KR101325094B1 - A composition comprising extract of Pleurotus eryngii for treating or preventing atopic dermatitis - Google Patents
A composition comprising extract of Pleurotus eryngii for treating or preventing atopic dermatitis Download PDFInfo
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- KR101325094B1 KR101325094B1 KR1020110019281A KR20110019281A KR101325094B1 KR 101325094 B1 KR101325094 B1 KR 101325094B1 KR 1020110019281 A KR1020110019281 A KR 1020110019281A KR 20110019281 A KR20110019281 A KR 20110019281A KR 101325094 B1 KR101325094 B1 KR 101325094B1
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- extract
- pleurotus eryngii
- atopic dermatitis
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Abstract
본 발명은 아토피 피부염의 예방 또는 치료용 조성물에 관한 것으로서, 더욱 상세하게는 아토피 피부염과 같은 각종 피부질환의 증세를 치료 또는 경감시키는 활성이 탁월한 새송이버섯 추출물에 관한 것이다.
본 발명에서는 상기 새송이버섯 추출물이 아토피 피부염에서 증가된다고 알려진 사이토카인 및 단백질인 TARC/CCL17, MDC/CCL22, CTACK/CCL27, ICAM-1 등의 발현을 감소시키는 것을 나타냄으로, 상기 추출물이 아토피 피부염의 치료제로 사용될 수 있음을 확인할 수 있다. The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, and more particularly, to a Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.
In the present invention, the Pleurotus eryngii extract is shown to reduce the expression of cytokines and proteins known to be increased in atopic dermatitis, TARC / CCL17, MDC / CCL22, CTACK / CCL27, ICAM-1, the extract of atopic dermatitis It can be confirmed that it can be used as a therapeutic agent.
Description
본 발명은 아토피 피부염의 예방 또는 치료용 조성물에 관한 것으로서, 더욱 상세하게는 아토피 피부염과 같은 각종 피부질환의 증세를 치료 또는 경감시키는 활성이 탁월한 새송이버섯 추출물에 관한 것이다. The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, and more particularly, to a Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.
아토피 피부염(atopic dermatitis)은 천식과 알레르기성 질환의 발달 전에 발생하는 만성적인 염증질환으로, 케모카인의 농도, 양적변화나 활성 정도에 따라 그 증상에 차이가 있으며(Pivarcsi and Homey, 2005), Th2 타입의 림프구가 병변부(lesional skin)로 침입하는 현상을 말하기도 한다(Grew et al., 1998).Atopic dermatitis is a chronic inflammatory disease that occurs before the development of asthma and allergic diseases. The symptoms vary depending on the level of chemokine, quantitative changes, and activity (Pivarcsi and Homey, 2005). Lymphocytes invade the lesional skin (Grew et). al ., 1998).
Th2 케모카인 중의 하나인 TARC/CCL17(thymus and activation-regulated chemokine)은 흉선(thymus)에서 지속적으로 발현되며 각질형성세포(keratinocytes)에서도 생성된다(Vestergaard et al., 1999). 상기 TARC 케모카인은 림프구를 끌어들이는 역할을 하며, Th2 림프구(Th2 lymphocytes), 호염기성 세포(basophils), 자연살해세포(natural killer cells)에서 우선적으로 발현된다(Sallusto et al., 1998; Nickel et al., 1999; Godisk et al., 1997). TARC와 밀접한 관계를 맺고 있는 케모카인으로 MDC/CCL22(macrophage-derived chemokine)가 있는데, 상기 MDC/CCL22도 각질형성세포에서 생성되는 것으로 알려져 있으며(Hino et al., 2007), 아토피 피부염 환자들의 혈청에는 TARC와 MDC가 과량으로 발현되는 것이 특징이다(Shimada et al., 2004; Hashimoto et al., 2006). 또한, CTACK/CCL27(cutaneous T cell-attracting chemokine)은 피부에서 발현되는 케모카인으로(Reiss et al., 2001) 피부 염증 질환에 관련이 되어 있다고 알려져 있다(Kakinuma et al., 2003). One of Th2 chemokines, TARC / CCL17 (thymus and activation-regulated chemokine), is constantly expressed in the thymus and is also produced in keratinocytes (Vestergaard et. al ., 1999). The TARC chemokine plays a role in attracting lymphocytes and is preferentially expressed in Th2 lymphocytes, basophils, and natural killer cells (Sallusto et. al ., 1998; Nickel et al ., 1999; Godisk et al ., 1997). A chemokine that is closely related to TARC is a macrophage-derived chemokine (MDC / CCL22), which is known to be produced in keratinocytes (Hino et. al ., 2007), which are characterized by excessive expression of TARC and MDC in the serum of patients with atopic dermatitis (Shimada et al., 2004; Hashimoto et. al ., 2006). In addition, CTACK / CCL27 (cutaneous T cell-attracting chemokine) is a chemokine expressed in the skin (Reiss et al ., 2001) are known to be involved in skin inflammatory diseases (Kakinuma et. al ., 2003).
아토피 피부염의 다른 증상으로, ICAM-1(intercellular adhesion molecule-1)이 과발현되는 것이 확인되는데(Campbell et al., 1999), ICAM-1은 림프구의 표면 항원(leukocyte surface antigen)을 위한 수용체(receptor)로 작용함으로써, 세포와 세포의 고착(adhesion)을 매개하는 역할을 한다. Another symptom of atopic dermatitis is the overexpression of ICAM-1 (intercellular adhesion molecule-1) (Campbell et. al ., 1999), ICAM-1 acts as a receptor for the lymphocyte surface antigen, thereby mediating the adhesion of cells to cells.
아토피 피부염은 면역학적 요인에 의해 각질형성세포의 증식이 비정상적으로 일어나는 현상이라고 할 수 있는데, 각질형성세포를 IFN-γ(interferon-γ)나 TNF-α(tumor necrosis factor-α)에 노출시키면 TARC, MDC, CTACK 등의 케모카인이 비정상적으로 발현하는 것을 확인할 수 있다. 이러한 현상은 염증반응이 일어나면서 단핵구/T세포(monocytes/T cells)가 피부로 침투되는 현상이 증가되기 때문이다(Sebastiani et al., 2002). Atopic dermatitis is a phenomenon in which keratinocytes proliferate abnormally due to immunological factors. When keratinocytes are exposed to IFN-γ (tumor necrosis factor-α), TARC It can be confirmed that abnormal expression of chemokines, such as MDC, CTACK. This is due to an increase in the infiltration of monocytes / T cells into the skin as an inflammatory reaction occurs (Sebastiani et al. al ., 2002).
아토피 피부염의 치료는 그 근원을 찾아 치료하는 것이 쉽지 않기 때문에, 완치를 목표로 하기보다는 유발인자를 피하고 적절한 치료를 통해 아토피 피부염의 증상을 조절해 나가는 것이 일반적이다. 아토피 피부염에 대한 처방은 스테로이드제, 항히스타민제, 항생제 등과 같은 약물요법이 주로 이루어지고 있는데, 스테로이드제(부신피질호르몬제)는 크게 소염작용과 면역억제 작용이 있으며 효과가 우수하지만, 장기간 바르면 피부약화, 전신 호르몬증상, 중독성 등의 부작용이 나타날 수 있다. 항히스타민제는 비만세포에서 히스타민이 유리되지 못하도록 하여 가려운 증상을 경감시키나 임시방편으로 사용하는 것으로 장기간 복용시 불면, 불안, 식욕감퇴 등의 부작용이 있을 수 있다. 따라서 아토피 피부염에 효과가 있으면서도 부작용이 없는 새로운 개념의 아토피 피부염 치료 조성물이 요청되고 있다.Since the treatment of atopic dermatitis is difficult to find and treat its source, it is common to avoid causing factors and to control the symptoms of atopic dermatitis through proper treatment rather than aiming at cure. Prescriptions for atopic dermatitis are mainly pharmacotherapy such as steroids, antihistamines, and antibiotics. The steroids (adrenal cortex hormones) have anti-inflammatory and immunosuppressive effects and are excellent, but they can weaken the skin after long-term application. Side effects such as systemic hormonal symptoms and addictive symptoms may occur. Antihistamines help prevent the release of histamine in mast cells, thus alleviating the itchy symptoms, but they are used as a temporary measure. There are side effects such as burning, anxiety and loss of appetite when taken over a long period of time. Therefore, there is a need for a new concept of atopic dermatitis treatment composition that is effective against atopic dermatitis and has no side effects.
새송이버섯(Pleurotus eryngii)은 한국에서 식재료와 전통의약품으로 폭 넓게 사용되고 있는 버섯 종류이다. 새송이버섯에는 지방이나 탄수화물은 거의 없지만 대부분의 채소류보다 단백질의 함량이 높은 편이다. 또한 비타민 B, D, K, A, C가 풍부하며, 저열량 식사에 적합하다(Kurtzman, 1997; Manzi and Aguzzi, 2001; Mattila et al., 2001; Yildiz et al., 1998). 새송이버섯의 활성과 관련되어서 새송이버섯이 항미생물 작용, 항산화 작용(Kalyoncu et al., 2010), 항염증, 항알레르기 효과 등이 있다고 알려져 있기는 하지만 아토피 피부염에 대한 치료 효과나 그 외의 기능은 다양하게 알려져 있는 편이 아니다. Pleurotus eryngii is a type of mushroom widely used as a food ingredient and traditional medicine in Korea. Pleurotus eryngii contains little fat or carbohydrates, but is higher in protein than most vegetables. It is also rich in vitamins B, D, K, A and C and suitable for low calorie meals (Kurtzman, 1997; Manzi and Aguzzi, 2001; Mattila et. al ., 2001; Yildiz et al ., 1998). The antimicrobial and antioxidative effects of Pleurotus eryngii (Kalyoncu et al. al ., 2010), anti-inflammatory and anti-allergic effects are known, but the therapeutic effect and other functions for atopic dermatitis are not widely known.
이에 대해, 본 발명자들은 새송이버섯의 에탄올 추출물(PEE)이 아토피 피부염의 완화 및 억제에 현저한 효과가 있다는 것을 발견함으로써 본 발명을 완성할 수 있었다. In contrast, the present inventors have been able to complete the present invention by discovering that the ethanol extract (PEE) of Pleurotus eryngii has a remarkable effect on the alleviation and inhibition of atopic dermatitis.
본 발명의 목적은 새송이버섯 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물을 제공하는 데에 있다. An object of the present invention to provide a composition for the prevention or treatment of atopic dermatitis containing Pleurotus eryngii extract.
본 발명은 아토피 피부염과 같은 각종 피부질환의 증세를 치료 또는 경감시키는 활성이 탁월한 새송이버섯 추출물을 함유하는 아토피 피부염의 예방 또는 치료용 조성물에 관한 것이다. The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, which contains Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.
상기 새송이버섯 추출물은 상기 새송이버섯을 에탄올 수용액, 프로판올, 부탄올, 이소프로판올, 에틸아세테이트, 에테르, 아세톤, 클로로포름 등과 같은 극성용매 및 이들의 혼합용매로 추출한 후, 이를 헥산과 물의 분배, 디아이온 HP-20 레진과 같은 흡착수지 사용방법, 칼럼 크로마토그래피에 의한 방법 등 식물체 성분의 분리 추출에 이용되는 공지의 방법을 단독 또는 적합하게 조합하여 용이하게 얻을 수가 있다. 조추출물은 필요에 따라서 상법에 따라서 더욱 정제할 수 있다.The Pleurotus eryngii extract is extracted with a polar solvent such as ethanol aqueous solution, propanol, butanol, isopropanol, ethyl acetate, ether, acetone, chloroform, and a mixed solvent thereof, and then the hexane and water distribution, diion HP-20 A well-known method used for separation extraction of plant components, such as a method of using an adsorption resin such as resin, or a method by column chromatography, can be easily obtained by combining alone or suitably. The crude extract can be further purified according to the conventional method, if necessary.
본 발명에서 사용하는 크로마토그래피에는 실리카겔 칼럼 크로마토그래피(silica gel column chromatography), 엘에이취-20 칼럼 크로마토그래피(LH-20 column chromatography), 박층 크로마토그래피(TLC; thin layer chromatography) 및 고성능 액체 크로마토그래피(high performance liquid chromatography) 등이 이용될 수 있다.Chromatography used in the present invention includes silica gel column chromatography, LH-20 column chromatography, thin layer chromatography (TLC) and high performance liquid chromatography high performance liquid chromatography, etc.) can be used.
또한 본 발명은 새송이버섯 추출물 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 아토피 피부염의 예방 또는 치료용 약학 조성물을 제공한다. In another aspect, the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis, containing Pleurotus eryngii mushroom extract or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명에 따른 새송이버섯 추출물을 포함하는 아토피 피부염의 예방 또는 치료용 약학 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 상기 새송이버섯 추출물을 포함하는 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화 할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 새송이버섯 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 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. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. Pharmaceutical compositions for the prevention or treatment of atopic dermatitis comprising the Pleurotus eryngii extract according to the present invention, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. It can be formulated in the form of suppositories and sterile injectable solutions. Carriers, excipients and diluents that may be included in the composition comprising the Pleurotus eryngii extract are lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin in the Pleurotus eryngii extract. The mixture is prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like.
상기 새송이버섯 추출물의 투여량은 치료 받을 대상의 연령, 성별, 체중과, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01㎎/㎏/일 내지 대략 2000㎎/㎏/일의 범위이다. 더 바람직한 투여량은 0.1㎎/㎏/일 내지 500㎎/㎏/일이다. 투여는 하루에 한 번 투여할 수도 있고, 수 회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명의 새송이버섯 추출물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다. The dosage of the Pleurotus eryngii extract will vary depending on the age, sex and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 0.1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or several times. The dose is not intended to limit the scope of the invention in any way. Pleurotus eryngii mushroom extract of the present invention can be administered to various mammals such as rats, livestock, humans. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.
특히, 새송이버섯은 예로부터 식품 또는 약재로 사용해 왔던 것으로 본 발명의 새송이버섯 추출물을 인체에 투여할 경우 다른 합성 의약품에 비해 부작용의 염려가 없을 것으로 사료되며, 실제로도 동물 실험을 통한 독성 시험 결과 생체 내 아무런 영향이 없는 것으로 판명되었다.In particular, the Pleurotus eryngii mushroom has been used as a food or medicinal herb for a long time. When the Pleurotus eryngii extract of the present invention is administered to the human body, it is considered that there is no concern of side effects compared to other synthetic medicines. No effect was found.
또한, 본 발명은 새송이버섯 추출물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 아토피 피부염의 예방 또는 개선용 건강기능식품을 제공한다. 본 발명의 건강기능식품은 정제, 캡슐제, 환제 또는 액제 등의 형태를 포함하며, 상기 건강기능식품으로는 드링크제, 육류, 소세지, 빵, 캔디류, 스넥류, 면류, 아이스크림을 포함한 낙농제품, 스프, 이온음료를 포함한 음료수, 알코올 음료 및 비타민 복합제를 포함한 영양 공급용 제품이 포함될 수 있다. In addition, the present invention provides a health functional food for the prevention or improvement of atopic dermatitis, including Pleurotus eryngii mushroom extract and a food supplement acceptable food supplement. The health functional food of the present invention includes tablets, capsules, pills or liquids, and the like, and the health functional foods include dairy products including soups, meat, sausages, breads, candy, snacks, noodles, ice cream, soups, Beverages containing ionic beverages, nutritional products including alcoholic beverages and vitamin complexes may be included.
본 발명은 아토피 피부염의 예방 또는 치료용 조성물에 관한 것으로서, 더욱 상세하게는 아토피 피부염과 같은 각종 피부질환의 증세를 치료 또는 경감시키는 활성이 탁월한 새송이버섯 추출물에 관한 것이다. The present invention relates to a composition for the prophylaxis or treatment of atopic dermatitis, and more particularly, to a Pleurotus eryngii extract with excellent activity for treating or alleviating the symptoms of various skin diseases such as atopic dermatitis.
본 발명에서는 상기 새송이버섯 추출물이 아토피 피부염에서 증가된다고 알려진 사이토카인 및 단백질인 TARC/CCL17, MDC/CCL22, CTACK/CCL27, ICAM-1 등의 발현을 감소시키는 것을 나타냄으로, 상기 추출물이 아토피 피부염의 치료제로 사용될 수 있음을 확인할 수 있었다. In the present invention, the Pleurotus eryngii extract is shown to reduce the expression of cytokines and proteins known to be increased in atopic dermatitis, TARC / CCL17, MDC / CCL22, CTACK / CCL27, ICAM-1, the extract of atopic dermatitis It could be confirmed that it can be used as a therapeutic agent.
도 1은 본 발명의 새송이버섯 추출물(PEE)이 세포독성이 없음을 나타내는 WST-1 어세이 결과이다.
도 2는 본 발명의 새송이버섯 추출물(PEE)이 TNF-α/IFN-γ로 증가된 TARC/CCL17의 발현을 억제하는 효과가 있음을 나타내는 면역측정법 결과이다.
도 3은 새송이버섯의 열수추출물(PEW)이 본 발명의 새송이버섯 추출물(PEE)과 달리, TNF-α/IFN-γ로 증가된 TARC/CCL17의 발현억제능력이 없음을 나타내는 면역측정법 결과이다.
도 4는 본 발명의 새송이버섯 추출물(PEE)이 TNF-α/IFN-γ로 증가된 TARC/CCL17, MDC/CCL22 및 CTACK/CCL27의 발현을 억제하는 효과가 있음을 나타내는 RT-PCR 결과이다.
도 5는 본 발명의 새송이버섯 추출물(PEE)이 TNF-α/IFN-γ로 증가된 ICAM-1의 발현을 억제하는 효과가 있음을 나타내는 웨스턴 블로팅 결과이다.1 is a WST-1 assay result showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention is not cytotoxic.
2 is an immunoassay result showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention has an effect of inhibiting the expression of TARC / CCL17 increased to TNF-α / IFN-γ.
Figure 3 is a result of immunoassay showing that the hot water extract of Pleurotus eryngii (PEW), unlike the Pleurotus eryngii mushroom extract (PEE) of the present invention, does not inhibit the expression of TARC / CCL17 that is increased to TNF-α / IFN-γ.
4 is a result of RT-PCR showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention has an effect of inhibiting the expression of TARC / CCL17, MDC / CCL22 and CTACK / CCL27, which are increased to TNF-α / IFN-γ.
5 is a Western blotting result showing that the Pleurotus eryngii mushroom extract (PEE) of the present invention has an effect of inhibiting the expression of ICAM-1 increased by TNF-α / IFN-γ.
이하 본 발명의 바람직한 실시예를 상세히 설명하기로 한다. 그러나, 본 발명은 여기서 설명되는 실시예에 한정되지 않고 다른 형태로 구체화될 수도 있다. 오히려, 여기서 소개되는 내용이 철저하고 완전해질 수 있도록 그리고 당업자에게 본 발명의 사상이 충분히 전달될 수 있도록 하기 위해 제공되는 것이다. Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.
<실시예 1. 새송이 버섯의 에탄올 추출물(PEE) 제조><Example 1. Preparation of Ethanol Extract (PEE) of Pleurotus eryngii>
새송이 버섯의 추출방법은 [Seo et al ., 2005]의 방법을 이용하였다.The extraction method of Pleurotus eryngii is [Seo et. al ., 2005].
새송이버섯은 육안으로 선별된 새송이버섯 부산물을 수돗물 또는 음용수로 낙하 세척하여, 가로와 세로가 각각 2±1㎝, 두께 0.5±1㎝ 정도의 크기로 세절한 다음, 전기열풍건조기를 사용하여 45℃에서 36시간 건조시켰다. 이 후 상기 건조된 새송이버섯(130g)을 에탄올 500㎖를 이용하여 60℃에서 4시간 동안 2회 추출하였다. 이 후, 상기 추출액을 회전농축기(EYELA, SB-1000)로 55℃에서 30rpm으로 농축시켜 동결한 후, 이를 동결건조하여 분말(PEE, Pleurotus eryngii ethanol extracts, 분말 회수율 0.23%)로 제조하였고, 상기 분말을 DMSO(dimethyl sulfoxide)에 녹여서 사용하였다. Pleurotus eryngii drops are washed and washed by tap or drinking water, and then cut into 2 ± 1cm in width and 0.5 ± 1cm in thickness, respectively, and then using an electric hot air dryer. Dried for 36 hours. Thereafter, the dried Pleurotus eryngii mushroom (130 g) was extracted twice with 60 ml of ethanol for 4 hours at 60 ° C. Thereafter, the extract was concentrated in a rotary condenser (EYELA, SB-1000) at 55 ° C. at 30 rpm and frozen, and then lyophilized to obtain a powder (PEE, Pleurotus). eryngii ethanol extracts, powder recovery rate 0.23%), and the powder was dissolved in DMSO (dimethyl sulfoxide).
또한 본 발명의 새송이버섯의 에탄올 추출물의 효과를 비교하기 위해 새송이버섯의 열수추출물을 제조하였다. 이를 위해 새송이버섯(130g)을 물 1000㎖를 이용하여 90℃에서 8시간 동안 추출하였다. 이 후, 상기 추출액을 회전농축기(EYELA, SB-1000)로 55℃에서 30rpm으로 농축시켜 동결한 후, 이를 동결건조하여 분말(PEW, Pleurotus eryngii water extracts, 분말 회수율 0.22%)로 제조하였고, 상기 분말을 DMSO(dimethyl sulfoxide)에 녹여서 사용하였다. In addition, to compare the effect of the ethanol extract of Pleurotus eryngii of the present invention was prepared hot water extract of Pleurotus eryngii. For this purpose, Pleurotus eryngii mushroom (130 g) was extracted for 8 hours at 90 ℃ using water 1000ml. Thereafter, the extract was concentrated in a rotary condenser (EYELA, SB-1000) at 55 ° C. at 30 rpm and frozen, and then freeze-dried to obtain a powder (PEW, Pleurotus). eryngii water extracts, powder recovery rate 0.22%), and the powder was used by dissolving in dimethyl sulfoxide (DMSO).
<실시예 2. 새송이버섯 추출물의 세포독성 확인> <Example 2. Checking cytotoxicity of Pleurotus eryngii extract>
세포독성은 WST-1 어세이 키트를 사용하였으며, 본 발명의 새송이 버섯 추출물의 처리로 인해 어떠한 독성도 확인되지 않았다. 이를 위해 먼저 인간각질형성세포주(human keratinocyte cell line)인 HaCaT 세포를 10%의 FBS(fetal bovine serum), 2mM L-글루타민(L-glutamine), 100U/㎖ 페니실린(penicillin), 및 100㎍/㎖ 스트렙토마이신(streptomycin)이 포함된 DMEM(Dulbeco's Modified Eagle's Medium)에서 배양하였다. 이 후의 모든 세포 배양은 상기 방법으로 배양하여 이용하였다. 본 발명의 새송이버섯 추출물은 DMSO에 녹여서 세포에 처리하였으며, 대조군에는 DMSO만을 처리하였다. 세포에 처리되는 DMSO는 0.1% 미만의 농도로 처리되었다. 상기 WST-1 어세이 결과는 도 1에 나타내었다.Cytotoxicity was used for the WST-1 assay kit, and no toxicity was confirmed due to the treatment of the Pleurotus eryngii extract of the present invention. To this end, HaCaT cells, a human keratinocyte cell line, were first prepared using 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U / ml penicillin, and 100 µg / ml. Cultured in Dulbeco's Modified Eagle's Medium (DMEM) containing streptomycin. All subsequent cell cultures were cultured and used in the above manner. The Pleurotus eryngii extract of the present invention was dissolved in DMSO and treated with cells, and the control was treated with only DMSO. DMSO treated in the cells was treated at a concentration of less than 0.1%. The WST-1 assay results are shown in FIG. 1.
도 1을 확인하면, 본 발명의 새송이버섯 추출물(PEE)이 세포독성이 없음을 확인할 수 있다. 1, it can be confirmed that the Pleurotus eryngii mushroom extract (PEE) of the present invention is not cytotoxic.
<실시예 3. 아토피 피부염 관련 케모카인과 단백질의 발현 확인><Example 3. Confirmation of the expression of chemokines and proteins related to atopic dermatitis>
3-1. TARC/CCL17 발현 확인 - 면역 측정법 이용 3-1. Confirmation of TARC / CCL17 Expression-Using Immunoassay
사이토카인 TARC/CCL17의 발현 확인을 위해, HaCaT 세포에 본 발명의 새송이버섯 추출물을 10~50㎍/㎖로 농도별로 30분간 처리하였고, 이후 TNF-α(20ng/㎖)/IFN-γ(20ng/㎖)를 24시간 동안 처리하였다. 24시간 이후에는, 세포 처리액의 상등액만을 수거하여 TARC의 발현을 면역 측정법(immunoassay, R&D Systems)으로 확인하였으며 이에 대한 결과는 도 2에 나타내었다. In order to confirm the expression of cytokine TARC / CCL17, HaCaT cells were treated with the Pleurotus eryngii extract of the present invention at a concentration of 10-50 μg / ml for 30 minutes, and then TNF-α (20 ng / ml) / IFN-γ (20 ng). / Ml) was treated for 24 hours. After 24 hours, only the supernatant of the cell treatment solution was collected, and the expression of TARC was confirmed by immunoassay (immunoassay, R & D Systems), and the results are shown in FIG. 2.
또한 동일한 조건으로 새송이버섯 열수추출물(PEW)도 HaCaT 세포에 처리하여 이에 대한 효과를 본 발명의 새송이버섯 추출물과 비교하였고, 상기 결과는 도 3에 나타내었다.Also, under the same conditions, Pleurotus eryngii extract (PEW) was also treated with HaCaT cells, and the effects thereof were compared with that of Pleurotus eryngii extract of the present invention, and the results are shown in FIG. 3.
도 2의 결과는, TNF-α/IFN-γ의 처리로 인해 증가되었던 TARC/CCL17이, 본 발명의 새송이버섯 추출물(PEE)의 처리로 인해 농도 의존적으로 억제되는 것을 나타내며, 이로 인해 상기 추출물이 아토피 피부염의 억제 효과가 있음을 확인할 수 있다. The results of FIG. 2 indicate that TARC / CCL17, which was increased due to the treatment of TNF-α / IFN-γ, was suppressed concentration-dependently due to the treatment of the Pleurotus eryngii extract (PEE) of the present invention, whereby the extract was It can be seen that there is an inhibitory effect of atopic dermatitis.
도 3의 결과는 새송이버섯 열수추출물(PEW)이 본 발명의 새송이버섯 추출물(PEE)과 달리, TNF-α/IFN-γ의 처리로 인해 증가되었던 TARC/CCL17에 대해 억제효과가 거의 없음을 알 수 있으며, 따라서, TARC/CCL17에 대한 억제효과는 새송이버섯의 에탄올 추출물만의 특징인 것으로 확인된다. 3 shows that the Pleurotus eryngii water extract (PEW) has little inhibitory effect on TARC / CCL17 that was increased due to the treatment of TNF-α / IFN-γ, unlike the Pleurotus eryngii extract (PEE) of the present invention. Therefore, the inhibitory effect on TARC / CCL17 is confirmed to be characteristic of the ethanol extract of Pleurotus eryngii.
3-2. MDC/CCL22와 CTACK/CCL27 확인 - RT-PCR 이용3-2. Check MDC / CCL22 and CTACK / CCL27-using RT-PCR
사이토카인 MDC/CCL22와 CTACK/CCL27의 발현 확인을 위해, RT-PCR을 수행하였다. RT-PCR was performed to confirm expression of cytokines MDC / CCL22 and CTACK / CCL27.
이를 위해 HaCaT 세포에 본 발명의 새송이버섯 추출물을 10~50㎍/㎖로 농도 별로 30분간 전처리하고, 이 후, TNF-α(20ng/㎖)/IFN-γ(20ng/㎖)를 24시간 동안 처리하였다. To this end, HaCaT cells were pretreated with the Pleurotus eryngii mushroom extract of the present invention at a concentration of 10-50 μg / ml for 30 minutes, and then TNF-α (20 ng / ml) / IFN-γ (20 ng / ml) for 24 hours. Treated.
총 RNA는 RNAiso Reagent(Takara)를 이용하여 추출하였다. RT-PCR을 위한 프라이머(primer)의 조건은 다음과 같으며, S16r 유전자(S16 ribosomal protein)는 하우스키핑 유전자(house keeping gene)로서, 전체 유전자의 증감을 비교하기 위한 비교군으로 사용되었다. Total RNA was extracted using RNAiso Reagent (Takara). Primer conditions for RT-PCR were as follows, and the S16r gene (S16 ribosomal protein) was a housekeeping gene, and was used as a comparison group to compare the increase and decrease of all genes.
MDC/CCL22 forward : 5’-AGG ACA GAG CAT GGC TCG CCT ACA GA-3’MDC / CCL22 forward: 5’-AGG ACA GAG CAT GGC TCG CCT ACA GA-3 ’
MDC/CCL22 reverse : 5’-TAA TGG CAG GGA GGT AGG GCT CCT GA-3’MDC / CCL22 reverse: 5’-TAA TGG CAG GGA GGT AGG GCT CCT GA-3 ’
CTACK/CCL27 forward : 5’-CTC AGC TCT ACC GAA AGC C-3’CTACK / CCL27 forward: 5’-CTC AGC TCT ACC GAA AGC C-3 ’
CTACK/CCL27 reverse : 5’-GCC CAT TTT CCT TAG CAT CC-3’CTACK / CCL27 reverse: 5’-GCC CAT TTT CCT TAG CAT CC-3 ’
S16 ribosomal protein (S16r) genes forward : 5’-TCC AAG GGT CCG CTG CAG TC-3’S16 ribosomal protein (S16r) genes forward: 5’-TCC AAG GGT CCG CTG CAG TC-3 ’
S16 ribosomal protein (S16r) genes reverse : 5’-CGT TCA CCT TGA TGA GCC CAT T-3’S16 ribosomal protein (S16r) genes reverse: 5′-CGT TCA CCT TGA TGA GCC CAT T-3 ’
PCR은 하기 조건으로 35 싸이클을 수행하였으며, 증폭된 DNA의 확인은 SYBRSafeDNA 젤 염색 키트를 이용하였고, 상기 결과는 도 4에 나타내었다.PCR was performed for 35 cycles under the following conditions, and the amplified DNA was identified using the SYBRSafeDNA gel staining kit, and the results are shown in FIG. 4.
PCR 조건 : [denaturation at 98°C for 10 sec, annealing at 60°C for 30 sec, elongation at 72°C for 60 sec]PCR conditions: [denaturation at 98 ° C for 10 sec, annealing at 60 ° C for 30 sec, elongation at 72 ° C for 60 sec]
도 4의 결과는 TNF-α/IFN-γ의 처리로 인해 증가되었던 MDC/CCL22와 CTACK/CCL27이, 본 발명의 새송이버섯 추출물(PEE)의 처리로 인해 농도 의존적으로 억제되는 것을 나타내며, 이로 인해 상기 추출물이 아토피 피부염의 억제 효과가 있음을 확인할 수 있다. The results of FIG. 4 show that MDC / CCL22 and CTACK / CCL27, which were increased due to the treatment of TNF-α / IFN-γ, are suppressed concentration-dependently due to the treatment of the Pleurotus eryngii extract (PEE) of the present invention. It can be seen that the extract has an inhibitory effect on atopic dermatitis.
3-3. ICAM-1의 발현 확인 - 웨스턴 블로팅 이용3-3. Confirmation of Expression of ICAM-1-Using Western Blotting
림프구의 표면 항원(leukocyte surface antigen)을 위한 수용체(receptor) 단백질인 ICAM-1의 발현 확인을 위해, 웨스턴 블로팅을 수행하였다. Western blotting was performed to confirm the expression of ICAM-1, a receptor protein for lymphocyte surface antigen.
HaCaT 세포에 본 발명의 새송이버섯 추출물을 10~50㎍/㎖로 30분간 전처리하고, TNF-α(20ng/㎖)/IFN-γ(20ng/㎖)를 24시간 동안 처리하였고, 각각 80㎍의 단백질을 10% SDS-PAGE 및 PVDF(polyvinylidene difluoride) 멤브레인으로 전이시켰다. 멤브브레인 블로킹 이후에 1차 항체를 반응시켰으며, 2차 항체(horseradish peroxidase-conjugated anti-IgG secondary antibody)를 반응시켰다. 최종적인 발현 비교는 ECL reagent를 이용하였으며, β-actin(house keeping gene)을 비교군으로 하였고, 이에 대한 결과는 도 5에 나타내었다.HaCaT cells were pretreated with Pleurotus eryngii mushroom extract of the present invention at 10-50 μg / ml for 30 minutes, and treated with TNF-α (20 ng / ml) / IFN-γ (20 ng / ml) for 24 hours, each of 80 μg. Proteins were transferred to 10% SDS-PAGE and polyvinylidene difluoride (PVDF) membranes. After membrane blocking, the primary antibody was reacted, and a secondary antibody (horseradish peroxidase-conjugated anti-IgG secondary antibody) was reacted. Final expression comparison was performed using ECL reagent, β-actin (house keeping gene) as a comparison group, the results are shown in FIG.
상기 도 5를 확인하면, TNF-α/IFN-γ의 처리로 인해 증가되었던 ICAM-1이 본 발명의 새송이버섯 추출물(PEE)의 처리로 인해 농도 의존적으로 억제되는 것을 나타내며, 이로 인해 상기 추출물이 아토피 피부염의 억제 효과가 있음을 확인할 수 있다. 5 shows that ICAM-1, which has been increased due to the treatment of TNF-α / IFN-γ, is suppressed concentration-dependently due to the treatment of the Pleurotus eryngii extract (PEE) of the present invention, whereby the extract is It can be seen that there is an inhibitory effect of atopic dermatitis.
<< 실시예Example 4 : 독성시험> 4: Toxicity Test>
4-1 : 경구투여4-1: Oral administration
ICR계 마우스(25g)를 10마리씩 5개의 군으로 나누어 본 발명에 따른 새송이버섯 추출물을 각각 0, 2, 10, 20 및 50㎍/kg의 용량으로 경구 투여한 후 2주간 독성여부를 관찰한 결과, 5개의 군에서 모두 생존하였다.ICR-based mice (25 g) were divided into 5 groups of 10 animals, and the worm mushroom extract according to the present invention was orally administered at doses of 0, 2, 10, 20 and 50 ㎍ / kg, respectively, and observed for 2 weeks. In all cases, all of them survived.
4-2 : 복강투여4-2: intraperitoneal administration
ICR계 마우스(25g)를 10마리씩 5개의 군으로 나누어 본 발명에 따른 새송이버섯 추출물을 각각 0, 2, 10, 20 및 50㎍/kg의 용량으로 복강 투여한 후 24시간 동안 독성여부를 관찰한 결과 5개의 군 모두 생존하였다.ICR-based mice (25 g) were divided into 5 groups of 10 rats, followed by intraperitoneal administration of the Pleurotus eryngii mushroom extracts according to the present invention at doses of 0, 2, 10, 20 and 50 ㎍ / kg, respectively, for 24 hours. Results All five groups survived.
이상의 결과에서 본 발명의 새송이버섯 추출물은 급성독성이 거의 없음을 확인하였다.From the above results, it was confirmed that the Pleurotus eryngii mushroom extract of the present invention has little acute toxicity.
<< 사용예Examples 1 : 약학적 1: Pharmaceutical 제제예Formulation example >>
1-1 : 정제의 제조1-1: Preparation of Tablet
본 발명의 새송이버섯 추출물 20g을 락토오스 175.9g, 감자전분 180g 및 콜로이드성 규산 32g과 혼합하였다. 이 혼합물에 10% 젤라틴 용액을 첨가시킨 후, 분쇄해서 14 메쉬체를 통과시켰다. 이것을 건조시키고 여기에 감자전분 160g, 활석 50g 및 스테아린산 마그네슘 5g을 첨가해서 얻은 혼합물을 정제로 만들었다. 20 g of Pleurotus eryngii extract of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.
1-2 : 주사액제의 제조1-2: Preparation of Injection Solution
본 발명의 새송이버섯 추출물 2g을 증류수에 용해시켜서 100㎖을 만들었다. 이 용액을 병에 넣고 20℃에서 30분간 가열하여 멸균시켰다.2 g of Pleurotus eryngii extract of the present invention was dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.
<< 사용예Examples 2 : 식품 2: Food 제조예Manufacturing example >>
2-1 : 조리용 양념의 제조2-1: Preparation of Cooking Seasonings
본 발명의 새송이버섯 추출물을 0.2~10 중량%로 하여 건강 증진용 조리용 양념을 제조하였다.Fresh pine mushroom extract of the present invention to 0.2 to 10% by weight to prepare a cooking seasoning for health promotion.
2-2 : 밀가루 식품의 제조2-2: Preparation of Wheat Flour Food
본 발명의 새송이버섯 추출물을 0.1~5.0 중량%로 하여 밀가루에 첨가하고, 이 혼합물을 이용하여 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.The Pleurotus eryngii mushroom extract of the present invention is added to flour at 0.1 to 5.0% by weight, and bread, cake, cookies, crackers and noodles are prepared using the mixture to prepare foods for health promotion.
2-3 : 2-3: 스프soup 및 육즙( And juicy ( graviesgravies )의 제조Manufacturing
본 발명의 새송이버섯 추출물을 0.1~1.0 중량%로 하여 스프 및 육즙에 첨가하여 건강 증진용 육가공 제품, 면류의 수프 및 육즙을 제조하였다.The Pleurotus eryngii mushroom extract of the present invention was added to the soup and gravy by 0.1 to 1.0% by weight to prepare meat products for health promotion, soup of noodles and gravy.
2-4 : 유제품(2-4: Dairy Products ( dairydairy productsproducts )의 제조Manufacturing
본 발명의 새송이버섯 추출물을 0.1~1.0 중량%로 하여 우유에 첨가하고, 상기 우유를 이용하여 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.The Pleurotus eryngii mushroom extract of the present invention is added to milk at 0.1 to 1.0 wt%, and various dairy products such as butter and ice cream are prepared using the milk.
<< 사용예Examples 3 : 음료 3: Drink 제조예Manufacturing example >>
3-1 : 3-1: 야채쥬스Vegetable juice 제조 Produce
본 발명의 새송이버섯 추출물 0.5g을 토마토 또는 당근 쥬스 1,000㎖에 가하여 건강 증진용 야채쥬스를 제조하였다.0.5 g of the Pleurotus eryngii extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.
3-2 : 3-2: 과일쥬스Fruit juice 제조 Produce
본 발명의 새송이버섯 추출물 0.1g을 사과 또는 포도 쥬스 1,000㎖에 가하여 건강 증진용 과일쥬스를 제조하였다.0.1 g of Pleurotus eryngii extract of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice for health promotion.
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Inhibition of thymus- and activation-regulated chemokine (TARC/CCL17) production by Pleurotus eryngil extract in the HaCaT cells. The 6th MEAMS, 포스터발표-57, 156쪽. (2010) * |
Inhibition of thymus- and activation-regulated chemokine (TARC/CCL17) production by Pleurotus eryngil extract in the HaCaT cells. The 6th MEAMS, 포스터발표-57, 156쪽. (2010)* |
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