KR20180057753A - Method for preparing fermented guava extract and composition incluing the fermented guava extract for improving atopic dermatitis symptom - Google Patents
Method for preparing fermented guava extract and composition incluing the fermented guava extract for improving atopic dermatitis symptom Download PDFInfo
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- KR20180057753A KR20180057753A KR1020160154839A KR20160154839A KR20180057753A KR 20180057753 A KR20180057753 A KR 20180057753A KR 1020160154839 A KR1020160154839 A KR 1020160154839A KR 20160154839 A KR20160154839 A KR 20160154839A KR 20180057753 A KR20180057753 A KR 20180057753A
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- fermentation
- fermented
- guava leaf
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Abstract
Description
본 발명은 고체발효 및 액체발효의 2단 발효에 의한 구아바잎 발효대사체의 제조방법과 이를 유효성분으로 하는 아토피 피부염 증상 개선용 조성물에 관한 것이다.The present invention relates to a method for producing a guava leaf fermented metabolite by two-stage fermentation of solid fermentation and liquid fermentation, and a composition for improving the symptoms of atopic dermatitis comprising the same as an active ingredient.
구아바(Psidium guajava Linn)는 옛 잉카인들이 고산지대에서 기르고 즐겨 먹었던 것으로 도금양과(Myrtaceae)의 상록활엽 관목 또는 소교목으로 아메리카 열대지방이 원산이며, 아열대에까지 널리 분포되어 있다. 구아바는 영문으로 Guava, Guayabo 또는 Kuawa로 표시되며, 과일, 뿌리 및 잎은 오랫동안 민간의약으로 이용되어 왔다. 구아바는 관목 또는 교목으로 높이가 3~7m이며 가지를 많이 치고 잎은 마주나며 잎을 누르면 강한 향기가 나는 특성이 있다. 꽃은 지름 3cm정도로 꽃잎이 4개이며 잎겨드랑이에 달리고 대부분 하나씩 피지만 가끔 2~3개의 흰 꽃이 피는 경우가 있다. 열매는 공 모양 또는 달걀을 거꾸로 세운 모양이며 길이 5~12cm, 지름 5~7cm 정도이고 연한 붉은빛으로 익어 향기를 풍기며 작고 단단한 종자가 여러 개 들어 있다. 과육은 즙이 많고 달콤하며 비타민 C가 오렌지 보다 5배 이상 많고 비타민 A, B도 풍부해 과일 중에 "비타민 제왕"이라 불린다. 또한 구아바는 재배하기 쉽고 기온 및 토양 조건에 대한 적응 범위가 비교적 넓으나 열대에서 아열대의 중간 조건이 생육에 가장 적당한 것으로 알려져 있다.Guadava ( Psidium guajava Linn) is an evergreen broad-leaved shrub or bush of the Myrtaceae, which the former Incaans raised and enjoyed in the highlands, native to the American tropics and widely distributed in the subtropical zone. Guava is expressed in English as Guava, Guayabo or Kuawa, and fruits, roots and leaves have long been used as private medicines. Guava is a shrub or tree with a height of 3 ~ 7m. It has many branches, leaves face to face, and leaves have a strong smell. The flower is about 3cm in diameter with 4 petals. It runs on the axilla and most of it is bloomed, but occasionally 2 ~ 3 white flowers sometimes bloom. Fruits are ball shaped or egg shaped upside down. They are 5 ~ 12cm long and 5 ~ 7cm in diameter. They are cooked with light reddish red color and contain small and hard seeds. Flesh is juicy and sweet, vitamin C is more than 5 times more than orange, and vitamin A and B are abundant and called "vitamin king" in fruit. It is also known that guava is easy to cultivate and has a wide range of adaptation to temperature and soil conditions, while intermediate conditions in the tropics are most suitable for growth.
구아바의 재배 환경적 특성과 생리적 특성은 지구 온난화로 인한 기후변화에 대처할 수 있는 매우 적합한 대체작물이며, 또한 고비용 시설하우스, 가온 등의 추가적인 시설을 전혀 필요로 하지 않는 무가온 식물로서 고비용 농산물의 생산 환경을 획기적으로 개선할 수 있는 저탄소 녹색성장에 적합한 친환경 식물이라 할 수 있다. 현재 우리나라에서 1991년부터 본격적으로 재배법이 연구되어 제주도, 의령 및 경기도 일대에서 재배되고, 현재 우리나라에 구아바의 토착화는 성공을 하였지만 대부분 관상용으로 사용되거나 열매만을 이용하여 소비되고 있으며, 구아바 잎을 이용한 가공제품은 미미한 수준이다. 구아바의 열매와 잎은 식용 및 기능성 소재로서 활용가치가 매우 높은 고부가가치 식물로서, 현재 국내에서는 식약청으로부터 구아바 잎 열수추출물이 식후혈당조절에 도움을 줄 수 있는 건강기능식품으로 개별인정이 되어 있고, 일본에서는 구아바차 음료가 혈당조절용 특정 보건용 식품으로서 시판되고 있는 등 향후 고소득 작물로서 재배면적이 확대될 것으로 예측된다.Guava's cultivating environmental and physiological characteristics are very suitable substitute crops to cope with climate change due to global warming. Also, it is a non-dwelling plant that does not require any additional facilities such as high-cost facility houses and heating facilities. Can be said to be an environmentally friendly plant suitable for low-carbon green growth that can dramatically improve the environment. Currently, the cultivation method has been studied in Korea since 1991, and it has been cultivated in Jejudo, Gyeonggi and Gyeonggi provinces. Currently, the inculturation of guava in Korea has succeeded, but most of them are used for ornamental use or only fruit, The product is minimal. Guava's fruit and leaves are high value-added plants with high value for use as edible and functional materials. In Korea, the Guava leaf hot water extract from the KFDA is recognized as a health functional food that can help control postprandial blood glucose. In Japan, Guava tea beverages are marketed as a specific health food for controlling blood sugar, and it is predicted that the cultivation area will be expanded as a high-income crop in the future.
특히, 의령군에서 특화작물로 육성중인 구아바는 저탄소, 친환경, 고소득 녹색성장, 무가온 대체식물로서 2008년부터 잎과 열매가 생산되고 있고, 매년 생산량이 증가되고 있으나, 이를 활용한 기능성 가공제품의 개발과 상품화사례는 극히 미진한 실정이다.In particular, guava cultivated as a specialized crop in the army group is low-carbon, environment-friendly, high-income green growth, and a non-GDP alternative plant. Since 2008, leaf and fruit have been produced and the production volume has been increasing every year. Commercialization is extremely limited.
아토피 피부염(atopic dermatitis)은 알레르기 질환의 일종으로 특히 유아기와 아동기에 주로 발생하며 심한 소양감을 동반하는 만성 염증성 피부질환이다. 구미, 유럽에서의 유병률은 20~35%로 매우 높으며 치료와 예방 방법이 개선되고 있음에도 불구하고 계속 증가하고 있다. 한국에서도 초등학생까지 아동의 아토피피부염 진단비율이 1995년 13.7%에서 2005년 29%로 두배 이상의 큰 폭으로 증가하였고 중증 환자의 비율도 증가하는 추세에 있는 것으로 보고되고 있다.Atopic dermatitis is a type of allergic disease that occurs mainly in infancy and childhood and is a chronic inflammatory skin disease with severe pruritus. The prevalence rate in Europe, North America and Europe is very high at 20 ~ 35% and continues to increase despite the improvement of treatment and prevention methods. The rate of diagnosis of atopic dermatitis in children from Korea to elementary school students was more than doubled from 13.7% in 1995 to 29% in 2005, and the proportion of severe patients is also increasing.
아토피(atopy)는 그리스어인 a-topos가 어원으로 '특이한', '부적당한' 또는 '비정상적인 반응' 등의 뜻을 가지고 있으며, 알레르기 반응을 일으키게 하는 면역물질(IgE, immunoglobulin E)을 쉽게 형성함으로써 천식, 알레르기성 비염, 아토피성 피부염 등을 잘 일으키는 유전적 경향을 나타낸다. 아토피피부염의 원인이나 발생기전은 아직 확실히 알려져있지 않으나 유전적, 환경적 요인, 여러 가지 면역학적 이상 등이 복잡하게 연관되어 작용한다고 알려져있다. 아토피 피부염의 임상적 특징은 크게 소양증(국한성 또는 전신성 가려움), 발진, 만성적 재발 등을 들 수 있다. 혈청 내 IgE 수치 향상, eosinophilia, basophilia의 자발적인 히스타민 분비 증가, CD23 발현 증가, CD8 suppressor 감소, Th2에 의한 IL-4, IL-5 분비 증가 등의 현상을 동반하기도 한다. 아토피 피부염은 연령대에 따라 유아형(생후 2~24개월), 소아형(3~4세에서 10세 전후의 소아), 사춘기 및 성인형(만 12세 이후)으로 분류되며 아토피 피부염은 대부분 IgE와 연관된 면역기전에 의해 발병되는데 특정 알러젠에 대한 즉시형 면역반응보다는 T 세포 이상에 의한 지연형 면역 반응이 관여하는 것으로 보고되어있다.Atopy is the Greek word for a-topos which has the meaning of 'unusual', 'improper' or 'abnormal reaction' and easily forms an immunological substance (IgE, immunoglobulin E) Asthma, allergic rhinitis, atopic dermatitis, and the like. The causes and mechanisms of atopic dermatitis are not known yet, but genetic, environmental factors and various immunological abnormalities are known to work in a complex manner. Clinical features of atopic dermatitis include pruritus (localized or systemic itching), rash, and chronic recurrence. Serum levels of IgE, eosinophilia, spontaneous histamine release from basophilia, increased CD23 expression, decreased CD8 suppressor, increased IL-4 and IL-5 secretion by Th2, and the like. Atopic dermatitis is classified into infant type (2 ~ 24 months of age), childhood type (3 ~ 4 years old children around 10 years old), adolescent type and adult type (after 12 years old) It is reported that a delayed immune response due to T-cell abnormality is involved rather than an immediate immune response to specific allergens, which is caused by a related immune mechanism.
현재 아토피 피부염의 증상 완화를 위하여 사용되고 있는 치료방법으로는 환경개선, 스테로이드 외용제 처방, 보습제, 항히스타민제, 항생제 등이 사용되고 있으나 항히스타민제를 장기간 사용할 경우 내성이나 저혈압, 피부염, 소화기장애 등의 부작용을 일으킬 수 있어 지속적인 사용은 어려우며, 특히 스테로이드 외용제를 장기간 복용할 경우에는 고혈압, 심부전, 피부위축 또는 소아 환자의 성장 지연 등의 심각한 부작용이 일어날 수 있으므로 사용이 더욱 제한된다. 이처럼 발병환자의 삶의 질을 떨어뜨리며 근본적인 치료방법이 없는 아토피 피부염의 증상 완화를 위하여 부작용을 최소화하고 장기간 사용할 수 있는 천연물 자원을 탐색하고 개발하는 것이 시급한 것으로 판단된다.Currently, treatment methods for relieving symptoms of atopic dermatitis include environmental improvement, prescription of steroid external medicine, moisturizer, antihistamine, and antibiotics. However, long-term use of antihistamines causes side effects such as resistance, hypotension, dermatitis and digestive disorders And it is difficult to continue to use it. In particular, long-term use of steroid external preparations is further restricted because serious side effects such as hypertension, heart failure, skin atrophy or delayed growth of pediatric patients may occur. In order to alleviate the symptoms of atopic dermatitis, which does not have a fundamental treatment method, it is urgent to minimize the adverse effects and to search for and develop natural resources that can be used for a long time.
본 발명은 2단 발효 구아바잎 발효대사체 제조방법을 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a method for producing a two-stage fermented guava leaf fermented metabolite.
본 발명은 곰팡이에 의한 1단 고체발효와 유산균혼합균주에 의한 2단 액체발효로 구성되는 2단 발효 구아바잎 발효대사체 제조방법을 제공하는 것을 목적으로 한다.It is an object of the present invention to provide a method for producing a two-stage fermented Guava leaf fermented metabolism comprising a two-stage liquid fermentation by a mixture of a solid fermentation with a fungus and a lactic acid bacterium.
또한, 본 발명은 상기 2단 발효 구아바잎 발효대사체 추출물을 유효성분으로 하는 아토피 피부염 증상 개선용 조성물을 제공하는 것을 목적으로 한다.It is another object of the present invention to provide a composition for improving the symptoms of atopic dermatitis comprising the above-mentioned two-stage fermented Guava leaf fermented metabolite extract as an active ingredient.
본 발명의 2단 발효 구아바잎 발효대사체 제조방법은 하기 단계를 포함한다:The two-stage fermentation guava leaf fermentation metabolism preparation method of the present invention comprises the following steps:
구아바잎을 세척 후 건조하여 수분함량 10% 이하로 준비한 다음 2-3cm 길이로 분쇄하여 분말화하는 발효기질 제조단계; Preparing a fermented substrate by washing the guava leaf and drying it to prepare a powder having a water content of 10% or less and pulverizing it to a length of 2-3 cm and pulverizing it;
상기 발효기질에 곰팡이 생육용 영양성분 혼합물을 구아바분말 발효기질 중량에 대해 5중량% 첨가 혼합하고 121℃ 및 1.5기압 조건에서 15분간 멸균하여 1단발효배지를 준비하는 단계;Preparing a first stage fermentation medium by adding 5% by weight of a nutrient mixture for growth of fungi to the fermentation substrate and adding to the weight of the guava powder fermentation substrate and sterilizing the mixture at 121 ° C and 1.5 atm for 15 minutes;
상기 1단 발효배지 준비단계와 동시 또는 전후에 곰팡이를 PDB 배지에서 종배양하는 곰팡이 배양액 준비단계;Preparing a fungal culture liquid for culturing the fungus in the PDB medium simultaneously or after the preparation of the first stage fermentation medium;
상기 1단 발효배지에 상기 곰팡이 배양액을 5%를 접종하고 수분함량을 40-60%로 조절한 후, 상대습도 80%로 유지하고 30℃ 온도에서 5일간 정치하여 배양하는 고체발효 단계;A solid fermentation step in which 5% of the above-mentioned fungal culture liquid is inoculated into the 1-stage fermentation medium, the moisture content is adjusted to 40-60%, the relative humidity is maintained at 80%, and the mixture is allowed to stand at 30 DEG C for 5 days;
상기 고체발효 단계에서 얻어진 구아바잎 1단 고체발효물에 가수, 환류추출, 여과, 및 감압농축을 거쳐 15-25브릭스 농도의 구아바잎 고체발효액 추출물을 얻는 단계;Obtaining a guava leaf solid fermentation broth having a concentration of 15-25 Bricks by hydrolysis, reflux extraction, filtration, and concentration under reduced pressure in a first step fermentation product of guava leaf obtained in the solid fermentation step;
상기 구아바잎 고체발효액 추출물에 탄소원으로서 설탕 및 질소원으로서 효모추출액을 첨가하여 2단 발효배지를 준비하는 단계;Preparing a two-stage fermentation medium by adding sugar as a carbon source and yeast extract as a nitrogen source to the Guava leaf solid fermentation broth extract;
상기 2단 발효배지 준비단계와 동시 또는 전후에 유산균혼합균주를 MRS 배지에서 종배양하는 유산균혼합균주 배양액 준비단계;Preparing a culture solution of a mixed culture of a lactic acid bacterium in which a mixed culture of a lactic acid bacterium is sown in a MRS medium simultaneously with or before and after the two-stage fermentation medium preparation step;
상기 2단 발효배지에 상기 유산균혼합균주 배양액을 10% 접종한 다음 37℃ 온도에서 5일간 정치하여 배양하는 액체발효단계;A liquid fermentation step of incubating the two-stage fermentation broth with 10% of the culture solution of the mixed lactic acid bacteria and incubating at 37 DEG C for 5 days;
상기 액체발효 단계에서 얻어진 구아바잎 발효물을 살균, 여과 및 감압농축을 거쳐 25-30 브릭스 농도의 구아바잎 발효대사체를 얻는 단계.The fermented guava leaf obtained in the liquid fermentation step is sterilized, filtered and concentrated under reduced pressure to obtain a Guava leaf fermented metabolite having a concentration of 25-30 Bricks.
상기 발효기질의 구아바잎 분말은 2-3cm로 분쇄되는 것이 바람직하였는데 곰팡이 생육에 가장 적합한 분말 크기인 것으로 확인하였다. 곰팡이 생육용 영양성분 혼합물은 전분 35.25중량%, 식이섬유 35.44중량%, β-글루칸 4.96중량%, 폴리페놀 2.25 중량%, 극미량의 전분분해효소와 조단백질 16.64중량%, 조회분 2.78 중량% 및 유리당 2.68 중량%으로 구성되며, 이러한 조성의 곰팡이 생육용 영양성분 혼합물은 맥강분말로 일컬어지기도 한다. 본 발명의 1단 발효배지용 곰팡이는 아스퍼질러스 오리재(Aspergillus oryzae, A. oryzae)인 것이 바람직하다. The guava leaf powder of the fermenter was preferably pulverized to 2-3 cm, and it was confirmed that it was the most suitable powder size for growing mold. The nutrient mixture for fungal growth was prepared by mixing 35.25% by weight of starch, 35.44% by weight of dietary fiber, 4.96% by weight of beta -glucan, 2.25% by weight of polyphenol, 16.64% by weight of starch decomposing enzyme and crude protein, 2.78% %, And the mixture of nutrients for the growth of fungi of this composition is sometimes referred to as a manganese powder. The fungus for the first stage fermentation medium of the present invention is preferably Aspergillus oryzae (A. oryzae).
맥강분말은 아스퍼질러스 오리재의 생육에 최적화된 영양성분일 뿐만아니라 아스퍼질러스 오리재의 기능성을 배가시키는 장점이 있다. 맥강만을 발효기질로 하여 아스퍼질러스 오리재의 생육도를 조사한 결과 103CFU/g 정도의 균이 검출되었고, 항산화활성도 대조구에 비해 증가하여 아스퍼질러스 오리재에 대한 우수한 발효기질임을 확인하였다. MacGrown powder is not only an optimal nutrient for the growth of Aspergillus oryzae but also has the advantage of doubling the functionality of Aspergillus oryzae. The growth of Aspergillus oryzae as a fermentation substrate was investigated. The antioxidant activities of the strains were increased to 10 3 CFU / g, which was found to be an excellent fermentation substrate for Aspergillus oryzae.
상기 곰팡이 배양액은 PDB 배지에서 30℃에서 5일간 진탕배양하여 준비되는 것이 바람직하다. 곰팡이 배양액을 5% 접종한 후 수분함량이 곰팡이 생육에 또한 중요한 요소인데, 1단 발효배지의 수분함량은 40-60%인 것이 곰팡이 생육에 가장 유리하다. 1단 발효배지의 수분함량을 유지하고 증발을 방지하기 위해 상대습도 약 80%로 유지하는 것이 중요하였다.The fungus culture solution is preferably prepared by shaking culture in PDB medium at 30 ° C for 5 days. Moisture content is also an important factor for fungal growth after 5% inoculation of the fungal culture. The moisture content of the first stage fermentation medium is 40-60%, which is most advantageous for fungal growth. It was important to maintain the moisture content of the first stage fermentation medium and to maintain the relative humidity at about 80% to prevent evaporation.
상기 구아바잎 고체발효액 추출물은 구아바잎 1단 고체발효물의 중량비 10배의 물을 가수한 후 95℃에서 30분간 환류추출한 다음 여과 및 감압농축을 거쳐 15-25브릭스 농도로 얻는 것이 바람직하다. 얻어진 추출물의 pH는 6.5-7.0이었다.The guava leaf solid fermentation broth extract is preferably obtained by subjecting water having a weight ratio of guava leaf 1-stage solid fermentation product of 10 times to water, refluxing at 95 ° C for 30 minutes, filtering and concentrating under reduced pressure to obtain a concentration of 15-25 Bricks. The pH of the obtained extract was 6.5-7.0.
상기 2단 발효배지는 탄소원으로서 설탕 5중량% 및 질소원으로서 효모추출액 0.5중량%을 첨가하여 준비하는 것이 바람직하다.The two-stage fermentation medium is preferably prepared by adding 5% by weight of sugar as a carbon source and 0.5% by weight of yeast extract as a nitrogen source.
상기 유산균혼합균주는 락토바실러스 플란타룸, 락토바실러스 아시도필러스, 락토바실러스 람노수스, 락토바실러스 카제이 및 비피도박테리움 롱검으로 구성되는 것이 가장 바람직한데, 상기 혼합균주들은 모두 식용으로서의 기능성과 무독성이 인정된 균이다. 이와 같이 구성된 유산균혼합균주를 MRS 배지에서 37℃의 온도 조건으로 24시간 정치 배양하여 얻어진 배양액을 2단 발효배지에 사용한다.It is most preferable that the lactic acid bacteria mixed strain is composed of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus lambatus, Lactobacillus casei, and Bifidobacterium longum, It is recognized as non-toxic. The lactic acid bacteria-mixed strain thus constructed is cultured for 24 hours at 37 ° C in an MRS medium, and the resulting culture is used for a two-stage fermentation medium.
공개특허 제1020020032429호 및 공개특허 제1020030059916호에서 대두의 2단 발효 기술에 대해 개시하고 있으나 이들 모두 단일균주를 사용하여 발효하는 기술이 공지되어 있는바 본 발명의 혼합균주에 의한 발효의 효능에 대해 현재까지 알려진바 없다.Open Patent Publication Nos. 1020020032429 and 1020030059916 disclose a two-stage fermentation technology of soybeans. However, all of these techniques are known to ferment using a single strain, and the effect of the fermentation by the mixed strain of the present invention It is not known to date.
상기 2단 액체발효 단계에서 얻어진 구아바잎 발효물은 100℃에서 10분간 살균한 후 여과 및 감압농축을 거쳐 25-30 브릭스 농도로 조절한 다음 동결건조하여 구아바잎 발효대사체를 얻는다.The guava leaf fermented product obtained in the above two-step liquid fermentation step is sterilized at 100 ° C for 10 minutes, filtered and concentrated under reduced pressure, adjusted to a concentration of 25-30 bricks, and lyophilized to obtain a guava leaf fermented metabolite.
본 발명은 아스퍼질러스 오리재에 의한 1단 고체발효 및 유산균혼합균주에 의한 2단 액체발효로 얻어지는 상기 제조방법에 따른 구아바잎 발효대사체 추출물을 유효성분으로 하는 아토피 피부염 증상 개선용 조성물을 제공한다.The present invention provides a composition for improving atopic dermatitis symptoms comprising, as an active ingredient, an extract of Guava leaf fermented metabolite obtained by the above-mentioned production method obtained by a two-stage liquid fermentation with a first stage solid fermentation by Aspergillus oryzae and a mixed culture of lactic acid bacteria do.
본 발명의 구아바잎 발효대사체 제조에 사용되는 유산균혼합균주는 락토바실러스 플란타룸, 락토바실러스 아시도필러스, 락토바실러스 람노수스, 락토바실러스 카제이 및 비피도박테리움 롱검으로 구성된 유산균혼합균주인 것이 바람직하다. The lactic acid bacteria mixed strain used in the production of the guava leaf fermented metabolite of the present invention is a lactic acid bacteria mixed strain composed of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus lambatus, Lactobacillus casei, and Bifidobacterium longum .
본 발명의 구아바잎 발효대사체 추출물은 공지의 천연물 추출방법에 의하여 추출될 수 있는데, 추출용매로는 바람직하게는 물, C 1 내지 C 6 의 유기용매에서 선택된 하나 이상의 용매로 추출될 수 있으며, 상기 C 1 내지 C 6 의 유기용매는 C 1 내지 C 6 의 알코올(alcohol), 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌 클로라이드(methylene chloride), 헥산(hexane), 시클로헥산(cyclohexane) 및 석유에테르(petroleum ether)로 이루어진 군 중에서 선택된 것일 수 있다.The guava leaf fermented metabolite extract of the present invention can be extracted by a known natural product extraction method. The extractant may be extracted with at least one solvent selected from water, C 1 to C 6 organic solvents, The C 1 to C 6 organic solvent may be at least one selected from the group consisting of C 1 to C 6 alcohols, acetone, ether, benzene, chloroform, ethyl acetate, and may be selected from the group consisting of methylene chloride, hexane, cyclohexane, and petroleum ether.
본 발명의 추출물 제조에 사용되는 용매는 더욱 바람직하게는 물, C1 내지 C6 의 알코올 또는 이들의 혼합용매일 수 있으며, 상기 알코올은 에탄올인 것이 가장 바람직하다. 가장 바람직하게는 물과 에탄올을 혼합하여 60-80% 에탄올을 사용할 수 있다.The solvent used in the preparation of the extract of the present invention may more preferably be water, a C 1 to C 6 alcohol or a mixture thereof, and most preferably, the alcohol is ethanol. Most preferably, 60-80% ethanol can be used by mixing water and ethanol.
상기 용매의 양은 추출대상인 2단 발효 구아바잎 발효대사체 시료의 양에 따라 달라질 수 있으며, 바람직하게는 재료 총 중량의 3배-10배의 부피량으로 사용되고, 추출용매로서 60-80% 에탄올을 사용할 경우 가장 바람직하게는 8배-10배의 부피량이다.The amount of the solvent may vary depending on the amount of the two-stage fermented Guava leaf fermented metabolite sample to be extracted, preferably 3 to 10 times the total weight of the material, and 60 to 80% Most preferably 8 to 10 times the volume.
본 발명의 추출물 추출온도는 특별히 제한되지 아니하나, 바람직하게는 50℃ 내지 100℃일 수 있고, 60-80% 에탄올을 사용할 경우 가장 바람직하게는 60-80℃일 수 있다. The extraction temperature of the extract of the present invention is not particularly limited, but may be preferably 50 ° C to 100 ° C, and most preferably 60-80 ° C when 60-80% ethanol is used.
본 발명의 추출물 추출시간은 특별히 제한되지 아니하나, 2시간 내지 6시간일 수 있으며, 60-80% 에탄올을 사용할 경우 가장 바람직하게는 3-4시간일 수 있다.The extraction time of the extract of the present invention is not particularly limited, but may be 2 hours to 6 hours, and most preferably 3-4 hours when 60-80% ethanol is used.
본 발명 추출물은 공지의 천연물 추출법으로 추출될 수 있다. 예를 들어 초임계추출, 아임계추출, 고온추출, 고압추출 또는 초음파추출법 등의 추출장치를 이용한 방법 또는 XAD 및 HP-20을 포함한 흡착수지를 이용하는 방법 등 당업계의 통상적인 추출방법을 사용할 수 있으며, 바람직하게는 가열추출 또는 환류추출법으로추출할 수 있다. 본 발명의 추출단계는 1회 내지 7회, 바람직하게는 2회 내지 7회 반복 추출할 수 있고, 60-80% 에탄올을 사용할 경우 가장 바람직하게는 1회 내지 3회 반복 추출할 수 있다.The extract of the present invention can be extracted by a known natural substance extraction method. For example, conventional extraction methods such as supercritical extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, ultrasonic extraction, or using adsorbent resins including XAD and HP-20 can be used And preferably, it can be extracted by heating extraction or reflux extraction. The extraction step of the present invention can be repeated one to seven times, preferably two to seven times, and when 60 to 80% ethanol is used, it is most preferable to repeatedly extract one to three times.
일례로 본 발명에 따른 추출물은, 2단발효 구아바잎 발효대사체를 투입한 후 환류 냉각장치가 부착된 추출용기에 넣고 분말무게의 3 내지 10배의 부피의 추출용매를 넣고 용액을 끓이며 환류 추출한 후, 일정시간 방치한 다음 거름종이 등으로 여과하여 알코올 추출물을 얻을 수 있다. 추출시간은 2 내지 6시간인 것이 바람직하며, 이후에 농축 및 살균 단계를 거칠 수 있고, 농축 및 살균 단계 후 추출물은 액상으로 사용할 수 있으며, 본 발명에서는 분무건조 또는 동결건조의 건조 공정을 통하여 분말화하여 사용하는 것이 바람직하다.For example, the extract according to the present invention may be prepared by adding a fermented metabolite of Guava leaf fermentation in a two-stage fermentation broth, placing it in an extraction container equipped with a reflux condenser, adding an extraction solvent having a volume of 3 to 10 times the weight of the powder, boiling the solution, After that, it is allowed to stand for a predetermined time and then filtered with a filter paper or the like to obtain an alcoholic extract. It is preferable that the extraction time is 2 to 6 hours. After the concentration and sterilization step, the extract can be used as a liquid phase after the concentration and sterilization step. In the present invention, It is preferable to use it in the form of a mixture.
본 발명의 2단발효 구아바잎 발효대사체 추출물은 아토피 피부염 증상을 개선하는데 효능이 있다. The two-stage fermented Guava leaf fermented metabolite extract of the present invention is effective for improving atopic dermatitis symptoms.
본 발명은 구아바잎 추출물의 항아토피 활성 검증을 위해 먼저 발효를 거치지 않은 구아바잎으로부터 추출한 열수 추출물(GW), 2단발효 구아바잎 발효대사체 열수추출물(GFW), 그리고 2단발효 구아바잎 발효대사체 에탄올 추출물(GFE)을 준비하여, 시험관내(in vitro) 아토피 피부염의 억제활성, 즉 TARC/CCL17의 발현능, 염증 억제활성, 그리고 알러지 억제활성을 조사하였다. In order to test the anti-atopy activity of guava leaf extract, firstly, hot water extract (GW) extracted from non-fermented guava leaves, 2-stage fermented guava leaf fermented metabolism hot water extract (GFW), and 2-stage fermented guava leaf fermentation metabolism (GFE) was investigated in vitro for the inhibitory activity of atopic dermatitis, namely, TARC / CCL17 expression, inflammation inhibitory activity, and allergic inhibitory activity.
인체 피부 각질세포인 HaCaT세포에 TNF-a를 처리하여 아토피 지표인 TARC/CCL17을 유도시킨 후, 구아바 잎 열수추출물(GW), 2단발효 구아바 잎 열수추출물(GFW) 및 2단발효 구아바 잎 에탄올 추출물(GFE) 을 10,50,100,200μg/mL씩 처리하여 TARC/CCL17의 생성능을 측정하였다.HaCaT cells, which are human keratinocytes, were treated with TNF-a to induce TARC / CCL17, which is an atopic indicator. Then, guava leaf hot water extract (GW), 2-stage fermented guava leaf hot water extract (GFW) and 2-stage fermented guava leaf ethanol Extracts (GFE) were treated at 10, 50, 100, and 200 μg / mL for TARC / CCL17 production.
그 결과 TNF-a에 의해 증가된 TARC/CCL17의 유전자 발현량과 단백질 생성량이 GW, GFW, GFE의 처리 농도에 의존적으로 감소됨을 확인하였다. 또한 TNF-a에 의해 증가된 RANTES/CCL5의 생성량이 GW, GFW, GFE 처리에 따라 농도 의존적으로 감소하였고, 항아토피 반응 관련 전사조절인자 NF-κB 및 상위신호전달 체계인 MAP kinases의 활성도 감소하였다.As a result, it was confirmed that gene expression amount and protein production amount of TARC / CCL17 increased by TNF-a were decreased depending on the treatment concentration of GW, GFW and GFE. In addition, the amount of RANTES / CCL5 produced by TNF-a decreased by GW, GFW, and GFE treatment, and the activity of anti-atopy reaction-related transcription factor NF-κB and MAP kinases .
구아바 잎 열수추출물(GW), 2단발효 구아바 잎 열수추출물(GFW), 2단발효 구아바 잎 에탄올 추출물(GFE)의 염증 억제활성을 평가하기 위하여 염증 지표인 NO (Nitric Oxide) 생성량을 측정하였다. 구축된 실험법을 통해 GW(50~200μg/mL), GFW(10~50μg/mL), GFE(10~50μg/mL)와 LPS를 동시에 처리하여 NO 생성능을 측정한 결과 GW, GFW, GFE의 처리농도에 의존적으로 LPS에 의해 증가된 NO 생성량이 감소하였다.In order to evaluate the inflammation inhibitory activity of guava leaf hot water extract (GW), 2 stage fermented Guava leaf hot water extract (GFW) and 2 stage fermented Guava leaf ethanol extract (GFE), NO (Nitric Oxide) production level was measured. GW, GFW, GFE (10 ~ 50μg / mL), GFE (10 ~ 50μg / mL) and LPS were simultaneously treated with GW (50-200μg / mL) The amount of NO production increased by LPS was decreased in a concentration-dependent manner.
그리고 NO 생성을 매개하는 유전자인 iNOS의 발현 역시 GW, GFW, GFE의 처리농도에 의존적으로 감소하여, 2단발효 구아바 잎 추출물의 항염증 활성을 확인하였다. The expression of iNOS, a gene that mediates NO production, also decreased depending on the treatment concentration of GW, GFW, and GFE, confirming the anti-inflammatory activity of the two-stage fermented Guava leaf extract.
또한 염증 반응에 관여하는 프로스타글란딘 E2(PGE2)의 생성량을 측정하기 위해 264.7 세포에 LPS와 구아바 잎 추출물 GW(50~200μg/mL), GFW(10~50μg/mL), GFE(10~50μg/mL)를 24시간 처리하였을 때 GW, GFW, GFE의 처리농도 의존적으로 PGE2 생성량이 감소하였다. PGE2 생성을 매개하며 염증반응을 유발하는 유전자인 사이클로콕시나아제-2(COX-2) 유전자의 발현과 단백질 생성량 및 COX-2 프로모터 활성도 2단발효 구아바 잎 추출물의 처리로 또한 감소하였다.To determine the amount of prostaglandin E 2 (PGE 2 ) involved in the inflammatory response, 264.7 cells were treated with LPS, guava leaf extract GW (50-200 μg / mL), GFW (10-50 μg / mL), GFE / mL) for 24 hours, the amount of PGE 2 production was decreased depending on the treatment concentration of GW, GFW and GFE. Expression of COX-2 gene, protein production and COX-2 promoter activity, which mediate PGE 2 -mediated production of inflammatory response, also decreased with the treatment of the second stage fermented Guava leaf extract.
COX-2 및 iNOS 외에 염증반응을 매개하는 사이토카인(cyokine) 생성량과 유전자 발현에 대한 2단발효 구아바 잎 추출물(GW, GFW, GFE)의 영향을 조사하였다. 2단발효 구아바 잎 추출물의 처리시에 대표적인 염증성 사이토카인인 TNF-a, IL-6, IL-1β의 유전자 발현과 단백질 생성량이높게 나타났다. COX-2 발현을 조절하는 전사조절인자인 NF-κB3의 활성도 억제되었음을 확인하였다.In addition to COX-2 and iNOS, the effects of two-stage fermentation guava leaf extract (GW, GFW, GFE) on cytokine production and gene expression mediating inflammatory responses were investigated. The expression of TNF-a, IL-6, and IL-1β, which are typical inflammatory cytokines, and protein production were high in the treatment of 2-stage fermented Guava leaf extract. And that the activity of NF-κB3, a transcriptional regulator controlling COX-2 expression, was suppressed.
비만 세포주인 RBL-2H3 세포에 알러지 반응의 지표인 탈과립(degranulation) 방출량과 히스타민 생성량을 조사하기 위하여 DNP-IgE 세포를 감작시키고 알러젠인 DNP-BSA로 처리하는 실험법을 구축한 다음, 2단발효 구아바 잎 추출물(GW, GFW, GFE)을 처리하였을 때 처리농도에 의존적으로 탈과립 방출량과 히스타민 생성량이 감소하였다. 또한 과민성 알러지 반응을 매개하는 cytokine IL-4 및 IL-5의 유전자 발현량, 단백질 생성량 및 promoter 활성들이 모두 농도의존적으로 감소함을 확인하였다. 또한 알러지 반응 관련 전사조절인자(IL-4 및 IL-5)의 활성도 억제되었다.In order to investigate degranulation and histamine production, which is an index of allergic reaction, RBL-2H3 cells were sensitized with DNP-IgE cells and treated with allergen DNP-BSA. Then, 2-stage fermentation guava When leaf extracts (GW, GFW, GFE) were treated, the amount of degranulation and histamine production decreased depending on the treatment concentration. In addition, we confirmed that the levels of gene expression, protein production, and promoter activities of cytokine IL-4 and IL-5, which mediate the hypersensitivity allergic response, are all dose-dependent. In addition, allergic response-related transcription factors (IL-4 and IL-5) were inhibited.
따라서 본 발명은 2단발효 구아바잎 발효대사체 추출물을 유효성분으로 포함하는 아토피 피부염 증상 개선용 조성물을 제공한다.Accordingly, the present invention provides a composition for improving the symptoms of atopic dermatitis comprising an extract of a two-stage fermented Guava leaf fermented metabolite as an active ingredient.
또한 본 발명은 2단발효 구아바잎 발효대사체 추출물을 유효성분으로 포함하는 아토피 피부염 증상 예방 및 경감용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing and alleviating symptoms of atopic dermatitis comprising an extract of a fermented Guava leaf fermented metabolite of two stages as an active ingredient.
본 발명의 식품조성물은 식품, 기능성 식품, 영양 보조제, 건강식품 및 식품 첨가제 등의 모든 천연소재의 가공 형태를 포함한다. 상기 유형의 식품 조성물은 당 업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 예를 들면, 차, 주스, 엑기스 파우치 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. The food composition of the present invention includes all forms of natural materials such as foods, functional foods, nutritional supplements, health foods and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art. For example, they may be prepared in the form of tea, juice, extract pouches, and drinks, and may be ingested, granulated, encapsulated and powdered.
또한, 본 발명의 추출물은 아토피 피부염 증상의 예방 또는 개선의 효과가 있다고 알려진 공지의 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다. In addition, the extract of the present invention can be prepared in the form of a composition by mixing with known active ingredients known to be effective for preventing or improving atopic dermatitis symptoms.
또한, 본 발명의 2단발효 구아바잎 발효대사체 추출물을 식품 첨가제의 형태로 사용하기 위해서는 액상, 분말 형태로 제조하여 사용할 수 있다. 식품 첨가물로 사용시에 원료에 대하여 5 내지 30중량%, 바람직하게는 10 내지 20중량%의 양으로 첨가할 수 있다. 유효 성분의 혼합양은 사용목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 그러나, 건강 및 위생을 목적으로 하거나 건강 조절을 목적으로 하는 장기간의 섭취의 경우에 상기 양은 상기 범위 이하일 수 있으며, 안정성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. 상기 식품의 종류에는 특별한 제한이 없다. 상기 추출물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함하는 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.In order to use the two-stage fermented Guava leaf fermented metabolite extract of the present invention in the form of a food additive, it may be prepared in the form of liquid or powder. When used as a food additive, it may be added in an amount of 5 to 30% by weight, preferably 10 to 20% by weight based on the raw material. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). However, in the case of long-term consumption intended for health and hygiene purposes or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount exceeding the above range. There is no particular limitation on the kind of the food. Examples of the food to which the extract can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, , An alcoholic beverage and a vitamin complex, and includes all the health foods in a conventional sense.
상기 외에 본 발명에 따른 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 중점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명에 따른 조성물은 천연 과일 주스 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있으며, 첨가제의 비율은 크게 중요하진 않지만, 본 발명에 따른 조성물의 100중량부당 30중량% 이하인 것이 바람직하다.In addition to the above, the composition according to the present invention may further comprise various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid concentrating agents, pH adjusting agents, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the composition according to the invention may contain flesh for the production of natural fruit juices and vegetable drinks. These components can be used independently or in combination, and the proportion of the additives is not critical, but is preferably 30% by weight or less per 100 parts by weight of the composition according to the present invention.
본 발명에 따른 2단발효 구아바잎 발효대사체 추출물을 유효성분으로 함유하는 아토피 피부염 증상 개선용 약학적 조성물은 하기의 다양한 경구 또는 비경구 투여 형태로 제형화할 수 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition for improving the atopic dermatitis symptom containing the two-stage fermented Guava leaf fermented metabolite extract according to the present invention as an active ingredient can be formulated into various oral or parenteral administration forms described below, but the present invention is not limited thereto.
경구 투여용 제형으로는 예를 들면 정제, 환제, 경/연질 캅셀제, 액제, 현탁제, 유화제, 시럽제, 과립제, 엘릭시르제 등이 있는데, 이들 제형은 상기 유효성분 이외에 통상적으로 사용되는 충진제, 증량제, 습윤제, 붕해제, 활택제, 결합제, 계면활성제 등의 희석제 또는 부형제를 1종 이상 사용할 수 있다. 붕해제로는 한천, 전분, 알긴산 또는 이의 나트륨염, 무수인산일수소 칼슘염 등이 사용될 수 있고, 활택제로는 실리카, 탈크, 스테아르산 또는 이의 마그네슘염 또는 칼슘염, 폴리에틸렌 글리콜 등이 사용될 수 있으며, 결합제로는 마그네슘 알루미늄 실리케이트, 전분 페이스트, 젤라틴, 트라가칸스, 메틸셀룰로오스, 나트륨카복시메틸셀룰로오스, 폴리비닐피롤리딘, 저치환도 하이드록시프로필셀룰로오스 등이 사용될 수 있다. 이외에도 락토즈, 덱스트로오스, 수크로오스, 만니톨, 소르비톨, 셀룰로오스, 글리신 등을 희석제로 사용할 수 있으며, 경우에 따라서는 일반적으로 알려진 비등 혼합물, 흡수제, 착색제, 향미제, 감미제 등을 함께 사용할 수 있다.Examples of formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules and elixirs. These formulations include fillers, extenders, A wetting agent, a disintegrant, a lubricant, a binder, a surfactant, and the like may be used. As the disintegrant, agar, starch, alginic acid or its sodium salt, anhydrous calcium monohydrogenphosphate, etc. may be used. As the lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol and the like may be used , Magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, and low-substituted hydroxypropylcellulose can be used as the binding agent. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine and the like can be used as a diluent. In general, a commonly known boiling mixture, an absorbent, a colorant, a flavoring agent and a sweetening agent can be used together.
또한 2단발효 구아바잎 발효대사체 추출물을 유효성분으로 함유하는 약학적 조성물은 비경구 투여할 수 있으며, 비경구 투여는 피하주사제, 정맥주사제, 근육 내 주사제 또는 흉부 내 주사제를 주입하는 방법에 의한다. 이때, 비경구 투여용 제형으로 제제화하기 위하여 상기 추출물을 안정제 또는 완충제와 함께 물에서 혼합하여 용액 또는 현탁액으로 제조하고, 이를 앰플 또는 바이알의 단위 투여형으로 제조할 수 있다. In addition, the pharmaceutical composition containing the fermented Guava leaf fermented metabolite extract of the two-stage fermentation can be administered parenterally, and the parenteral administration can be carried out by injecting subcutaneous injection, intravenous injection, intramuscular injection, All. At this time, in order to formulate the formulation for parenteral administration, the extract may be mixed with a stabilizer or a buffer in water to prepare a solution or suspension, which may be prepared into a unit dosage form of ampoule or vial.
상기 조성물은 멸균되거나 또는 방부제, 안정화제, 수화제 또는 유화촉진제, 삼투압 조절을 위한 염, 완충제 등의 보조제 및 기타 치료적으로 유용한 물질을 함유할 수 있으며, 통상적인 방법인 혼합, 과립화 또는 코팅방법에 따라 제제화할 수 있다. The composition may be sterilized or contain adjuvants such as preservatives, stabilizers, wettable or emulsifying accelerators, salts for controlling osmotic pressure, buffering agents and other therapeutically useful substances and may be prepared by conventional methods such as mixing, granulating or coating . ≪ / RTI >
본 발명의 2단발효 구아바잎 발효대사체 추출물을 유효성분으로 함유하는 약학적 조성물을 단위 용량 형태로 제형화하는 경우, 유효성분으로서 2단발효 구아바잎 발효대사체 추출물을 약 2 내지 20㎎의 단위용량으로 함유되는 것이 바람직하다. 투여량은 환자의 체중, 나이 및 질병의 특수한 성질과 심각성과 같은 요인에 따라 의사의 처방에 따른다. 그러나, 성인 치료에 필요한 투여량은 투여의 빈도와 강도에 따라 하루에 약 10 내지 100㎎ 범위가 보통이다. When the pharmaceutical composition containing the two-stage fermented Guava leaf fermented metabolite extract of the present invention as an active ingredient is formulated in a unit dosage form, it is preferable to use a two-stage fermented Guava leaf fermented metabolite extract as an active ingredient in an amount of about 2 to 20 mg And it is preferably contained in a unit dose. The dosage depends on the physician's prescription depending on factors such as the patient's weight, age and the particular nature and severity of the disease. However, the dosage required for adult therapy is usually in the range of about 10 to 100 mg per day, depending on the frequency and intensity of administration.
본 발명의 구아바잎 발효대사체 제조방법에 따라 제조된 2단발효 구아바잎 발효대사체 추출물을 유효성분으로 포함하는 식품 및 약제를 적용함으로써 부작용 없는 성분으로 아토피 피부염 증상의 완화 및 치료에 획기적인 효과를 거둘 수 있다.By applying the food and medicament containing the extract of the fermented Guava leaf fermented metabolite of the two-stage fermented Guava leaf fermented by the method of the present invention, which is produced by the method of the present invention, as an effective ingredient, it is a side effect-free ingredient and has a remarkable effect on relieving and treating atopic dermatitis symptoms You can.
도 1은 본 발명의 2단 발효에 의한 구아바잎 발효대사체 제조방법에 대한 모식도이고,
도2는 HaCaT 세포내 TARC 유전자 발현능에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도3은 HaCaT 세포내 TARC 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도4는 HaCaT 세포내 RANTES/CCL5 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도5는 로 264.7 세포내 세포독성에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도6은 로 264.7 세포내 NO 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도7은 로 264.7 세포내 NO생성 매개 유전자인 iNOS 발현정도에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며, (도7이 도8과 동일합니다. 중복인듯합니다. )
도8는 로 264.7 세포내 COX-2 유전자 발현 정도에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도9은 로 264.7 세포내 COX-2 단백질 발현량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도10은 로 264.7 세포내 COX-2 루시퍼라아제 활성에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도11은 로 264.7 세포내 TNF-α 유전자 발현정도에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도12는 로 264.7 세포내 IL-6 유전자 발현정도에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도13은 로 264.7 세포내 IL-1β 유전자 발현정도에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도14는 로 264.7 세포내 TNF-α 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도15는 로 264.7 세포내 IL-6 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도16은 로 264.7 세포내 IL-1β 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도17은 로 264.7 세포내 NF-B 루시퍼라아제 활성에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도18은 로 264.7 세포내 MAP 키나아제 활성화에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도19는 RBL-2H3 세포내 세포독성에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도20은 RBL-2H3 세포내 β-헥소사미니다아제 활성에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도21은 RBL-2H3 세포내 히스타민 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도22는 RBL-2H3 세포내 IL-4 유전자 발현정도에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도23은 RBL-2H3 세포내 IL-4 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도24는 RBL-2H3 세포내 IL-4 루시퍼라아제 활성에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도25는 RBL-2H3 세포내 IL-5 유전자의 단백질 생성량에 대한 GW(A), GFW(B), GFE(C)의 영향에 대한 결과 그래프이며,
도26은 열수추출방법에 대한 모식도이다.FIG. 1 is a schematic view of a method for producing a guava leaf fermented metabolite by two-stage fermentation of the present invention,
FIG. 2 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on TARC gene expression in HaCaT cells,
FIG. 3 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the amount of TARC produced in HaCaT cells,
FIG. 4 is a graph showing the effect of GW (A), GFW (B) and GFE (C) on the amount of RANTES / CCL5 produced in HaCaT cells,
Figure 5 is a graph of the effect of GW (A), GFW (B), and GFE (C)
FIG. 6 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the NO production amount in 264.7 cells,
7 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the degree of expression of iNOS, which is a NO production mediator gene in 264.7 cells (Fig. 7 is the same as Fig. It seems to be.
FIG. 8 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the degree of expression of COX-2 gene in ROS 264.7 cells,
FIG. 9 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the expression level of COX-2 protein in 264.7 cells,
Figure 10 is a graph of the effect of GW (A), GFW (B), and GFE (C) on COX-2 luciferase activity in RO 264.7 cells,
FIG. 11 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the degree of expression of TNF-?
FIG. 12 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the degree of IL-6 gene expression in ROS 264.7 cells,
FIG. 13 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the degree of IL-1β gene expression in ROS 264.7 cells,
Figure 14 is a graph of the effect of GW (A), GFW (B), and GFE (C) on the amount of TNF-
Figure 15 is a graph of the effect of GW (A), GFW (B), and GFE (C) on IL-6 production in L264.7 cells,
FIG. 16 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the amount of IL-1?
Figure 17 is a graph of the effect of GW (A), GFW (B), and GFE (C) on NF-B luciferase activity in L264.7 cells,
Figure 18 is a graph of the effect of GW (A), GFW (B), and GFE (C) on MAP kinase activation in L264.7 cells,
FIG. 19 is a graph showing the effect of GW (A), GFW (B) and GFE (C) on cytotoxicity in RBL-2H3 cells,
20 is a graph showing the effect of GW (A), GFW (B) and GFE (C) on the activity of? -Hexosaminidase in RBL-2H3 cells,
FIG. 21 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on histamine production in RBL-2H3 cells,
22 is a graph showing the effect of GW (A), GFW (B) and GFE (C) on the degree of IL-4 gene expression in RBL-2H3 cells,
23 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on IL-4 production in RBL-2H3 cells,
24 is a graph showing the effect of GW (A), GFW (B) and GFE (C) on IL-4 luciferase activity in RBL-2H3 cells,
25 is a graph showing the effect of GW (A), GFW (B), and GFE (C) on the amount of protein produced by the IL-5 gene in RBL-
26 is a schematic diagram of a hot water extraction method.
본 발명을 실시예를 통하여 상세히 설명한다. 단, 하기의 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기의 실시예에 의해 한정되는 것은 아니다. The present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited by the following examples.
실시예 1 <2단 발효 구아바잎 발효대사체의 제조>Example 1 < Preparation of fermented guava leaf fermented metabolite of 2-stage fermentation >
수분함량이 10%이하인 건조 구아바잎을 2~3cm 크기로 분쇄한후 구아바 중량 대비 맥강분말(곰팡이 생육 영양성분)을 5%첨가하여 골고루 혼합한 다음 121℃, 1.5기압, 15분간 멸균한다. 상기 곰팡이 배지(1단발효배지)에 PDB배지에서 종배양한 액(30℃, 5일간 진탕배양) 10%를 접종하면서 수분함량을 40~60%(곰팡이 생육용이)로 조절하였다. 이것을 상대습도가 80% (배지 수분 유지 및 증발 방지)유지되는 배양기에서 30℃, 5일간 정치배양하였다. 1단 고체발효물의 중량 대비 10배 가수하여 95℃, 30분간 환류 추출한 후 여과, 감압농축하여 최종농도가 20brix로 조절하였다. 이 농축액에 설탕(탄소원) 5%와 효모추출액(질소원) 0.5%을 첨가하여 2단 발효배지를 제조하였다. 배지에 식용으로서 기능성과 무독성이 인정된 프로바이오틱스 혼합균주 5종(락토바실러스 플란타룸, 락토바실러스 아시도필러스, 락토바실러스 람노수스, 락토바실러스 카제이 및 비피도박테리움 롱검)을 MRS에서 종배양(37℃, 24시간 정치배양)한 액 10%를 접종한 후 37℃, 5일간 정치배양하였다. Dried guava leaves having a moisture content of 10% or less are pulverized to 2 to 3 cm in size and mixed with 5% of mugwort powder (fungus growth nutrient) relative to the weight of guava, uniformly mixed and sterilized at 121 ° C and 1.5 atm for 15 minutes. The water content was adjusted to 40 to 60% (easy growth of the fungus) by inoculating 10% of the liquid of the seed culture (30 ° C, shaking culture for 5 days) in the PDB medium to the fungal medium (1 stage fermentation medium). This was incubated for 5 days at 30 DEG C in an incubator maintained at a relative humidity of 80% (to keep the medium moisture and prevent evaporation). Stage fermentation product was refluxed at 95 ° C for 30 minutes, filtered, and concentrated under reduced pressure to a final concentration of 20 brix. A 2-stage fermentation broth was prepared by adding 5% of sugar (carbon source) and 0.5% of yeast extract (nitrogen source) to this concentrate. Five kinds of probiotic mixed strains (Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus lambatus, Lactobacillus casei and Bifidobacterium long sword), which were recognized as functional and non-toxic for food, (37 占 폚, 24 hours of stationary culture) was inoculated and cultured for 5 days at 37 占 폚.
상기 액을 100℃, 10분간 살균한 후 여과, 감압농축하여 25~30brix로 조절한 다음 동결건조하여 발효대사체를 제조하였다(도 1 참조)The solution was sterilized at 100 ° C for 10 minutes, filtered and concentrated under reduced pressure to adjust the pH to 25 to 30, followed by lyophilization to prepare a fermented metabolite (see FIG. 1)
제조예 <구아바잎 추출물의 제조>Preparation Example < Preparation of guava leaf extract >
발효를 거치지 않은 구아바잎 분말을 열수추출 방법은 도27과 같이 추출하여 대조군으로서 GW 추출물을 준비하고, 실시예 1에 따라 제조된 발효대사체를 상기 GW 추출물 제조와 동일한 조건의 열수추출방법으로 추출하여 GFW 추출물을 준비하였고, 실시예 1에 따른 발효대사체 에탄올 추출물은 열수추출물에 동량의 에탄올을 가하여 24시간 저온 침지시킨 후 원심분리한 후 동결건조하여 GFE 추출물을 준비하였다.The guava leaf powder not subjected to fermentation was extracted as shown in FIG. 27 to prepare a GW extract as a control. The fermented metabolism prepared according to Example 1 was extracted with a hot water extraction method having the same conditions as the preparation of the GW extract The ethanol extract of the fermented metabolite according to Example 1 was prepared by the same method as that of Example 1 except that ethanol was added to the hot water extract for 24 hours at low temperature, followed by centrifugation and lyophilization to prepare GFE extract.
실험예 1Experimental Example 1
인체 각질 세포에서 여러 가지 사이토카인 중 TNF-a와 IFN-γ에 의해 TARC/CCL17에 의해 생성이 증가하므로 인체 피부 각질 세포인 HaCaT 세포에 TNF-a를 처리하여 아토피 지표인 TARC/CCL17을 유도시킨 후 구아바 잎 열수추출물(GW), 2단발효 구아바 잎 열수 추출물(GFW)및 2단발효 구아바 잎 에탄올 추출물(GFE)을 각각 처리하여 각 추출물의 TARC/CCL17의 생성에 대한 영향을 측정하였다.. TNF-a에 의해 증가된 TARC의 유전자 발현과 단백질 생성량이 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50 ug/ml)처리에 따라 농도 의존적으로 감소하였다(도2 및 도3).Since TARC / CCL17 production is increased by TNF-a and IFN-γ among various cytokines in human keratinocytes, HaCaT cells, which are human keratinocytes, are treated with TNF-a to induce TARC / CCL17 The effect of each extract on the production of TARC / CCL17 was investigated by treating Fuguaba leaf hot water extract (GW), 2 stage fermented Guava leaf hot water extract (GFW) and 2 stage fermented Guava leaf ethanol extract (GFE) . . The expression of TNF-a-induced TARC and protein production were decreased in a concentration-dependent manner by treatment with GW (50-200 ug / ml), GFW (10-50 ug / ml) and GFE (10-50 ug / ml) (Figs. 2 and 3).
실험예 2Experimental Example 2
HacaT 세포에 TNF-a를 처리하여 구아바 열수 추출물 및 2단발효 구아바 잎 열수 및 에탄올 추출물의 RANTES/CCL5 생성량에 대한 영향을 조사한 결과 TNF-a에 의해 증가된 RANTES의 생성량이 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)처리에 따라 농도 의존적으로 감소하였다(도 4). The effect of TNF-a treatment on the production of RANTES / CCL5 by guava hot water extract and 2-stage fermentation guava leaf hydrothermal extract and ethanol extract was investigated. The amount of RANTES produced by TNF-a was increased by GW (50-200 ug / ml), GFW (10-50 ug / ml) and GFE (10-50 ug / ml) treatment (Fig.
실험예 3Experimental Example 3
마우스 대식세포주인 로(raw) 264.7 세포에 염증유발원인 리포폴리사카라이드(lipopolysaccaride, LPS)를 이용하여 구축된 실험법으로, 구아바 열수 추출물 (GW), 2단발효 구아바 잎 열수추출물(GFW)와 에탄올추출물(GFE)을 농도별로 처리하여 유발된 세포독성을 측정한 다음, 세포독성이 없는 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)의 처리농도를 결정하였다(도 5).(GW), 2-stage fermented Guava leaf hot-water extract (GFW) and ethanol (GFW) were prepared by using lipopolysaccaride (LPS) as an inducer of inflammation in mouse macrophage line 264.7 cells. (GFE), GFW (10-50 ug / ml), and GFE (10-50 ug / ml) without cell cytotoxicity. (Figure 5).
또한, 상기 로 264.7 세포를 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)와 LPS를 동시 처리하여 NO (Nitric Oxide) 생성량을 측정한 결과, GW, GFW, GFE의 처리농도에 의존적으로 LPS에 의해 증가된 NO 생성량이 감소하였다(도 6).As a result of measuring the amount of NO (nitric oxide) production by simultaneously treating GW (50-200 ug / ml), GFW (10-50 ug / ml) and GFE (10-50 ug / ml) The amount of NO production increased by LPS was decreased depending on the treatment concentration of GW, GFW and GFE (Fig. 6).
실험예 4Experimental Example 4
NO 생성을 매개하는 유전자인 iNOS의 발현을 조사하기 위해, 로 264.7 세포에 LPS와 GW(50-200 ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)를 6시간 처리하여 cDNA를 합성하여 유전자 발현 정도를 측정한 결과 GW, GFW, GFE의 처리농도에 의존적으로 LPS에 의해 증가된 iNOS의 발현이 감소하였다(도 7).LPS and GW (50-200 ug / ml), GFW (10-50 ug / ml) and GFE (10-50 ug / ml) were added to 264.7 cells in order to investigate the expression of iNOS, As a result of measuring the degree of gene expression by synthesizing cDNA by time-processing, the expression of iNOS increased by LPS was decreased depending on the treatment concentration of GW, GFW and GFE (FIG. 7).
실험예 5Experimental Example 5
염증 억제 효능평가를 위하여 염증 반응에 의해 생성되는 PGE2의 생성량을 측정하였다. 로 264.7 세포에 LPS와 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)를 24시간 처리하여 PGE2 생성량을 측정한 결과, GW, GFW, GFE의 처리농도 의존적으로 LPS에 의해 증가된 PGE2 생성량이 감소하였다(도 8).The amount of PGE 2 produced by the inflammatory reaction was measured to evaluate the anti - inflammatory effect. With LPS and GW (50-200ug / ml) to the 264.7 cells, GFW (10-50ug / ml), GFE (10-50ug / ml) to a 24 hours treatment was measured for PGE 2 production, GW, GFW, GFE The amount of PGE 2 produced increased by LPS in a concentration-dependent manner (FIG. 8).
PGE2의 생성을 매개하며, 염증 반응을 유발하는 유전자인 COX-2의 유전자 및 단백질 발현을 조사하였다. 로 264.7 세포에 LPS와 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)를 2시간(COX-2 유전자 발현) 및 16시간 (COX-2 단백질 발현수준) 처리하여 확인한 결과, GW, GFW, GFE의 처리농도 의존적으로 LPS에 의해 증가된 COX-2의 유전자 및 단백질 발현이 감소하였다(도 9 및 도 10). The gene and protein expression of COX-2, a gene that mediates the production of PGE 2 and induces an inflammatory response, was examined. (COX-2 gene expression) and 16 hours (COX-2 protein expression) in LPS and GW (50-200 ug / ml), GFW (10-50 ug / ml) Expression level). As a result, COX-2 gene expression and protein expression increased by LPS were decreased in dependence of treatment concentration of GW, GFW and GFE (FIGS. 9 and 10).
COX-2 발현에 관여하는 promoter의 활성을 확인하기 위해 COX-2 프로모터를 세포에 형질전환시킨 뒤 LPS와 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)를 처리하여 확인한 결과, GW, GFW, GFE의 처리농도 의존적으로 LPS에 의해 증가된 COX-2의 프로모터 활성이 감소하였다(도 11). In order to confirm the activity of promoter involved in COX-2 expression, COX-2 promoter was transformed into LPS and GW (50-200 ug / ml), GFW (10-50 ug / ml), GFE / ml), the promoter activity of COX-2 increased by LPS was decreased depending on the treatment concentration of GW, GFW and GFE (FIG. 11).
실험예 6Experimental Example 6
COX-2와 iNOS 외에 염증 반응을 매개하는 사이토카인들의 생성량 및 유전자 발현에 대한 구아바 잎 열수(GW), 2단발효 구아바 잎 열수(GFW)와 에탄올(GFE) 추출물 영향을 조사하였다.In addition to COX-2 and iNOS, the effects of guava leaf hydrolysis (GW), two-stage fermentation Guava leaf hydrolysis (GFW) and ethanol (GFE) extracts on the production and gene expression of cytokines mediating inflammatory responses were examined.
LPS와 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)를 시간별로 처리하여 대표적인 염증성 사이토카인으로 TNF-a, IL-6, IL-1?에 대한 영향을 측정하였다. GW, GFW, GFE의 처리농도 의존적으로 LPS에 의해 증가된 TNF-a, IL-6, IL-1β 유전자 발현이 일괄적으로 감소하였다(도 12, 도 13, 도 14). IL-6, IL-1, and IL-6 as typical inflammatory cytokines were treated with LPS and GW (50-200 ug / ml), GFW (10-50 ug / ml) and GFE Were measured. TNF-a, IL-6, and IL-1β gene expression increased by LPS in a dose-dependent manner in the treatment concentration of GW, GFW, and GFE was collectively decreased (FIGS. 12, 13, and 14).
이들 염증성 사이토카인의 유전자 발현에 대한 영향을 측정한 결과 GFE에서 특이적으로 더욱 효과가 높은 것을 확인하였다. The effect of these inflammatory cytokines on the gene expression was evaluated and it was confirmed that GFE was more specifically effective.
또한, GW, GFW, GFE의 처리농도 의존적으로 LPS에 의해 증가된 TNF-a, IL-6, IL-1β 단백질 생성량이 일괄적으로 감소하였다(도15, 도16 및 도 17). 이들 염증성 사이토카인 단백질 생성량에 대한 영향을 측정한 결과 GFE에서 특이적으로 더욱 효과가 높은 것을 확인하였다. In addition, the amounts of TNF-a, IL-6, and IL-1β protein increased by LPS were collectively decreased depending on the treatment concentration of GW, GFW, and GFE (FIGS. 15, 16, and 17). As a result of measuring the effect on the production of inflammatory cytokine protein, it was confirmed that GFE was more effective specifically.
실험예 7Experimental Example 7
구아바 잎 열수추출물(GW), 2단발효 구아바 잎 열수(GFW)와 에탄올(GFE)추출물의 COX-2 발현 조절은 대표적으로 NF-κB 전사조절인자에 의해 영향을 받으므로 이들 추출물의 NF-κB 전사활성에 대한 영향을 조사하였다. NF-κB luciferase vector를 세포에 형질전환시킨 뒤 LPS와 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)를 처리하여 NF-κB 전사활성을 확인한 결과 GW, GFW, GFE의 처리농도 의존적으로 LPS에 의해 증가된 NF-κB 전사활성이 감소하였다(도 18 및 도 19).The expression of COX-2 in the extracts of guava leaves (GW), 2-stage fermented guava leaves (GFW) and ethanol (GFE) is typically influenced by NF-κB transcription factors. Therefore, NF-κB The effect on transcriptional activity was investigated. NF-κB luciferase vector was transformed into LPS and GW (50-200 μg / ml), GFW (10-50 μg / ml) and GFE (10-50 μg / ml) Results The NF-κB transcriptional activity increased by LPS was decreased in a concentration-dependent manner in the treatment concentration of GW, GFW, and GFE (FIGS. 18 and 19).
실험예 8Experimental Example 8
비만세포주인 RBL-2H3 세포에 알러지 반응의 지표인 탈과립 (degranulation) 방출량과 히스타민 생성량을 측정하기 위하여 DNP-IgE로 세포를 감작시키고 알러젠인 DNP-BSA 처리하는 실험법을 구축하였다. 구아바 열수 추출물(GW), 2단발효 구아바 잎 열수(GFW)와 에탄올(GFE)추출물을 RBL-2H3 세포에 농도별로 처리하여 세포독성을 측정한 뒤 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)의 처리농도를 결정하였다(도 20). In order to measure the degree of degranulation and histamine production, which is an index of allergic reaction, RBL-2H3 cells were sensitized with DNP-IgE and tested for allergic DNP-BSA treatment. GW (50-200 μg / ml), GFW (10 μg / ml), and GFW (10 μg / ml) were prepared by measuring the cytotoxicity of Guava hot water extract (GW), two step fermented Guava leaf water (GFW) and ethanol -50 ug / ml), and the treatment concentration of GFE (10-50 ug / ml) (Fig. 20).
구축된 실험법을 이용하여 비만세포에서의 탈과립 방출량 및 히스타민 생성량 변화를 조사한 결과, GW, GFW, GFE의 처리농도에 의존적으로 DNP-BSA에 의해 증가된 탈과립 방출량(도 21)과 히스타민 생성량 (도 22)이 감소하였다. As a result of examining the changes in the amount of degranulation and histamine production in mast cells using the established experimental method, it was found that the amount of degranulation (Figure 21) and histamine production (Figure 22) increased by DNP-BSA depending on the treatment concentration of GW, GFW, ) Decreased.
실험예 9Experimental Example 9
구아바 잎 열수추출물(GW), 2단발효 구아바 잎 열수(GFW)와 에탄올(GFE)추출물의 알러지 반응을 매개하는 사이토카인(cytokine)인 IL-4 및 에오시노필(eosinophil)의 증식 및 분화를 촉진시켜 에오시노필리아(eosinophilia)를 초래하는 사이토카인인 IL-5에 대한 영향을 조사하였다. 비만세포에 DNP-IgE로 세포를 감작시키고 GW(50-200ug/ml), GFW(10-50ug/ml), GFE (10-50ug/ml)와 알러젠인 DNP-BSA를 처리하여 IL-4의 유전자 발현, 단백질 생성량 및 프로모터 활성에 대한 영향을 측정하였다. DNP-BSA에 의해 증가된 IL-4의 유전자 발현, 단백질 생성량 및 프로모터 활성이 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml) 농도 의존적으로 감소하였으며, GFE의 효과가 가장 강하였다(도 23, 도 24, 도 25). The proliferation and differentiation of IL-4 and eosinophil, which are cytokines mediating the allergic reaction of guava leaf hot water extract (GW), two stage fermented Guava leaf hydrothermal (GFW) and ethanol (GFE) 5 was investigated for its effect on IL-5, a cytokine that results in eosinophilia. Mast cells were sensitized with DNP-IgE and treated with GW (50-200 ug / ml), GFW (10-50 ug / ml), GFE (10-50 ug / ml) and allergen DNP- The effects on gene expression, protein production and promoter activity were measured. The amount of IL-4 gene expression, protein production, and promoter activity increased by DNP-BSA decreased in a dose-dependent manner in GW (50-200 ug / ml), GFW (10-50 ug / ml) and GFE (10-50 ug / ml) , And the effect of GFE was the strongest (Figs. 23, 24, 25).
그리고 비만세포에 DNP-IgE로 세포를 감작시키고 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml)와 알러젠인 DNP-BSA를 처리하여 IL-5의 유전자 발현에 대한 영향을 측정하였다. DNP-BSA에 의해 증가된 IL-5의 유전자 발현이 GW(50-200ug/ml), GFW(10-50ug/ml), GFE(10-50ug/ml) 농도 의존적으로 감소하였으며, GFE의 효과가 가장 강하게 나타났다(도 26). Cells were sensitized with DNP-IgE to mast cells and treated with GW (50-200 ug / ml), GFW (10-50 ug / ml), GFE (10-50 ug / ml) and allergen DNP- Were examined for their effect on gene expression. The effect of GFE (50-200 μg / ml), GFW (10-50 μg / ml), and GFE (10-50 μg / ml) was decreased by DNP-BSA-induced IL-5 gene expression (Fig. 26).
본 발명에 따르면 아토피 피부염 증상에 대해 억제 및 개선 효능을 갖는 천연성분 조성물을 제공함으로써 아토피 피부염 관련 건강식품분야 및 의약분야 산업에 획기적인 기여를 할 것으로 예상된다.It is expected that the present invention will make a significant contribution to atopic dermatitis related health food industry and medicine industry industry by providing a natural ingredient composition having an inhibitory and improving effect against atopic dermatitis symptoms.
GW: 구아바잎의 열수 추출물
GFW: 2단발효 구아바잎 발효대사체의 열수 추출물
GFE: 2단발효 구아바잎 발효대사체의 에탄올 추출물GW: Hot water extract of Guava leaf
GFW: Hot Water Extract of Fermented Metabolites from Two-Stage Fermented Guava Leaves
GFE: Ethanol Extract of Fermented Metabolite of Guava Leaf from Two-Stage Fermented Guava
Claims (9)
상기 발효기질에 곰팡이 생육용 영양성분 혼합물을 구아바분말 발효기질 중량에 대해 5중량% 첨가 혼합하고 121℃ 및 1.5기압 조건에서 15분간 멸균하여 1단발효배지를 준비하는 단계;
상기 1단 발효배지 준비단계와 동시 또는 전후에 곰팡이를 PDB 배지에서 종배양하는 곰팡이 배양액 준비단계;
상기 1단 발효배지에 상기 곰팡이 배양액을 10%를 접종하고 수분함량을 40-60%로 조절한 후, 상대습도 80%로 유지하고 30℃ 온도에서 5일간 정치하여 배양하는 고체발효 단계;
상기 고체발효 단계에서 얻어진 구아바잎 1단 고체발효물에 가수, 환류추출, 여과, 및 감압농축을 거쳐 15-25브릭스 농도의 구아바잎 고체발효액 추출물을 얻는 단계;
상기 구아바잎 고체발효액 추출물에 탄소원으로서 설탕 및 질소원으로서 효모추출액을 첨가하여 2단 발효배지를 준비하는 단계;
상기 2단 발효배지 준비단계와 동시 또는 전후에 유산균혼합균주를 MRS 배지에서 종배양하는 유산균혼합균주 배양액 준비단계;
상기 2단 발효배지에 상기 유산균혼합균주 배양액을 10% 접종한 다음 37℃ 온도에서 5일간 정치하여 배양하는 액체발효단계;
상기 액체발효 단계에서 얻어진 구아바잎 발효물을 살균, 여과 및 감압농축을 거쳐 25-30 브릭스 농도의 구아바잎 발효대사체를 얻는 단계;
를 포함하는 2단 발효 구아바잎 발효대사체 제조방법.
Preparing a fermented substrate by washing the guava leaf and drying it to prepare a powder having a water content of 10% or less and pulverizing it to a length of 2-3 cm and pulverizing it;
Preparing a first stage fermentation medium by adding 5% by weight of a nutrient mixture for growth of fungi to the fermentation substrate and adding to the weight of the guava powder fermentation substrate and sterilizing the mixture at 121 ° C and 1.5 atm for 15 minutes;
Preparing a fungal culture liquid for culturing the fungus in the PDB medium simultaneously or after the preparation of the first stage fermentation medium;
A solid fermentation step in which 10% of the above-mentioned fungal culture liquid is inoculated into the 1-stage fermentation medium, the moisture content is adjusted to 40 to 60%, the relative humidity is maintained at 80%, and the mixture is allowed to stand at 30 DEG C for 5 days;
Obtaining a guava leaf solid fermentation broth having a concentration of 15-25 Bricks by hydrolysis, reflux extraction, filtration, and concentration under reduced pressure in a first step fermentation product of guava leaf obtained in the solid fermentation step;
Preparing a two-stage fermentation medium by adding sugar as a carbon source and yeast extract as a nitrogen source to the Guava leaf solid fermentation broth extract;
Preparing a culture solution of a mixed culture of a lactic acid bacterium in which a mixed culture of a lactic acid bacterium is sown in a MRS medium simultaneously with or before and after the two-stage fermentation medium preparation step;
A liquid fermentation step of incubating the two-stage fermentation broth with 10% of the culture solution of the mixed lactic acid bacteria and incubating at 37 DEG C for 5 days;
Obtaining a guava leaf fermented metabolite having a concentration of 25-30 Bricks by sterilizing the guava leaf fermentation product obtained in the liquid fermentation step, filtering and concentrating the fermented product under reduced pressure;
/ RTI > of fermenting a guava leaf fermented by a fermentation process.
The method according to claim 1, wherein the nutrient mixture for fungal growth comprises 35.25% by weight of starch, 35.44% by weight of dietary fiber, 4.96% by weight of beta -glucan, 2.25% by weight of polyphenol, 16.64% by weight of starch decomposition enzyme and crude protein in trace amounts, 2.78% by weight and 2.68% by weight per glass-body, and the fungus is Aspergillus oryzae (A. oryzae).
The method according to claim 1, wherein the fungus is cultured in a PDB medium at 30 DEG C for 5 days with shaking to prepare a culture medium for fungi.
2. The method of claim 1, wherein water of 10 times the weight of the first fermented product of guava leaves is subjected to reflux extraction at 95 DEG C for 30 minutes, followed by filtration and concentration under reduced pressure to obtain a Guava leaf solid fermentation broth having a concentration of 15-25 Bricks, A method for producing a fermented metabolite of a two - stage fermented Guava leaf.
The method for producing a two-stage fermented Guava leaf fermented metabolism according to claim 1, wherein the two-stage fermentation medium is prepared by adding 5% by weight of sugar as a carbon source and 0.5% by weight of a yeast extract as a nitrogen source.
The method according to claim 1, wherein the lactic acid bacteria mixed culture is a lactic acid bacteria mixed strain consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus lambatus, Lactobacillus casei, and Bifidobacterium long sword, In the presence of a culture medium for 24 hours.
The method according to claim 1, wherein the guava leaf fermented product obtained in the liquid fermentation step is sterilized at 100 ° C for 10 minutes, and then subjected to filtration and concentration under reduced pressure to treat at a concentration of 25-30 bricks.
[Claim 9] The method according to claim 8, wherein the lactic acid bacteria mixed strain for two-stage liquid fermentation is a lactic acid bacterial mixed strain consisting of Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus lambatus, Lactobacillus casei and Bifidobacterium longbum, A composition for improving the symptoms of atopic dermatitis comprising guava leaf fermented metabolite extract as an active ingredient.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151067A (en) * | 2021-03-31 | 2021-07-23 | 绽妍生物科技有限公司 | Extraction and separation process of lactobacillus fermentation extract |
| KR102303400B1 (en) * | 2021-04-06 | 2021-09-24 | (주)브이에스신비 | Preparation Methods of Fermentation Products Using JEJU Lava Seawater, JEJU Barley Yeast and Natural Plant and Cosmetic Compositions Having Thereof |
| CN116019747A (en) * | 2023-02-01 | 2023-04-28 | 广州优科生物科技有限公司 | Preparation method of guava fermentation product, product and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113151067A (en) * | 2021-03-31 | 2021-07-23 | 绽妍生物科技有限公司 | Extraction and separation process of lactobacillus fermentation extract |
| KR102303400B1 (en) * | 2021-04-06 | 2021-09-24 | (주)브이에스신비 | Preparation Methods of Fermentation Products Using JEJU Lava Seawater, JEJU Barley Yeast and Natural Plant and Cosmetic Compositions Having Thereof |
| CN116019747A (en) * | 2023-02-01 | 2023-04-28 | 广州优科生物科技有限公司 | Preparation method of guava fermentation product, product and application thereof |
| CN116019747B (en) * | 2023-02-01 | 2023-11-14 | 广州优科生物科技有限公司 | Preparation method of guava fermentation product, product and application thereof |
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