KR102198964B1 - Composition comprising essential oil extract derived anthoxylum coreanum Nakaiis for prevention or treatment of allergic diseases - Google Patents
Composition comprising essential oil extract derived anthoxylum coreanum Nakaiis for prevention or treatment of allergic diseases Download PDFInfo
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- KR102198964B1 KR102198964B1 KR1020180119232A KR20180119232A KR102198964B1 KR 102198964 B1 KR102198964 B1 KR 102198964B1 KR 1020180119232 A KR1020180119232 A KR 1020180119232A KR 20180119232 A KR20180119232 A KR 20180119232A KR 102198964 B1 KR102198964 B1 KR 102198964B1
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- Prior art keywords
- essential oil
- oil extract
- extract derived
- allergic
- cells
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Abstract
본 발명은 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환의 예방 또는 치료용 약학적 조성물에 관한 것으로, 더욱 상세하게는 왕초피나무 유래 정유 추출물은 베타-헥소사미니데이즈 방출을 억제하고, 염증성 사이토카인 및 IgE를 억제하는 효과가 있어 알레르기 질환의 치료효과가 있고, 나아가 아토피 피부염을 유도한 동물에게 처리한 결과 염증반응을 억제하는 효과가 있으므로, 상기 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for the prevention or treatment of allergic diseases comprising an essential oil extract derived from C. C., more specifically, an essential oil extract derived from C. C., inhibits beta-hexosaminidase release, and inflammatory cytokines and Since it has the effect of inhibiting IgE, it has the effect of treating allergic diseases, and further, it has the effect of suppressing the inflammatory response as a result of treatment on the animal that induces atopic dermatitis. It relates to a pharmaceutical composition for use.
Description
본 발명은 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환의 예방 또는 치료용 약학적 조성물에 관한 것으로, 더욱 상세하게는 왕초피나무 유래 정유 추출물은 베타-헥소사미니데이즈 방출을 억제하고, 염증성 사이토카인 및 IgE를 억제하는 효과가 있어 알레르기 질환의 치료효과가 있고, 나아가 아토피 피부염을 유도한 동물에게 처리한 결과 염증반응을 억제하는 효과가 있으므로, 상기 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환의 예방 또는 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for the prevention or treatment of allergic diseases comprising an essential oil extract derived from C. C., more specifically, an essential oil extract derived from C. C., inhibits beta-hexosaminidase release, and inflammatory cytokines and Since it has the effect of inhibiting IgE, it has the effect of treating allergic diseases, and further, it has the effect of suppressing the inflammatory response as a result of treatment on the animal that induces atopic dermatitis.Therefore, the prevention or treatment of allergic diseases including the essential oil extract derived from C. It relates to a pharmaceutical composition for use.
대부분의 알레르기 환자는 유전적으로 IgE 생성에 취약하다. 비만 세포는 전 염증성 알레르기 반응에 중요한 고친화성 IgE 수용체를 세포막에 표출한다. IgE 항원이 FcεRI (면역 글로블린 E의 Fc 영역에 대한 고친화성 수용체)와 결합하면 수용체가 활성화되고 염증 질환을 비롯한 알레르기 반응을 일으키는 복잡한 생물학적 반응이 발생한다. 또한, PMA/A23187과 병용 처리는 염증성 사이토카인의 생성을 유도하는 것으로 알려졌기 때문에 비만 세포의 활성화에 널리 사용되어왔다. 비만 세포주인 RBL-2H3 세포는 쥐의 호염기구성 백혈병에서 유래하였으며, IgE-FcγRI 상호 작용 및 탈과립화를 연구하는 데 사용되어왔다. 또한, RBL-2H3 세포는 In vitro 상에서 항알레르기제 후보 물질을 선별하는데 유용한 모델이다. 과립-연합 엑소글리코시다제인 베타-헥소사미니다아제 (β-hexosaminidase)는 비만세포의 분비과립에 저장되며, 히스타민이 사용된 것처럼 비만세포의 탈과립을 모니터 하는 데 사용되어 왔다. IL-4는 알레르기 질환의 발병에 있어 IgE 합성과 비만 세포의 발달에 중요한 역할을 한다. 알레르기는 전 세계적으로 임상 건강 문제이며, 알레르기 환자는 매년 다양한 요인으로 인해 매년 증가하고 있고 전 세계 인구의 약 10 ~ 20%가 알레르기에 의해 영향을 받는다. 따라서 많은 연구자가 항알레르기 효과를 가지는 천연물에 대하여 연구하였다. 예를 들면, 한국특허공개번호 제10-2017-0110067호에는 연 자방 및 연 수술 추출물을 유효성분으로 포함하는 알레르기 질환의 예방 및 치료용 조성물이 개시되어 있고, 한국특허등록번호 제10-1402599호에 각시취 추출물을 유효성분으로 함유하는 알레르기성 질환의 예방 및 치료용 조성물에 개시되어 있다.Most allergy sufferers are genetically susceptible to IgE production. Mast cells express high-affinity IgE receptors, which are important for proinflammatory allergic reactions, on the cell membrane. When IgE antigen binds to FcεRI (a high-affinity receptor for the Fc region of immunoglobulin E), the receptor is activated and a complex biological reaction that causes allergic reactions, including inflammatory diseases, occurs. In addition, treatment in combination with PMA/A23187 has been widely used for activation of mast cells because it is known to induce the production of inflammatory cytokines. The mast cell line, RBL-2H3 cells, was derived from murine basophil leukemia and has been used to study IgE-FcγRI interactions and degranulation. In addition, RBL-2H3 cells are a useful model for selecting candidate anti-allergic agents in vitro . Beta-hexosaminidase, a granule-associated exoglycosidase, is stored in secreted granules in mast cells and has been used to monitor mast cell degranulation as histamine has been used. IL-4 plays an important role in IgE synthesis and mast cell development in the onset of allergic diseases. Allergy is a clinical health problem worldwide, and allergy sufferers are increasing each year due to a variety of factors, and about 10 to 20% of the world's population is affected by allergies. Therefore, many researchers have studied natural products with anti-allergic effects. For example, Korean Patent Publication No. 10-2017-0110067 discloses a composition for the prevention and treatment of allergic diseases containing extracts of lotus leaf and lotus surgery as active ingredients, and Korean Patent Registration No. 10-1402599 It is disclosed in a composition for the prevention and treatment of allergic diseases containing the extract of Gakshichwi as an active ingredient.
염증은 면역 세포의 활성화 및 비활성화를 수반하는 복잡한 기전(機轉)으로, 만성질환이 유발되는 세포질 및 조직 손상을 가져올 수 있다. 대식세포는 염증 반응을 시작하고 유지하는 주요 면역 세포이다. 그람음성균(Gram-negative bacteria) 외막의 주성분인 지질다당류 (Lipopolyssacharide (LPS))는 대식세포의 염증 반응을 유도하고, 산화질소 (NO), 종양 괴사 인자 (TNF-α) 및 IL-6와 같은 염증성 매개체의 생성을 자극할 수 있다. 이것은 샘플의 항염증 활성을 평가하는 데 사용될 수 있다. NF-κB는 염증에 관여하는 전염증성 매개체의 발현을 조절하는 보편적인 전사 인자이다. NF-κB는 핵으로 전이되고 염증성 신호 분자에 반응하는 cyclooxygenase 2 (COX-2) 및 iNOS와 같은 전염증성 효소의 발현을 이끄는 다양한 전이인자의 발현을 조절한다.Inflammation is a complex mechanism involving the activation and inactivation of immune cells, and can lead to cellular and tissue damage that causes chronic diseases. Macrophages are the main immune cells that initiate and maintain the inflammatory response. Lipopolyssacharide (LPS), the main component of the outer membrane of Gram-negative bacteria, induces the inflammatory response of macrophages, and nitric oxide (NO), tumor necrosis factor (TNF-α) and IL-6 It can stimulate the production of inflammatory mediators. It can be used to evaluate the anti-inflammatory activity of the sample. NF-κB is a universal transcription factor that regulates the expression of pro-inflammatory mediators involved in inflammation. NF-κB modulates the expression of a variety of transfer factors leading to the expression of pro-inflammatory enzymes such as cyclooxygenase 2 (COX-2) and iNOS, which metastasize to the nucleus and respond to inflammatory signaling molecules.
한국의 라임나무인 왕초피 나무(Zanthoxylum coreanum Nakai; ZCO)는 한국과 중국에서만 자라는 희귀한 관목이다. 한국에서는 왕초피나무라고 불린다. 이 수종은 제주도의 해발 700-1100m에 서식하는 희귀 수종이며, 많은 목적으로 사용되고 있다. 한국에서 Zanthoxylum piperitum는 초피나무라고 불린다. 왕초피나무의 과피는 한국에서 위축성 비염, 류마티스, 비강염, 인후통 등의 생약 치료제(crude medicine)로 사용되어왔다. 또한, 최화정에 의해 수행된 선행 연구에 따르면 왕초피나무는 피코르나바이러스에 대한 항바이러스 효과를 가지고 있는 것이 입증되었다. 그러나 왕초피나무의 정유에 대한 항알레르기성 염증 효과는 입증되지 않았다.The Korean lime tree, Zanthoxylum coreanum Nakai (ZCO), is a rare shrub that grows only in Korea and China. It is called Wangchopi tree in Korea. This species is a rare species that lives at 700-1100m above sea level in Jeju Island and is used for many purposes. In Korea, Zanthoxylum piperitum is called chopidae . The pericarp of the barberry has been used in Korea as a crude medicine for atrophic rhinitis, rheumatism, rhinitis, and sore throat. In addition, according to a previous study conducted by Hwa-Jung Choi, it was proved that Pseudomonas C. has antiviral effect against picornavirus. However, the anti-allergic inflammatory effect on essential oils of C.
이에, 본 발명자의 발명자들은 알레르기성 피부질환, 아토피성 피부염, 알레르기성 비염 등 인간에게 이에, 본 발명의 발명자들은 알레르기성 피부질환, 아토피성 피부염, 알레르기성 비염 등 인간에게 불편함을 주거나 때로는 치명적인 다양한 알레르기성 질환을 유발하는 비만세포로부터 알레르기 유발물질이 분비되는 것을 차단함으로써 알레르기성 질환을 치료할 수 있는 물질을 확보하고자 예의 노력한 결과, 왕초피나무의 정유가 RBL-2H3 비만 세포에서 농도 의존적으로 IgE-항원 복합체나 PMA/A23187-유도된 베타-헥소사미니다아제 (β-hexosaminidase) 방출 및 IL-4 생산을 억제한다는 것을 확인하였고, LPS-유도된 쥐(murine) 대식세포에서 NF-κB의 전사를 억제함으로써 염증 인자 (NO, TNF-α 및 IL-6) 생산을 감소시킨다는 것을 확인함으로써, 본 발명을 완성하였다. Therefore, the inventors of the present inventors have allergic skin diseases, atopic dermatitis, allergic rhinitis, and the like to humans, and the inventors of the present invention provide discomfort or sometimes fatal to humans such as allergic skin diseases, atopic dermatitis, allergic rhinitis, etc. As a result of careful efforts to secure substances that can treat allergic diseases by blocking the secretion of allergens from mast cells that cause various allergic diseases, the essential oil of the Japanese chinensis is IgE-in a concentration-dependent manner in RBL-2H3 mast cells. It was confirmed that antigen complex or PMA/A23187-induced beta-hexosaminidase release and IL-4 production were inhibited, and the transcription of NF-κB in LPS-induced murine macrophages By confirming that it reduces the production of inflammatory factors (NO, TNF-α and IL-6) by suppressing, the present invention was completed.
본 발명의 목적은 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공하는 데 목적이 있다. It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases, including the essential oil extract derived from C.
본 발명에서는 또한 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환 또는 염증성 질환의 예방 또는 개선용 건강기능식품 조성물을 제공하는데 목적이 있다. Another object of the present invention is to provide a health functional food composition for the prevention or improvement of allergic diseases or inflammatory diseases comprising an essential oil extract derived from C.
본 발명은 왕초피나무 (Zanthoxylum coreanum Nakai) 유래 정유 추출물을 알레르기 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases by extracting essential oils derived from Zanthoxylum coreanum Nakai.
상기 왕초피나무 유래 정유 추출물은 왕초피나무의 잎, 뿌리, 줄기 및 열매로 이루어진 군으로부터 선택되는 어느 하나 이상을 선택하여 추출할 수 있다. The essential oil extract derived from Wangchopi tree may be extracted by selecting any one or more selected from the group consisting of leaves, roots, stems, and fruits of Wangchopi tree.
상기 왕초피나무 유래 정유 추출물은 오시멘 (β-ocimene), 알파 피넨 ((-)-α-pinene), 카르보멘테놀 (4-carvomenthenol), 사비넨 (sabinene), 리나롤(linalool), 사이멘 (ο-cymene), 펠란드렌 (β-phellandrene), 리모넨 (limonene) 또는 테르피네올 (α-terpineol)을 포함할 수 있다. The essential oil extract derived from C. chinensis is osimene (β-ocimene), alpha pinene ((-)-α-pinene), carvomenthenol, sabinene, linalool, and cymene (ο-cymene), phellandrene (β-phellandrene), limonene (limonene) or terpineol (α-terpineol).
상기 왕초피나무 유래 정유 추출물은 왕초피나무의 부피의 1 내지 1.5배 부피의 물을 가하고, 클레벤저형 추출장치(Clevenger type apparatus)를 이용하여 90 ℃ 내지 110℃ 온도에서 1 시간 내지 72시간 동안 추출할 수 있다. The essential oil extract derived from C. C. C. is added 1 to 1.5 times the volume of water and extracted for 1 to 72 hours at a temperature of 90 to 110 C using a Clevenger type apparatus. I can.
상기 알레르기 질환은 아토피성 피부염, 알레르기성 비염, 알레르기성 결막염, 알레르기성 천식, 또는 아나필락틱 쇼크(anaphylactic shock)일 수 있다. The allergic disease may be atopic dermatitis, allergic rhinitis, allergic conjunctivitis, allergic asthma, or anaphylactic shock.
상기 염증성 질환은 알레르기, 피부염, 아토피, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증 또는 급성 및 만성 염증 질환일 수 있다. The inflammatory diseases are allergy, dermatitis, atopic, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis. , Osteoarthritis, rheumatoid arthritis, peri-shoulderitis, tendinitis, tendonitis, peritonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, or acute and chronic inflammatory diseases.
상기 왕초피나무 유래 정유 추출물은 전체 조성물 중량에 대하여 0.0001 내지 1 중량%일 수 있다. The essential oil extract derived from C. chopiaceae may be 0.0001 to 1% by weight based on the total weight of the composition.
본 발명은 또한 왕초피나무 (Zanthoxylum coreanum Nakai) 유래 정유 추출물을 포함하는 알레르기 질환 또는 염증성 질환의 예방 또는 개선용 건강기능식품 조성물을 제공할 수 있다. The present invention can also provide a health functional food composition for the prevention or improvement of allergic diseases or inflammatory diseases comprising the essential oil extract derived from the tree ( Zanthoxylum coreanum Nakai).
상기 왕초피나무 유래 정유 추출물은 왕초피나무의 잎, 뿌리, 줄기 및 열매로 이루어진 군으로부터 선택되는 어느 하나 이상을 선택하여 추출할 수 있다. The essential oil extract derived from Wangchopi tree may be extracted by selecting any one or more selected from the group consisting of leaves, roots, stems, and fruits of Wangchopi tree.
상기 왕초피나무 유래 정유 추출물은 오시멘 (β-ocimene), 알파 피넨 ((-)-α-pinene), 카르보멘테놀 (4-carvomenthenol), 사비넨 (sabinene), 리나롤(linalool), 사이멘 (ο-cymene), 펠란드렌 (β-phellandrene), 리모넨 (limonene) 또는 테르피네올 (α-terpineol)을 포함할 수 있다. The essential oil extract derived from C. chinensis is osimene (β-ocimene), alpha pinene ((-)-α-pinene), carvomenthenol, sabinene, linalool, and cymene (ο-cymene), phellandrene (β-phellandrene), limonene (limonene) or terpineol (α-terpineol).
상기 왕초피나무 유래 정유 추출물은 왕초피나무의 부피의 1 내지 1.5배 부피의 물을 가하고, 클레벤저형 추출장치(Clevenger type apparatus)를 이용하여 90 ℃ 내지 110 ℃ 온도에서 1 시간 내지 72 시간 동안 추출할 수 있다. The essential oil extract derived from C. chopi tree is added with 1 to 1.5 times the volume of water and extracted for 1 hour to 72 hours at a temperature of 90° C. to 110° C. using a Clevenger type apparatus. I can.
상기 알레르기 질환은 아토피성 피부염, 알레르기성 비염, 알레르기성 결막염, 알레르기성 천식, 또는 아나필락틱 쇼크(anaphylactic shock)일 수 있다. The allergic disease may be atopic dermatitis, allergic rhinitis, allergic conjunctivitis, allergic asthma, or anaphylactic shock.
상기 염증성 질환은 알레르기, 피부염, 아토피, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증 또는 급성 및 만성 염증 질환일 수 있다. The inflammatory diseases are allergy, dermatitis, atopic, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis. , Osteoarthritis, rheumatoid arthritis, peri-shoulderitis, tendinitis, tendonitis, peritonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, or acute and chronic inflammatory diseases.
상기 왕초피나무 유래 정유 추출물은 전체 조성물 중량에 대하여 0.0001 내지 1 중량%일 수 있다. The essential oil extract derived from C. chopiaceae may be 0.0001 to 1% by weight based on the total weight of the composition.
본 발명은 또한 왕초피나무 유래 정유 추출물을 포함하는 항염증용 화장료 조성물을 제공할 수 있다. The present invention can also provide an anti-inflammatory cosmetic composition comprising the essential oil extract derived from the C.
본 발명에 따른 왕초피나무 유래 정유 추출물은 비만세포로부터 알레르기 유발물질이 분비되는 것을 억제한다. 따라서 본 발명에 따른 왕초피나무 유래 정유 추출물은 종래 증상의 완화 중심의 치료제와는 달리 비만세포에서 알레르기 유발물질이 분비되는 것을 저해 내지 차단함으로써 알레르기성 질환의 근본적인 치료가 가능하다. 아토피 피부염을 유도한 동물모델에 왕초피나무 유래 정유 추출물을 처리한 결과 염증반응이 감소하는 효과가 있다. 이와 함께 왕초피나무 유래 정유 추출물은 세포독성이 없으며, 부작용이 적어 안전한 의약품, 건강기능식품으로 유용하게 사용될 수 있다. The essential oil extract derived from C. chopidae according to the present invention inhibits the secretion of allergens from mast cells. Therefore, unlike the conventional treatment-centered treatments for symptomatic relief, the essential oil extract derived from P. chopi tree according to the present invention inhibits or blocks the secretion of allergens from mast cells, thereby enabling fundamental treatment of allergic diseases. As a result of treatment with essential oil extract derived from the Atopic dermatitis in an animal model that induces atopic dermatitis, there is an effect of reducing the inflammatory response. Along with this, the essential oil extract derived from C. chopiaceae has no cytotoxicity and has few side effects, so it can be usefully used as a safe medicine and health functional food.
도 1은 왕초피나무 유래 정유 추출물을 처리한 (a) RBL-2H3 세포, (b) RAW 264.7 세포 및 (c) 293T 세포의 세포독성을 측정한 결과를 나타낸 그래프이다.
도 2는 왕초피나무 유래 정유 추출물을 농도 별로 처리하였을 때 (a) DNP/BSA로 자극 시킨 RBL-2H3 세포 및 (b) PMA/A23187로 자극 한 RBL-2H3 세포로부터 베타- 헥소사미니다아제 (β-hexosaminidase)의 분비를 억제하는 효과를 측정한 결과를 나타낸 것이다.
도 3은 왕초피나무 유래 정유 추출물을 농도 별로 처리하였을 때 (a) DNP/BSA로 자극 시킨 RBL-2H3 세포 및 (b) PMA/A23187로 자극 한 RBL-2H3 세포로부터 IL-4의 분비를 억제하는 효과를 측정한 결과를 나타낸 것이다.
도 4는 왕초피나무 유래 정유 추출물을 처리하였을 때 PMA로 자극 시킨 293T 세포로부터 NF-κB 활성화를 억제하는 효과를 나타낸 결과이다.
도 5는 왕초피나무 유래 정유 추출물을 농도 별로 처리하였을 때 LPS로 자극 한 Raw 264.7 세포로부터 (a) TNF-α, (b) IL-6 및 (c) NO의 발생량을 억제하는 효과를 나타낸 결과이다.
도 6은 왕초피나무 유래 정유 추출물을 농도 별로 처리하였을 때 LPS로 자극 시킨 Raw 264.7 세포로부터 (a) iNOS 및 (b) COX-2의 단백질의 발현량을 억제하는 효과를 나타낸 결과이다.
도 7은 왕초피나무 유래 정유 추출물을 농도 별로 처리하였을 때 DNCB로 아토피피부염을 유도한 동물모델로부터 (a) 실제 귀의 표습 (b) 귀의 두께 및 (c) 조직학적 결과에서 아토피피부염을 억제하는 효과를 나타낸 것이다. 1 is a graph showing the results of measuring the cytotoxicity of (a) RBL-2H3 cells, (b) RAW 264.7 cells, and (c) 293T cells treated with an essential oil extract derived from C.
2 shows beta-hexosaminidase (β) from RBL-2H3 cells stimulated with DNP/BSA and (b) RBL-2H3 cells stimulated with PMA/A23187 when the essential oil extracts derived from C. -hexosaminidase) secretion inhibitory effect was measured.
Figure 3 is when the concentration-specific treatment of essential oil extract derived from C. (A) inhibits the secretion of IL-4 from RBL-2H3 cells stimulated with DNP/BSA and (b) RBL-2H3 cells stimulated with PMA/A23187. It shows the result of measuring the effect.
Figure 4 is a result showing the effect of inhibiting the activation of NF-κB from 293T cells stimulated with PMA when treated with an essential oil extract derived from C.
Figure 5 is a result showing the effect of inhibiting the amount of (a) TNF-α, (b) IL-6 and (c) NO from Raw 264.7 cells stimulated with LPS when the essential oil extract derived from C. .
6 is a result showing the effect of inhibiting the expression level of proteins of (a) iNOS and (b) COX-2 from Raw 264.7 cells stimulated with LPS when the essential oil extract derived from C.
Figure 7 shows the effect of inhibiting atopic dermatitis in (a) actual ear skin (b) ear thickness and (c) histological results from an animal model inducing atopic dermatitis with DNCB when treated with an essential oil extract derived from C. Is shown.
이하, 본 발명을 자세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명자들은 알레르기에 치료 효과가 있는 천연물질을 연구하던 중 왕초피나무 유래 정유 추출물이 항 알레르기 및 항 염증 효과가 있는 것을 확인하였다. 왕초피나무 유래 정유 추출물은 비만세포 및 염증세포인 RBL-2H3 세포, RAW 264.7 세포 및 293T 세포에서 0.0025, 0.005, 0.01%의 농도에서 세포독성을 나타내지 않았다 (도 1). 왕초피나무 유래 정유 추출물을 사용하여 전 처리하면 농도 의존적으로 IgE-항원 복합체 또는 PMA/A23187-자극된 RBL-2H3 세포의 탈과립을 억제하였다 (도 2). 또한, 왕초피나무 유래 정유 추출물은 PMA/A23187에 의해 자극된 민감화 RBL-2H3 세포에서 염증성 사이토카인 IL-4의 분비를 농도 의존적으로 억제하였다 (도 3). 왕초피나무 유래 정유 추출물은 PMA에 의해 자극된 293T 세포에서 NF-κB 전사 활성의 전사를 효과적으로 억제하였다 (도 4). 왕초피나무 유래 정유 추출물은 LPS-활성화된 Raw 264.7 세포에서 염증 매개체인 TNF-a, IL-6 및 NO의 발생량을 농도 의존적으로 줄였다 (도 5). 왕초피나무 유래 정유 추출물은 LPS-활성화된 Raw 264.7 세포에서 iNOS와 COX-2 단백질 발현을 효과적으로 억제하였다 (도 6). 나아가, 아토피 피부염을 유도한 동물에게 처리한 결과 염증반응을 억제하는 효과가 있었다 (도 7). The inventors of the present invention confirmed that while studying a natural substance that has a curative effect on allergies, the essential oil extract derived from C. chopidae has anti-allergic and anti-inflammatory effects. The essential oil extract derived from C. chopidae did not show cytotoxicity at concentrations of 0.0025, 0.005, and 0.01% in mast cells and inflammatory cells, RBL-2H3 cells, RAW 264.7 cells, and 293T cells (FIG. 1). When pretreatment using the essential oil extract derived from C. chopidae inhibited the degranulation of IgE-antigen complex or PMA/A23187-stimulated RBL-2H3 cells in a concentration-dependent manner (FIG. 2). In addition, the essential oil extract derived from P. chopidae inhibited the secretion of the inflammatory cytokine IL-4 in sensitized RBL-2H3 cells stimulated by PMA/A23187 in a concentration-dependent manner (FIG. 3). The essential oil extract derived from P. chopidae effectively inhibited the transcription of NF-κB transcriptional activity in 293T cells stimulated by PMA (FIG. 4). The essential oil extract derived from C. chopidae reduced the amount of inflammatory mediators TNF-a, IL-6, and NO in LPS-activated Raw 264.7 cells in a concentration-dependent manner (FIG. 5). The essential oil extract derived from C. chopidae effectively inhibited the expression of iNOS and COX-2 proteins in LPS-activated Raw 264.7 cells (FIG. 6). Furthermore, as a result of treatment with animals inducing atopic dermatitis, there was an effect of suppressing the inflammatory reaction (FIG. 7).
본 발명은 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환 또는 염증성 질환의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for the prevention or treatment of allergic diseases or inflammatory diseases comprising the essential oil extract derived from C.
상기 욍초피나무의 학명은 Zanthoxylum coreanum Nakai이며, 왕산초나무라고도 한다. 왕초피나무는 제주도의 저지대 해변에서 자라는 나무로 산초나무와 달리 초피나무나 개산초나무처럼 가시가 마주나고 잎이 좀 더 크다. 가시의 생김새는 날개처럼 넓게 가지에 붙어 있다. 잎에서 짙은 향기가 나지만 초피나무보다는 못한 편이다. 개산초와 달리 잎줄기에 날개가 거의 발달하지 않고 작은 잎의 수가 많은 것이 특징이다.The scientific name of the Yopchopi tree is Zanthoxylum coreanum Nakai, and is also called Wangsancho tree. The Wangchopi tree is a tree that grows on the low-lying beaches of Jeju Island, and unlike the Sancho tree, the thorns are facing each other and the leaves are larger like the grasshopper tree or the Kaesancho tree. The appearance of thorns is attached to the branch as wide as wings. The leaves have a deep scent, but they are worse than the chopi trees. Unlike Gaesancho, the leaf stem has few wings and has a large number of small leaves.
본 발명에서 “치료”는 본 발명의 약학적 조성물을 알레르기성 질환에 적용한 결과로서 알레르기성 질환의 완치는 물론 알레르기성 질환 증세의 부분적 완치, 호전 및 경감을 포함한다.In the present invention, "treatment" is a result of applying the pharmaceutical composition of the present invention to an allergic disease, and includes partial cure, amelioration and alleviation of allergic disease symptoms as well as cure of allergic diseases.
본 발명에서 “예방”은 본 발명의 약학적 조성물을 알레르기성 질환에 적용하여 알레르기성 질환 증세를 억제 또는 차단함으로써, 아토피 피부와 같은 알레르기성 질환이 사전에 발생하지 않도록 하는 것을 의미한다.In the present invention, "prevention" refers to preventing or preventing allergic diseases such as atopic skin from occurring in advance by applying the pharmaceutical composition of the present invention to an allergic disease to suppress or block the symptoms of allergic diseases.
상기 왕초피나무는 왕초피나무의 잎, 뿌리, 줄기 및 열매로 이루어진 군으로부터 선택되는 어느 하나를 선택하여 추출할 수 있으며, 바람직하게는 열매일 수 있다. The Wangchopi tree may be extracted by selecting any one selected from the group consisting of leaves, roots, stems, and fruits of the Wangchopi tree, and preferably, it may be a fruit.
“정유”는 식물의 표피나 엽육 조직에서 분화된 세포 외 특정 공간에 저장되어있는 저비점의 기름성 물질들로 공기 중에 쉽게 휘발되어 인간의 후각을 통해 인지될 수 있는 저 분자량의 액상 혼합체로 식물의 생명 유지에 있어서 필수적인 2차 대사산물이다. 이러한 정유는 식물생장과 발아를 효과적으로 억제하며, 방충제(insect-repellent), 그리고 해충제(anti-feedant)로서 작용한다. 정유는 상기된 함유 물질에 의해서 항미생물 활성, 항산화 효과, 항암효과, 진정작용 등을 가지고 있는 것으로 알려져 있다.“Essential oil” is a low-boiling, oily substance stored in a specific space other than cells differentiated from the epidermis or mesophyll tissue of plants. It is a liquid mixture of low molecular weight that can be easily volatilized in the air and recognized through the human sense of smell. It is an essential secondary metabolite in maintaining life. These essential oils effectively inhibit plant growth and germination, and act as an insect-repellent and an anti-feedant. Essential oils are known to have antimicrobial activity, antioxidant effect, anticancer effect, sedative effect, etc. by the above-described substances.
상기 왕초피나무 유래 정유 추출물은 오시멘 (β-ocimene), 알파 피넨 ((-)-α-pinene), 카르보멘테놀 (4-carvomenthenol), 사비넨 (sabinene), 리나롤(linalool), 사이멘 (ο-cymene), 펠란드렌 (β-phellandrene), 리모넨 (limonene) 또는 테르피네올 (α-terpineol)을 포함할 수 있다. The essential oil extract derived from C. chinensis is osimene (β-ocimene), alpha pinene ((-)-α-pinene), carvomenthenol, sabinene, linalool, and cymene (ο-cymene), phellandrene (β-phellandrene), limonene (limonene) or terpineol (α-terpineol).
상기 왕초피나무 유래 정유 추출물은 왕초피나무를 재료 부피의 1 내지 1.5 배에 달하는 부피의 물, C1 내지 C4의 저급 알콜 또는 이들의 혼합용매, 바람직하게는 물을 가하고, 클레벤저형 추출장치(Clevenger type apparatus)를 이용하여 90 ℃ 내지 110℃ 온도, 바람직하게는 100 ℃ 내지 105℃ 온도에서, 1 내지 72시간, 바람직하게는 8 시간 내지 48 시간 동안 추출할 수 있다. The essential oil extract derived from C. C. C. tree is added with a volume of water, C 1 to C 4 of lower alcohol or a mixed solvent thereof, preferably water, which is 1 to 1.5 times the volume of the material, and a Clevenzer-type extraction device ( Clevenger type apparatus) can be extracted at a temperature of 90°C to 110°C, preferably 100°C to 105°C for 1 to 72 hours, preferably 8 to 48 hours.
상기의 클레벤저형 추출장치는 왕초피 나무의 열매를 추출기에 넣고, 추출기 하부로부터 기화 수증기를 100℃에서 6시간 이상 계속 공급하여 정유 성분들이 수증기와 함께 기화되게 하고 냉각 콘덴서를 통과시켜 액화된다. 물 및 오일은 다른 밀도를 가지기 때문에 정유는 별도의 관으로 모이고, 물은 다시 플라스크에 모이게 된다. The Clevenzer-type extraction device puts the fruit of the chinensis tree into an extractor, and continuously supplies vaporized water vapor from the bottom of the extractor at 100° C. for 6 hours or more so that essential oil components are vaporized together with water vapor and passed through a cooling condenser to liquefy. Because water and oil have different densities, the essential oil is collected in separate tubes, and the water is collected in the flask again.
상기 제조방법을 통하여 얻어진 왕초피나무 유래 정유 추출물의 수득율(yield)은 1 내지 10%, 바람직하게는 1 내지 5%(w/w)이며, 더 바람직하게는 2.1%(w/w)일 수 있다. The yield (yield) of the essential oil extract obtained through the above preparation method is 1 to 10%, preferably 1 to 5% (w/w), and more preferably 2.1% (w/w). .
상기 알레르기 질환은 아토피성 피부염, 알레르기성 비염, 알레르기성 결막염, 알레르기성 천식, 또는 아나필락틱 쇼크(anaphylactic shock)일 수 있다. The allergic disease may be atopic dermatitis, allergic rhinitis, allergic conjunctivitis, allergic asthma, or anaphylactic shock.
상기 염증성 질환은 알레르기, 피부염, 아토피, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증 또는 급성 및 만성 염증 질환일 수 있다. The inflammatory diseases are allergy, dermatitis, atopic, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis. , Osteoarthritis, rheumatoid arthritis, peri-shoulderitis, tendinitis, tendonitis, peritonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, or acute and chronic inflammatory diseases.
상기 왕초피나무 유래 정유 추출물은 전체 조성물 중량에 대하여 0.0001 내지 1 중량%일 수 있으며 바람직하게는 0.0025 내지 0.01 중량%일 수 있다. The essential oil extract derived from C. chopidae may be 0.0001 to 1% by weight, and preferably 0.0025 to 0.01% by weight, based on the total weight of the composition.
상기 약학적 조성물은 정제, 과립제, 환제, 캅셀제, 액제 및 산제로 구성된 그룹으로부터 선택되는 어느 하나의 제형으로 제형화 될 수 있으나, 이로 한정되는 것은 아니다.The pharmaceutical composition may be formulated in any one formulation selected from the group consisting of tablets, granules, pills, capsules, liquids, and powders, but is not limited thereto.
상기 본 발명의 약학적 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용 가능한 담체를 포함하는 상기 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로오스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용될 수 있다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테로 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. The composition including a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations contain at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. in one or more compounds. It is prepared by mixing. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, and syrups.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, and preservatives may be included. have. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
상기 본 발명의 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 용어 “약학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 약물의 활성, 약물에 대해 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 그러나 바람직한 효과를 위해서, 본 발명의 왕초피나무 유래 정유 추출물은 1일 0.0001 내지 50mg/kg으로, 구체적으로는 0.001 내지 50 mg/kg으로 투여될 수 있으나, 이로 한정되는 것은 아니다.The composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type and severity of the subject, age, sex, and activity of the drug. , Sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. However, for a desirable effect, the essential oil extract derived from the C. chopidae of the present invention may be administered at 0.0001 to 50 mg/kg per day, specifically 0.001 to 50 mg/kg, but is not limited thereto.
본 발명의 조성물은 개별 치료제로 투여하거나 항알레르기 효과를 나타내는 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention may be administered as an individual therapeutic agent or administered in combination with another therapeutic agent exhibiting an anti-allergic effect, and may be administered sequentially or simultaneously with a conventional therapeutic agent. And can be administered single or multiple. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all the above factors, and can be easily determined by a person skilled in the art.
상기 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 본 발명의 조성물은 목적하는 바에 따라 복강내 투여, 정맥내 투여, 근육내 투여, 피하 투여, 피내 투여, 경구 투여, 비내 투여, 폐내 투여, 직장내 투여될 수 있으나, 이에 제한되지는 않는다. 또한, 상기 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다.The administration route of the composition may be administered through any general route as long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermal, oral, intranasal, intrapulmonary, or rectal, as desired, but is not limited thereto. In addition, the composition can be administered by any device capable of moving the active substance to the target cell.
본 발명은 왕초피나무 유래 정유 추출물을 포함하는 알레르기 질환 또는 염증성 질환의 예방 또는 개선용 건강기능식품 조성물을 제공한다. The present invention provides a health functional food composition for the prevention or improvement of allergic diseases or inflammatory diseases, including the essential oil extract derived from C.
상기 욍초피나무의 학명은 Zanthoxylum coreanum Nakai이며, 왕산초나무라고도 한다. 왕초피나무는 제주도의 저지대 해변에서 자라는 나무로 산초나무와 달리 초피나무나 개산초나무처럼 가시가 마주나고 잎이 좀 더 크다. 가시의 생김새는 날개처럼 넓게 가지에 붙어 있다. 잎에서 짙은 향기가 나지만 초피나무보다는 못한 편이다. 개산초와 달리 잎줄기에 날개가 거의 발달하지 않고 작은 잎의 수가 많은 것이 특징이다.The scientific name of the Yopchopi tree is Zanthoxylum coreanum Nakai, and is also called Wangsancho tree. The Wangchopi tree is a tree that grows on the low-lying beaches of Jeju Island, and unlike the Sancho tree, the thorns are facing each other and the leaves are larger like the grasshopper tree or the Kaesancho tree. The appearance of thorns is attached to the branches as wide as wings. The leaves have a deep scent, but they are worse than the chopi trees. Unlike Kaesancho, the leaf stem has few wings and has a large number of small leaves.
상기 왕초피나무는 왕초피나무의 잎, 뿌리, 줄기 및 열매로 이루어진 군으로부터 선택되는 어느 하나를 선택하여 추출할 수 있으며, 바람직하게는 열매일 수 있다. The Wangchopi tree may be extracted by selecting any one selected from the group consisting of leaves, roots, stems, and fruits of the Wangchopi tree, and preferably, it may be a fruit.
상기 왕초피나무 유래 정유 추출물은 오시멘 (β-ocimene), 알파 피넨 ((-)-α-pinene), 카르보멘테놀 (4-carvomenthenol), 사비넨 (sabinene), 리나롤(linalool), 사이멘 (ο-cymene), 펠란드렌 (β-phellandrene), 리모넨 (limonene) 또는 테르피네올 (α-terpineol)을 포함할 수 있다. The essential oil extract derived from C. chinensis is osimene (β-ocimene), alpha pinene ((-)-α-pinene), carvomenthenol, sabinene, linalool, and cymene (ο-cymene), phellandrene (β-phellandrene), limonene (limonene) or terpineol (α-terpineol).
상기 왕초피나무 유래 정유 추출물은 왕초피나무를 재료 부피의 1 내지 1.5 배에 달하는 부피의 물, C1 내지 C4의 저급 알콜 또는 이들의 혼합용매, 바람직하게는 물을 가하고, 클레벤저형 추출장치(Clevenger type apparatus)를 이용하여 90 ℃ 내지 110℃ 온도, 바람직하게는 100 ℃ 내지 105℃ 온도에서, 1 내지 72시간, 바람직하게는 8 시간 내지 48 시간 동안 추출할 수 있다. The essential oil extract derived from C. C. C. tree is added with a volume of water, C 1 to C 4 of lower alcohol or a mixed solvent thereof, preferably water, which is 1 to 1.5 times the volume of the material, and a Clevenzer-type extraction device ( Clevenger type apparatus) can be extracted at a temperature of 90°C to 110°C, preferably 100°C to 105°C for 1 to 72 hours, preferably 8 to 48 hours.
상기 알레르기 질환은 아토피성 피부염, 알레르기성 비염, 알레르기성 결막염, 알레르기성 천식, 또는 아나필락틱 쇼크(anaphylactic shock)일 수 있다. The allergic disease may be atopic dermatitis, allergic rhinitis, allergic conjunctivitis, allergic asthma, or anaphylactic shock.
상기 염증성 질환은 알레르기, 피부염, 아토피, 결막염, 치주염, 비염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 통풍, 강직성 척추염, 류마티스 열, 루푸스, 섬유근통(fibromyalgia), 건선관절염, 골관절염, 류마티스 관절염, 견관절주위염, 건염, 건초염, 건주위염, 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증 또는 급성 및 만성 염증 질환일 수 있다. The inflammatory diseases are allergy, dermatitis, atopic, conjunctivitis, periodontitis, rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, fibromyalgia, psoriatic arthritis. , Osteoarthritis, rheumatoid arthritis, peri-shoulderitis, tendinitis, tendonitis, peritonitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis, or acute and chronic inflammatory diseases.
상기 왕초피나무 유래 정유 추출물은 전체 조성물 중량에 대하여 0.0001 내지 1 중량%일 수 있으며 바람직하게는 0.0025 내지 0.01 중량%일 수 있다. The essential oil extract derived from C. chopidae may be 0.0001 to 1% by weight, and preferably 0.0025 to 0.01% by weight, based on the total weight of the composition.
본 발명의 건강기능식품 조성물은 상기 왕초피나무 유래 정유 추출물을 포함하되, 적절한 식품조첨가제가 포함될 수 있다. 본 발명에서 용어 "식품보조첨가제”란 식품에 보조적으로 첨가될 수 있는 구성요소를 의미하며, 각 제형의 건강기능식품을 제조하는데 첨가되는 것으로서 당업자가 적절히 선택하여 사용할 수 있다. 식품보조첨가제의 예로는 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산음료에 사용되는 탄산화제 등이 포함되지만, 상기 예들에 의해 본 발명의 식품보조첨가제의 종류가 제한되는 것은 아니다.The health functional food composition of the present invention includes the essential oil extract derived from the apricot tree, but may contain an appropriate food additive. In the present invention, the term "food supplementary additive" refers to a component that can be added to food, and is added to the manufacture of health functional foods of each formulation, and can be appropriately selected and used by those skilled in the art. Is a variety of nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stable The types of food additives of the present invention are not limited by the above examples, although they include an agent, a preservative, glycerin, alcohol, and a carbonating agent used in carbonated beverages.
본 발명에서 용어 "건강기능식품”이란 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 정제, 캅셀, 분말, 과립, 액상 및 환 등의 형태로 제조 및 가공한 식품을 말한다. 여기서 “기능성”이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다. 본 발명에 따른 건강기능식품은 당업계에서 통상적으로 사용되는 방법에 의하여 제조 가능하며, 상기 제조 시에는 당업계에서 통상적으로 첨가하는 원료 및 성분을 첨가하여 제조할 수 있다. 또한, 일반 약품과는 달리 식품을 원료로 하여 약품의 장기 복용 시 발생할 수 있는 부작용 등이 없는 장점이 있고, 휴대성이 뛰어나, 본 발명의 건강기능식품은 항알레르기 효과를 증진시키기 위한 보조제로 섭취가 가능하다.In the present invention, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Means to control nutrients on the structure and function of the human body or obtain useful effects for health purposes such as physiological actions, etc. The health functional food according to the present invention can be manufactured by a method commonly used in the art. , In the above manufacturing, it can be prepared by adding raw materials and ingredients commonly added in the art In addition, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as raw materials. , Excellent portability, the health functional food of the present invention can be ingested as an adjuvant for enhancing the anti-allergic effect.
유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품의 제조 시에 본 발명의 왕초피나무 유래 정유 추출물은 원료 조성물 중 0.001 내지 5 중량%, 또는 0.001 내지 3 중량%의 양으로 포함될 수 있다. 건강음료의 경우 100 mL를 기준으로 0.01 내지 2 g, 구체적으로 0.02 내지 2 g, 보다 구체적으로 0.3 내지 1 g 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취는 상기 양은 상기 범위 이하로도 사용될 수 있다.The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, in the manufacture of food, the essential oil extract derived from C. chopidae of the present invention may be included in an amount of 0.001 to 5% by weight, or 0.001 to 3% by weight of the raw material composition. In the case of a health drink, 0.01 to 2 g, specifically 0.02 to 2 g, more specifically 0.3 to 1 g, based on 100 mL may be added. However, for long-term intake for the purpose of health and hygiene or for the purpose of health control, the above amount may be used even below the above range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and it includes all health foods in the usual sense.
상기 외에 본 발명의 왕초피나무 유래 정유 추출물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 및 천연 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다.In addition to the above, the essential oil extract derived from P. chopidae of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and heavy agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid and salts thereof, Organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, etc. may be contained.
본 발명은 왕초피나무 유래 정유 추출물을 포함하는 항염증용 화장료 조성물을 제공할 수 있다.The present invention can provide an anti-inflammatory cosmetic composition comprising the essential oil extract derived from the C.
본 발명의 상기 화장료 조성물의 제형은 용액, 현탁액, 에멀전, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클렌징, 오일, 분말 파운데이션, 에멀전 파운데이션, 왁스 파운데이션 및 스프레이로 이루어지는 군으로부터 선택된 어느 하나의 조성 형태로 제형화 될 수 있다. The formulation of the cosmetic composition of the present invention is selected from the group consisting of solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray. It can be formulated in any one composition form.
이하, 본 발명에 따르는 실시예 및 실험예를 통하여 본 발명을 보다 상세히 설명하나, 본 발명의 범위가 하기 제시된 실시예에 의해 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples and experimental examples according to the present invention, but the scope of the present invention is not limited by the examples presented below.
실시예 1Example 1
1-1. 왕초피나무 유래 정유의 추출1-1. Extraction of essential oils derived from chinensis tree
본연구에서 왕초피나무 (Zanthoxylum coreanum Nakaiis)의 열매가 사용되었다. 열매는 2017년 8월 한국 진주시에 위치한 국립산림과학원의 실험림에서 채집되었다. Z. coreanum의 열매는 clevenger형 장치를 사용하여 대기압에서 하이드로-증류되었다. 수집된 에센셜 오일을 무수 황산나트륨 상에서 건조하고, 0.45 ㎛ 막 디스크 필터를 통해 여과하였다. 얻어진 에센셜 오일을 밀폐된 어두운 바이알에 옮기고 추후 분석을 위해 4℃에서 보관하였다. 에센셜 오일 함량은 오븐 건조물을 기준으로 결정되었으며 측정은 3회로 수행되었습니다. 에센셜 오일의 수율(%)은 하기의 식을 사용하여 계산하였다. 왕초피나무 유래 정유의 추출물의 수율은 약 2.1%이었다. The fruit of Zanthoxylum coreanum Nakaiis was used in this study. Fruits were collected in an experimental forest of the National Institute of Forest Science in Jinju City, Korea in August 2017. The fruits of Z. coreanum were hydro-distilled at atmospheric pressure using a clevenger type apparatus. The collected essential oil was dried over anhydrous sodium sulfate and filtered through a 0.45 μm membrane disk filter. The obtained essential oil was transferred to a sealed dark vial and stored at 4° C. for later analysis. The essential oil content was determined based on the oven dry matter and measurements were taken in 3 times. The yield (%) of essential oil was calculated using the following formula. The yield of the extract of the essential oil derived from C. chopidae was about 2.1%.
에센셜 오일 수율 (%) = (수득 된 에센셜 오일 질량 (g)) / (오븐 건조 물질 질량 (g)) ×100Essential oil yield (%) = (obtained essential oil mass (g)) / (oven dry mass (g)) × 100
1-2. 왕초피나무 유래 정유 추출물의 가스 크로마토 그래피 - 질량 분석 (GC-MS) 분석1-2. Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Essential Oil Extracts from C.
불꽃 이온화 검출기 (FID)와 질량 분석기 (MS)가 장착된 Thermo Scientific Model ISQ LT에서 결합 가스 크로마토그래피 - 질량 분석 (GC-MS) 분석을 수행했다. GC/MS 분석 용액은 1.0ml의 디클로로 메탄 용액 (methyl undecanote 100ppm을 함유) 1.0ml에 오일 4ml를 녹인 후 1㎕를 주입하여 제조하였다. VF-5MS GC 칼럼 (60 m ×0.25 mm ×0.25 μm 필름 두께, Agilent Technologies)을 사용했다. 캐리어 가스는 1.0 mL/min의 일정한 유속에서 헬륨이었다. 사출 온도는 250℃, 분할비 1 : 20로 하였다. 오븐 온도는 50℃에서 5분간 유지 한 다음 10℃/min에서 65℃로 30 분간 유지 한 다음 5℃/min에서 120℃로 증가시켰다. 10분, 5℃/min, 210℃, 10분, 최종적으로 20℃/min에서 325℃로 10분간 유지하였다. FID 검출을 위해 온도는 300 ℃, 기류는 350.0㎖/min, 수소 유량은 35.0㎖/min, 보충 가스(헬륨)는 40.0㎖/min으로 흐르게 하였다. 매스 인터페이스 온도는 250 ℃이고 이온 소스 온도는 250 ℃이다. 매스 스캔 데이터는 35 amu 내지 550 amu 범위의 스캔 범위에서 0.2 초 스캔 속도로 EI 모드에서 얻었다. 피크의 확인은 피크의 평균 질량 스펙트럼을 전자 라이브러리 데이터베이스 (NIST / EPA / NIH 질량 스펙트럼 라이브러리, 버전 2.0 g)와 비교하여 수행했다. 또한, kovats 지수 (KI)는 n-alkane (C7-C30) 표준 주입에 의해 계산되었고 피크 식별을 위한 보충 데이터로 설명되었다. 왕초피나무 유래 정유 추출물의 화학적 조성은 하기 표 1과 같다. Z. coreanum의 열매의 에센셜 오일에서 37가지 성분이 확인되었으며, 주성분은 β-Ocimene으로 전체의 24.48%, 그 뒤를 이어 (-)-α-pinene(16.56 %), 4-carvomenthenol(11.61%), sabinene(10.81%), linalool(10.09%), ο-cymene(3.56%), β-phellandrene(3.15%), limonene(2.63.%) α-terpineol(1.74%)의 성분이 확인되었다. Combined gas chromatography-mass spectrometry (GC-MS) analysis was performed on a Thermo Scientific Model ISQ LT equipped with a flame ionization detector (FID) and mass spectrometer (MS). The GC/MS analysis solution was prepared by dissolving 4 ml of oil in 1.0 ml of 1.0 ml of dichloromethane solution (containing 100 ppm of methyl undecanote) and then injecting 1 µl. A VF-5MS GC column (60 m×0.25 mm×0.25 μm film thickness, Agilent Technologies) was used. The carrier gas was helium at a constant flow rate of 1.0 mL/min. The injection temperature was 250°C and the split ratio was 1:20. The oven temperature was maintained at 50° C. for 5 minutes, then maintained at 10° C./min to 65° C. for 30 minutes, and then increased from 5° C./min to 120° C. 10 minutes, 5 ℃ / min, 210 ℃, 10 minutes, and finally held at 325 ℃ 10 minutes at 20 ℃ / min. For FID detection, the temperature was 300°C, the air flow was 350.0 ml/min, the hydrogen flow rate was 35.0 ml/min, and the make-up gas (helium) was flowed at 40.0 ml/min. The mass interface temperature is 250°C and the ion source temperature is 250°C. Mass scan data were obtained in EI mode with a scan rate of 0.2 seconds in the scan range ranging from 35 amu to 550 amu. The identification of the peak was performed by comparing the average mass spectrum of the peak with an electronic library database (NIST/EPA/NIH mass spectrum library, version 2.0 g). In addition, the kovats index (KI) was calculated by standard injection of n-alkane (C7-C30) and described as supplementary data for peak identification. The chemical composition of the essential oil extract derived from C. chopi tree is shown in Table 1 below. 37 kinds of ingredients were identified in the essential oil of the fruit of Z. coreanum, the main ingredient was β-Ocimene, 24.48% of the total, followed by (-)-α-pinene (16.56%), 4-carvomenthenol (11.61%), and Components of sabinene (10.81%), linalool (10.09%), ο-cymene (3.56%), β-phellandrene (3.15%), limonene (2.63.%) and α-terpineol (1.74%) were identified.
실시예 2. 왕초피나무 유래 정유 추출물의 항알레르기 효과 확인Example 2. Confirmation of anti-allergic effect of essential oil extract derived from C.
2-1. 세포배양 및 시약2-1. Cell culture and reagents
호염기구성 백혈병 세포주 RBL-2H3은 한국세포주은행 (서울, 한국)으로부터 공급받았다. RBL-2H3 세포는 5% CO2 및 가습 된 37℃ 배양기에서 10 % FBS (Gibco, Rockville, MD, USA)와 1% penicillin-streptomycin이 함유된 Minimum Essential Medium (MEM) (Welgene, Korea)에서 배양되었다. RAW 264.7 대식세포, IgEL b4 세포 및 293 T 세포를 American Type Culture Collection (Manassas, VA, USA)에서 구입하였고, 10 % FBS 및 1 % 페니실린-스트렙토마이신을 함유한 Dulbecco Modified Eagle 배지 (DMEM)에서 배양 하였다. Calcium ionophore A23187과 phorbol 12-myristate13-acetate (PMA)는 Sigma-Aldrich (St. Louis, MO, USA), 퀘르세틴은 Sigma-Aldrich (St. Louis, MO, USA)에서 구입하였다. 알부민, 2,4-dinitrophenylated (DNP-BSA)는 Thermo Fisher Scientific (Eugene, OR, USA)에 의해, rIL-4와 mTNF-α는 R&D system Inc (Minneapoils, MN, USA)에서 구입했고, mIL-6은 Biolegend (San Diego, CA, USA)로부터 구입하였다.Basophilic leukemia cell line RBL-2H3 was supplied from Korea Cell Line Bank (Seoul, Korea). RBL-2H3 cells were cultured in Minimum Essential Medium (MEM) (Welgene, Korea) containing 10% FBS (Gibco, Rockville, MD, USA) and 1% penicillin-streptomycin in a humidified 37°C incubator with 5% CO 2. Became. RAW 264.7 macrophages, IgEL b4 cells, and 293 T cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco Modified Eagle's medium (DMEM) containing 10% FBS and 1% penicillin-streptomycin. I did. Calcium ionophore A23187 and phorbol 12-myristate13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA), and quercetin from Sigma-Aldrich (St. Louis, MO, USA). Albumin, 2,4-dinitrophenylated (DNP-BSA) was purchased by Thermo Fisher Scientific (Eugene, OR, USA), rIL-4 and mTNF-α were purchased from R&D system Inc (Minneapoils, MN, USA), and mIL- 6 was purchased from Biolegend (San Diego, CA, USA).
2-2. 세포 생존력의 측정2-2. Measurement of cell viability
RBL-2H3 세포, RAW 264.7 세포 및 293T 세포는 1.5 ×105 cells/ mL의 농도로 96 well plate (SPL Life Sciences Co., Pocheon, Korea) 에 분주한 후 온도 37 ℃, 5 % CO2 배양기에서 밤새 배양하였으며, 세포에 0.0025, 0.005, 0.01 %의 농도의 왕초피나무 유래 정유 추출물을 각각 처리하여 24h 동안 배양하였다. 세포 증식은 제조사의 지시에 따라 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega, Madison, WI, USA)를 이용하여 분석하였고, 마이크로 플레이트 리더기 (BioTek, Winooski, VT, USA)를 사용하여 490 nm에서 흡광도를 측정하였다. 세포 생존 능력에 대한 왕초피나무 유래 정유 추출물의 알레르기성 염증 억제 효과가 각 조건에서의 세포 사멸로 인한 것이 아닌 것을 확인하기 위해 RBL-2H3, RAW 264.7 및 293T 세포에서 각각 MTS를 이용해 평가했다. 24시간 동안 0.0025, 0.005, 0.01 중량%의 농도의 왕초피나무 유래 정유 추출물 처리 결과 유의한 세포 독성 효과를 나타내지 않았다 (도 1).RBL-2H3 cells, RAW 264.7 cells, and 293T cells were dispensed in a 96 well plate (SPL Life Sciences Co., Pocheon, Korea) at a concentration of 1.5 × 10 5 cells/mL in an incubator at 37°C and 5% CO 2 . The cells were cultured overnight, and the cells were treated with essential oil extracts derived from A. serrata at concentrations of 0.0025, 0.005, and 0.01%, respectively, and cultured for 24 h. Cell proliferation was performed according to the manufacturer's instructions 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) (Promega, Madison, WI). , USA), and absorbance was measured at 490 nm using a microplate reader (BioTek, Winooski, VT, USA). In order to confirm that the inhibitory effect of allergic inflammation of the essential oil extract derived from C. on cell viability was not due to apoptosis in each condition, RBL-2H3, RAW 264.7, and 293T cells were evaluated using MTS, respectively. As a result of treatment with essential oil extracts derived from serrata chinensis at concentrations of 0.0025, 0.005, and 0.01% by weight for 24 hours, a significant cytotoxic effect was not shown (FIG. 1).
2-3. RBL-2H3 세포에서 β-hexosaminidase 분비에 대한 왕초피나무 유래 정유 추출물의 영향2-3. Effect of Essential Oil Extracts from C. chinensis on the Secretion of β-hexosaminidase in RBL-2H3 Cells
베타-헥소사미니다아제는 비만 세포 탈과립의 지표(marker)이다. RBL-2H3 세포는 10 % FBS와 IgE anti-DNP가 함유된 MEM를 사용하여 48 well plate (2 ×105 세포 / mL)에 서 배양하였다. Siraganian 완충액 (SB) (pH 7.2, 119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2 .6H2O, 25 mM PIPES (piperazine-N,N') 으로 2회 씻은 뒤(washing), 세포를 5.6 mM glucose, 1 mM CaCl2 및 0.1% BSA (SBC 완충액)를 포함하는 Siraganian 완충액과 함께 배양하였다. 1시간 동안 0.0025, 0.005, 0.01 % 농도의 왕초피나무 유래 정유 추출물과 양성 대조군 퀘르세틴 (20 μM)을 각각 전처리하고, 그런 다음 세포를 37℃ 30분간 DNP-BSA (1 μg/mL) (Thermo Fisher Science, OR, USA)로 자극한 후에 얼음 위에서 5 분간 냉각시킨 후, 원심 분리에 의해 수집된 세포 상등액 (50 μL)을 1 mM 4-nitrophenyl-N-acetyl-β-D-glucosaminide (Sigma, St Louis, MO, USA)을 포함하는 0.1M sodium citrate (pH 4.5)에 37℃에서 1시간 동안 배양하였다. 반응은 0.1M Na2CO3 및 0.1M NaHCO3 (pH 10)을 함유하는 200㎕/well의 탄산염 완충액으로 종결시키고 마이크로 플레이트 리더 (BioTek, Winooski, VT, USA)로 405nm에서 흡광도를 측정하였다. 왕초피나무 유래 정유 추출물을 사용한 전처리는 IgE-항원 복합체 또는 PMA/A23187-자극된 RBL-2H3 세포의 탈과립을 억제했다. 또한, 왕초피나무 유래 정유 추출물 처리는 RBL-2H3 세포의 탈과립을 용량 의존적으로 감소시켰다. PMA/A23187-자극 RBL-2H3 세포에서 0.01 % 농도의 왕초피나무 유래 정유 추출물의 베타-헥소사미니다아제 방출 억제 효과는 양성대조군 퀘르세틴의 베타-헥소사미니다아제 방출 억제 효과와 유사하였다 (도 2).Beta-hexosaminidase is a marker of mast cell degranulation. RBL-2H3 cells were cultured in 48 well plates (2 × 10 5 cells/mL) using MEM containing 10% FBS and IgE anti-DNP. Siraganian buffer (SB) (pH 7.2, 119 mM NaCl, 5 mM KCl, 0.4
2-4. RBL-2H3 세포에서 IL-4 분비에 대한 왕초피나무 유래 정유 추출물의 영향2-4. Effect of Essential Oil Extracts from C. chinensis on IL-4 Secretion in RBL-2H3 Cells
배양 배지에서 IL-4 농도를 측정하기 위해 RBL-2H3 세포를 10 % FBS와 IgE anti-DNP를 37℃ 조건으로 48-well plate (2 X 105 Cells/ML) MEM에서 밤새 배양했다. MEM으로 2회 세척한 후, 0.0025, 0.005, 0.01 % 농도의 왕초피나무 유래 정유 추출물과 퀘르세틴(20μM)에서 각각 2시간 동안 전 처리한 후에 37℃에서 16시간 동안 DNP-BSA (1 μg/ml)로 처리하였다. 무세포 상층 액(cell-free supernant)을 수집하고 IL-4 농도는 제조자의 지시에 따라 ELISA 키트 (R&D, Minneapolis, MN, USA)를 사용하여 측정했다. 또한, RBL-2H3 세포를 10% FBS가 함유된 MEM에서 48 well plate로 밤새 배양하고, 각각 0.0025, 0.005, 0.01 % 농도의 왕초피나무 유래 정유 추출물과 퀘르세틴 (20μM)에서 2 시간 동안 전처리하였다. 그다음 세포는 30 ng/mL의 PMA 및 350 ng/mL의 A23187로 16 시간 동안 자극시켰다. 상층액을 수집하여 IL-4 수준을 측정하였다. DNP-BSA로 자극된 RBL-2H3 또는 PMA/A23187에 의해 자극된 RBL-2H3 세포에 왕초피나무 유래 정유 추출물을 처리하면 농도의존적으로 IL-4의 방출량이 억제되는 것을 확인하였다 (도 3). To measure the IL-4 concentration in the culture medium, RBL-2H3 cells were cultured overnight in 48-well plate (2
실시예 3. 왕초피나무 유래 정유 추출물의 항염증 효과 확인Example 3. Confirmation of anti-inflammatory effect of essential oil extract derived from C.
3-1. 293T 세포에서 NF-κB 활성화에 대한 왕초피나무 유래 정유 추출물의 영향3-1. Effect of Essential Oil Extracts from C. 293T Cells on NF-κB Activation
293T 세포를 poly-D-lysine hydrobromide (Sigma, MO, USA)로 코팅 한 96 well plate에 1.5 x 105 cells/mL로 처리한 후, 리포터 플라스미드 pNF-κB-SEAP (Clontech Laboratories, Palo Alto, CA, USA)의 일시적인 세포 감염을 4시간 동안 HilyMax에서 세포에 수행하였다. 형질 감염 후, 세포 배지를 새로운 DMEM으로 교체한 후 밤새 두었다. 그 다음, 세포를 0.0025, 0.005, 0.01 % 농도의 왕초피나무 유래 정유 추출물과 양성 대조군 Bay11-7082 (20μM)로 2시간 동안 처리한 후 24시간 동안 3ng/mL PMA (Sigma, St. Louis, MO, USA)로 자극하였다. 상층액 (10μL)을 Quanti-Blue (100 μL) (Invitrogen, Carlsbad, MA, USA)와 1시간 동안 배양하고 흡광도를 ELISA 마이크로 플레이트 판독기 (BioTek, Winooski, VT, USA)로 630 nm에서 판독하였다. PMA는 NF-κB 전사를 증가 시켰고, 이는 왕초피나무 유래 정유 추출물에 의해 용량 의존적으로 감소 되었다. 0.01 %의 농도에서의 왕초피나무 유래 정유 추출물은 NF-κB 전사 활성을 가장 많이 억제하였다 (도 4A).293T cells were treated with 1.5 x 10 5 cells/mL in a 96 well plate coated with poly-D-lysine hydrobromide (Sigma, MO, USA), and the reporter plasmid pNF-κB-SEAP (Clontech Laboratories, Palo Alto, CA) , USA) was performed on cells in HilyMax for 4 hours. After transfection, the cell medium was replaced with new DMEM and left overnight. Then, the cells were treated with essential oil extracts derived from C. chinensis at concentrations of 0.0025, 0.005, and 0.01% and positive control Bay11-7082 (20 μM) for 2 hours, and then 3 ng/mL PMA (Sigma, St. Louis, MO, for 24 hours). USA). The supernatant (10 μL) was incubated with Quanti-Blue (100 μL) (Invitrogen, Carlsbad, MA, USA) for 1 hour and the absorbance was read at 630 nm with an ELISA microplate reader (BioTek, Winooski, VT, USA). PMA increased NF-κB transcription, which was dose-dependently reduced by the essential oil extract derived from C. The essential oil extract derived from C. chopidae at a concentration of 0.01% inhibited the NF-κB transcription activity the most (FIG. 4A).
3-2. Raw264.7 세포에서 NF-κB 활성화에 대한 왕초피나무 유래 정유 추출물의 영향3-2. Effect of Essential Oil Extracts from C. chinensis on NF-κB Activation in Raw264.7 Cells
밤새 RAW 264.7 세포를 8-well glass chamber plate (Thermo Fisher Scientific, USA)에 1 ×105 cells/mL에 놓았다. 자극 전에 2시간 동안 세포를 왕초피나무 유래 정유 추출물 (0.01 %)와 Bay11-7082 (20μM)로 전처리 후 1 μg/mL의 LPS로 2시간 동안 자극을 준 다음 4% paraformaldehyde (Molecular Probes, Inc., Eugene, OR, USA)로 고정시키고 15분 동안 0.1% 트리톤으로 투과하였다. NF-κB p65 단백질은 polyclonal-anti-NF-κB p65 항체 (Invitrogen, Carlsbad, MA, USA)와 Alexa Fluor 488-접합 항 토끼 IgG 항체 (Molecular Probes Invitrogen, MA, USA)를 사용하여 면역 염색에 의해 검출되었다. Actin은 Alexa Fluor 594 conjugated phalloidin으로 염색하여 검출하였다. 세포는 DAPI (Molecular Probes)가 있는 Prolong gold anti-fade 시약을 사용하여 세포를 안착했다. Bay 11-7082 (Sigma, MO, USA)를 NF-κB 억제제에 대해 양성 대조군으로 사용하였다. 형광 이미지는 레이저 스캐닝 공초점 현미경 시스템 (Leica TCS SP5 / AOBS / Tandem, 독일)을 사용하여 한국 기초 과학 지원연구원 광주 센터에서 획득했다. LPS-NF-κB 경로에 대한 왕초피나무 유래 정유 추출물의 효과를 시험했다. 왕초피나무 유래 정유 추출물에 의해 억제된 LPS는 세포기질 (cytosol)에서 세포핵까지의 NF-κB의 전사를 억제 하였다 (도 4B).RAW 264.7 cells were placed overnight at 1 × 10 5 cells/mL in an 8-well glass chamber plate (Thermo Fisher Scientific, USA). Before stimulation, the cells were pretreated with essential oil extract (0.01%) and Bay11-7082 (20μM) derived from C. chinensis, and then stimulated with 1 μg/mL LPS for 2 hours and then 4% paraformaldehyde (Molecular Probes, Inc., Eugene, OR, USA) and permeated with 0.1% Triton for 15 minutes. NF-κB p65 protein was obtained by immunostaining using polyclonal-anti-NF-κB p65 antibody (Invitrogen, Carlsbad, MA, USA) and Alexa Fluor 488-conjugated anti-rabbit IgG antibody (Molecular Probes Invitrogen, MA, USA). Was detected. Actin was detected by staining with Alexa Fluor 594 conjugated phalloidin. Cells were settled using Prolong gold anti-fade reagent with DAPI (Molecular Probes). Bay 11-7082 (Sigma, MO, USA) was used as a positive control for the NF-κB inhibitor. Fluorescence images were acquired at the Gwangju Center, Korea Basic Science Institute, using a laser scanning confocal microscope system (Leica TCS SP5 / AOBS / Tandem, Germany). The effect of the essential oil extract derived from C. chopidae on the LPS-NF-κB pathway was tested. LPS, which was inhibited by the essential oil extract derived from C. chopidae, inhibited the transcription of NF-κB from the cytosol to the cell nucleus (Fig. 4B).
3-3. LPS-활성화된 RAW264.7 세포에서 산화질소 생성에 대한 왕초피나무 유래 정유 추출물의 영향3-3. Effect of Essential Oil Extracts from Cerciaceae on Nitric Oxide Production in LPS-activated RAW264.7 Cell
RAW 264.7 세포를 48-well plate(SPL Life Sciences Co., Pocheon, Korea)에 2 x 105cells/mL로 놓고, 2 시간 동안 왕초피나무 유래 정유 추출물 (0.0025, 0.005, 0.01 %)와 양성 대조군 Bay11-7082 (20μM)로 처리한 후 500 ng/mL 의 LPS로 자극 하였다. 배양 24 시간 후, 배양 상층액에서 산화 질소 (NO) 생산은 Griess 시약 (1% sulfanilamide in 5% H3PO4,0.1%N-(1-naphthyl)-ethylendiaminedihydrochloride)을 이용해 측정하고 실온에서 30 분간 배양 하였다. 흡광도는 ELISA 마이크로 플레이트 판독기(BioTek, Winooski, VT, USA)를 사용하여 570 nm에서 판독하였다. LPS-활성화된 RAW 264.7 세포에서의 산화질소 생산 또한 왕초피나무 유래 정유 추출물의 처리에 의해 유의하게 감소하였다 (도 5C).RAW 264.7 cells were placed in a 48-well plate (SPL Life Sciences Co., Pocheon, Korea) at 2 x 10 5 cells/mL, and the essential oil extract (0.0025, 0.005, 0.01%) derived from C. chinensis and positive control Bay11 for 2 hours After treatment with -7082 (20 μM), it was stimulated with 500 ng/mL of LPS. After 24 hours of incubation, nitric oxide (NO) production in the culture supernatant was measured using Griess reagent (1% sulfanilamide in 5% H 3 PO 4 ,0.1%N-(1-naphthyl)-ethylendiaminedihydrochloride) and at room temperature for 30 minutes. Cultured. Absorbance was read at 570 nm using an ELISA microplate reader (BioTek, Winooski, VT, USA). Nitric oxide production in LPS-activated RAW 264.7 cells was also significantly reduced by treatment with essential oil extract derived from C. chopidae (Fig. 5C).
3-4. LPS-활성화된 RAW264.7 세포에서 염증 매개체 생성에 대한 왕초피나무 유래 정유 추출물의 영향3-4. Effect of Essential Oil Extracts from C. C. on the Production of Inflammatory Mediators in LPS-activated RAW264.7 Cells
RAW 264.7 세포를 48-well plate (SPL Life Sciences, Pocheon, Korea)에 2 x 105 cells/mL로 배양하고, 2시간 동안 왕초피나무 유래 정유 추출물 (0.0025, 0.005, 0.01 %)와 양성 대조군 Bay11-7082 (20μM)로 처리한 후 500 ng/mL의 LPS로 자극하였다. TNF-α (R&D system, Minneapolis, MN, USA) 및 IL-6 (Biolegend, CA, USA)의 측정을 위해 24시간 동안 배양한 후 상층액을 수집하여 ELISA 키트로 분석했다. 왕초피나무 유래 정유 추출물은 농도 의존적으로 TNF-α 방출에 대해 현저한 활성을 저해하는 것을 보여주었다 (도 5A). 게다가 왕초피나무 유래 정유 추출물은 IL-6 분비를 유의하게 억제하였으며 대조군과 매우 큰 차이를 보였다. 0.01 중량%의 왕초피나무 유래 정유 추출물의 억제 효과는 20 μ에서의 기준 화합물 Bay11-7082의 효과에 근접했다. RAW 264.7 cells were cultured in a 48-well plate (SPL Life Sciences, Pocheon, Korea) at 2 x 10 5 cells/mL for 2 hours, and the essential oil extract (0.0025, 0.005, 0.01%) and positive control Bay11- After treatment with 7082 (20 μM), it was stimulated with 500 ng/mL of LPS. After incubation for 24 hours for the measurement of TNF-α (R&D system, Minneapolis, MN, USA) and IL-6 (Biolegend, CA, USA), the supernatant was collected and analyzed with an ELISA kit. It was shown that the essential oil extract derived from C. chopidae inhibited the remarkable activity of TNF-α release in a concentration-dependent manner (FIG. 5A). In addition, the essential oil extract derived from C. chopidae significantly inhibited the secretion of IL-6 and showed a very large difference from the control group. The inhibitory effect of 0.01% by weight of the essential oil extract derived from C. chinensis was close to that of the reference compound Bay11-7082 at 20 μ.
3-5. LPS-활성화된 RAW264.7 세포에서 COX-2 및 iNOS의 발현에 대한 왕초피나무 유래 정유 추출물의 영향3-5. Effect of essential oil extracts derived from chinensis chinensis on the expression of COX-2 and iNOS in LPS-activated RAW264.7 cells
6 well plate에서 1 ×106 cells/mL로 밤새 배양 한 RAW 264.7 세포를 0.01 %, 0.005 % 농도의 왕초피나무 유래 정유 추출물과 celecoxib (10 μM) (Sigma Co., MO, USA)로 2시간 동안 처리한 후 16 시간 동안 200 ng/mL의 LPS (Sigma Co., MO, USA)로 자극하였다. 용해 완충액에서 동일한 양의 단백질 추출물 (20 μg)을 10 % SDS-PAGE 분석을 한 후 polyvinylidene fluoride막 (PVDF) (Millipore, Bedford, MA, USA)에 전기 전사시켰다. Western blot 분석은 특이성 다클론항체(polyclonal antibodies)를 COX-2 (1: 1000, Cell Signaling Tech., Danvers, MA, USA)와 iNOS (1 : 1000, Santa Cruz, CA, USA), β-actin (1 : 1000, Santa Cruz, MA, USA)에 수행 하였다. western blot 분석을 통해 LPS를 가진 RAW 264.7 세포의 자극이 iNOS 및 COX-2 단백질의 축적을 일으킨 것을 확인했고, 왕초피나무 유래 정유 추출물은 iNOS 단백질의 수준을 상당히 감소시켰다. 또한, 왕초피나무 유래 정유 추출물은 LPS-활성화된 RAW 264.7 세포에서의 COX-2 단백질 발현을 감소시켰고, 특히 0.01 % 농도의 왕초피나무 유래 정유 추출물은 COX-2와 iNOX 발현의 억제에 두드러진 효과를 나타내었다 (도 6).RAW 264.7 cells cultured overnight at 1 × 10 6 cells/mL in a 6 well plate with 0.01% and 0.005% concentrations of essential oil extracts derived from Chopidae and celecoxib (10 μM) (Sigma Co., MO, USA) for 2 hours. After treatment, it was stimulated with 200 ng/mL of LPS (Sigma Co., MO, USA) for 16 hours. The same amount of protein extract (20 μg) in the lysis buffer was subjected to 10% SDS-PAGE analysis and then electrotransferred to a polyvinylidene fluoride membrane (PVDF) (Millipore, Bedford, MA, USA). Western blot analysis was performed using COX-2 (1: 1000, Cell Signaling Tech., Danvers, MA, USA), iNOS (1: 1000, Santa Cruz, CA, USA), β-actin and specific polyclonal antibodies. (1: 1000, Santa Cruz, MA, USA). Western blot analysis confirmed that stimulation of RAW 264.7 cells with LPS caused the accumulation of iNOS and COX-2 proteins, and essential oil extracts derived from Japanese barberry significantly reduced the level of iNOS protein. In addition, the essential oil extract derived from C. chopidae reduced the expression of COX-2 protein in LPS-activated RAW 264.7 cells, and in particular, the essential oil extract derived from Chopidae at a concentration of 0.01% showed a remarkable effect on the inhibition of COX-2 and iNOX expression. (Fig. 6).
실시예 4. 왕초피나무 유래 정유 추출물의 아토피피부염의 치료 효과 확인Example 4. Confirmation of the therapeutic effect of atopic dermatitis of essential oil extract derived from C.
본 발명에서는 7주령의 Female BALB/c 마우스 20마리를 다물사이언스에서 구입하여, 1일간 적응시킨 다음 실험에 사용하였다. 실험동물실의 환경은 표준적인 사육조건으로서 온도는 23±3℃, 습도는 40-60%, 명암 주기는 12시간으로 유지되며, 실험동물전용사료와 음수는 제한 없이 공급하였다. 본 발명의 모든 동물실험은 전남대학교 동물실험윤리위원회의 승인 하에 수행되었다. BALB/c 마우스는 배쪽의 털을 넓게 제모하고 2% 1-Chloro-2,4-dinitrobenzene (DNCB)을 olive oil/acetone 혼합물(1:4)에 용해시킨 후, 150μL를 배쪽의 제모한 피부에 도포하여 감작시켰다. 감작 후 5일 뒤에 1% DNCB을 olive oil/acetone 혼합물(1:4)에 용해시킨 후, 마우스의 양쪽 귀에 각각 20μL를 처리하는데 왕초피나무 유래 정유(ZCO)를 30분 전처리 후 1% DNCB을 처리하고 3시간 뒤에 왕초피나무 유래 정유를 후 처리하여 2일동안 피부염을 유발하였다. 그 후 하루에 한번씩 왕초피나무 유래 정유를 3일 동안 반복적으로 처리하여 왕초피나무 유래 정유(ZCO)의 약물 효과를 확인하였다. 그 결과, 2%, 1% 왕초피나무 유래 정유 처리군은 1% DNCB을 처리한 군보다 귀 두께가 감소하였고, 염증반응을 억제하는 것을 확인하였다. 왕초피나무 유래 정유를 처리한 군의 염증 억제 효과는 양성 대조군으로 사용한 1% Dexamethasone (Dex)처리한 군과 비슷하였다 (도 7). In the present invention, 20 7-week-old female BALB/c mice were purchased from Damul Science, acclimated for 1 day, and then used in the experiment. The environment of the laboratory animal room is standard breeding conditions, the temperature is 23±3℃, the humidity is 40-60%, and the light and dark cycle is maintained for 12 hours, and feed and drinking water for experimental animals are supplied without restrictions. All animal experiments of the present invention were performed under the approval of Chonnam National University Animal Experimental Ethics Committee. In BALB/c mice, hair on the abdomen was removed widely, and 2% 1-Chloro-2,4-dinitrobenzene (DNCB) was dissolved in an olive oil/acetone mixture (1:4), and 150 μL was added to the epilated skin on the abdomen. It was applied and sensitized. 5 days after sensitization, 1% DNCB was dissolved in an olive oil/acetone mixture (1:4), and 20 μL of each was treated in both ears of the mouse. After 30 minutes of pretreatment with essential oil (ZCO) derived from Panax chinensis, 1% DNCB was treated. After 3 hours, dermatitis was induced for 2 days by post-treatment with essential oils derived from the C. After that, once a day, the essential oil derived from C. C. was repeatedly treated for 3 days to confirm the drug effect of the essential oil from C. C. tree (ZCO). As a result, it was confirmed that the 2% and 1% essential oil-derived groups treated with 1% DNCB decreased the ear thickness and suppressed the inflammatory response. The inhibitory effect of inflammation of the group treated with essential oil derived from C. chopiaceae was similar to that of the group treated with 1% Dexamethasone (Dex) used as a positive control (Fig. 7).
Claims (15)
The health functional food composition according to claim 8, wherein the essential oil extract derived from C. chopidae is 0.0001 to 1% by weight based on the total weight of the composition.
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