KR20220103641A - Composition for immune regulation or enhancement comprising natural complex extracts as an active ingredient - Google Patents
Composition for immune regulation or enhancement comprising natural complex extracts as an active ingredient Download PDFInfo
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- KR20220103641A KR20220103641A KR1020220004420A KR20220004420A KR20220103641A KR 20220103641 A KR20220103641 A KR 20220103641A KR 1020220004420 A KR1020220004420 A KR 1020220004420A KR 20220004420 A KR20220004420 A KR 20220004420A KR 20220103641 A KR20220103641 A KR 20220103641A
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Abstract
Description
본 발명은 천연 복합 추출물을 유효성분으로 포함하는 면역조절 또는 면역증강효과를 가지는 건강기능성 식품 조성물에 관한 것이다. The present invention relates to a health functional food composition having an immunomodulatory or immune enhancing effect comprising a natural complex extract as an active ingredient.
면역 반응은 체내에 존재하는 자기방어체계로서 인체가 독소와 같은 외부물질 또는 박테리아, 바이러스 등과 같은 병원체(pathogen)를 자기가 아닌 이물질로 인식하여 제거하고 대사시키는 과정이다. 면역 반응은 면역 기능의 변화를 조절하여 정상상태로 회복시키거나 변화의 폭을 줄여주는 작용으로 면역 기능 증진과 면역 기능 억제로 구분된다. The immune response is a self-defense system that exists in the body, and is a process in which the human body recognizes foreign substances such as toxins or pathogens such as bacteria and viruses as foreign substances, removes them and metabolizes them. The immune response modulates changes in immune function to restore it to a normal state or reduces the width of the change, and is divided into enhancement of immune function and suppression of immune function.
면역 기능 증진은 외부 물질이나 병원체로부터 자신을 보호하는 기능을 말하며, 기본적으로 신체 내 면역세포의 활성화에 기인한 염증반응에 의해 이루어진다. 신체 내에 외부 물질이나 병원체가 침입하는 경우 신체는 염증반응을 통해 외부 물질 또는 병원체를 제거한다. 따라서, 외부 물질에 대한 염증 반응이 제대로 이루어지지 않는 경우 세균, 바이러스 등의 병원체로부터 신체를 효율적으로 보호하지 못하게 된다. Immune function enhancement refers to the function of protecting oneself from foreign substances or pathogens, and is basically achieved by an inflammatory response caused by the activation of immune cells in the body. When a foreign substance or pathogen enters the body, the body removes the foreign substance or pathogen through an inflammatory reaction. Accordingly, when an inflammatory response to an external substance is not properly performed, the body cannot be effectively protected from pathogens such as bacteria and viruses.
또한, 외부 물질 또는 병원체가 제거된 이후, 면역 반응으로 활성화된 염증반응은 다시 억제되어야 한다. 지나친 염증반응은 신체 조직을 손상시킬 수 있기 때문이다. 이러한 지나친 염증반응의 대표적인 예는 만성염증성 질환, 자가면역질환 등이 있다. In addition, after the foreign substance or pathogen is removed, the inflammatory response activated by the immune response must be suppressed again. This is because an excessive inflammatory response can damage body tissues. Representative examples of such an excessive inflammatory response include chronic inflammatory diseases and autoimmune diseases.
따라서, 면역 기능 억제는 외부 물질 또는 병원체를 방어하기 위해 증가된 염증반응 또는 자기 항원 또는 변형된 자기항원에 대한 반응 등 바람직하지 않게 증가된 면역 반응을 억제시키는 것을 말한다.Therefore, the suppression of immune function refers to suppressing an undesirably increased immune response, such as an increased inflammatory response or a response to a self-antigen or a modified self-antigen to defend against a foreign substance or pathogen.
최근 사스(Severe Acute Respiratory Syndrome: SRAS), 조류독감(Avian Influenza: AI), 메르스(Middle East Respiratory Syndrome, MERS), 신종플루, 코로나바이러스-19(Severe Acute Respiratory syndrome coronavirus 2: SARS-CoV-2) 등 신규 유행성 질환의 빈번한 발생은 세계적으로 심각한 경제적 손실과 국민 건강의 위해 요인이 되고 있다. 이러한 질환들은 외부 세균이나 바이러스로부터 신체를 보호하지 못하고 체내의 면역 작용이 원활히 이루어지지 않아 발생하는 것으로 알려져 있다. Recent Severe Acute Respiratory Syndrome (SRAS), Avian Influenza (AI), Middle East Respiratory Syndrome (MERS), H1N1 flu, Severe Acute Respiratory syndrome coronavirus 2: SARS-CoV- 2) The frequent occurrence of new epidemic diseases is causing serious economic loss and harm to public health worldwide. These diseases are known to occur because the body does not protect the body from external bacteria or viruses and the immune function in the body does not work smoothly.
이에 따라 마땅한 치료제와 백신이 없는 상황에서 세계적으로 예방의학적 차원의 면역관련 기능성 제품 및 일반의약품 판매가 급증하고 있다.As a result, in the absence of suitable treatments and vaccines, Sales of immune-related functional products and over-the-counter drugs for preventive medicine are rapidly increasing worldwide.
종래부터 인삼 또는 인삼 가공품을 면역조절 또는 면역증강용으로 복용해왔으나, 인삼 또는 인삼 가공품 외에 특별히 허가받은 기능성 식품 원료는 아직까지는 없어, 면역조절 또는 면역증강 효과가 있는 기능성 식품 원료를 개발하는 것이 필요하다. In the past, ginseng or processed ginseng products have been taken for immune regulation or immunity enhancement, but there are no functional food ingredients specially approved other than ginseng or ginseng processed products. do.
이에 본 발명자들은 상기 문제를 해결하고자 다각적으로 연구를 수행한 결과, 금은화, 황금, 내복자 및 강황의 복합 추출물이 우수한 NO 생성 억제효과 및 염증성 사이토카인의 발현 억제효과를 가진다는 것을 확인하고, 면역억제 동물모델에서 면역 반응을 증강시키는 결과를 확인하여 본 발명을 완성하였다. Accordingly, the present inventors have conducted multifaceted research to solve the above problem, and as a result, it is confirmed that the complex extract of gold and silver coins, gold, naitakeja and turmeric has an excellent NO production inhibitory effect and an inflammatory cytokine expression inhibitory effect, The present invention was completed by confirming the results of enhancing the immune response in the suppressed animal model.
본 발명의 목적은 상기 복합 추출물을 포함하는 면역조절 또는 면역증강용 조성물을 제공하는 데 있다. It is an object of the present invention to provide a composition for immunomodulatory or immune enhancement comprising the complex extract.
본 발명의 다른 목적은 상기 조성물을 포함하는 건강 기능성 식품을 제공하는 데 있다. Another object of the present invention is to provide a health functional food comprising the composition.
본 발명의 다른 목적은 상기 조성물을 포함하는 식품 조성물을 제공하는 데 있다. Another object of the present invention is to provide a food composition comprising the composition.
본 발명의 다른 목적은 상기 조성물을 포함하는 약학적 조성물을 제공하는 데 있다. Another object of the present invention is to provide a pharmaceutical composition comprising the composition.
본 발명의 다른 목적은 상기 복합 추출물을 포함하는 면역 기능 증진용 식품 조성물을 제공하는 데 있다.Another object of the present invention is to provide a food composition for enhancing immune function comprising the complex extract.
본 발명의 다른 목적은 상기 복합 추출물을 포함하는 항염 조성물을 제공하는 데 있다.Another object of the present invention is to provide an anti-inflammatory composition comprising the complex extract.
상기 목적을 달성하기 위해, 본 발명은 금은화, 황금, 내복자 및 강황의 복합 추출물을 포함하는 면역조절 또는 면역증강용 조성물을 제공한다. In order to achieve the above object, the present invention provides a composition for immunomodulatory or immune enhancement comprising a complex extract of gold ginseng, gold, Naibokja and turmeric.
상기 복합 추출물은 자소엽 추출물을 추가로 포함할 수 있다. The complex extract may further include perilla leaf extract.
상기 복합 추출물은 고삼 추출물, 황련 추출물, 황백 추출물 및 연교 추출물 중에서 하나 이상의 추출물을 추가로 포함할 수 있다. The complex extract may further include one or more extracts from ginseng extract, hwangryeon extract, hwangbaek extract and yeonkyo extract.
아울러, 본 발명은 상기 면역조절 또는 면역증강용 조성물을 포함하는 건강 기능성 식품, 식품 조성물, 또는 약학적 조성물을 제공한다. In addition, the present invention provides a health functional food, food composition, or pharmaceutical composition comprising the composition for immunomodulatory or immune enhancement.
본 발명에 따른 복합 추출물은 세포 독성을 갖지 않고, 염증 모델인 LPS로 자극한 대식세포에서 NO 생성 및 염증성 사이토카인 생성을 억제하는 효과를 가질 뿐 아니라, 면역억제 동물모델에서 면역 기능을 증진시키는 효과를 가지므로, 면역 기능 조절 또는 증진용 건강기능성 식품, 항염증성 건강 기능성 식품, 항염증성 식품 조성물, 또는 항염증성 약학적 조성물로 사용될 수 있다. The complex extract according to the present invention has no cytotoxicity, has an effect of inhibiting NO production and inflammatory cytokine production in macrophages stimulated with LPS, an inflammatory model, as well as enhancing immune function in immunosuppressed animal models Since it has, it can be used as a health functional food for regulating or enhancing immune function, an anti-inflammatory health functional food, an anti-inflammatory food composition, or an anti-inflammatory pharmaceutical composition.
도 1은 금은화, 황금, 내복자, 강황 및 자소엽 각각의 함량을 달리한 복합 추출물의 세포생존율에 미치는 영향을 분석한 결과이다.
도 2, 3은 금은화, 황금, 내복자, 강황 및 자소엽 각각의 함량을 달리한 복합 추출물의 NO 생성에 미치는 영향을 분석한 결과이다.
도 4는 본 발명의 복합 추출물 및 각각의 단일 추출물의 NO 생성에 미치는 영향을 분석한 결과이다.
도 5는 본 발명의 복합 추출물이 염증성 사이토카인 발현에 미치는 영향을 분석한 결과이다.
도 6은 면역억제 동물모델에 본 발명의 복합 추출물을 처리했을 때 비장지수를 분석한 결과이다.
도 7은 면역억제 동물모델에 본 발명의 복합 추출물을 처리했을 때 비장세포의 증식능을 분석한 결과이다.
도 8, 9는 면역억제 동물모델에 본 발명의 복합 추출물을 처리했을 때 복강 내 대식세포에서 면역관련 유전자의 발현양을 분석한 결과이다.
도 10은 면역억제 동물모델에 본 발명의 복합 추출물을 처리했을 때 혈청 내 NO 농도를 분석한 결과이다. 1 is a result of analyzing the effect on cell viability of a complex extract with different contents of gold and silver coins, gold, Naebokja, turmeric and perilla leaf.
2 and 3 are results of analysis of the effect on NO production of complex extracts with different contents of gold and silver coins, gold, Naibokja, turmeric, and perilla leaf.
4 is a result of analyzing the effect on NO production of the complex extract of the present invention and each single extract.
5 is a result of analyzing the effect of the complex extract of the present invention on the expression of inflammatory cytokines.
6 is a result of analyzing the spleen index when the complex extract of the present invention is treated in an immunosuppressed animal model.
7 is a result of analyzing the proliferation capacity of splenocytes when the complex extract of the present invention is treated in an immunosuppressed animal model.
8 and 9 are results of analyzing the expression level of immune-related genes in intraperitoneal macrophages when the complex extract of the present invention is treated in an immunosuppressed animal model.
10 is a result of analyzing the concentration of NO in serum when the complex extract of the present invention is treated in an immunosuppressed animal model.
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시 형태를 설명한다. 그러나, 본 발명의 실시 형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다.Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명에 의하면 금은화, 황금, 내복자의 복합 추출물을 포함하는 면역조절용 또는 면역증강용 조성물 또는 항염 조성물이 제공된다. 또한, 본 발명에 있어서, 복합 추출물에는 필요한 경우 자소엽 추출물을 추가로 포함할 수 있다. 또한, 본 발명에 있어서, 추가로 필요한 경우 고삼 추출물, 황련 추출물, 황백 추출물 및 연교 추출물 중 선택되는 하나 이상의 추출물을 추가로 포함할 수 있다. According to the present invention, there is provided a composition for immunomodulatory or immune enhancement or an anti-inflammatory composition comprising a complex extract of ginseng, gold, and Naibokki. In addition, in the present invention, the complex extract may further include perilla leaf extract if necessary. In addition, in the present invention, if necessary, it may further include one or more extracts selected from ginseng extract, hwangryeon extract, hwangbaek extract and yeonkyo extract.
금은화(Lonicerae Flos)는 인동과(Caprifoliaceae)에 속하는 인동 덩굴(Lonicera japonica Thunb.)의 꽃으로, 한방이나 민간에서는 이뇨, 건위, 관절염, 화농성 피부염, 기관지염에 사용되어 왔다. 본 발명에서 복합 추출물 제조를 위하여 상업적으로 판매되는 것을 구입해서 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수도 있다. Lonicerae Flos is a flower of Lonicera japonica Thunb. belonging to the Caprifoliaceae family. For the preparation of the complex extract in the present invention, commercially available ones may be purchased and used, or those harvested or grown in nature may be used.
황금(Scutellariae Radix)은 꿀풀과(Labiatae)에 속한 다년생 본초인 황금을 의미하며, 특히 황금의 뿌리를 거피하여 건조한 것을 의미한다. 황금은 제습열, 지혈 등의 효능이 있어 고혈압, 유행성 뇌척수막염의 치료에 사용되어 왔다. 지금까지 밝혀진 약리작용으로는 항암, 항암보조작용, 항바이러스 및 항균 작용이 있다. 본 발명에서 복합 추출물 제조를 위하여 뿌리 부분을 사용하며 상업적으로 판매되는 것을 구입해서 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수도 있다.Gold ( Scutellariae Radix ) means gold, which is a perennial herb belonging to the Lamiaceae ( Labiatae ), and in particular, it means that the root of gold is peeled and dried. Gold has effects such as dehumidifying heat and hemostasis, and has been used in the treatment of hypertension and epidemic meningitis. The pharmacological actions revealed so far include anticancer, anticancer adjuvant action, antiviral and antibacterial action. In the present invention, the root part is used for the preparation of the complex extract, and commercially available ones may be purchased and used, or those harvested or grown in nature may be used.
내복자(Raphani Semen)는 기를 통하게 하고 쳇기를 내려가게 하는 효능이 있어서 복부창만, 트림, 위산과다, 설사 등에 사용하였으며, 식욕부진을 치료하는 효과가 있다고 알려져 있다. 본 발명에서 복합 추출물 제조를 위하여 씨앗을 사용하며 상업적으로 판매되는 것을 구입해서 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수도 있다. Raphani Semen has the effect of increasing the qi and lowering the qi, so it is used only for stomach upset, belching, acid hyperacidity, diarrhea, etc. It is known to be effective in treating anorexia. In the present invention, seeds are used to prepare the complex extract, and commercially available ones may be purchased and used, or those harvested or grown in nature may be used.
강황(Curcuma longa)은 생강과 쿠르쿠마속의 여러해살이풀이며, 뿌리줄기를 주로 이용한다. 한방에서는 어혈 제거, 소화기능 개선 등의 용도로 사용되어 왔으며, 한방뿐 아니라 동서양을 막론하고 약재, 향신료 등으로 오래 전부터 사용되어 왔다. 본 발명에서 복합 추출물 제조를 위하여 강황의 뿌리줄기를 사용할 수도 있고, 상업적으로 판매되는 것을 구입해서 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수도 있다.Turmeric ( Curcuma longa ) is a perennial herb belonging to the genus Ginger and Curcuma, and its rhizome is mainly used. In oriental medicine, it has been used for the purpose of removing eohyeol and improving digestive function, and has been used for a long time as medicine and spice, not only in oriental medicine, but also in East and West. In the present invention, the rhizome of turmeric may be used for the preparation of the complex extract, commercially available ones may be purchased and used, or those harvested or grown in nature may be used.
자소엽(Perillae Folium)은 진해거담, 감기, 해열제로 사용되며 주성분은 perillaldehyde(55%), perillaketone, elsholtziaketone, rosmarinic acid 등이 알려져 있다. 본 발명에서 복합 추출물 제조를 위하여 자소엽 잎 및 끝가지를 사용할 수 있고, 상업적으로 판매되는 것을 구입해서 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수도 있다.Perillae Folium ( Perillae Folium ) is used as an expectorant for cough, cold, and fever, and its main components are perillaldehyde (55%), perillaketone, elsholtziaketone, rosmarinic acid, and the like. In the present invention, perilla leaves and tips may be used for the preparation of the complex extract, commercially available ones may be purchased and used, or those harvested or grown in nature may be used.
고삼(Sophorae Radix)은 고삼의 주피를 벗긴 뿌리로, 특이한 냄새가 있고 잔류성이며 약성은 매우 쓰고 찬 것이 특징이며, 하초습열로 인한 이질, 대하, 음부소양증, 피부가려움증 등에 사용하고 방광열로 인하여 통증이 있을 때 사용한다고 알려져 있다. 본 발명에서 복합 추출물 제조를 위하여 상업적으로 판매되는 것을 구입해서 사용하거나, 자연에서 채취 또는 재배된 것을 사용할 수도 있다. Sophorae Radix is a root with peeled skin of old ginseng. It has a characteristic odor, is persistent, and has a very bitter and cold weakness. It is known to be used when For the preparation of the complex extract in the present invention, commercially available ones may be purchased and used, or those harvested or grown in nature may be used.
황련(Coptidis rhizoma)은 항균작용, 혈압강하작용, 소염작용 등의 약리 효과가 있는 것으로 알려져 있으며, 한방에서는 건위제, 중추 신경계 작용제, 자궁, 방광, 기관지 및 신경계 작용제로 사용되고 있다. 주로 약리 성분으로서 알칼로이드(alkaloid)인 베르베린(berberine), 마그노플로린(magnoflorine) 등을 함유하고 있는 것으로 알려져 있다.Yellow lotus ( Coptidis rhizoma ) is known to have pharmacological effects such as antibacterial action, blood pressure lowering action, and anti-inflammatory action. It is known to contain mainly alkaloids, such as berberine and magnoflorine, as pharmacological components.
황백(Phellodendron amurense Ruprecht)은 약재로 사용되는 황벽나무의 껍질을 지칭하는 것으로, 황경피라고도 불리운다. 상기 황백은 하지 전후에 나무줄기에서 껍질을 벗겨내어 조피(粗皮: 거친 껍질)를 제거하거나 썰어서 햇볕에 말려 약재로 사용된다. 상기 황백은 혈당저하 작용을 하며, 폐렴쌍구균, 인형결핵균, 포도상구균 등의 발육 및 종양세포의 번식을 저지시키고, 살균작용을 하는 것으로 알려져 있다. Hwangbaek ( Phellodendron amurense Ruprecht ) refers to the bark of the Hwangbyeok tree used for medicinal purposes, and is also called Hwanggyeongpi. The hwangbaek is used as a medicine by peeling the bark from the tree trunk before and after the summer solstice, removing or cutting the rough skin (粗皮: rough bark) and drying it in the sun. It is known that the yellow white has a blood sugar lowering action, inhibits the growth of pneumococcus, tuberculosis, staphylococcus, and the like and the proliferation of tumor cells, and has a sterilizing action.
연교(Forsythiae Fructus)는 물푸레나무과에 속하는 의성개나리 또는 당개나리의 건조시킨 열매를 의미한다. 연교의 주요성분은 리그난(lignan) 화합물과 페닐프로파노이드(phenylpropanoid) 유도체 등이며 항균작용, 항염작용, 혈압강하, 해열 등의 효능이 있는 것으로 알려져 있으며, 한방에서는 심장의 열을 내리고 고열에 쓰며, 종기에 소염에 사용하고 이뇨약으로 쓰인다. 본 발명에서, 연교는 당 분야에서 통상적으로 입수할 수 있는 연교를 이용할 수 있으며, 상업적으로 판매되는 것을 사용할 수 있다.Yeongyo ( Forsythiae Fructus ) refers to the dried fruits of Uiseong Forsythia or Forsythia belonging to the family Aceae. The main ingredients of Yeongyo are lignan compounds and phenylpropanoid derivatives, which are known to have antibacterial, anti-inflammatory, blood pressure lowering and antipyretic effects. , used in boils for anti-inflammation and as a diuretic. In the present invention, soft bridges that are commonly available in the art may be used, and commercially available ones may be used.
앞서 언급한 금은화, 황금, 내복자, 강황, 자소엽은 식품원료로서 사용가능한 원료로 이들의 복합 추출물은 세포 독성을 보이지 않았다. 또한, 고삼, 황련, 황백, 연교는 모두 종래부터 이용해온 약제 원료이다.The above-mentioned ginseng, gold, jasmine, turmeric, and perilla leaf are available as food raw materials, and their complex extracts did not show cytotoxicity. In addition, ginseng, hwangryeon, hwangbaek, and yeongyo are all pharmaceutical raw materials that have been used in the past.
본 발명의 복합 추출물은 Raw 246.7 마우스 대식세포에서 LPS에 의한 NO(Nitric Oxide) 발생을 억제하였을 뿐 아니라, 염증성 사이토카인인 TNF-α, IL-1β 및 IL-6 유전자의 발현을 농도 의존적으로 억제하였다. 또한, 금은화, 황금, 내복자, 강황 및 자소엽의 함량비가 각각 전체 중량 대비 5~10 중량%, 40~70 중량%, 10~40 중량%, 5~10 중량%, 및 10~40 중량%인 복합 추출물의 경우 효과적인 항산화 및 항염효과를 나타내는 것을 확인할 수 있었다. The complex extract of the present invention not only inhibited NO (nitric oxide) generation by LPS in Raw 246.7 mouse macrophages, but also inhibited the expression of inflammatory cytokines TNF-α, IL-1β and IL-6 genes in a concentration-dependent manner did. In addition, the content ratios of gold and silver coins, gold, jasmine, turmeric and perilla leaves are 5-10 wt%, 40-70 wt%, 10-40 wt%, 5-10 wt%, and 10-40 wt%, respectively, based on the total weight In the case of the phosphorus complex extract, it was confirmed that it exhibited effective antioxidant and anti-inflammatory effects.
TNF-α, IL-1β 및 IL-6는 모두 대표적인 염증성 사이토카인으로 TNF-α(Tumor necrosis factor- α)는 미성숙 면역세포를 성숙된 형태의 세포로 전환시켜 항원에 노출된 세포의 살해능력을 증진시켜 초기 면역 반응에 중요한 역할을 하는 것으로 알려져 있다. TNF-α는 항원에 자극된 T 림프구, 자연살해세포(NK cell), 비만세포(mast cell), 활성화된 대식세포(macrophage)에서 생성된다. IL-1β(Interleukin-1β)는 TNF와 비슷하게 감염 및 다른 자극에 대한 숙주의 염증반응 매개자로서 작용하며, 대식세포, 호중구(neutrophil) 등에서 생산한다. IL-6(Interleukin-6)는 염증성 사이토카인 생산의 주 자극원으로서, 대식세포, T 세포 등에서 생성된다. TNF-α, IL-1β, and IL-6 are all representative inflammatory cytokines, and TNF-α (tumor necrosis factor-α) converts immature immune cells into mature cells, thereby enhancing the killing ability of cells exposed to antigen. It is known to play an important role in the early immune response by enhancing it. TNF-α is produced by antigen-stimulated T lymphocytes, natural killer cells (NK cells), mast cells, and activated macrophages. IL-1β (Interleukin-1β) acts as a mediator of the host's inflammatory response to infection and other stimuli, similar to TNF, and is produced by macrophages and neutrophils. IL-6 (Interleukin-6) is a major stimulator of inflammatory cytokine production, and is produced by macrophages, T cells, and the like.
NO는 대표적인 염증 매개 물질로서, 신경전달, 혈관의 이완 및 세포 매개성 면역 반응에 관여하는 등의 높은 반응성을 가지는 것으로 알려져 있다. LPS 등과 같은 자극 물질에 의해 NO 생성이 증가되는 경우, 생성된 NO는 대식세포를 활성화시킴으로써 IL-1β, IL-6, IL-8, TNF-α와 같은 염증성 사이토카인을 생산하게 된다. NO, as a representative inflammatory mediator, is known to have high reactivity, such as being involved in neurotransmission, relaxation of blood vessels, and cell-mediated immune response. When NO production is increased by a stimulant such as LPS, the generated NO activates macrophages to produce inflammatory cytokines such as IL-1β, IL-6, IL-8, and TNF-α.
또한, 본 발명의 복합 추출물은 사이클로포스파마이드(cyclophosphamide) 유도 면역억제 동물모델에서 비장지수를 높이고, 비장 유래의 T 세포 및 B 세포의 증식능을 향상시키고, 복강 내 대식 세포에서 염증성 사이토카인인 TNF-α, IL-1β 및 IL-6의 mRNA 발현을 농도 의존적으로 높이고, 면역관련 효소인 COX-2 및 iNOS 유전자의 발현 역시 농도의존적으로 높였다. 특히 본 발명의 복합 추출물은 양성 대조군인 홍삼 추출물보다 더 높은 수준으로 억제된 면역 반응을 회복시키는 결과를 보였다. 또한, 본 발명의 복합 추출물은 혈청 내 NO 농도를 정상 대조군 수준으로까지 회복시키는 결과를 보였다. In addition, the complex extract of the present invention increases the spleen index in an animal model of cyclophosphamide-induced immunosuppression, improves the proliferative capacity of spleen-derived T cells and B cells, and TNF, an inflammatory cytokine, in intraperitoneal macrophages The mRNA expression of -α, IL-1β and IL-6 was increased in a concentration-dependent manner, and the expression of COX-2 and iNOS genes, which are immune-related enzymes, was also increased in a concentration-dependent manner. In particular, the complex extract of the present invention showed a result of restoring the suppressed immune response to a higher level than the positive control, red ginseng extract. In addition, the complex extract of the present invention showed a result of restoring the serum NO concentration to the normal control level.
면역 반응은 체내에 존재하는 자기방어체계로서 인체가 외부로부터 침입해오는 각종 물질이나 병원체를 자기 자신에 대한 이물질로 인식하여 제거하고 대사시키는 과정이다. 면역 반응은 외부 자극에 의한 손상이나 병원체의 침입으로부터 자신을 방어하는 기전이지만, 염증반응 등과 같이 자기 자신의 조직에 손상을 줄 수도 있다. 면역 반응의 기본적인 작용기전이 염증반응이기 대문이다. 면역 반응은 면역 기능의 변화를 조절하여 정상으로 회복시키거나 변화의 폭을 줄여 주는 작용으로 면역 기능 억제와 면역 기능 증진으로 구분된다. The immune response is a self-defense system that exists in the body, and is a process in which the human body recognizes and removes and metabolizes various substances or pathogens that invade from the outside as foreign substances against itself. The immune response is a mechanism to defend itself from damage caused by external stimuli or invasion of pathogens, but it can also damage one's own tissues, such as an inflammatory response. This is because the basic mechanism of action of the immune response is the inflammatory response. The immune response modulates changes in immune function to restore normalcy or reduce the width of the change, and is divided into suppression of immune function and enhancement of immune function.
본 발명에서 면역 기능 증진이란 면역 반응을 조절하여 생체방어 능력을 증강시키는 기능을 의미한다. 상기 면역 기능 증진의 기능성을 확인할 수 있는 바이오 마커로는 NK 세포, 사이토카인, 탐식능, NO, iNOS, COX-2, 보체계, 림프구 아집단 비율, 면역글로블린, 세포 생존율, 비장세포 증식, 비장, 흉선 등의 조직 무게 등을 들 수 있고, 본 발명에서는 면역억제 동물 모델을 사용하여 비장 지수, 비장세포의 증식능 평가, 염증성 사이토카인 및 COX-2, iNOS 유전자 발현양 분석, 혈청 내 NO 농도 분석 등을 통해 본 발명의 복합 추출물이 면역이 억제된 개체에서 면역 기능을 회복시킨다는 것을 확인하였을 뿐 아니라, 대표적인 면역 증강 식품인 홍삼추출물에 비해 우수한 면역 기능 증진효과가 있다는 것을 확인하였다. In the present invention, the enhancement of immune function refers to the function of enhancing biological defense ability by regulating an immune response. Biomarkers that can confirm the functional enhancement of immune function include NK cells, cytokines, phagocytic capacity, NO, iNOS, COX-2, complement system, lymphocyte subpopulation ratio, immunoglobulin, cell viability, splenocyte proliferation, spleen, thymus and the like, and in the present invention, spleen index, evaluation of spleen cell proliferation ability, inflammatory cytokines, COX-2, iNOS gene expression level analysis, serum NO concentration analysis, etc. are performed using an immunosuppressed animal model. Through this, it was confirmed that the complex extract of the present invention restores immune function in immunosuppressed individuals, as well as that it has an excellent immune function enhancing effect compared to red ginseng extract, which is a representative immune enhancing food.
본 발명에서 면역 기능 억제란 증가된 면역 기능을 억제하여 정상상태로 되돌리는 기능을 의미한다. 상기 면역 기능 억제의 기능성을 확인할 수 있는 바이오마커로는 앞서 언급한 면역 기능 증진의 지표의 감소를 의미한다. 본 발명에서는 대표적인 면역세포인 Raw 246.7 마우스 대식세포에 면역증강제인 LPS를 처리하여 면역 기능을 높인 후, 본 발명의 복합 추출물을 처리하여 증가된 면역 관련 유전자 발현이 억제되는 것을 확인하였다. In the present invention, suppression of immune function refers to the function of suppressing increased immune function and returning it to a normal state. As a biomarker that can confirm the functionality of the immune function suppression, it means a decrease in the above-mentioned index of immune function enhancement. In the present invention, it was confirmed that the immune function was increased by treating Raw 246.7 mouse macrophages, which are representative immune cells, as an immune enhancer, and then, by treating the complex extract of the present invention, the increased expression of immune-related genes was suppressed.
생체 내에서 적절한 조건 하에서는 염증 초기 상태가 지난 후 정상 기능을 되찾게 되지만, 염증을 자극하는 자극제가 없어지지 않거나 계속해서 만들어질 경우에는 결과적으로 만성 염증이 일어나게 되고 이 경우, 만성 염증성 질환을 일으키게 된다. Under appropriate conditions in vivo, normal function is restored after the initial state of inflammation has passed, but if the stimulant that stimulates inflammation does not disappear or is continuously made, chronic inflammation occurs as a result, and in this case, chronic inflammatory disease occurs.
본 발명에 있어서 상기 만성 염증성 질환은 천식, 만성폐쇄성폐질환(COPD), 알레르기 비염, 피부염, 관절염, 알레르기 및 염증성 장질환으로 이루어진 그룹으로부터 선택되는 것이지만, 이에 제한되지 않는다. In the present invention, the chronic inflammatory disease is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), allergic rhinitis, dermatitis, arthritis, allergy and inflammatory bowel disease, but is not limited thereto.
본 발명에 있어서, 상기 식물의 복합 추출물을 포함하는 면역 조절용 또는 면역 증강용 조성물의 제조 방법은 금은화, 황금, 내복자, 강황, 자소엽 등의 식물을 혼합하여 추출하여 복합 추출물을 획득하는 단계; 및In the present invention, the method for producing a composition for immune regulation or immune enhancement comprising the complex extract of the plant comprises the steps of: obtaining a complex extract by mixing and extracting plants such as gold and silver ginseng, gold, Naebokja, turmeric, perilla leaf; and
획득된 복합 추출물을 농축 및 동결건조하는 단계를 포함한다. Concentrating and lyophilizing the obtained complex extract.
상기 복합 추출물을 추출하는 방법은 열수 추출, 주정추출, 알코올 추출, 분획법 등의 일반적인 추출방법이 사용될 수 있으며, 본 발명의 일 실시예에서는 열수 추출법을 사용한다. As a method of extracting the complex extract, a general extraction method such as hot water extraction, alcohol extraction, alcohol extraction, fractionation method, etc. may be used, and in an embodiment of the present invention, hot water extraction method is used.
본 발명의 복합 추출물은 면역 조절용 또는 면역 증강용 건강기능성 식품 조성물에 포함될 수 있다. The complex extract of the present invention may be included in a health functional food composition for immune modulation or immune enhancement.
또한, 본 발명의 면역 조절용 또는 면역 증강용 조성물은 건강 기능성 식품 조성물, 식품 조성물 또는 약학적 조성물에 포함될 수 있다. In addition, the composition for immune modulation or immune enhancement of the present invention may be included in a health functional food composition, a food composition or a pharmaceutical composition.
본 발명에 있어서, 상기 기술된 식품 조성물 또는 건강기능성 식품 조성물은 상기 유효성분 이외에, 식품 제조시에 통상적으로 첨가되는 성분을 포함할 수 있으며, 예를 들어, 단백질, 탄수화물, 지방, 영양소, 조미제, 감미제 및 향미제를 포함할 수 있으나, 이에 한정되는 것은 아니다. 상기 탄수화물의 예로는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어, 말토스, 슈크로스, 올리고당 등; 및 폴리사카라이드, 예를 들어, 덱스트린, 사이클로 덱스트린 등; 과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜을 들 수 있다. 상기 감미제로는 천연 감미제(타우마틴, 스테비아 추출물, 레바우디오시드 A, 글리시리진 등) 및 합성 감미제(사카린, 아스파르탐 등)를 사용할 수 있다. In the present invention, the above-described food composition or health functional food composition may include, in addition to the active ingredient, ingredients commonly added during food production, for example, proteins, carbohydrates, fats, nutrients, and seasonings. , sweeteners and flavoring agents, but are not limited thereto. Examples of the carbohydrate include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides such as dextrin, cyclodextrin, and the like; and sugar alcohols such as conventional sugars such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners (taumatin, stevia extract, rebaudioside A, glycyrrhizin, etc.) and synthetic sweeteners (saccharin, aspartame, etc.) may be used.
그러나 이들로 한정되는 것은 아니며, 이 분야에서 공지된 성분들로서 본 발명의 효과를 저해하지 않는 성분들은 모두 사용될 수 있다.However, the present invention is not limited thereto, and any component that does not impair the effects of the present invention as components known in the art may be used.
상기 식품 조성물 또는 건강기능성 식품 조성물의 예로는 환자영양식, 육류, 곡류, 카페인 음료, 일반음료, 유제품, 초콜렛, 빵류, 스넥류, 과자류, 피자, 젤리, 면류, 껌류, 아이스크림류, 알코올성 음료, 술, 비타민 복합제, 그 밖의 건강보조식품류 등을 들 수 있으나, 이들로 한정되는 것은 아니다. 상기와 같은 식품 조성물 또는 건강기능성 식품 조성물의 형태로 제조될 경우, 편하고 쉽게 복용할 수 있어서 바람직하다.Examples of the food composition or health functional food composition include patient nutrition, meat, grains, caffeinated beverages, general drinks, dairy products, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice cream, alcoholic beverages, alcohol, and vitamin complexes and other health supplements, but is not limited thereto. When prepared in the form of a food composition or health functional food composition as described above, it is preferable because it is convenient and easy to take.
본 발명에 있어서 상기 건강기능성 식품 조성물 및 약학 조성물은 과립제(granules), 리모나데제(lemonades), 산제(powders), 시럽제(syrups), 액제(liquids and solutions), 엑스제(extracts), 엘릭실제(elixirs), 유동엑스제(fluidextracts), 현탁제(suspesions), 전제(decoctions), 침제(infusions), 정제(tablets), 주정제(spirits), 캅셀제(capsules), 트로키제(troches), 환제(pills), 연질 또는 경질 젤라틴 캅셀제 형태로 제조될 수 있으나, 이들로 한정되는 것은 아니다. In the present invention, the health functional food composition and pharmaceutical composition are granules, limonades, powders, syrups, liquids and solutions, extracts, and elixirs. (elixirs), fluidextracts, suspensions, decoctions, infusions, tablets, spirits, capsules, troches, pills (pills), may be prepared in the form of soft or hard gelatin capsules, but is not limited thereto.
상기 약학 조성물은 약제학적으로 허용되는 담체를 더 포함할 수 있다. 본 발명의 약학 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. The pharmaceutical composition may further include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, silicic acid. calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it is not
본 발명의 약학 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally.
본 발명의 약학 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. Alternatively, it may be prepared by being introduced into a multi-dose container.
또한, 상기 성분들을 포함하는 약학적 조성물, 식품 조성물, 건강기능성 식품 조성물은 1일 1회 또는 2회 이상으로 분할하여 투여할 수 있다. In addition, the pharmaceutical composition, food composition, and health functional food composition comprising the above components may be administered once a day or divided into two or more times.
그러나 상기의 투여량은 일 예에 불과하며, 복용자의 상태에 따라 의사의 처방에 의해 변화될 수 있다.However, the above dosage is only an example, and may be changed by a doctor's prescription depending on the user's condition.
이하, 본 발명을 하기 실시예에 의거하여 상세하게 설명하고자 한다. 단, 하기 실시예는 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail based on the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.
실시예 1. 금은화, 황금, 내복자, 강황 및 자소엽의 복합물의 추출Example 1. Extraction of a complex of ginseng, gold, sagebrush, turmeric and perilla leaf
건조 금은화, 건조 황금, 건조 내복자, 건조 강황 및 건조 자소엽은 태풍농산(서울)에서 입수하였다. 금은화, 황금, 내복자, 강황 및 자소엽을 아래 표 1에 나타낸 각각의 비율에 따라 혼합하여 10~15배의 물로 열수 추출(80~100℃, 30~90분) 후 농축 및 동결건조하였다. Dried gold and silver coins, dried gold, dried jasmine, dried turmeric and dried perilla were obtained from Typhoon Nongsan (Seoul). Gold ginseng, gold, sagebrush, turmeric and perilla leaf were mixed according to the respective ratios shown in Table 1 below, followed by hot water extraction (80-100° C., 30-90 minutes) with 10 to 15 times water, followed by concentration and freeze-drying.
실시예 2: 금은화, 황금, 내복자, 강황 및 자소엽 복합 추출물의 세포생존율에 미치는 영향 분석Example 2: Analysis of the effect on cell viability of the extracts of gold and silver coins, gold, naitake, turmeric and perilla leaf complex extract
금은화, 황금, 내복자, 강황 및 자소엽 복합 추출물이 세포 생존에 미치는 영향을 확인하기 위하여 마우스 대식세포인 Raw 264.7 세포에 복합 추출물을 처리한 후 세포 생존율을 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide(MTT) 분석법으로 측정하였다.In order to check the effect of the complex extracts of gold, silver, goldenrod, turmeric, and perilla leaf on cell survival, the cell viability of Raw 264.7 cells, a mouse macrophage, was treated with 3-[4,5-Dimethylthiazol-2 -yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay was used.
Raw 264.7 세포를 24 well plate에 5×104cell/well의 밀도로 분주하여 24시간 동안 37℃, 5% CO2 배양기에서 배양한 후 시료 1 내지 7의 복합 추출물(200 ug/mL), LPS(Positive control, 1 ug/mL)를 세포에 처리하고 37℃, 5% CO2 배양기에서 24 시간 동안 배양하였다. Raw 264.7 cells were aliquoted in a 24 well plate at a density of 5×10 4 cells/well and cultured at 37° C., 5% CO 2 in an incubator for 24 hours, and then the complex extract of
배양이 완료된 후 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 용액을 배지에 1/10 비율로 첨가한 후 37℃, 5% CO2 배양기에서 3 시간 동안 추가 배양한 후 DMSO(Dimethyl sulfoxide) 700ul를 이용하여 Formazan 염료를 용출하였다. 96 well plate에 각 well당 40ul씩 Duplication하였고 550nm에서 흡광도를 측정하였다. After the culture was completed, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution was added to the medium at a ratio of 1/10, and then incubated at 37°C, 5% CO 2 for 3 hours. After additional incubation for a while, Formazan dye was eluted using 700ul of DMSO (dimethyl sulfoxide). In a 96-well plate, 40ul of each well was duplicated, and absorbance was measured at 550nm.
그 결과를 도 1에 나타내었다. 세포 생존율 측정 결과 시료 1 내지 7의 복합 추출물은 모두 세포 독성을 나타내지 않는 것을 확인할 수 있었다(N은 무처리군, LPS는 LPS 처리군, 숫자는 시료명임). (**는 N과 대비하여 P<0.05 임)The results are shown in FIG. 1 . As a result of cell viability measurement, it was confirmed that all of the complex extracts of
실시예 3: LPS로 활성화된 Raw 264.7 세포에서 복합 추출물의 NO 생성 저해 작용Example 3: NO production inhibitory effect of the complex extract in Raw 264.7 cells activated with LPS
금은화, 황금, 내복자, 강황 및 자소엽 복합 추출물의 항염증 효능을 분석하기 위하여 염증 유발 인자인 LPS에 의한 NO 생산을 각 복합 추출물이 억제할 수 있는지 분석하였다. In order to analyze the anti-inflammatory efficacy of the extracts of gold ginseng, gold, jasmine, turmeric and perilla leaf, it was analyzed whether each complex extract could inhibit NO production by LPS, an inflammatory factor.
Raw 264.7 세포를 5×104 cell/well의 밀도로 24 well plate에 분주하고 37℃, 5% CO2 배양기에서 24시간 동안 배양하였다. 시료 1 내지 7의 복합 추출물을 200 ug/mL 농도로 세포에 처리하고 37℃, 5% CO2 배양기에서 1시간 동안 배양한 후 LPS(Lipopolysaccharides)를 최종 농도 1ug/ml로 분주하며, 37℃, 5% CO2 배양기에서 24시간 동안 배양함. 배양이 완료된 후 상등액을 96 well plate에 100 ul씩 옮기고 Griess reagent A와 B(1% Sulfanilic acid 용액, 1% N-(1-naphthyl)ethylene diamine 용액)를 1:1의 비율로 혼합하여 well당 100 ul로 처리한 후 540 nm에서 흡광도를 측정하였다.Raw 264.7 cells were aliquoted in a 24 well plate at a density of 5×10 4 cells/well and cultured at 37° C., 5% CO 2 in an incubator for 24 hours. The complex extracts of
그 결과를 도 2에 나타내었다. 금은화, 황금, 내복자, 강황 및 자소엽 복합 추출물의 시료 중 시료명 5, 6, 7이 의미 있는 NO 생성 억제효과를 보였고, 특히 시료명 7은 LPS 대비 50% 이상의 NO 저해 효과를 보였다(N은 무처리군, LPS는 LPS 처리군, 숫자는 시료명임). (**는 LPS와 대비하여 P<0.05 임) The results are shown in FIG. 2 .
따라서, 시료명 7의 복합 추출물이 NO 생성 억제에 있어 가장 우수한 효과를 가진다는 것을 알 수 있다. Therefore, it can be seen that the complex extract of
추가적으로 금은화, 황금, 내복자, 강황, 자소엽의 함량비의 범위를 분석하기 위해 시료명 7-1, 7-2. 7-3의 추출물을 실시예 1의 방법으로 제조하였다. In addition, sample names 7-1 and 7-2 to analyze the range of content ratios of gold and silver coins, gold, sagebrush, turmeric, and perilla leaf. The extract of 7-3 was prepared by the method of Example 1.
시료명 7-1, 7-2, 7-3의 복합 추출물의 세포 생존에 미치는 영향을 확인하기 위해 실시예 2의 방법에 따라 분석한 결과 시료명 7-1 내지 7-3의 복합 추출물은 모두 세포 독성을 나타내지 않는 것을 확인하였다. As a result of analysis according to the method of Example 2 to determine the effect of the complex extracts of sample names 7-1, 7-2, and 7-3 on cell survival, the complex extracts of sample names 7-1 to 7-3 were all cytotoxic. It was confirmed that it does not show .
또한, 각 복합 추출물에 의한 NO 생성 저해 효과를 분석하기 위해 앞서 시료명 1 내지 7을 분석한 방법과 동일한 방법으로 NO 생성 억제 효과를 분석한 결과 시료명 7과 유사한 결과를 확인하였다(도 3). (**는 LPS와 대비하여 P<0.05 임)In addition, in order to analyze the NO production inhibitory effect of each complex extract, the NO production inhibitory effect was analyzed in the same manner as in the previous analysis of
따라서, 금은화, 황금, 내복자, 강황 및 자소엽의 함량비가 각각 전체 중량 대비 5~10 중량%, 40~70 중량%, 10~40 중량%, 5~10 중량%, 및 10~40 중량%인 경우 염증 유발 모델에서 효과적인 항산화 및 항염효과를 나타내는 것을 알 수 있다. Accordingly, the content ratios of gold and silver coins, gold, jasmine, turmeric and perilla leaf are 5 to 10% by weight, 40 to 70% by weight, 10 to 40% by weight, 5 to 10% by weight, and 10 to 40% by weight, respectively, based on the total weight. In the case of , it can be seen that it exhibits effective antioxidant and anti-inflammatory effects in the inflammation-inducing model.
실시예 4: 단일원료 대비 복합물의 시너지 효과 확인Example 4: Confirmation of the synergistic effect of the composite compared to a single raw material
금은화, 황금, 내복자, 강황 및 자소엽 복합 추출물의 시너지 효과를 확인하기 위해 금은화, 내복자, 강황 및 자소엽 각각의 단일 추출물을 실시예 1의 방법으로 제조하였다. In order to confirm the synergistic effect of the complex extracts of Geumgeumhwa, Geumgeumhwa, Naebokja, turmeric and perilla leaf, single extracts of each of Geumgeumhwa, Naebokja, turmeric and perilla leaf were prepared by the method of Example 1.
금은화, 내복자, 강황 및 자소엽 각각의 단일 추출물 및 시료명 7의 복합 추출물 500ug/mL처리하였을 때의 NO 생성 억제 효과를 실시예 3의 방법으로 분석하였다. The NO production inhibitory effect of 500 ug/mL of a single extract of each single extract of Geumgeumhwa, Naebokja, turmeric and perilla leaf and the complex extract of
그 결과를 도 3에 나타내었다. 금은화, 내복자, 강황, 및 자소엽 각각의 단일 추출물은 LPS에 의해 유도된 NO 생성을 억제하지 못했다. 하지만, 시료명 7의 복합 추출물은 공지된 iNOS 억제제인 L-NIL(20uM)보다 우수한 정도로 NO 생성을 억제하는 것을 확인할 수 있었다. The results are shown in FIG. 3 . Single extracts of each of ginseng chrysanthemum, ginseng, turmeric, and perilla leaf did not inhibit LPS-induced NO production. However, it was confirmed that the complex extract of
따라서, 본 발명의 복합 추출물(시료명 7)은 각각의 개별 추출물에 비해 우수한 시너지 효과를 가지는 것을 확인할 수 있었다. (**는 LPS와 대비하여 P<0.05 임)Therefore, it was confirmed that the composite extract of the present invention (Sample Name 7) had an excellent synergistic effect compared to each individual extract. (** is P<0.05 versus LPS)
실시예 5: 각 복합 추출물에서 필수 성분의 선정Example 5: Selection of essential components in each complex extract
금은화, 황금, 내복자, 강황 및 자소엽 복합 추출물에서 필수 구성성분을 분석하기 위해 각 성분을 하나씩 뺀 복합 추출물을 아래의 표 3과 같이 시료명 8 내지 12의 복합 추출물을 실시예 1의 방법으로 제조하였다. In order to analyze the essential components in the complex extracts of gold, gold, gold, turmeric and perilla leaf, as shown in Table 3 below, the complex extract of sample names 8 to 12 was prepared by the method of Example 1. did.
시료명 8 내지 12의 복합 추출물의 NO 생성 억제능을 시료명 7의 복합 추출물과 대비한 결과, 시료명 10의 경우 시료명 7과 유사한 억제능을 보였으나, 시료명 8, 9, 11, 12는 NO 생성 억제능이 감소한 결과를 보였다. As a result of comparing the NO production inhibitory ability of the complex extracts of sample names 8 to 12 with the complex extract of
따라서, 본 발명의 금은화, 황금, 내복자, 강황 및 자소엽 복합 추출물에서 필수 성분은 금은화, 황금, 강황 및 자소엽인 것을 알 수 있었다. Therefore, it was found that essential components in the complex extract of gold and silver coins, gold, turmeric, and perilla leaf of the present invention were gold and silver coins, gold, turmeric and perilla leaf.
실시예 6: 복합 추출물 처리에 의한 염증 관련 사이토카인 발현 분석Example 6: Inflammation-related cytokine expression analysis by complex extract treatment
복합 추출물이 실제 면역 증강 모델(염증 모델)에서 면역 억제 효과(항염 효과)가 있는지 확인하기 위해 시료명 7의 복합 추출물 처리 후 세포에서의 염증성 사이토카인 유전자의 발현을 분석하였다. In order to confirm whether the complex extract has an immunosuppressive effect (anti-inflammatory effect) in an actual immune enhancement model (inflammatory model), the expression of inflammatory cytokine genes in cells after treatment with the complex extract of
Raw 264.7 세포를 3x105cell/plate의 밀도로 60mm dish에 분주하고 37oC 5% CO2 배양기에서 24시간 동안 배양한 후, 복합 추출물을 각 농도별로(125ug/ml, 250ug/ml, 500ug/ml, 750ug/ml) 세포에 처리하고 37oC 5% CO2 배양기에서 1시간 동안 배양한 후 LPS (Lipopolysaccharides)를 최종 농도 1μg/ml로 분주하였다. 37oC, 5% CO2 배양기에서 24시간 동안 배양한 후 qRT-PCR을 통해 대표적인 염증성 사이토카인으로 알려진 TNF-α, IL-1β및 IL-6 유전자의 발현을 분석하였다. Raw 264.7 cells were dispensed into a 60mm dish at a density of 3x10 5 cell/plate and cultured in a 37 o
그 결과를 도 4에 결과를 나타내었다. 본 발명의 복합 추출물은 LPS에 의해 증가된 염증성 사이토카인인 TNF-α, IL-1β 및 IL-6 유전자의 발현을 처리 농도 의존적으로 효과적으로 억제하였고, 이러한 결과로부터 본 발명의 복합 추출물은 과도한 염증반응을 억제하는 항염효과를 가질 것이라는 것을 알 수 있다(**는 LPS(+)와 대비하여 P<0.05 임). The results are shown in FIG. 4 . The complex extract of the present invention effectively inhibited the expression of TNF-α, IL-1β and IL-6 genes, which are inflammatory cytokines increased by LPS, in a treatment concentration-dependent manner. It can be seen that it will have an anti-inflammatory effect that inhibits (** is P<0.05 compared to LPS(+)).
실시예 7: 면역억제 동물모델에서 복합 추출물의 면역활성 증진효과 분석Example 7: Analysis of immune activity enhancement effect of complex extract in immunosuppressed animal model
본 발명의 복합 추출물이 실제 면역 기능 증진 효과가 있는지 확인하기 위해 면역억제 동물모델을 제작하여 복합 추출물 처리 후 다양한 면역 기능을 분석하였다.In order to check whether the complex extract of the present invention has an actual immune function enhancing effect, an immunosuppressed animal model was prepared and various immune functions were analyzed after treatment with the complex extract.
특정 병원체가 없는(specific pathogen free; SPF) 5주령의 암컷 Balb/c mouse 35두를 두열바이오텍에서 제공받아 강원대학교 동물실험윤리위원회의 승인 후 (승인번호: KW211021-2) 1주일 동안 일반사료로 적응 후 약물을 투여하였다. 35 Specific pathogen free (SPF), 5-week-old female Balb/c mice were provided by Dooyeol Biotech and were approved by the Animal Experimental Ethics Committee of Kangwon National University (Approval No.: KW211021-2) as general feed for one week. After adaptation, the drug was administered.
면역억제를 위해 사이클로포스파마이드(cyclophosphamide; CPA) 150 mg/kg(체중)의 농도로 멸균된 PBS(phosphate buffered saline, pH 7.2)에 용해하여 1회 복강 주사한 후, 48시간 후 110 mg/kg(체중)의 농도로 2차 복강 주사하였고, 24시간 이후 복합 추출물을 투여하였다. For immunosuppression, cyclophosphamide (CPA) was dissolved in sterile PBS (phosphate buffered saline, pH 7.2) at a concentration of 150 mg/kg (body weight) and injected intraperitoneally once, and after 48 hours, 110 mg/kg/kg The second intraperitoneal injection was performed at a concentration of kg (body weight), and the complex extract was administered after 24 hours.
정상 대조군(Normal)은 동량의 PBS를 복강주사하였다. The normal control group (Normal) was intraperitoneally injected with the same amount of PBS.
T1 및 T2의 각 실험군은 아래의 표 4와 같이 복합 추출물(시료명 7)을 1주일 동안 50 mg/kg, 100 mg/kg의 용량으로 경구투여하였고, 양성 대조군으로는 홍삼 추출물(한국인삼유통공사)을, 음성 대조군으로는 PBS를 경구투여하였다. For each experimental group of T1 and T2, the complex extract (sample name 7) was orally administered at doses of 50 mg/kg and 100 mg/kg for 1 week as shown in Table 4 below, and as a positive control group, red ginseng extract (Korea Ginseng Distribution Corporation) ), and as a negative control, PBS was orally administered.
(n=4)army
(n=4)
(CPA)cyclophosphamide
(CPA)
(일 단위 투여량)Volume
(daily unit dose)
실시예 7-1: 비장지수(Spleen Index) 산정Example 7-1: Spleen Index calculation
비장은 면역 반응과 밀접한 관련이 있는 장기로, 그 크기나 세포의 수가 면역 반응의 직접적인 지표로 사용된다. 시험물질을 섭취시킨 실험동물에서 적출한 비장을 생리식염수(PBS)로 세척한 후 여과지로 수분을 제거하고 무게를 측정하였다. 실험동물의 체중간 차이에 따른 오차를 없애고 이를 표준화하기 위하여 적출한 비장의 무게와 마우스의 체중을 바탕으로 아래 공식에 따라 비장지수를 구하였다. (조직무게: 절대무게(측정무게), 조직무게를 체중으로 나눈 값: 상대무게)The spleen is an organ closely related to the immune response, and its size or number of cells is used as a direct indicator of the immune response. The spleen extracted from the experimental animals ingested with the test substance was washed with physiological saline (PBS), water was removed with filter paper, and the weight was measured. The spleen index was calculated according to the formula below based on the weight of the extracted spleen and the weight of the mouse in order to eliminate the error due to the difference between the body weights of the experimental animals and to standardize it. (Tissue weight: absolute weight (measured weight), tissue weight divided by body weight: relative weight)
상대무게 (Relative weight) = 
비장지수 (Spleen index) = 
측정 결과 양성 대조군 대비 본 발명의 복합 추출물을 농도별로 처리한 T1, T2에서 모두 비장 지수가 증가하였다. 비장지수의 증가는 면역 증진의 지표로 사용되므로, 본 발명의 복합추출물은 면역 증진 효능이 있음을 확인하였다(도 5). (**는 Negative control와 대비하여 P<0.05 임)As a result of the measurement, the spleen index increased in both T1 and T2 treated with the complex extract of the present invention by concentration compared to the positive control group. Since the increase in the spleen index is used as an indicator of immune enhancement, it was confirmed that the complex extract of the present invention has an immune enhancing effect (FIG. 5). (** is P<0.05 compared to negative control)
실시예 7-2: 비장세포의 증식능 평가Example 7-2: Evaluation of proliferative capacity of splenocytes
앞서 실시예 6-1에서 무게측정이 끝난 비장을 1.5ml 마이크로튜브에 옮긴 후 RPMI1640 배지와 조직 균질기(Cell Strainer)를 이용하여 비장조직 현탁액을 제조한 후, 세포 스트레이너(Cell strainer)를 이용하여 여과한 후 적혈구 용혈 완충액(Red 일반적으로 사용되는 것 같아 수정하였습니다. 확인 부탁드립니다. Blood Cell lysing Buffer)를 이용하여 적혈구를 제거하여 단일세포를 얻었다.After transferring the spleen weighed in Example 6-1 to a 1.5ml microtube, a spleen tissue suspension was prepared using RPMI1640 medium and a tissue homogenizer (Cell Strainer), and then using a cell strainer. After filtration, red blood cell lysis buffer (Red seems to be commonly used, so it was modified. Please check. Blood Cell lysing Buffer) was used to remove red blood cells to obtain single cells.
수득한 비장 단일세포에 10% FBS와 1% 페니실린/스트렙토마이신이 함유된 complete RPMI 1640 배지를 첨가한 단일세포 현탁액은 2×105cell/well의 농도로 24 well 플레이트에 접종하였다(seeding). T 세포 증식유도를 위해 5 μg/mL의 콘카나발린(concanavalin A;ConA)를, B 세포 증식유도를 위해 1 μg/mL의 LPS를 각각 처리하여 48시간동안 배양한 후, 이지-사이톡스(EZ-Cytox)를 배지의 1/10만큼 첨가한 이후 4 시간 동안 반응시키고 450nm에서 흡광도를 측정하였다. A single cell suspension in which complete RPMI 1640 medium containing 10% FBS and 1% penicillin/streptomycin was added to the obtained spleen single cells was inoculated in a 24-well plate at a concentration of 2×10 5 cells/well (seeding). 5 μg/mL of concanavalin A; ConA for inducing T cell proliferation, and 1 μg/mL of LPS for inducing B cell proliferation, respectively, and culturing for 48 hours, EZ-Cytox ( EZ-Cytox) was added as much as 1/10 of the medium, followed by reaction for 4 hours and absorbance was measured at 450 nm.
도 6에 나타난 바와 같이 양성 대조군, 음성 대조군, 및 양성 대조군에 비해 T1 및 T2군에서 T 세포 및 B 세포 모두 2배 이상의 우수한 증식능을 가지는 것을 확인하였다. (**는 Negative control 대비하여 P<0.05 임)As shown in FIG. 6 , it was confirmed that both T cells and B cells in the T1 and T2 groups had an excellent proliferative capacity of two or more times compared to the positive control, the negative control, and the positive control. (** is P<0.05 compared to negative control)
실시예 7-3: 복강내 대식세포의 유전자 발현 평가Example 7-3: Evaluation of gene expression in intraperitoneal macrophages
시험물질을 섭취한 실험동물을 희생한 후, 복강 내 주사를 통해 5 mL의 PBS를 주입하였다. 복강 마사지를 통해 복부로 대식세포가 모이도록 유도한 후 대식세포 현탁액을 회수하여 원심분리로 세포 펠렛(Cell pellet)을 얻었다. 상기 세포 펠렛에서 RNA를 분리한 후 정량 RT-PCR 법을 통해 면역 반응에 관계된 TNF-α, IL-1β, IL-6, COX-2 및 iNOS의 유전자 발현을 분석하였다. After sacrificing the experimental animals ingesting the test substance, 5 mL of PBS was injected through the intraperitoneal injection. After induced to collect macrophages to the abdomen through abdominal massage, the macrophage suspension was recovered and a cell pellet was obtained by centrifugation. After RNA was isolated from the cell pellet, gene expression of TNF-α, IL-1β, IL-6, COX-2 and iNOS related to the immune response was analyzed through quantitative RT-PCR.
각각의 유전자를 증폭하기 위한 프라이머 서열은 다음과 같다. The primer sequences for amplifying each gene are as follows.
Reverse: 5' - GGG AGT TGC TGT TGA AGT - 3' (18 mer)Forward: 5' - AAC CTG CCA AGT ATG ATG AC - 3' (20 mer)
Reverse: 5' - GGG AGT TGC TGT TGA AGT - 3' (18 mer)
Reverse: 5' - TTG CTA CGA CGT GGG CTA CA - 3' (20 mer)Forward: 5' - AGA CCC TCA CAC TCA GAT CAT C - 3' (22 mer)
Reverse: 5' - TTG CTA CGA CGT GGG CTA CA - 3' (20 mer)
Reverse: 5' - TTG GTA TTG CTT GGG ATC C - 3' (19 mer)Forward: 5' - ACC TGT CCT GTG TAA ATG AAA GAC G - 3' (25 mer)
Reverse: 5' - TTG GTA TTG CTT GGG ATC C - 3' (19 mer)
Reverse: 5' - GAA GTA GGG AAG GCC GTG G - 3' (19 mer)Forward: 5' - CTC TGC AAG AGA CTT CCA TCC AGT - 3' (24 mer)
Reverse: 5' - GAA GTA GGG AAG GCC GTG G - 3' (19 mer)
Reverse: 5' - CTT GAA GTG GGT CAG GAT GTA G - 3' (22 mer)Forward: 5' - CGG ACT GGA TTC TAT GGT GAA A - 3' (22 mer)
Reverse: 5' - CTT GAA GTG GGT CAG GAT GTA G - 3' (22 mer)
Reverse: 5' - GCT GTG GTC ACA GAA GTC TCG AAC TC - 3' (26 mer)Forward: 5' - AAT GGC AAC ATC AGG TCG GCC TAC ATC - 3' (27 mer)
Reverse: 5' - GCT GTG GTC ACA GAA GTC TCG AAC TC - 3' (26 mer)
분석 결과 TNF-α 유전자는 CPA 처리에 의해 면역활성이 저해된 처리군(음성 대조군)에서는 정상군에 비해 10% 정도의 발현양을 보이고, 양성 대조군(홍삼 추출물 처리군)에서는 음성 대조군의 면역 억제효과를 크게 개선하지는 못했지만, 본 발명의 복합 추출물 처리에 의해 농도 의존적으로 증가하는 양상을 보였으나(T1, T2), 정상군에는 미치지 못했다. As a result of the analysis, the TNF-α gene showed an expression level of about 10% in the treatment group (negative control group) whose immune activity was inhibited by CPA treatment compared to the normal group, and in the positive control group (red ginseng extract treatment group), the immune suppression of the negative control group Although the effect was not significantly improved, it showed a concentration-dependent increase by the treatment of the complex extract of the present invention (T1, T2), but did not reach the normal group.
IL-1β 유전자와 IL-6 유전자는 모두 본 발명의 복합 추출물 처리로 양성 대조군보다 높은 수준으로 발현하였고, 특히 정상 대조군 정도로 유전자 발현양이 회복하는 양상을 보였다(도 7). (*은 Negative control와 대비하여 P<0.05 이고, **는 P<0.05 임)Both the IL-1β gene and the IL-6 gene were expressed at a higher level than the positive control by the complex extract treatment of the present invention, and in particular, the gene expression level was restored to the level of the normal control ( FIG. 7 ). (* is P<0.05 compared to negative control, ** is P<0.05)
PGE2 등의 신호전달물질을 생산하는 COX-2와 NO를 생산하는 iNOS 유전자는 모두 본 발명의 복합 추출물 처리에 의해 농도의존적으로 발현양이 증가하였다(도 8). (*은 Negative control와 대비하여 P<0.05 임)Both COX-2 producing signal transmitters such as PGE 2 and iNOS gene producing NO were increased in concentration-dependently by the complex extract treatment of the present invention (FIG. 8). (* is P<0.05 compared to negative control)
실시예 7-4: 혈청내 NO 농도 분석Example 7-4: Analysis of Serum NO Concentration
시험물질을 섭취시킨 실험동물을 희생시키고 심장 채혈로 혈액을 채취하여 응고시킨 후, 원심분리를 통해 혈청을 획득하였다. 혈청을 PBS로 희석하여 96 well 플레이트에 100 ul씩 분주한 이후 그리스 용액(Griess reagent) A와 B(1% Sulfanilic acid 용액, 1% N-(1-naphthyl)ethylene diamine 용액)를 1:1의 비율로 혼합하여 well당 100 ul로 처리한 후 540 nm에서 흡광도를 측정하였다. After sacrificing the experimental animals ingesting the test substance, blood was collected by cardiac bleed and coagulated, and serum was obtained by centrifugation. After diluting the serum with PBS and dispensing 100 ul each in a 96-well plate, a 1:1 ratio of Griess reagent A and B (1% sulfanilic acid solution, 1% N-(1-naphthyl)ethylene diamine solution) After mixing at a ratio of 100 ul per well, absorbance was measured at 540 nm.
측정 결과 음성 대조군에 비해 정상 대조군에서 혈청 내 NO의 양이 증가하였다는 것을 확인하였다. 또한, T2군에서 혈중 NO의 양이 크게 증가한 것을 확인할 수 있었다. As a result of the measurement, it was confirmed that the amount of NO in the serum was increased in the normal control group compared to the negative control group. In addition, it was confirmed that the amount of NO in the blood was significantly increased in the T2 group.
NO는 여러 조직에서 신경전달물질의 역할을 하면서, 혈관확장도를 조절하고 내림프액(endolymph)와 이온의 항상성을 유지하며, 면역 반응에 주요한 역할을 하는 것으로 알려져 있으므로, 혈중 NO의 증가는 면역력이 증가하였다고 할 수 있다. 즉, 본 발명의 복합 추출물은 면역이 저하된 경우, 면역 기능을 상승시킬 수 있다고 할 수 있다. (*은 Negative control와 대비하여 P<0.05 임) NO plays a role as a neurotransmitter in various tissues, regulates vasodilation, maintains endolymph and ion homeostasis, and is known to play a major role in immune response. can be said to have increased. That is, it can be said that the complex extract of the present invention can increase immune function when immunity is lowered. (* is P<0.05 compared to negative control)
본 발명의 모든 실시예에서의 각 실험은 3 회 이상 반복하였다. 결과는 평균 ± SD로 표시된다. 대조군과 샘플 그룹 간의 통계적 유의성은 SPSS (v.23, Windows 용)를 사용하여 one way-ANOVA와 Dunett test 검정을 실시하였다.Each experiment in all Examples of the present invention was repeated three or more times. Results are presented as mean ± SD. For statistical significance between control and sample groups, one way-ANOVA and Dunett test were performed using SPSS (v.23, for Windows).
이상에서 설명한 본 발명은 전술한 실시예 및 첨부한 도면에 의해 한정되지 않으며, 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 치환, 변형 및 변환이 가능하다는 것을 본 발명이 속하는 기술 분야에서, 통상의 지식을 가진 자에게 있어 명백할 것이다.The present invention described above is not limited by the above-described embodiments and the accompanying drawings, and it is common in the technical field to which the present invention pertains that substitutions, modifications and transformations are possible within the scope without departing from the technical spirit of the present invention. It will be clear to those who have knowledge.
Claims (17)
상기 조성물은 자소엽 추출물을 추가로 포함하는, 면역조절 또는 면역증강용 조성물.According to claim 1,
The composition further comprises a perilla leaf extract, immunomodulatory or immune enhancing composition.
상기 조성물은 고삼 추출물, 황련 추출물, 황백 추출물 및 연교 추출물에서 하나 이상의 추출물을 추가로 포함하는, 면역조절 또는 면역증강용 조성물. 3. The method of claim 2,
The composition further comprises one or more extracts from ginseng extract, hwangryeon extract, hwangbaek extract and yeonkyo extract, immunomodulatory or immune-enhancing composition.
상기 조성물의 금은화, 황금, 내복자, 강황 및 자소엽의 함량비는 복합 추출물 전체 중량을 기준으로 5~10 중량%, 40~70 중량%, 10~40 중량%, 5~10 중량%, 및 10~40 중량%인, 면역조절 또는 면역증강용 조성물.3. The method of claim 2,
The content ratios of gold and silver coins, gold, Naebokja, turmeric and perilla leaf in the composition are 5 to 10% by weight, 40 to 70% by weight, 10 to 40% by weight, 5 to 10% by weight, and 10 to 40% by weight, immunomodulatory or immune enhancement composition.
상기 면역 기능 증진용 건강기능성 식품 조성물은 자소엽 추출물을 추가로 포함하는 면역 기능 증진용 건강기능성 식품 조성물.8. The method of claim 7,
The functional health food composition for enhancing immune function is a health functional food composition for enhancing immune function, further comprising a perilla leaf extract.
상기 면역 기능 증진용 건강기능성 식품 조성물은 고삼 추출물, 황련 추출물, 황백 추출물 및 연교 추출물에서 하나 이상의 추출물을 추가로 포함하는 면역 기능 증진용 건강기능성 식품 조성물. 9. The method of claim 8,
The functional health food composition for enhancing immune function is a health functional food composition for enhancing immune function, further comprising one or more extracts from ginseng extract, hwangryeon extract, hwangbaek extract and yeonkyo extract.
상기 면역 기능 증진용 건강기능성 식품 조성물의 금은화, 황금, 내복자, 강황 및 자소엽의 함량비는 복합 추출물 전체 중량을 기준으로 5~10 중량%, 40~70 중량%, 10~40 중량%, 5~10 중량%, 및 10~40 중량%인, 면역 기능 증진용 건강기능성 식품 조성물.9. The method of claim 8,
The content ratio of gold, silver, gold, turmeric and perilla leaf in the health functional food composition for enhancing immune function is 5 to 10% by weight, 40 to 70% by weight, 10 to 40% by weight, based on the total weight of the complex extract, 5 to 10% by weight, and 10 to 40% by weight, a health functional food composition for enhancing immune function.
상기 상염증용 조성물은 자소엽 추출물을 추가로 포함하는, 항염증용 조성물.12. The method of claim 11,
The anti-inflammatory composition further comprises a perilla leaf extract.
상기 조성물은 고삼 추출물, 황련 추출물, 황백 추출물 및 연교 추출물에서 하나 이상의 추출물을 추가로 포함하는, 항염증용 조성물.13. The method of claim 12,
The composition further comprises one or more extracts from ginseng extract, hwangryeon extract, hwangbaek extract and yeonkyo extract, anti-inflammatory composition.
상기 조성물의 금은화, 황금, 내복자, 강황 및 자소엽의 함량비는 복합 추출물 전체 중량을 기준으로 5~10 중량%, 40~70 중량%, 10~40 중량%, 5~10 중량%, 및 10~40 중량%인, 항염증용 조성물.13. The method of claim 12,
The content ratios of gold and silver coins, gold, Naebokja, turmeric and perilla leaf in the composition are 5 to 10% by weight, 40 to 70% by weight, 10 to 40% by weight, 5 to 10% by weight, and 10 to 40% by weight, anti-inflammatory composition.
An anti-inflammatory agent comprising the composition for anti-inflammatory according to any one of claims 11 to 14.
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