acid] 100㎕를 96-웰 플레이트에서 혼합하여 10분간 암반응 시킨 후 540nm에서 흡광도를 측정하였다. RAW 264.7 cells were seeded at a concentration of 8.0 Pre-cultured for 18 hours in an incubator under 2 conditions. Afterwards, black tea extract or black tea bioconversion extract and LPS (1 μg/ml) were simultaneously treated and cultured for 24 hours. The culture was then centrifuged at 10,000 rpm for 3 minutes to remove precipitates, and the supernatant was recovered. 100 ㎕ of the recovered supernatant and 100 ㎕ of Greek reagent [1% (w/v) sulfanilamide, 0.1% (w/v) naphylethylenediamine in 2.5% (v/v) phosphoric acid] were mixed in a 96-well plate for 10 minutes. After dark reaction, absorbance was measured at 540 nm.
그 결과, 도 6에 개시한 바와 같이 아무것도 처리하지 않은 대조군 대비 LPS 단독처리군에서 NO 생성이 현저하게 증가되었다. 반면, 홍차 추출물 처리군 및 홍차 생물전환 추출물 처리군에서 LPS 단독처리군과 비교하여 NO 생성이 농도의존적으로 감소되었으며, 홍차 생물전환 추출물은 기존 홍차 추출물에 대비하여 NO 생성이 현저하게 더 감소되었다.As a result, as shown in Figure 6, NO production was significantly increased in the LPS-only treated group compared to the untreated control group. On the other hand, NO production was reduced in a concentration-dependent manner in the black tea extract treatment group and the black tea bioconversion extract treatment group compared to the LPS-only treatment group, and the black tea bioconversion extract significantly reduced NO production compared to the existing black tea extract.
(2) PGE(2) PGE 22 (Prostaglandin E(Prostaglandin E 22 )) 및 전염증성 사이토카인의 생성 억제and inhibition of production of pro-inflammatory cytokines.
10% FBS(fetal bovine serum)가 첨가된 DMEM(Dulbecco's Modified Eagle Medium)을 이용하여 24-웰 플레이트에 RAW 264.7 세포를 8.0×104 cells/well의 농도로 분주하여 37℃, 5% CO2 조건의 인큐베이터에서 18시간 동안 전 배양한 뒤, 홍차 추출물 또는 홍차 생물전환 추출물과 LPS(1㎍/㎖)를 동시 처리하여 24시간 동안 배양하였다. 이후 배양액을 12,000rpm에서 3분 동안 원심분리하여 침전물을 제거한 뒤, 상등액을 회수하였다. 회수한 상등액에서 PGE2의 함량은 ELISA 키트(Mouse PGE2, R&D Systems, USA)를 이용하여 측정하였고, 전염증성 사이토카인 IL-6(Interleukin-6)의 함량은 마우스 IL-6 ELISA 키트(San diego, USA)를 이용하여 측정하였다.Using DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS (fetal bovine serum), RAW 264.7 cells were distributed at a concentration of 8.0 After pre-culturing in an incubator for 18 hours, black tea extract or black tea biotransformation extract and LPS (1 μg/ml) were simultaneously treated and cultured for 24 hours. Afterwards, the culture was centrifuged at 12,000 rpm for 3 minutes to remove precipitates, and the supernatant was recovered. PGE 2 in the recovered supernatant The content was measured using an ELISA kit (Mouse PGE 2 , R&D Systems, USA), and the content of proinflammatory cytokine IL-6 (Interleukin-6) was measured using a mouse IL-6 ELISA kit (San diego, USA). Measured.
그 결과, 도 7 및 도 8에 개시한 바와 같이 아무것도 처리하지 않은 대조군 대비 LPS 단독처리군에서 PGE2 및 IL-6 생성이 현저하게 증가되었다. 반면, 홍차 추출물 처리군 및 홍차 생물전환 추출물 처리군에서 LPS 단독처리군과 비교하여 PGE2 및 IL-6 생성이 농도의존적으로 억제되었으며, 홍차 생물전환 추출물은 기존 홍차 추출물에 대비하여 PGE2 및 IL-6 생성이 현저하게 더 감소되었다. As a result, as shown in Figures 7 and 8, the production of PGE 2 and IL-6 was significantly increased in the LPS-only treated group compared to the untreated control group. On the other hand, in the black tea extract treatment group and the black tea bioconversion extract treatment group, the production of PGE 2 and IL-6 was suppressed in a concentration-dependent manner compared to the LPS-only treatment group, and the black tea bioconversion extract produced PGE 2 and IL-6 compared to the existing black tea extract. -6 production was significantly reduced.
제형예 1-3. 유연화장수(스킨) Formulation Example 1-3. Soft lotion (skin)
하기 표 1에 개시된 조성에 따라, 균일한 겔(Gel)상이 되도록 충분히 분산 및 습윤하여 혼합한 후 중화제를 통해 첨가하여 중화하고 미용성분을 첨가하여 균일 교반하여 가용화시킨 후 균일하게 분포되도록 교반하였으며, 용기에 담아 본 발명의 홍차 생물전환 추출물을 포함하는 유연화장수(스킨)를 제조하였다.According to the composition disclosed in Table 1 below, the mixture was sufficiently dispersed, moistened and mixed to form a uniform gel, then neutralized by adding a neutralizer, and then added with cosmetic ingredients and stirred to solubilize, and then stirred to ensure uniform distribution. Soft lotion (skin) containing the black tea bioconversion extract of the present invention was prepared in a container.
제형예 4-6. 영양유액(밀크로션)Formulation Example 4-6. Nutritional emulsion (milk lotion)
하기 표 2에 개시된 조성에 따라, 균일한 겔(Gel)상이 되도록 충분히 분산 및 습윤하여 혼합한 후 중화제를 통해 첨가하여 중화하고 미용성분을 첨가하여 균일 교반하여 가용화시킨 후 균일하게 분포되도록 교반하였으며, 용기에 담아 본 발명의 홍차 생물전환 추출물을 포함하는 영양유액(밀크로션)를 제조하였다.According to the composition disclosed in Table 2 below, the mixture was sufficiently dispersed, moistened and mixed to form a uniform gel, then neutralized by adding a neutralizer, and then added with cosmetic ingredients and stirred to solubilize, and then stirred to ensure uniform distribution. A nutritional emulsion (milk lotion) containing the black tea bioconversion extract of the present invention was prepared in a container.
제형예 7-9. 크림Formulation Example 7-9. cream
하기 표 3에 개시된 조성에 따라, 균일한 겔(Gel)상이 되도록 충분히 분산 및 습윤하여 혼합한 후 중화제를 통해 첨가하여 중화하고 미용성분을 첨가하여 균일 교반하여 가용화시킨 후 균일하게 분포되도록 교반하였으며, 용기에 담아 본 발명의 홍차 생물전환 추출물을 포함하는 크림을 제조하였다.According to the composition disclosed in Table 3 below, the mixture was sufficiently dispersed, moistened and mixed to form a uniform gel, then neutralized by adding a neutralizer, and then added with cosmetic ingredients and stirred to solubilize, and then stirred to ensure uniform distribution. A cream containing the black tea bioconversion extract of the present invention was prepared in a container.
Claims (8)
(1) 홍차에 추출용매를 가하여 추출하는 단계;
(2) 단계 (1)에서 획득한 홍차 추출물과 미생물을 혼합하여 생물전환 반응을 유도하는 단계; 및
(3) 단계 (2)에서 획득한 생물전환 반응물을 원심분리한 후, 상등액을 수득하는 단계;를 포함하여 제조되는 것을 특징으로 하는 피부 염증의 예방 또는 개선, 자외선에 대한 피부 보호 및 항산화용 화장료 조성물.The method of claim 1, wherein the black tea bioconversion extract is
(1) Extracting black tea by adding an extraction solvent;
(2) mixing the black tea extract obtained in step (1) with microorganisms to induce a bioconversion reaction; and
(3) centrifuging the bioconversion reactant obtained in step (2) and obtaining a supernatant; a cosmetic for preventing or improving skin inflammation, protecting the skin against ultraviolet rays, and providing antioxidant properties, comprising: Composition.
(2) 단계 (1)에서 획득한 홍차 추출물과 바실러스 속(Baillus sp.) 균주를 혼합하여 생물전환 반응을 유도하는 단계; 및
(3) 단계 (2)에서 획득한 생물전환 반응물을 원심분리한 후, 상등액을 수득하는 단계;를 포함하여 제조되는 것을 특징으로 하는 피부 염증의 예방 또는 개선, 자외선에 대한 피부 보호 및 항산화 효과를 갖는 홍차 생물전환 추출물의 제조 방법.(1) Extracting black tea by adding an extraction solvent;
(2) mixing the black tea extract obtained in step (1) with a Baillus sp. strain to induce a bioconversion reaction; and
(3) centrifuging the bioconversion reactant obtained in step (2) and then obtaining a supernatant; preventing or improving skin inflammation, protecting the skin against ultraviolet rays, and providing antioxidant effects. Method for producing black tea bioconversion extract.
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