KR102366763B1 - Food composition for improvement of respiratory health - Google Patents
Food composition for improvement of respiratory health Download PDFInfo
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- KR102366763B1 KR102366763B1 KR1020210052469A KR20210052469A KR102366763B1 KR 102366763 B1 KR102366763 B1 KR 102366763B1 KR 1020210052469 A KR1020210052469 A KR 1020210052469A KR 20210052469 A KR20210052469 A KR 20210052469A KR 102366763 B1 KR102366763 B1 KR 102366763B1
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- food composition
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- food
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Abstract
Description
본 발명은 식물 추출물을 활용한 호흡기 건강 개선용 식품 조성물에 관한 것이다.The present invention relates to a food composition for improving respiratory health using a plant extract.
호흡기 질환은 미세먼지, 진드기, 꽃가루, 황사 등 유해 환경에 노출된 현대인에게 가장 흔한 질병 중 하나이다. 건강보험공단 자료에 따르면, 주요 환경성 호흡기 질환인 알레르기비염 환자와 천식 환자수는 2017년 기준 인구 1만 명당 각각 1,353명 및 288명으로 집계되었으며, 특히 알레르기성 비염 환자수는 매년 급증세를 보이고 있다. 최근들어 미세먼지로 인한 대기의 질이 급격히 악화되면서, 미세먼지에 의해 유발된 호흡기 질환이 많은 주목을 받고 있다.Respiratory diseases are one of the most common diseases among modern people exposed to harmful environments such as fine dust, mites, pollen, and yellow dust. According to data from the National Health Insurance Corporation, the number of patients with allergic rhinitis and asthma, a major environmental respiratory disease, was counted as 1,353 and 288 people per 10,000 population, respectively, as of 2017, and the number of patients with allergic rhinitis is increasing rapidly every year. Recently, as the air quality has rapidly deteriorated due to fine dust, respiratory diseases induced by fine dust are receiving a lot of attention.
이와 관련하여 세계보건기구(WHO)는 미세먼지(PM10, PM2.5)에 대한 대기질 가이드라인을 1987년부터 제시해 왔고, 2013년 세계보건기구 산하의 국제암연구소(IARC)에서 미세먼지를 사람에게 발암이 확인된 1군 발암물질(Group 1)로 지정하였다. 1군(Group 1) 발암물질은 인간에서 발암성이 있는 것으로 확인된 물질로, 석면, 벤젠, 미세먼지 등을 들 수 있다.In this regard, the World Health Organization (WHO) has been presenting air quality guidelines for fine dust (PM10, PM2.5) since 1987, and in 2013, the International Agency for Research on Cancer (IARC) under the World Health Organization (WHO) It was designated as a
한편, 삼백초(Saururus chinensis)는 한국, 중국, 일본 등지의 동북아시아에 자생하는 다년생 초본으로, 삼백초의 꽃을 포함한 잎과 줄기 뿌리가 전통의학에서 약재로 사용된 바 있고, 삼백초의 알코올 추출물에서 기관지 천식 등의 호흡기 질환 효과가 있음이 알려져있다(대한민국 공개특허 제10-2006-0111790호). 그러나, 상기 문헌은 삼백초 추출물과 미세먼지 등 유해환경으로 인해 유발된 호흡기 질환의 상관관계를 언급하고 있지는 않다.On the other hand, Saururus chinensis is a perennial herb that is native to Northeast Asia, such as Korea, China, and Japan. It is known that there is an effect on respiratory diseases such as asthma (Korean Patent Publication No. 10-2006-0111790). However, the above document does not mention the correlation between the extract of ginseng plant and respiratory diseases induced by harmful environments such as fine dust.
또한, 기존에 호흡기 질환을 대상으로 하는 삼백초 추출물은 유효성분으로 여겨진 사우세르네올 B를 분리하기 위해 메탄올 또는 에탄올을 추출용매로 사용한 것들인데(대한민국 등록특허 제10-0739399호 등), 이때 인체 독성(마나산틴 A의 LD50=5.4 mg/kg)을 나타내는 마나산틴(manassantin) A 및 마나산틴 B가 필연적으로 함유되는바, 호흡기 개선을 위한 상업적 사용을 위해서는 위 독성 물질을 제거하기 위한 헥산 분획 공정이 추가적으로 요구된다(대한민국 공개특허 제10-2013-0142638호). 그러나 헥산은 호흡기계 자극이나 장기 손상, 피부 및 눈 자극 등의 위험이 있고, 신경계 손상을 유발하는 위험물질에 해당하므로, 결과적으로 기존의 삼백초 유기용매 추출 또는 분획을 통해 식품으로 활용하기 위해서는 안전성 이슈가 여전히 제기되는 실정이다.In addition, the existing extracts of ginseng extract for respiratory diseases are those using methanol or ethanol as an extraction solvent to separate saucerneol B, which is considered an active ingredient (Korea Patent No. 10-0739399, etc.), at this time, toxic to the human body. (LD50 of manasanthin A = 5.4 mg/kg) Manassantin A and manasanthin B, which are inevitably contained, for commercial use for respiratory improvement, a hexane fractionation process to remove gastric toxic substances It is additionally required (Korean Patent Publication No. 10-2013-0142638). However, hexane has a risk of respiratory system irritation, organ damage, skin and eye irritation, etc., and is a dangerous substance that causes damage to the nervous system. is still being raised.
본 발명의 목적은 유해환경으로 인한 호흡기 개선 효과를 갖는 삼백초 유래 기능성 식품 조성물을 제공하는 것이다.It is an object of the present invention to provide a functional food composition derived from ginseng plant having a respiratory improvement effect due to a harmful environment.
본 발명자는 유해환경으로 인한 호흡기 질환을 개선할 수 있고 안전성이 보다 확보된 식물 추출물 활용 호흡기 건강 개선용 식품을 제공하기 위해 예의 노력한 결과, 종래 삼백초 알코올 추출물 내 사우세르네올을 대신한 신규 유효성분 조합을 찾아내고 이를 조성물에 일정 함량으로 함유하도록 조절함으로써, 본 발명에 따른 식품 조성물을 완성하였다. As a result of the present inventor's earnest efforts to provide food for improving respiratory health using plant extracts that can improve respiratory diseases caused by harmful environments and have more secured safety, a novel active ingredient combination instead of saucerneol in the conventional trifolium alcohol extract By finding and adjusting it to contain it in a certain amount in the composition, the food composition according to the present invention was completed.
구체적으로 본 발명은, 삼백초 잎을 유기용매가 포함되지 않은 물로 80 ℃ 이상에서 2 시간 이상 추출하여 수득한 추출물을 포함하되, 상기 조성물은 미??리아닌, 루틴 및 퀘르시트린의 조합을 조성물 총 중량 기준 3~30 mg/g로 함유하고, 상기 조합의 총 중량 기준으로 루틴을 3.0~10.0 중량%으로, 퀘르시트린을 11.0~20.0 중량%로 함유하는 것을 특징으로 하는 호흡기 질환 개선용 식품 조성물을 제공한다.Specifically, the present invention includes an extract obtained by extracting the leaves of trifolieae at 80° C. or higher with water not containing an organic solvent for 2 hours or more, wherein the composition contains a combination of myryanine, rutin, and quercitrin. A food composition for improving respiratory diseases, characterized in that it contains 3 to 30 mg/g by weight, rutin in 3.0 to 10.0% by weight, and quercitrin in 11.0 to 20.0% by weight, based on the total weight of the combination to provide.
상기 유효성분에 포함된 미??리아닌(miquelianin)은 하기 화학식 1로 표시된다:Miquelianin included in the active ingredient is represented by the following Chemical Formula 1:
[화학식 1][Formula 1]
상기 유효성분에 포함된 루틴(rutin)은 하기 화학식 2로 표시된다:Rutin contained in the active ingredient is represented by the following formula 2:
[화학식 2][Formula 2]
상기 유효성분에 포함된 퀘르시트린(quercitrin)은 하기 화학식 3으로 표시된다:Quercitrin included in the active ingredient is represented by the following Chemical Formula 3:
[화학식 3][Formula 3]
상기 유효성분에 포함된 퀘르세틴(quercetin)은 하기 화학식 4로 표시된다:Quercetin contained in the active ingredient is represented by the following Chemical Formula 4:
[화학식 4][Formula 4]
본 발명에서, 삼백초(Saururus chinensis Baill)는 삼백초과(Saururaceae)에 속하는 여러해살이풀로, 자연에서 자생하는 것, 구입하거나 재배한 것을 모두 제한 없이 사용할 수 있으며, 이의 전초, 지상부, 잎, 뿌리, 줄기, 꽃, 종자 등을 모든 부위를 제한 없이 포함하나 바람직하게는 이의 전초, 지상부, 잎 또는 꽃을 사용한다.In the present invention, three hundred seconds ( Saururus chinensis Baill ) is a perennial plant belonging to the family Saururaceae, and those that grow in nature, those that are purchased or cultivated can all be used without limitation, and its outposts, above-ground parts, leaves, roots, stems , flowers, seeds, etc. all parts are included without limitation, but the outpost, above-ground part, leaf or flower thereof is preferably used.
본 발명에서 "추출물"이란 삼백초로부터 분리하여 얻은 물질을 의미하며, 구체적으로 물 추출물, 조추출물 또는 이의 농축물일 수 있다. 바람직하게는, 추출 용매는 메탄올 또는 에탄올을 포함하지 않으며, 바람직하게는 알코올을 포함하지 않는다. 가장 바람직하게는, 추출 용매는 알코올 외 헥산, 아세트산에틸, 디클로로메탄, 에테르, 클로로포름, 아세톤 또는 이들의 조합과 같은 일체의 유기용매를 포함하지 않는다. 일 실시양태에서, 본 발명의 삼백초 물 추출물은 유기용매 추출 단계를 거치지 않는 것을 특징으로 한다.In the present invention, the term "extract" means a material obtained by separating from ginseng plant, and specifically, it may be a water extract, a crude extract, or a concentrate thereof. Preferably, the extraction solvent does not contain methanol or ethanol, and preferably does not contain alcohol. Most preferably, the extraction solvent does not contain any organic solvent other than alcohol, such as hexane, ethyl acetate, dichloromethane, ether, chloroform, acetone, or a combination thereof. In one embodiment, the water extract of Sambaekcho of the present invention is characterized in that it does not go through the organic solvent extraction step.
본 발명의 추출물은 물 추출 용매에 의한 추출물뿐만 아니라 이후 통상적인 정제과정을 거친 추출물도 제한없이 포함될 수 있다. 예를 들어, 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등, 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획도 본 발명의 추출물에 포함된다. 또한 본 발명의 추출물은 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가적인 과정에 의해 분말 상태로도 제조될 수 있다.The extract of the present invention may include, without limitation, not only the extract by the water extraction solvent, but also the extract that has undergone a conventional purification process thereafter. For example, separation using an ultrafiltration membrane having a constant molecular weight cut-off value, separation by various chromatography (prepared for separation according to size, charge, hydrophobicity, or affinity), etc. The fraction obtained through this is also included in the extract of the present invention. In addition, the extract of the present invention may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze-drying or spray-drying.
본 발명의 삼백초 물 추출물은 다음과 같은 공정을 통해 제조될 수 있으나, 이에 한정되지 않는다. The water extract of Sambaekcho of the present invention may be prepared through the following process, but is not limited thereto.
1) 삼백초 지상부를 음건하고 분쇄하는 단계; 1) Drying and grinding the above-ground part of 300 seconds;
2) 분쇄된 삼백초를 추출하여 추출물을 얻는 단계; 2) extracting the pulverized baekchoi to obtain an extract;
3) 추출물을 여과한 후 감압 농축하는 단계. 3) After filtering the extract, the step of concentration under reduced pressure.
상기 1) 단계에 사용되는 삼백초는 자연에서 자생하는 것, 직접 재배한 것 또는 시판되는 것을 제한 없이 사용할 수 있다. As for the three hundred plants used in step 1), those that are naturally grown, those that are directly grown, or those that are commercially available can be used without limitation.
상기 2) 단계의 추출은 침지(냉침, 온침) 추출, 열수 추출, 초음파 추출, 초임계 추출 또는 환류 냉각 추출법을 제한없이 사용할 수 있으나, 침지 추출, 초음파 추출, 환류 추출법을 사용하는 것이 바람직하다. 상기 추출의 추출온도는 60 내지 100℃, 바람직하게는 80 내지 100℃일 수 있고, 추출 시간은 1시간 내지 10시간, 바람직하게는 2시간 내지 6시간일 수 있다. 또한, 본 발명의 추출물은 추출 효율에 따라 상기 추출 횟수를 한정하지 않으나, 추출 횟수는 1 내지 5, 바람직하게는 2 또는 3회일 수 있다.For the extraction in step 2), immersion (cold-chim, warm-chim) extraction, hot water extraction, ultrasonic extraction, supercritical extraction, or reflux cooling extraction method can be used without limitation, but it is preferable to use immersion extraction, ultrasonic extraction, or reflux extraction method. The extraction temperature of the extraction may be 60 to 100 ℃, preferably 80 to 100 ℃, the extraction time may be 1 hour to 10 hours, preferably 2 hours to 6 hours. In addition, the extract of the present invention does not limit the number of extractions according to extraction efficiency, but the number of extractions may be 1 to 5, preferably 2 or 3 times.
삼백초 추출물은 재배 지역, 생육 조건, 산지, 채취시기 등의 다양성으로 인해 동일 추출 공정을 적용하더라도 추출물 내 성분의 함량에 편차가 발생한다. 본 발명의 조성물 제공을 위해, 추출물에 성분 분석을 실시하여 설정된 함량 기준을 충족하는 조성물을 선택하여 본 발명의 조성물을 제조할 수 있다. 설정된 함량에 미달될 경우 해당 조성물을 폐기할 수도 있고, 유효성분을 추가하여 대안적인 본 발명의 조성물을 제조할 수 있다.Even if the same extraction process is applied due to the diversity of cultivation regions, growth conditions, production areas, and harvesting period, the content of ingredients in the extract varies. In order to provide the composition of the present invention, the composition of the present invention can be prepared by performing component analysis on the extract to select a composition that meets the set content standards. If the content does not reach the set content, the composition may be discarded, or an alternative composition of the present invention may be prepared by adding an active ingredient.
일 실시양태에서, 본 발명의 식품 조성물은 In one embodiment, the food composition of the present invention comprises
(i) 삼백초 전초(또는 잎)을 유기용매가 포함되지 않은 물로 80 ℃ 이상에서 2 시간 이상 추출하는 단계;(i) extracting three hundred stalks (or leaves) with water that does not contain an organic solvent at 80 ° C. or higher for 2 hours or more;
(ii) 삼백초 물 추출물을 수득하는 단계;(ii) obtaining a water extract of Trifolium Root;
(iii) 상기 삼백초 물 추출물에서 HPLC를 수행하여 미??리아닌, 루틴, 퀘르시트린 및 퀘르세틴의 함량을 측정하는 단계;(iii) measuring the contents of myrcyanine, rutin, quercitrine and quercetin by performing HPLC on the water extract of Trifolium Root;
(iv) 본 발명의 유기성분 함량 구간에 포함되는 조성물을 선별하는 단계(iv) selecting a composition included in the organic component content section of the present invention
를 통해 수득할 수 있다.can be obtained through
대안적 실시양태에서, 상술한 단계 (iv) 대신, 미??리아닌, 루틴, 퀘르시트린 및 퀘르세틴을 화학양론에 따라 추가함으로써 본 발명의 식품 조성물을 수득한다.In an alternative embodiment, instead of step (iv) described above, the food composition of the present invention is obtained by stoichiometrically adding myrcyanine, rutin, quercitrin and quercetin.
일 실시양태에서, 본 발명의 식품 조성물은 유효성분으로서 미??리아닌, 루틴 및 퀘르시트린의 조합을 조성물 총 중량 기준으로 3 mg/g 이상, 구체적으로 5 mg/g 이상, 더욱 구체적으로 10 mg/g 이상 포함한다. 또한, 상기 식품 조성물은 유효성분으로 퀘르세틴을 더 포함할 수 있다.In one embodiment, the food composition of the present invention contains a combination of myrcyanine, rutin, and quercitrin as an active ingredient at 3 mg/g or more, specifically 5 mg/g or more, more specifically 10 mg/g or more. In addition, the food composition may further include quercetin as an active ingredient.
상기 실시양태에서, 본 발명의 식품 조성물은 유효성분 총 중량 기준으로 하기 유효성분을 포함할 수 있다:In the above embodiment, the food composition of the present invention may include the following active ingredients based on the total weight of the active ingredients:
(i) 70~85 중량%의 미??리아닌,(i) 70-85% by weight of microcyanine;
(ii) 3 내지 10 중량%의 루틴; 및(ii) 3 to 10% by weight of rutin; and
(iii) 11 내지 20 중량%의 퀘르시트린(iii) 11 to 20% by weight of quercitrin
(iv) 선택적으로 퀘르세틴(iv) optionally quercetin
일 실시양태에서, 본 발명의 식품 조성물은 총 중량 기준 마나산틴 A 및 마나산틴 B의 함량이 0~0.2 mg/g인 것을 특징으로 한다.In one embodiment, the food composition of the present invention is characterized in that the content of manaxanthin A and manaxanthin B based on the total weight is 0-0.2 mg/g.
일 실시양태에서, 본 발명의 식품 조성물은 미??리아닌, 루틴 및 퀘르시트린의 조합을 조성물 총 중량 기준 15~30 mg/g로 함유한다. 이 경우, 예컨대 본 발명의 식품 조성물은 조성물 총 중량 기준으로 하기 성분을 유효성분으로 포함할 수 있다:In one embodiment, the food composition of the present invention contains a combination of mycyanine, rutin, and quercitrin at 15-30 mg/g based on the total weight of the composition. In this case, for example, the food composition of the present invention may include the following ingredients as active ingredients based on the total weight of the composition:
(i) 11.75±0.092 mg/g의 미??리아닌(i) 11.75±0.092 mg/g microcyanine
(ii) 1.20±0.008 mg/g의 루틴(ii) 1.20±0.008 mg/g of rutin
(iii) 2.38±0.389 mg/g의 퀘르시트린; 및(iii) quercitrin at 2.38±0.389 mg/g; and
(iv) 0.068±0.017 mg/g의 퀘르세틴.(iv) 0.068±0.017 mg/g of quercetin.
본 발명의 식품 조성물은 호흡기 질환 개선 효과를 갖는 것을 특징으로 한다. 일 실시양태에서, 상기 호흡기 질환은 알레르기성 폐질환, 구체적으로 알레르기성 천식 또는 알레르기성 비염이다.The food composition of the present invention is characterized in that it has an effect of improving respiratory diseases. In one embodiment, the respiratory disease is allergic lung disease, specifically allergic asthma or allergic rhinitis.
일 실시양태에서, 상기 호흡기 질환은 미세먼지로 인해 유발된 것을 특징으로 한다.In one embodiment, the respiratory disease is characterized in that it is caused by fine dust.
미세먼지는 평균 입자 크기가 0.1 μm 이하, 0.1-2.5 μm, 2.5-10 μm인 미세먼지(분진)으로 분류할 수 있다. 입자의 크기가 10 μm 이상인 경우에는 코나 후인두에서 걸러지지만 입자의 크기가 이보다 작은 경우 폐 안쪽으로 미세먼지가 깊숙이 흡입된다. 이때 폐 내 호산구, 호중구 및 대식세포 등이 활성화되고 IL-13 등 염증성 사이토카인이 분비되어 기도와 폐포에 염증을 일으키며 기도 상피세포에 미세먼지를 노출하게 되고 섬모 운동에 의해서 미세먼지가 제거된다.Fine dust can be classified into fine dust (dust) with an average particle size of less than 0.1 μm, 0.1-2.5 μm, and 2.5-10 μm. If the particle size is 10 μm or more, it is filtered by the nose or larynx, but if the particle size is smaller than this, the fine dust is deeply sucked into the lungs. At this time, eosinophils, neutrophils, and macrophages in the lungs are activated, and inflammatory cytokines such as IL-13 are secreted, causing inflammation in the airways and alveoli, exposing fine dust to airway epithelial cells, and removing fine dust by ciliary movement.
미세먼지가 폐 조직 내에 노출되면 산화스트레스로 인한 방어기전, 세포활성화, 세포구조와 관련 단백질 발현이 증가하고, 기도 염증과 염증성 사이토카인 발현을 증가시켜 호흡기 질환을 악화시키게 된다. 특히 미세먼지는 호중구, 호산구, 대식세포 등 염증세포를 자극하여 TNF-α, TGF-β1, GM-CSF, PDGF, IL-6, IL-8, IL-4, IL-13 등 염증성 사이토카인의 분비를 증가시키고, 급성 및 만성 폐 염증을 유발한다. 특히, 미세먼지와 알레르기 유발물질(알러겐)을 동시에 투여하면 T세포 매개 염증이 더 심화된다.When fine dust is exposed in the lung tissue, the defense mechanism, cell activation, and cell structure and related protein expression due to oxidative stress increase, and airway inflammation and inflammatory cytokine expression increase, thereby exacerbating respiratory diseases. In particular, fine dust stimulates inflammatory cells such as neutrophils, eosinophils, and macrophages to reduce inflammatory cytokines such as TNF-α, TGF-β1, GM-CSF, PDGF, IL-6, IL-8, IL-4, and IL-13. It increases secretion and causes acute and chronic lung inflammation. In particular, when fine dust and an allergen (allergen) are administered at the same time, T-cell-mediated inflammation intensifies.
미세먼지로부터 유도된 호흡기 질환 개선용 식품을 개발하기 위해 본 발명자들은 미세먼지에 의해 유도된 질병 유발 기전과 유사한 증상이 유발되는 미세먼지 유도 동물모델을 제조하고자, 미세먼지와 오발부민을 기도 내로 흡입시켜 미세먼지 질환 마우스를 제작하였다.In order to develop a food for improving respiratory diseases induced by fine dust, the present inventors inhaled fine dust and ovalbumin into the airways to produce a fine dust-induced animal model that induces symptoms similar to the disease-inducing mechanism induced by fine dust. to produce mice with fine dust disease.
그 결과, 본 발명의 조성물 투여로 인해 미세먼지 질환 마우스에서 나타나는 폐 조직 내 염증 세포 증가, 염증성 사이토카인 증가 및 혈청 내 IgE 레벨 증가 현상이 효과적으로 억제되고, 실제 폐조직의 유익한 조직학적 변화를 이끌어낼 수 있음을 확인하였다(도 4 내지 도 11).As a result, the administration of the composition of the present invention effectively inhibits the increase in inflammatory cells in the lung tissue, the increase in inflammatory cytokines, and the increase in the serum IgE level, which appear in mice with fine dust disease, and leads to beneficial histological changes in the actual lung tissue. It was confirmed that it is possible ( FIGS. 4 to 11 ).
일 실시양태에서, 본 발명의 식품 조성물은 호흡기 조직 내 백혈구 및 호중구 수 감소 효과를 가진다.In one embodiment, the food composition of the present invention has the effect of reducing the number of leukocytes and neutrophils in the respiratory tissue.
일 실시양태에서, 본 발명의 식품 조성물은 폐세척액 내 백혈구 또는 호중구 수 억제 효과, 혈청 내 IgE 수치 감소 효과를 가진다.In one embodiment, the food composition of the present invention has the effect of inhibiting the number of leukocytes or neutrophils in the lung lavage fluid, and reducing the IgE level in the serum.
일 실시양태에서, 본 발명의 식품 조성물은 1종 이상의 식품 첨가제를 포함한다. In one embodiment, the food composition of the present invention comprises one or more food additives.
일 실시양태에서, 본 발명의 식품 조성물은 음료 형태이다.In one embodiment, the food composition of the present invention is in the form of a beverage.
본 발명은 또한, 미??리아닌, 루틴 및 퀘르시트린의 조합을 조성물 총 중량 기준 3~30 mg/g로 함유하고, 상기 조합의 총 중량 기준으로 루틴을 3.0~10.0 중량%으로, 퀘르시트린을 11.0~20.0 중량%로 함유하는 것을 특징으로 하는 호흡기 질환 건강 개선용 식품 조성물을 제공한다. 상기 조성물은 퀘르세틴을 더 포함할 수 있다. 보다 구체적인 실시양태에서 본 발명은 (i) 11.75±0.092 mg/g의 미??리아닌, (ii) 1.20±0.008 mg/g의 루틴, (iii) 2.38±0.389 mg/g의 퀘르시트린; 및 (iv) 0.068±0.017 mg/g의 퀘르세틴을 포함하는 식품 조성물을 제공한다.The present invention also contains a combination of myryanine, rutin and quercitrin in an amount of 3 to 30 mg/g based on the total weight of the composition, and rutin in an amount of 3.0 to 10.0% by weight based on the total weight of the combination, and quercitrin It provides a food composition for improving health of respiratory diseases, characterized in that it contains 11.0 to 20.0 wt%. The composition may further include quercetin. In a more specific embodiment the present invention provides a pharmaceutical composition comprising: (i) myrcyanine at 11.75±0.092 mg/g, (ii) rutin at 1.20±0.008 mg/g, (iii) quercitrin at 2.38±0.389 mg/g; and (iv) 0.068±0.017 mg/g of quercetin.
본 발명의 상기 삼백초 물 추출물은 고?t량의 미??리아닌(miquelianin), 루틴(rutin) 및 퀘르시트린(quercitrin)을 포함하는 것을 특징으로 한다. 상기 미??리아닌, 루틴 및 퀘르시트린은 삼백초 물 추출물에서 호흡기 질환에 대한 예방, 개선, 및 치료 효과를 나타내는 활성 물질들로서, 삼백초 물 추출물에 이들 활성 물질을 조합하여 포함함으로써, 우수한 호흡기 질환의 예방, 개선 및 치료 효과를 나타낸다. The water extract of trifolieae of the present invention is characterized in that it contains a high amount of my?? lianin (miquelianin), rutin (rutin) and quercitrin (quercitrin). The myrcyanine, rutin, and quercitrin are active substances that show prevention, improvement, and treatment effects for respiratory diseases in the water extract of Trifolieae. It exhibits preventive, ameliorating and therapeutic effects.
일 실시양태에서, 상기 삼백초 물 추출물은 추출물 총 중량 기준으로 미??리아닌, 루틴 및 퀘르시트린의 총합이 2.0 mg/g 이상이고, 바람직하게는 3.0 mg/g 이상이다. In one embodiment, the sum of myrcyanine, rutin, and quercitrine, based on the total weight of the extract, is 2.0 mg/g or more, and preferably 3.0 mg/g or more.
미??리아닌, 루틴 및 퀘르시트린 총합의 상한은 특별히 제한되지는 않으나, 100 mg/g 이하, 50 mg/g 이하일 수 있고, 예컨대 30 mg/g 이하일 수 있으나 이에 제한되지 않는다.The upper limit of the total of myrcyanine, rutin and quercitrine is not particularly limited, but may be 100 mg/g or less, 50 mg/g or less, for example, 30 mg/g or less, but is not limited thereto.
일 실시양태에서, 상기 삼백초 물 추출물은 퀘르세틴이 더 함유된 것을 특징으로 하는 조성물.In one embodiment, the composition characterized in that the water extract of Sambaekcho water contains more quercetin.
본 발명에 따른 식품 조성물은 마나산틴 A 및 마나산틴 B가 실질적으로 함유되지 않거나 저농도로 함유되어 있어, 기존의 삼백초 유기용매 추출물에서 발생하는 독성 문제를 해결한 것을 특징으로 한다.The food composition according to the present invention is characterized in that it does not substantially contain manasanthin A and manasanthin B or contains it at a low concentration, thereby solving the toxicity problem occurring in the existing organic solvent extract of Trifolium Root.
본 발명에서 “개선”은 본 발명의 식품 조성물 섭취로 인해 호흡기 질환을 예방, 억제 또는 지연시키거나, 질환의 증세를 호전하거나 이롭게 변경하는모든 행위를 의미한다.In the present invention, "improvement" refers to any action that prevents, suppresses, or delays respiratory diseases, improves symptoms of diseases, or changes beneficially due to ingestion of the food composition of the present invention.
본 발명의 식품 추출물은 단독 또는 호흡기 질환에 효과가 있는 것으로 공지된 1종 이상의 의약품, 생약제 등과 혼합하여 사용할 수 있다. 다른 식물의 추출물과 혼합하여 사용하는 경우, 해당 식물은 삼백초와 혼합한 뒤 추출될 수 있고, 개별적으로 추출된 후 혼합될 수 있다. The food extract of the present invention can be used alone or mixed with one or more pharmaceuticals, herbal medicines, etc. known to be effective for respiratory diseases. When used by mixing with extracts of other plants, the plants may be extracted after mixing with trifolieae, or may be individually extracted and then mixed.
본 발명에 따른 식품 추출물의 권장 복용량은 소비자의 연령, 체중, 성별, 건강상태, 식이, 복용시간, 복용방법 등에 따라 그 범위가 다양하며, 성인 기준으로 0.1 mg/kg 내지 1000 mg/kg을 1회 내지 수회에 나누어 복용할 수 있다.The recommended dosage of the food extract according to the present invention varies according to the age, weight, sex, health condition, diet, administration time, method of administration, etc. of the consumer, and 0.1 mg/kg to 1000 mg/kg per adult It can be taken in divided doses or several times.
본 발명의 식품 조성물은 호흡기 염증 세포의 비정상적인 증가를 억제하여 우수한 호흡기 건강 개선 효과를 나타낸다.The food composition of the present invention exhibits an excellent effect of improving respiratory health by suppressing an abnormal increase in respiratory inflammatory cells.
본 발명의 식품 조성물은 본 발명의 목적에 따라 기능성 식품일 수 있다. 상기 기능성 식품은 식품의 생체 조절 기능을 강조한 식품으로 물리적, 생화학적, 생물공학적인 방법을 이용하여 특정 목적에 작용 및 발현하도록 부가가치를 부여한 식품이다. 이러한 기능성 식품의 성분은 생체 방어와 신체 리듬의 조절, 질환의 방지 및 회복에 관계하는 신체 조절 기능을 생체에 대하여 충분히 발휘하도록 설계하여 가공하게 되며, 식품으로 허용 가능한 식품 보조 첨가제, 감미료 또는 기능성 원료를 함유할 수 있다. The food composition of the present invention may be a functional food for the purpose of the present invention. The functional food is a food that emphasizes the bioregulatory function of food, and is a food with added value to act and express for a specific purpose using a physical, biochemical, and bioengineering method. The ingredients of these functional foods are designed and processed to sufficiently exert the body control functions related to the body defense, regulation of body rhythm, prevention and recovery of diseases, and are food supplementary additives, sweeteners or functional raw materials that are acceptable as food. may contain.
본 발명에 따른 식품 조성물로는, 예를 들어, 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 건강보조 식품류 등이 있고 상기 음료에는 천연 과일주스, 과일주스 음료, 야채 음료 등이 포함된다. 본 발명의 식품 조성물은 공지의 제조방법에 따라 타정, 정제, 과립, 분말, 캡슐, 액상의 용액, 환 등의 제형으로 제조될 수 있다. The food composition according to the present invention includes, for example, various foods, such as beverages, gum, tea, vitamin complexes, and health supplements, and the beverage includes natural fruit juice, fruit juice beverage, vegetable beverage, and the like. Included. The food composition of the present invention may be prepared in the form of tablets, tablets, granules, powders, capsules, liquid solutions, pills, and the like according to known manufacturing methods.
또한, 본 발명의 식품 조성물은 통상의 여러 가지 향미제, 천연 탄수화물 등을 추가로 함유할 수 있다. 향미제로는 타우마틴, 스테비아 추출물과 같은 천연 향미제가 있으며 사카린, 아스파르탐 등의 합성 향미제를 사용하기도 한다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 수크로스와 같은 디사카라이드, 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨과 같은 당알콜 등을 포함할 수 있다. 이 외에도 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제나 천연 풍미제 등의 풍미제, 착색제, 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 무수 구연산과 같은 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제, 천연 과일 주스, 과일 주스 음료 및 야채 음료 제조를 위한 과육 등 식품학적으로 허용 가능한 식품 보조 첨가제를 추가로 함유할 수 있다. 이러한 첨가제는 독립적으로 또는 조합하여 사용될 수 있다.In addition, the food composition of the present invention may further contain various conventional flavoring agents, natural carbohydrates, and the like. As flavoring agents, natural flavoring agents such as thaumatin and stevia extract, and synthetic flavoring agents such as saccharin and aspartame are sometimes used. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. . In addition, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic or natural flavoring agents, coloring agents, thickeners (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its Salts, organic acids such as citric anhydride, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, natural fruit juices, fruit juices for beverages and fruit pulp for vegetable beverages, etc. may additionally contain acceptable food supplement additives. These additives may be used independently or in combination.
일례로, 본 발명의 식품 조성물은 건조 엑스 조성물 100 mg과 함께, 구연산 1,000 ㎎, 올리고당 100 g, 과실농축액 2 g, 타우린 1 g 및 정제수를 가하여 전체 900 ㎖ 용량의 건강기능 음료로 제품화될 수 있다. 이 경우, 통상의 건강기능성 음료 제조방법에 따라 상기의 성분을 혼합한 다음, 약 1시간동안 80℃에서 교반 가열한 후, 만들어진 용액을 여과하여 멸균된 2ℓ용기에 취득하여 밀봉 멸균한 뒤 냉장 보관한 다음 본 발명의 건강기능성 음료 조성물 제조에 사용될 수 있다.As an example, the food composition of the present invention is a health functional beverage with a total capacity of 900 ml by adding 1,000 mg of citric acid, 100 g of oligosaccharide, 2 g of fruit concentrate, 1 g of taurine and purified water together with 100 mg of dry extract composition. . In this case, the above ingredients are mixed according to a conventional health functional beverage manufacturing method, and then stirred and heated at 80° C. for about 1 hour. Then it can be used to prepare the health functional beverage composition of the present invention.
상기 조성비는 비교적 기호음료에 적합한 성분을 바람직한 실시예로 혼합 조성하였지만, 수요계층, 수요국가, 사용 용도 등 지역적, 민족적 기호도에 따라서 그 배합비를 임의로 변형 실시하여도 무방하다.Although the composition ratio is prepared by mixing ingredients suitable for relatively favorite beverages in a preferred embodiment, the mixing ratio may be arbitrarily modified according to regional and ethnic preferences such as demand class, demand country, and use purpose.
본 발명의 삼백초 물 추출물 유래 식품 조성물은 호흡기 건강 개선 효과가 우수하며, 특히 미세먼지 유발 등으로 인한 호흡기 질환 개선용 건강기능식품으로 활용될 수 있다.The water extract-derived food composition of the present invention has an excellent effect on improving respiratory health, and in particular, it can be used as a health functional food for improving respiratory diseases caused by fine dust, etc.
도 1은 본 발명의 식품 조성물 제조 과정 중 삼백초 물 추출물 내 유효성분 군에 대한 HPLC 측정 결과를 나타내고,
도 2는 실험 동물에 대한 PCA(Passive cutaneous anaphylaxis) 반응 실시 결과를 나타내고,
도 3은 미세먼지 유발 알레르기성 폐질환 동물에 대한 실험 스케쥴을 나타내고,
도 4는 미세먼지 유발 알레르기성 폐질환 동물의 백혈구 수 측정 결과를 나타내고,
도 5는 미세먼지 유발 알레르기성 폐질환 동물의 호중구 수 측정 결과를 나타내고,
도 6는 미세먼지 유발 알레르기성 폐질환 동물의 염증세포 수 관찰 결과를 나타내고(좌측 상단: 수돗물 처리군. 우측 상단: 오발부민 및 미세먼지 처리군, 좌측 하단: 오발부민, 미세먼지 및 덱사메타손 처리군, 우측 하단: 오발부민, 미세먼지 및 본 발명 조성물 300 mg/kg 처리군),
도 7은 미세먼지 유발 알레르기성 폐질환 동물의 혈청 내 IgE 레벨 측정 결과를 나타내고,
도 8 및 도 9는 미세먼지 유발 알레르기성 폐질환 동물의 조직학적 변화를 나타내고(화살표: 점액 과다분비, 화살표 머리: 폐 상피세포 과형성, 별 모양: 염증 세포 침투),
도 10 및 도 11은 미세먼지 유발 알레르기성 폐질환 조직 내 염증성 사이토카인의 발현 레벨을 나타낸다.1 shows the HPLC measurement results for the active ingredient group in the water extract of Sambaekcho during the manufacturing process of the food composition of the present invention,
Figure 2 shows the results of PCA (Passive cutaneous anaphylaxis) reaction to the experimental animals,
3 shows an experimental schedule for animals with fine dust-induced allergic lung disease,
4 shows the results of measuring the number of white blood cells in animals with fine dust-induced allergic lung disease;
5 shows the measurement results of the number of neutrophils in animals with fine dust-induced allergic lung disease,
6 shows the results of observation of the number of inflammatory cells in the animals with fine dust-induced allergic lung disease (top left: tap water treated group, top right: ovalbumin and fine dust treated group, bottom left: ovalbumin, fine dust and dexamethasone treated group , lower right: ovalbumin, fine dust and 300 mg/kg of the composition of the present invention treated group),
7 shows the measurement result of the IgE level in the serum of the animal with fine dust-induced allergic lung disease,
8 and 9 show histological changes in animals with fine dust-induced allergic lung disease (arrow: mucus hypersecretion, arrow head: lung epithelial hyperplasia, star shape: inflammatory cell infiltration);
10 and 11 show the expression level of inflammatory cytokines in the tissues of fine dust-induced allergic lung disease.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 본 발명의 내용이 이에 한정되는 것은 아니다.Hereinafter, preferred examples are presented to help the understanding of the present invention. However, the following examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited thereto.
실시예 1. 삼백초 식품 조성물의 준비Example 1. Preparation of three hundred seconds food composition
건조된 삼백초는 전남, 경북, 충북의 재배농가에서 직접 구입하거나 한약재시장에 유통되는 것으로 준비하였고, 미??리아닌, 루틴, 퀘르세틴은 시그마-알드리치(St. Louis, Mo, UA)에서, 퀘르시트린은 인터팜(Koyang-si, Korea)에서 구매했다. The dried ginseng plant was purchased directly from growers in Jeollanam-do, Gyeongbuk, and North Chungcheong Province or prepared for distribution in the herbal medicine market. was purchased from Interfarm (Koyang-si, Korea).
실험에 사용한 미세먼지(FD, Phyllite powder, PH-DG2000)는 홍익 바이오텍(Jeonam, Korea)에서 구매하였으며, 이때 미세먼지의 평균 입자크기는 2.6 μm였다.The fine dust (FD, Phyllite powder, PH-DG2000) used in the experiment was purchased from Hongik Biotech (Jeonam, Korea), and the average particle size of the fine dust was 2.6 μm.
삼백초의 지상부 100g 을 유기용매를 전혀 함유하지 않은 정제수 1,500 mL(삼백초의 15배수, 1 kg/15L)로 95℃ 에서 3시간 동안 2회 추출하여 삼백초 물 추출물을 수득하였다. 삼백초 물 추출물을 여과하여 회전 증발기를 사용해 진공에서 농축한 다음, 25% 덱스트린을 함유하게 분무건조했다. 100 g of the above-ground part of 300 seconds was extracted twice with 1,500 mL of purified water containing no organic solvent (15 multiples of 300 seconds, 1 kg/15 L) at 95° C. for 3 hours to obtain a water extract of 300 seconds. The trifoliea water extract was filtered, concentrated in vacuo using a rotary evaporator, and then spray-dried to 25% dextrin.
그 다음, 고속액체크로마토그래피(HPLC)를 하기와 같은 조건으로 수행하였다. Then, high performance liquid chromatography (HPLC) was performed under the following conditions.
- HPLC 분석기기: Waters e2695 alliance System 과 Empower 2 프로그램- HPLC analyzer: Waters e2695 alliance System and Empower 2 program
- 분석용 컬럼: Fortis C18 Column (4.6 × 150 mm, 5㎛)- Analytical column: Fortis C18 Column (4.6 × 150 mm, 5㎛)
삼백초 물 추출물의 3차원 HPLC크로마토그램 확인시 350 nm, 355 nm에서 루틴, 미??리아닌 검출을 확인할 수 있으며, 머무름시간은 18.3, 28.3 분 이었고, 퀘르시트린의 머무름시간은 29.3분이었고, 퀘르세틴은 365 nm에서 측정시 머무름시간이 35.8 분이었다(도 1).When confirming the three-dimensional HPLC chromatogram of the water extract of 300 sec., rutin and myryanine were detected at 350 nm and 355 nm, and the retention times were 18.3 and 28.3 minutes, the retention times of quercitrine were 29.3 minutes, and quercetin When measured at 365 nm, the retention time was 35.8 minutes (FIG. 1).
HPLC 분석 결과에 따라 추출물 내 미??리아닌, 루틴, 퀘르시트린 및 퀘르세틴 성분의 양을 아래 식에 따라 정량하였다.According to the HPLC analysis result, the amounts of myrcyanine, rutin, quercitrine and quercetin components in the extract were quantified according to the following formula.
[식 1][Equation 1]
추출물 내 미??리아닌, 루틴, 퀘르시트린 및 퀘르세틴 조합이 본 발명에서 설정한 유효성분 함량 범위에 속하는 추출물 배치를 선별 및 분리하여 본 발명의 식품 조성물을 준비하였다.A food composition of the present invention was prepared by selecting and separating a batch of extracts in which myryanine, rutin, quercitrin and quercetin combinations in the extract fall within the active ingredient content range set in the present invention.
그 결과, 본 발명의 예시적인 식품 조성물을 아래와 같이 준비하였다. 조성물 1, 2, 3은 미??리아닌, 퀘르시트린, 루틴의 함량을 조절한 본 발명의 삼백초 식품 조성물이고, 비교 조성물 1은 미??리아닌, 퀘르시트린, 루틴의 양이 본 발명에서 설정한 함량 기준에 미달하는 비교예이다.As a result, an exemplary food composition of the present invention was prepared as follows.
[표 1][Table 1]
또한, 종래 대한민국 공개특허 제10-2006-0111790호에 개시된 것과 같이 삼백초의 전초 100g을 30% 에탄올 1,500 mL(삼백초의 15배수)로 80 ℃에서 4시간 동안 추출하여 삼백초 30% 에탄올 추출물을 제조하였고, 이를 비교 조성물 2로 하였다.In addition, as disclosed in the prior Korean Patent Application Laid-Open No. 10-2006-0111790, 100 g of the whole plant of 300 seconds was extracted with 1,500 mL of 30% ethanol (15 times the number of 300 seconds) at 80 ° C. for 4 hours to prepare a 30% ethanol extract of 300 seconds. , this was used as
나아가, 삼백초 물 추출물을 대상으로 독성 물질인 마나산틴 A, 마나산틴 B 함량을 HPLC로 측정한 결과, 종래 삼백초 알코올 추출물과 달리 마나산틴 A 및 마나산틴 B이 검출되지 않음을 확인하였다.Furthermore, as a result of measuring the contents of toxic substances such as manaxanthin A and manaxanthin B in the water extract of Trifolium Root, it was confirmed that Manaxanthin A and Manaxanthin B were not detected, unlike the conventional extract of Trifolium cinnamon.
실시예 2. 알레르기성 폐질환 동물에서 호중구 억제 효과Example 2. Neutrophil Inhibitory Effect in Animals with Allergic Lung Disease
6주령 암컷 BALB/c 마우스에 오발부민 20 ㎍ (Sigma Aldrich) 및 알룸 1mg (Thermo Fisher Scientific)을 생리식염수 500 ㎕에 녹인 용액을 주 1회로 2주간 복강투여하여 감작한다. 15일째부터는 5% 오발부민 용액을 5일간 흡입시켜 야기한 후 본 발명의 식품 조성물을 섭취시켰다. 부검 후 인산염 완충액 염수(PBS) 용액을 투여하여 기관지 세정액을 얻은 후, 세정액을 5분간 3,000 rpm으로 원심분리하여 획득한 펠렛으로 혈구분석을 실시하였다. 본 실험에서는 양성 대조군으로는 덱사메타손(dexamethasone; 삼백초 추출물과 동일자에 1mg/kg 경구투여)을 사용했다.6-week-old female BALB/c mice are sensitized by intraperitoneal administration of a solution of
실험 결과, 본 발명의 실시예 조성물은 비교 조성물 2 대비 호중구(NE) 억제 효과가 50% 이상 개선된 것으로 나타났다.As a result of the experiment, the composition of the Example of the present invention showed an improvement of 50% or more in the neutrophil (NE) inhibitory effect compared to
[표 2][Table 2]
실시예 3. 염증성 사이토카인 IL-4 생성 억제 효과Example 3. Inhibitory effect on inflammatory cytokine IL-4 production
본 발명의 식품 조성물(조성물 4: 미??리아닌, 루틴 및 퀘르시트린 총량 12.95 mg/g)을 처리한 세포 실험에서 알레르기성 폐질환 관련 염증성 사이토카인인 IL-4의 생성에 미치는 영향을 측정하였다. Measuring the effect on the production of IL-4, an inflammatory cytokine associated with allergic lung disease, in a cell experiment treated with the food composition of the present invention (composition 4: myryanine, rutin and quercitrine total amount 12.95 mg/g) did
구체적으로, RBL-2H3 세포주를 10% FBS를 포함한 DMEM에 현탁시킨 후 24 웰 플레이트(Corning, USA)에 5 × 105cells/㎖의 세포수가 되도록 분주하였다. 그 후 항-DNP IgE (30ng/㎖)로 감작하고 37℃ 5% CO2 인큐베이터에서 24시간 배양하였다. 배지 교환 후 실시예 조성물 또는 비교 조성물을 각각 400 ug/mL 처리하여 1시간 동안 37℃ 5% CO2 인큐베이터에서 배양한 후 DNP-HSA (10 ug/mL)를 처리하여 4시간 동안 자극시키고 얼음조에 10분 동안 방치하여 반응을 종결시켰다. 상층액을 분리하여 5,000 rpm에서 10분 동안 원심 분리하여 분리된 상층액을 -70℃에 보관하였다. 염증성 사이토카인 수치를 IL-4 ELISA kit (BD Biosciences Pharmingen, USA)를 사용하여 측정하였다. Microplate reader (Molecular Devices Co. Ltd., USA)를 사용하여 450 nm에서 흡광도를 측정하여 누출된 IL-4의 양을 계산하였다.Specifically, the RBL-2H3 cell line was suspended in DMEM containing 10% FBS and then aliquoted to a cell number of 5 × 10 5 cells/ml in a 24-well plate (Corning, USA). Thereafter, it was sensitized with anti-DNP IgE (30 ng/ml) and incubated for 24 hours at 37° C. in a 5
실험 결과, 항-DNP IgE로 자극한 세포에서 IL-4 생성량은 21.93 pg/mL인 반면, 실시예 조성물을 처리한 세포에서 IL-4 생성량은 16.8 pg/mL (p<0.05)로 유의한 억제 효과를 나타냈다. 반면, 대조용 조성물 처리 후 IL-4 생성량은 18.8 pg/ml에 불과하여, 항원으로 자극한 세포에 비해 유의하지 않았다.As a result of the experiment, the amount of IL-4 production in cells stimulated with anti-DNP IgE was 21.93 pg/mL, whereas the amount of IL-4 production in cells treated with the composition of Example was 16.8 pg/mL (p<0.05), which was significantly inhibited. showed effect. On the other hand, the amount of IL-4 produced after treatment with the control composition was only 18.8 pg/ml, which was not significant compared to the cells stimulated with the antigen.
[표 3][Table 3]
실시예 4. PCA(Passive cutaneous anaphylaxis) 반응 분석Example 4. Passive cutaneous anaphylaxis (PCA) response analysis
5주령의 수컷 ICR 마우스를 샘타코로부터 공급받아 실험 전 1주 적응기 동안 사료와 물을 마음껏 섭취하도록 하였다. 사육실의 온도는 22±1℃, 습도는 55±10%를 유지하였고, 매일 명암주기가 각 12시간이 되도록 조절하여 사육하였다. Five-week-old male ICR mice were supplied from Samtaco and allowed to freely consume food and water during the adaptation period for one week before the experiment. The temperature of the breeding room was 22±1℃ and the humidity was maintained at 55±10%, and the daily light-dark cycle was adjusted to be 12 hours each.
IgE-dependent cutaneous reaction은 anti-DNP IgE를 피부 내에 주사하여 피부를 감작시켜 유도하였고, 24시간 뒤에 DNP-HAS를 생쥐 꼬리의 정맥에 주사하였다. 구체적으로 Anti-DNP-HSA 특이적 IgE 항체 0.5 ug을 마우스 한 쪽 귀에 피내주사 (intradermal injection)하고 24시간 경과 후 본 발명에 따른 조성물과 대조용 조성물을 경구 섭취시켰다. The IgE-dependent cutaneous reaction was induced by sensitization of the skin by injecting anti-DNP IgE into the skin, and 24 hours later, DNP-HAS was injected into the vein of the tail of the mouse. Specifically, 0.5 ug of the Anti-DNP-HSA-specific IgE antibody was intradermally injected into one ear of a mouse, and the composition according to the present invention and the control composition were orally ingested after 24 hours.
한 시간 뒤 250 ug 항원(DNP-HSA)과 1% Evans blue 색소를 용해한 인산완충식염수(PBS, phosphate-buffered saline) 250 uL를 마우스의 꼬리에 정맥주사(intravenous injection)하여 수동 피부 과민증 반응을 일으켰다. 면역반응에 의해 귀 혈관으로부터 누출된 Evans blue 염색정도를 관찰하였다.One hour later, 250 ug of antigen (DNP-HSA) and 250 uL of phosphate-buffered saline (PBS) in which 1% Evans blue was dissolved was intravenously injected into the tail of the mouse to induce a passive skin hypersensitivity reaction. . The degree of Evans blue staining leaked from the ear vessels by the immune response was observed.
수동 피부 아나필락시스 (passive cutaneous anaphylaxis, PCA) 반응은 국소 알레르기 반응에서 민감성을 측정하는 매우 유용한 동물모델 중 하나이다. 실험 결과, 실시예 조성물(조성물 4; 도 2의 A)은 대조용 조성물(비교 조성물 2; 도 2의 B) 대비 과민 반응 억제 효과가 우수한 것을 확인하였다(도 2).The passive cutaneous anaphylaxis (PCA) response is one of the most useful animal models for measuring sensitivity in local allergic reactions. As a result of the experiment, it was confirmed that the example composition (composition 4; FIG. 2A) had an excellent hypersensitivity reaction inhibitory effect compared to the control composition (
실시예 5. 미세먼지에 의해 유발된 알레르기성 폐질환 동물에서 호중구 및 IgE 억제 효과Example 5. Neutrophil and IgE Inhibition Effect in Animals with Allergic Lung Disease Induced by Fine Dust
미세먼지에 의해 유도된 알레르기성 폐질환에 대한 본 발명의 식품 조성물의 개선 효과를, 동물 모델에서 측정하였다.The improvement effect of the food composition of the present invention on the allergic lung disease induced by fine dust was measured in an animal model.
본 발명의 식품 조성물로, 실시예 1에 따라 제조된 조성을 사용하였으며, 이때 미??리아닌, 루틴, 퀘르시트린 및 퀘르세틴의 함량은 각각 11.75±0.092 mg/g, 1.20±0.008 mg/g, 2.38±0.389 mg/g 및 0.068±0.017 mg/g이었다(조성물 5).As the food composition of the present invention, the composition prepared according to Example 1 was used, wherein the contents of myrcyanine, rutin, quercitrin and quercetin were 11.75±0.092 mg/g, 1.20±0.008 mg/g, and 2.38, respectively. ± 0.389 mg/g and 0.068 ± 0.017 mg/g (composition 5).
먼저, BALB/c 마우스를 수돗물 처리군(CON), 오발부민 처리군(OVA), 오발부민 및 미세먼지 처리군(OVA+FD), 오발부민, 미세먼지 및 덱사메타손 처리군(OVA+FD+DEX) 및 오발부민, 미세먼지 및 본 발명의 조성물 처리군에 각각 6마리씩 할당하였다. 동물 실험은 동일한 방법으로 2회 반복하였다.First, BALB/c mice were treated with tap water (CON), ovalbumin treated group (OVA), ovalbumin and fine dust treated group (OVA+FD), ovalbumin, fine dust and dexamethasone treated group (OVA+FD+DEX). ) and ovalbumin, fine dust, and 6 animals were assigned to each treatment group of the present invention. Animal experiments were repeated twice in the same manner.
모든 그룹에 1주 간격으로 2회씩 오발부민(500 ul 식염수 내 20 ug 오발부민 및 1 mg 수화알루미늄 수화물)을 복강 투여하였다(CON 그룹 제외). 모든 마우스에 네불라이저 미스트로 오발부민 및 미세먼지를 매일 150 ug/m3 농도로 30분씩 5일 동안 노출시켰고, 2회 주사 후에는 7일 동안 노출시켰다(CON 그룹 제외). 모든 그룹에서 노출 당일 오발부민 및 미세먼지 미스트 흡입 전에 수돗물(CON 또는 OVA+FD) 또는 덱사메타손(OVA+FD+DEX)을 3회 섭취시켰다(도 3).Ovalbumin (20 ug ovalbumin and 1 mg aluminum hydrate hydrate in 500 ul saline) was administered intraperitoneally to all groups twice at intervals of 1 week (except for the CON group). All mice were exposed to ovalbumin and fine dust as a nebulizer mist daily at a concentration of 150 ug/m 3 for 30 minutes for 5 days, and after 2 injections, they were exposed for 7 days (except the CON group). All groups ingested tap water (CON or OVA+FD) or dexamethasone (OVA+FD+DEX) three times on the day of exposure before inhalation of ovalbumin and fine dust mist (FIG. 3).
실험 결과, 본 발명의 식품 조성물을 섭취한 마우스의 경우(조성물 5를 300 mg/kg로 섭취), 미세먼지에 의해 유발된 폐세척액(BALF) 내 백혈구 수 감소(도 4) 및 호중구(NE) 수 감소(도 5) 효과를 확인하였다. 폐 세척액 내 염증 세포의 수 역시 본 발명의 조성물 처리시 대조군에 비해 감소됨을 확인하였다(도 6 및 표 4).As a result of the experiment, in the case of mice ingesting the food composition of the present invention (composition 5 was consumed at 300 mg/kg), the number of white blood cells in the lung lavage fluid (BALF) induced by fine dust decreased ( FIG. 4 ) and neutrophils (NE) The number reduction (FIG. 5) effect was confirmed. It was confirmed that the number of inflammatory cells in the lung lavage was also reduced compared to the control when the composition of the present invention was treated ( FIGS. 6 and 4 ).
[표 4][Table 4]
실시예 6. 미세먼지에 의해 유발된 알레르기성 폐질환 동물에서 조직학적 변화 관찰Example 6. Observation of histological changes in animals with allergic lung disease induced by fine dust
미세먼지에 의해 유도된 알레르기성 폐질환에 대한 본 발명의 식품 조성물(조성물 5)의 개선 효과를 조직학적 측면에서 조사하였다.The improvement effect of the food composition (composition 5) of the present invention on the allergic lung disease induced by fine dust was investigated in terms of histology.
테스트 동물에서 폐 샘플을 EtOH로 탈수된 10%(v/v) 포름알데히드로 고정하고 파라핀에 포매했다. 샘플을 4 um 두께 절편으로 제작한 후 헤마톡실린 & 에오신 (H&E) 염색을 실시했다. 이때, 당단백질 변화를 포함한 폐 내 형태 변화 평가를 위해 PAS(periodic acid schiff) 염색을 함께 실시했다. 광학 현미경(Axioscope A1, Carl Zeiss, Germany)으로 병리학적 레벨을 0에서 3으로 평가하여, 점액 과다분비, 폐 상피 세포 과다형성, 및 염증세포 침투과 같은 호흡기 조직 내 형태 변화를 관찰하였다.Lung samples from test animals were fixed with 10% (v/v) formaldehyde dehydrated with EtOH and embedded in paraffin. After the samples were prepared in 4 um thick sections, hematoxylin & eosin (H&E) staining was performed. At this time, PAS (periodic acid schiff) staining was performed together to evaluate changes in lung morphology including glycoprotein changes. The pathological level was evaluated from 0 to 3 with an optical microscope (Axioscope A1, Carl Zeiss, Germany), and morphological changes in respiratory tissues such as mucus hypersecretion, lung epithelial cell hyperplasia, and inflammatory cell infiltration were observed.
점액 과다분비의 경우, 0점 (없음), 1점(점액 방출이 거의 일어나지 않음), 2점(점액이 전체 도관의 절반 수준임) 및 3점(점액으로 가득 참)으로 평가했다.Hypersecretion of mucus was rated on a scale of 0 (none), 1 (little mucus discharge), 2 (mucus is half of the total duct), and 3 (full of mucus).
상피 세포 과형성(hyperplasia)의 경우, 0점 (없음), 1점(corrugated wall), 2점 (folded epithelium), 3점(severe folded epithelium)으로 평가했다.For epithelial cell hyperplasia, scores of 0 (none), 1 (corrugated wall), 2 (folded epithelium), and 3 (severe folded epithelium) were evaluated.
염증 세포 침투의 경우 0점 (없음), 1점(백혈구가 거의 없음), 2점(보통량의 백혈구), 3점(많은 수의 백혈구)로 평가했다.Inflammatory cell infiltration was rated as 0 (none), 1 (few white blood cells), 2 (moderate amount of white blood cells), and 3 (high number of white blood cells).
대표적인 조직 사진은 도 8, 9에 기재하였으며, 전체 실험 결과는 하기 표에 기재된 바와 같다.Representative tissue photos are shown in FIGS. 8 and 9, and the overall experimental results are as shown in the table below.
[표 5][Table 5]
실시예 7. 미세먼지에 의해 유발된 알레르기성 폐질환에서 염증성 사이토카인 발현에 미치는 영향Example 7. Effect on inflammatory cytokine expression in allergic lung disease induced by fine dust
오발부민 마우스 모델에서, 미세먼지 처리에 의해 증가된 IL-13 발현량에 본 발명의 식품 조성물(조성물 5)이 미치는 영향을 조사하였다.In the ovalbumin mouse model, the effect of the food composition (composition 5) of the present invention on the IL-13 expression level increased by the fine dust treatment was investigated.
먼저, 알레르기성 폐질환 관련 사이토카인의 mRNA 발현을 확인하기 위해 RT-PCR을 수행하였다. 총 RNA를 RNeasy Mini Kit(Qiagen, Germany)를 이용해 폐 조직에서 추출하여, 100 ng의 RNA를 반응에 사용했다. IL-13에 대한 프라이머는 서열번호 1(Foward) 및 서열번호 2(Reverse)로 제공된 서열을 사용했다.First, RT-PCR was performed to confirm the mRNA expression of cytokines related to allergic lung disease. Total RNA was extracted from lung tissue using RNeasy Mini Kit (Qiagen, Germany), and 100 ng of RNA was used for the reaction. As primers for IL-13, sequences provided as SEQ ID NO: 1 (Forward) and SEQ ID NO: 2 (Reverse) were used.
- 서열번호 1: 5’-CAGCCCTCAGCCATGAAATA-3’- SEQ ID NO: 1: 5'-CAGCCCTCAGCCATGAAATA-3'
- 서열번호 2: 5’-CTTGAGTGTGTAACAGGCCATTCT-3’- SEQ ID NO: 2: 5'-CTTGAGTGTGTAACAGGCCATTCT-3'
RT-PCR에서 변성(denature) 단계는 95 ℃에서 5초, 어닐링/확장 단계는 65 ℃에서 30초씩 40 사이클로 수행하였으며, qTOWER2.2 (Analytic Jena, Gemany)를 이용하여 결과를 수득했다.In RT-PCR, the denaturation step was performed at 95° C. for 5 seconds, and the annealing/extension step was performed at 65° C. for 30 seconds in 40 cycles, and the results were obtained using qTOWER2.2 (Analytic Jena, Gemany).
한편, 단백질 발현량 확인을 위해 lysis 버퍼로 조직을 균질화하여 동량의 단백질을 수집하고 분석하여 IL-13에 대한 ELISA를 수행하였다.On the other hand, to check the protein expression level, the tissue was homogenized with a lysis buffer, the same amount of protein was collected and analyzed, and ELISA for IL-13 was performed.
실험 결과, 미세먼지 유발된 알레르기성 폐질환 마우스에서 IL-13의 mRNA 및 단백질 발현량은 대조군 대비 급격히 상승하였으나, 본 발명의 식품 조성물을 섭취한 마우스의 경우, 양성 대조군인 덱사메타손과 유사한 수준으로 IL-13의 발현 억제 효과를 나타내는 것으로 확인되었다(도 10 및 도 11).As a result of the experiment, the mRNA and protein expression levels of IL-13 in mice with fine dust-induced allergic lung disease increased sharply compared to the control group. -13 was confirmed to exhibit an expression inhibitory effect ( FIGS. 10 and 11 ).
Claims (8)
삼백초 지상부를 유기용매가 포함되지 않은 물로 80 ℃ 이상에서 2 시간 이상 추출하여 삼백초 물 추출물을 수득하는 단계;
상기 추출물에서 성분분석을 수행하여 미??리아닌, 루틴 및 퀘르시트린의 함량을 측정하는 단계; 및
건조 조성물 기준으로
(i) 11.75±0.092 mg/g의 미??리아닌
(ii) 1.20±0.008 mg/g의 루틴; 및
(iii) 2.38±0.389 mg/g의 퀘르시트린
을 포함하는 조성물을 선별하는 단계를 통해 수득되고,
상기 조성물은 독성물질인 마나산틴 A 및 마나산틴 B을 조성물 기준으로 0~0.2 mg/g 함유하고,
상기 조성물은 유기용매 추출 또는 분획 과정을 포함하지 않는 것을 특징으로 하고,
상기 조성물은 IL-13의 발현을 억제하고,
상기 호흡기 건강 개선은 평균 입자 크기가 0.1 μm 내지 10 μm인 미세먼지 흡입으로 인해 유발된 알레르기성 천식 또는 비염의 개선인 것을 특징으로 하는 호흡기 건강 개선용 식품 조성물.As a food composition for improving respiratory health,
extracting the above-ground part of 300 seconds at 80 ° C. or higher with water not containing an organic solvent for 2 hours or more to obtain a water extract of 300 seconds;
measuring the contents of myrcyanine, rutin and quercitrine by performing component analysis on the extract; and
based on dry composition
(i) 11.75±0.092 mg/g microcyanine
(ii) 1.20±0.008 mg/g of rutin; and
(iii) quercitrin at 2.38±0.389 mg/g
Obtained through the step of screening a composition comprising,
The composition contains 0 to 0.2 mg/g of toxic substances, manasanthin A and manaxanthin B, based on the composition,
The composition is characterized in that it does not include an organic solvent extraction or fractionation process,
The composition inhibits the expression of IL-13,
The respiratory health improvement is a food composition for improving respiratory health, characterized in that the improvement of allergic asthma or rhinitis induced by inhalation of fine dust having an average particle size of 0.1 μm to 10 μm.
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| KR20130142638A (en) * | 2012-06-20 | 2013-12-30 | 영남대학교 산학협력단 | A method for preparing a purified extract and the composition comprising the same for treating and preventing asthma and allergic disease |
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| KR20130142638A (en) * | 2012-06-20 | 2013-12-30 | 영남대학교 산학협력단 | A method for preparing a purified extract and the composition comprising the same for treating and preventing asthma and allergic disease |
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| 마스크로 인해 건조해진 호흡기 건강을 챙기세요! 네이버 블로그. [online], 2020.07.09., [2021.06.03. 검색], 인터넷: <URL: https://blog.naver.com/blogchunjiin/222025596316> 1부.* * |
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