KR101987821B1 - A composition comprising mixture of the extract of Cinnamomi Cortex and silkworm powder for improving immunity - Google Patents
A composition comprising mixture of the extract of Cinnamomi Cortex and silkworm powder for improving immunity Download PDFInfo
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- KR101987821B1 KR101987821B1 KR1020170044368A KR20170044368A KR101987821B1 KR 101987821 B1 KR101987821 B1 KR 101987821B1 KR 1020170044368 A KR1020170044368 A KR 1020170044368A KR 20170044368 A KR20170044368 A KR 20170044368A KR 101987821 B1 KR101987821 B1 KR 101987821B1
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- extract
- silkworm
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Abstract
본 발명은 육계와 누에의 혼합 추출물을 유효성분으로 포함하는 면역증강용 조성물에 관한 것이다. 본 발명의 조성물은 비장세포에서 IL-2, IL-4, IL-10, IL-12 및 IFN-γ의 분비를 촉진할 뿐만 아니라 T 세포의 분열을 촉진하여, 선천면역과 후천면역을 모두 증강시키는 효과를 나타낸다. 따라서, 본 발명의 조성물은 면역증강용 약학적 조성물 또는 식품 조성물의 개발에 매우 유용하게 활용될 수 있다. The present invention relates to a composition for enhancing immunity comprising a mixed extract of broiler chicks and silkworm as an active ingredient. The composition of the present invention not only promotes the secretion of IL-2, IL-4, IL-10, IL-12 and IFN-y in splenocytes but also promotes cleavage of T cells to enhance both innate and acquired immunity . Therefore, the composition of the present invention can be very usefully utilized in the development of a pharmaceutical composition or a food composition for immunostaining.
Description
본 발명은 육계와 누에의 혼합 추출물을 유효성분으로 포함하는 면역증강용 조성물에 관한 것으로, 보다 상세하게는 비장세포에서 IL-2, IL-4, IL-10, IL-12 및 IFN-γ의 분비를 촉진할 뿐만 아니라 T 세포의 분열을 촉진하는 효과를 나타내어 선천면역과 후천면역을 동시에 증강시키는 효과를 나타내는, 육계와 누에의 혼합 추출물을 유효성분으로 포함하는 약학적 조성물, 그리고 식품 조성물에 관한 것이다.
The present invention relates to a composition for enhancing an immune system comprising a mixed extract of broiler and silkworm as an active ingredient. More particularly, the present invention relates to a composition for enhancing an immune response comprising a mixture of IL-2, IL-4, IL-10, IL-12 and IFN- The present invention relates to a pharmaceutical composition comprising, as an active ingredient, a mixed extract of broiler chickens and silkworms, which exhibits an effect of promoting the secretion and promoting cleavage of T cells to simultaneously enhance innate immunity and acquired immunity. will be.
면역반응은 활성화된 면역세포가 외래성 및 내인성 물질(항원)에 대하여 일으키는 일련의 반응이다. 세균, 바이러스 등을 포함한 미생물 및 생체의 이물질 등이 생체로 유입되면, 숙주세포는 감염을 극복하기 위해 염증반응의 원인이 되는 사이토카인 등의 인자를 분비하여 염증반응을 유발시킨다. 그리고 이러한 미생물 및 생체의 이물질 등이 면역세포에 의해 인식이 되면 면역세포가 활성화되고, 활성화된 면역세포에서도 염증반응의 원인이 되는 많은 인자를 분비하여 염증반응을 유발한다고 알려져 있다.An immune response is a series of reactions caused by activated immune cells against adventitious and endogenous substances (antigens). When a microorganism including a bacterium, a virus, or a foreign body of a living body enters the living body, the host cell secretes a factor such as cytokine which causes the inflammation reaction to overcome the infection and induces an inflammatory reaction. When these microorganisms and living organisms are recognized by the immune cells, the immune cells are activated, and in the activated immune cells, many factors that cause the inflammation reaction are secreted to induce the inflammation reaction.
이러한 면역반응은 태어날 때부터 가지고 있는 선천면역 (innate immunity)과 후천적으로 생활 등에 적응되어 얻어지는 후천면역(acquired immunity)로 구분된다. 여기서, 선천면역은 일명 '자연면역'이라고도 하는 것으로, 항원에 대하여 비특이적으로 반응하며 특별한 기억작용은 보이지 않는다. 선천면역 체계로는 항원의 침입을 차단하는 피부, 점액조직, 상산성의 위산, 체내로 들어온 침입자들을 제거하는 식균작용을 담당하는 대식세포(macrophage), 다형핵 백혈구 등과 같은 식세포 등이 있다. 이러한 선천면역은 실제 대부분의 감염을 방어한다. 한편, 후천면역은 처음 침입한 항원에 대한 기억작용이 있어, 다시 침입이 발생하였을 때, 특이적으로 반응하여 효과적으로 항원을 제거하는 특징이 있다.
These immune responses are divided into innate immunity, which is acquired from birth, and acquired immunity, which is obtained by adaptation to life and the like. Here, congenital immunity is also called "natural immunity" and responds nonspecifically to the antigen and does not show any special memory function. The congenital immune system includes skin, mucus tissue, acid stomach acid that blocks the entry of the antigen, macrophage which is responsible for the phagocytosis of the body, phagocytes such as polymorphonuclear leukocytes and the like. This congenital immune defenses most infections in practice. On the other hand, acquired immunity has a memory effect on the first invaded antigen, and when the invaded again occurs, it specifically reacts to effectively remove the antigen.
이러한 면역기능이 저하되면, 천식, 계절성 또는 통년성 비염, 알러지성 비염, 결막염, 아토피성 피부염, 두드러기, 적혈구의 용혈, 급성 사구체 신염, 감기, 만성 피로 및 암 등 다양한 면역질환이 발생할 수 있다. 이러한 면역질환은 치료보다 예방이 중요한데, 현재 시판되고 있는 치료제는 예방을 위하여 복용하기에는 어려운 것이 현실이다. 이에 부작용의 가능성이 있는 면역증강제의 복용 없이, 면역기능 장애를 예방 및 치료할 수 있는 방법이 요구되었다.
When this immune function deteriorates, a variety of immune diseases such as asthma, seasonal or perennial rhinitis, allergic rhinitis, conjunctivitis, atopic dermatitis, urticaria, hemolysis of red blood cells, acute glomerulonephritis, cold, chronic fatigue and cancer may occur. Such immune diseases are more important to prevent than treatment, and currently commercial drugs are difficult to take for prevention. Therefore, there is a need for a method for preventing and treating immune dysfunction without taking an immunostimulant that has the potential for side effects.
이를 위하여, 부작용이 적은 천연물로부터 면역활성을 조절할 수 있는 성분을 발굴하고, 이를 이용하여 면역활성을 조절할 수 있는 제제를 개발하기 위한 연구가 활발하게 진행되고 있다. 예를 들어, 한국등록특허 제1141314호에는 오적산 추출물을 유효성분으로 포함하는 면역증강용 조성물이 개시되어 있고, 한국공개특허 제2009-0108208호에는 홍삼 추출물, 표고버섯균사체 추출물, 알로에 추출물 및 유산균을 포함하는 스트레스성 면역질환의 예방 또는 치료용 약학 조성물이 개시되어 있다. 그러나, 이러한 천연물 유래 성분은 천연물이 나타내는 효과보다도 면역조절 활성이 크게 감소되거나, 동일한 천연물로부터 얻어진 추출물이 서로 다른 면역조절 활성을 나타내거나, 천연물로부터 면역조절 활성을 나타내는 유효성분의 분리 동정이 용이하지 않다는 등의 다양한 어려움이 있어, 상기 천연물 유래 성분을 유효성분으로 포함하는 면역조절 제제의 개발이 지체되고 있는 실정이다.
For this purpose, studies have been actively carried out to develop a preparation capable of modulating immunological activity by using a natural substance having a low side effect and extracting a component capable of controlling the immune activity. For example, Korean Patent No. 1141314 discloses a composition for enhancing immunity comprising an extract of Ohtansan as an active ingredient. In Korean Patent Laid-Open No. 2009-0108208, red ginseng extract, shiitake mushroom mycelium extract, aloe extract, A pharmaceutical composition for preventing or treating a stress-related immune < RTI ID = 0.0 > disorder. ≪ / RTI > However, such a natural-origin-derived component has a problem that the immunomodulating activity is greatly reduced, the extract obtained from the same natural product exhibits different immunomodulating activity than the effect exhibited by the natural product, or the active ingredient exhibiting the immunomodulating activity from the natural product is easy to identify The development of an immunomodulating agent containing the natural-substance-derived component as an active ingredient has been delayed. SUMMARY OF THE INVENTION
이에, 본 발명자는 천연물 유래의 면역 증강용 조성물을 개발하기 위하여 예의 노력을 기울인 결과, 육계와 누에의 혼합 추출물이 각각의 단독 물질의 추출물과 비교하여 현저히 향상된 면역 증강작용을 나타낸다는 것을 발견하고 본 발명을 완성하게 되었다.
Accordingly, the present inventor has made a diligent effort to develop a composition for enhancing immunity derived from a natural product, and as a result, found that a mixed extract of broiler and silkworm exhibits a significantly improved immune enhancing effect as compared with the extract of each single substance, Thereby completing the invention.
따라서, 본 발명의 목적은 육계 및 누에의 혼합 추출물을 유효성분으로 포함하는 면역 증강용 약학적 조성물을 제공하는 것이다.
Accordingly, an object of the present invention is to provide a pharmaceutical composition for enhancing immunity which comprises a mixed extract of broiler and silkworm as an active ingredient.
본 발명의 다른 목적은 육계 및 누에의 혼합 추출물을 유효성분으로 포함하는 면역 증강용 식품 조성물을 제공하는 것이다.
Another object of the present invention is to provide a food composition for enhancing immunity comprising a mixed extract of broiler and silkworm as an active ingredient.
상기한 본 발명의 목적을 달성하기 위하여 본 발명은 육계 및 누에의 혼합 추출물을 유효성분으로 포함하는 면역 증강용 약학적 조성물을 제공한다.
In order to accomplish the object of the present invention, the present invention provides a pharmaceutical composition for enhancing immunity comprising a mixed extract of broiler chickens and silkworms as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여 본 발명은 육계 및 누에의 혼합 추출물을 유효성분으로 포함하는 면역 증강용 식품 조성물을 제공한다.
In order to accomplish another object of the present invention, there is provided a food composition for enhancing immunity comprising a mixed extract of broiler and silkworm as an active ingredient.
이하, 본 발명에 대해 보다 상세히 설명한다.
Hereinafter, the present invention will be described in more detail.
본 발명은 육계 및 누에의 혼합 추출물을 유효성분으로 포함하는 면역 증강용 약학적 조성물을 제공한다.
The present invention provides a pharmaceutical composition for enhancing immunity comprising a mixed extract of broiler chickens and silkworms as an active ingredient.
본 발명에서는 상기 육계 및 누에의 혼합 추출물이 비장세포에서 IL-2, IL-4, IL-10, IL-12 및 IFN-γ의 분비를 촉진할 뿐만 아니라 T 세포의 분열을 촉진하는 효과가 있음을 확인하였다. 한편, 상기 혼합물의 이러한 면역증강 효과는 각각의 단독 물질의 추출물에 비해 현저히 향상된 것으로 확인되었다. 이처럼, 육계 및 누에의 혼합 추출물이 면역 증강효과의 상승작용을 나타낸다는 것은 본 발명에서 처음으로 공개하는 것이다.
In the present invention, the broth and silkworm mixed extract promotes the secretion of IL-2, IL-4, IL-10, IL-12 and IFN-y in spleen cells as well as promotes the division of T cells Respectively. On the other hand, it was confirmed that the immune enhancing effect of the mixture was remarkably improved as compared with the extract of each single substance. As described above, it is disclosed for the first time in the present invention that the mixed extract of broiler and silkworm exhibits a synergistic effect of the immune enhancing effect.
상술한 바와 같이, 본 발명의 조성물은 다양한 사이토카인들의 분비를 촉진하고 T 세포의 분열을 촉진함으로써 선천면역과 후천면역을 동시에 향상시키는 효과를 나타낼 수 있다. 따라서, 본 발명에서 상기 면역 증강이란 선천면역과 후천면역을 모두 증강시키는 것을 의미한다. 또한, 면역증강은, 면역세포의 초기활성화 과정에서 비특이적으로 항원에 대해 면역반응을 촉진하는 기능, 또는 면역계의 세포의 활성을 증대시킴으로써 면역을 강화하는 기능을 의미한다.
As described above, the composition of the present invention promotes the secretion of various cytokines and promotes cleavage of T cells, thereby exhibiting an effect of simultaneously enhancing innate immunity and acquired immunity. Therefore, in the present invention, the above-mentioned immune enhancement means to enhance both innate immunity and acquired immunity. In addition, the term " immune enhancement " means a function of promoting an immune response to an antigen nonspecifically in the initial activation process of immune cells, or a function of enhancing immunity by increasing the activity of cells of the immune system.
본 발명에서 상기 육계(Cinnamomum cassia Blume)는 녹나무과(Lauraceae)에 속하는 상록교목인 육계나무(Cinnamomum loureirii) 또는 계피나무(Cinnamomum cassia)의 줄기 및 가지 수피로서, 주성분으로는 신남알데하이드(Cinnamic aldehyde), 캄펜(Camphene), 치네올(Cineol), 리날로올(Linalool), 유게놀(Eugenol) 등이 있으며 진정, 항균 등의 효능이 있다고 알려져 있다
In the present invention, the broiler (Cinnamomum cassia Blume) is a stem and a bark of Cinnamomum loureirii or Cinnamomum cassia, an evergreen tree belonging to Lauraceae. Cinnamomum cassia Blume is Cinnamic aldehyde, Camphene, Cineol, Linalool, Eugenol, etc., and it is known to have a sedative, antimicrobial, etc. effect
본 발명의 상기 누에는 알에서 깨어나 4번의 탈피를 거쳐 누에고치를 짓고 누에번데기로 변태한 후, 나방이 되어 알을 낳고 약 2주일을 경과하면 죽게 되는 일생의 과정을 가진 완전변태 곤충의 하나이다. 누에유충의 성장단계에 따른 생리학적 변화는 매우 크고, 탈피전후 유충체내의 효소를 비롯한 물질수준의 차이는 현저하다고 알려져 있다. 누에체내에는 주로 뽕잎성분의 누에똥과 각종 효소가 들어있는 중장소화액 그리고 혈액이 있다. 이중 중장소화액과 혈액은 강한 산화력을 가지고 있어, 공기와 접하게 되면 쉽게 산화되어 검게 변색되면서 불활화 된다. 본 발명에서 사용하는 누에는 그 자체를 이용하여 추출할 수 있으며, 누에 분말을 제조하여 추출할 수도 있다. 상기 누에분말의 제조방법은 특별히 한정되지 않으며, 당업계에서 공지된 방법에 따라 제조된 누에분말이라면 제한 없이 사용될 수 있다. 누에분말의 제조방법은 한국등록특허 12-46266호, 한국등록특허 11-96959호 등을 참고할 수 있다.
The silkworm of the present invention is one of the complete transformant insects having a lifelong process of being killed when it is awakened from an egg, has 4 claws, builds a cocoon, transforms into a silkworm pupa, gives birth to an egg, and lays about 2 weeks. Physiological changes due to the growth stages of silkworm larvae are very large, and the difference in the levels of substances including enzymes in larvae is known to be remarkable. In silkworm body, there are mulberry digestive juice mainly containing mulberry leaves and various enzymes and blood. Double medium digestive juices and blood have a strong oxidizing power. When they come into contact with air, they become easily oxidized and become blackened and inactivated. The silkworm used in the present invention can be extracted by itself, and the silkworm powder can also be produced and extracted. The method for producing the silkworm powder is not particularly limited, and silkworm powder prepared by a method known in the art can be used without limitation. Methods for producing silkworm powder can be found in Korean Patent No. 12-46266, Korean Patent No. 11-96959, and the like.
상기 육계와 누에의 혼합 추출물을 포함하는 조성물은 육계와 누에 단독 추출물을 혼합한 것일 수도 있고, 또는 육계와 누에를 혼합한 다음 추출한 복합 추출물일 수도 있다. 본 명세서에서 상기 단독 추출물의 혼합물 또는 복합 추출물을 모두 혼합 추출물로 통칭할 수 있다. 본 발명의 조성물은 유효성분으로 포함하는 육계와 누에 추출물의 혼합 방식에 제한받지 아니한다. 상기 추출물의 형태나 성상에는 제한이 없으며, 용액, 농축물일 수도 있고, 추출물 제조에 사용된 용매를 제거한 고형분 또는 분말일 수도 있다.
The composition containing the mixed extract of broiler and silkworm may be a mixture of broiler and silkworm alone extracts, or may be a combination of broth and silkworm mixed and then extracted. In the present specification, a mixture or a complex extract of the single extract may be collectively referred to as a mixed extract. The composition of the present invention is not limited to the mode of mixing the broiler and silkworm extracts as an active ingredient. There are no limitations on the form or properties of the extract, and it may be a solution, a concentrate, or a solid or powder from which the solvent used for the preparation of the extract is removed.
본 발명의 조성물은 육계와 누에의 단독 추출물을 1 : 0.5 ~ 3의 중량비로 혼합한 것일 수 있다. 본 발명의 조성물이 육계와 누에의 단독 추출물을 혼합하여 포함하는 경우, 각각에 대하여 두 가지 추출물을 1 : 0.5 ~ 3의 중량비로 혼합할 수 있다. 또한 본 발명의 조성물은 건조 중량 기준으로 육계와 누에를 1 : 0.5 ~ 3 의 중량비로 혼합한 다음 동시에 추출한 것일 수도 있다. 즉, 본 발명의 조성물이 육계와 누에의 복합 추출물을 포함하는 경우에는 육계와 누에를 1: 0.5~3의 중량비로 혼합한 다음 추출한 것일 수 있다. 가장 바람직하게는 상기 누에와 육계의 혼합 중량비는 1:1일 수 있으나 이에 제한되는 것은 아니다.
The composition of the present invention may be obtained by mixing broiler and silkworm extracts at a weight ratio of 1: 0.5-3. When the composition of the present invention contains a mixture of broiler and silkworm alone extracts, the two extracts may be mixed at a weight ratio of 1: 0.5 ~ 3. In addition, the composition of the present invention may be obtained by mixing broiler and silkworms at a weight ratio of 1: 0.5 ~ 3 on a dry weight basis and then extracting them simultaneously. That is, when the composition of the present invention includes a combination of broiler and silkworm extracts, broth and silkworm may be mixed at a weight ratio of 1: 0.5 to 3 and then extracted. Most preferably, the mixing weight ratio of the silkworm to broiler chickens may be 1: 1, but is not limited thereto.
본 발명에서의 생약 추출물은 물, 탄소수 1 내지 6인 알코올, 헥산, 에틸아세테이트 및 이들의 혼합용매로 이루어진 군에서 선택된 어느 하나의 용매로 추출한 것일 수 있다. 본 발명의 추출물은 바람직하게는 물, 탄소수 1 내지 6인 알코올 및 이들의 혼합용매에서 선택된 용매로 추출한 것일 수 있으며, 또는 탄소수 1 내지 6의 저급 알코올 수용액일 수 있다. 상기 알코올 수용액의 농도는 10 내지 100%(v/v)일 수 있으며, 바람직하게는 70 내지 100%(v/v)일 수 있다. 본 발명의 추출 용매는 가장 바람직하게는 물 또는 95%(v/v)의 에탄올 수용액일 수 있다.
The herbal medicine extract in the present invention may be extracted with any one solvent selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, hexane, ethyl acetate and a mixed solvent thereof. The extract of the present invention is preferably extracted with a solvent selected from water, an alcohol having 1 to 6 carbon atoms and a mixed solvent thereof, or a lower alcohol aqueous solution having 1 to 6 carbon atoms. The concentration of the aqueous alcohol solution may be 10 to 100% (v / v), preferably 70 to 100% (v / v). The extraction solvent of the present invention may most preferably be water or a 95% (v / v) aqueous solution of ethanol.
본 발명에서의 추출 방법은 당업계에 공지된 것이면 제한 없이 사용할 수 있으며, 그 예로 냉침, 열수 추출, 초음파 추출, 환류냉각 추출 등의 방법이 있으나 여기에 국한되는 것은 아니다. 바람직하게는 환류냉각 추출할 수 있다.
The extraction method in the present invention can be used without limitation as long as it is known in the art, and examples thereof include, but are not limited to, cold-pressing, hot water extraction, ultrasonic extraction, Preferably by reflux cooling and extraction.
상기 방법으로 제조된 추출물은 여과하여 여액을 모으고, 다시 잔사에 원료 중량의 4 내지 7배의 추출 용매, 예컨대 물을 가하여 환류냉각 추출 조건으로 4시간 동안 재추출하고 여과하여 이전의 여액과 혼합함으로써 추출 효율을 높일 수 있다. 본 발명의 추출물이 추출의 횟수에 한정되는 것은 아니다.
The extract prepared by the above method is collected by filtration, and the extract is re-extracted for 4 hours under reflux and cooling conditions by addition of an extraction solvent such as water of 4 to 7 times the weight of the raw material to the residue, filtered and mixed with the previous filtrate The extraction efficiency can be increased. The extract of the present invention is not limited to the number of times of extraction.
상기와 같이 추출한 후 합한 여액은 40 내지 60℃에서 감압농축하여 추출물 중에 잔존하는 용매를 제거하고, 감압농축된 농축물 중량의 25 내지 50배의 물로 2 내지 3회 공비 농축한 후 재차 동량의 물을 가하여 균질하게 현탁시킨 후 최종 추출물은 추가적으로 더욱 정제할 수도 있고, 동결건조 등의 방법에 의해 건조시켜 용매를 제거하여 고형화하거나 분말 형태로 제조할 수 있다.
After the extraction as described above, the combined filtrate is concentrated under reduced pressure at 40 to 60 ° C to remove the solvent remaining in the extract, and then azeotropically concentrated with
본 발명자들은 육계와 누에의 혼합 추출물이 비장세포에서 IL-2, IL-4, IL-10, IL-12 및 IFN-γ의 분비를 촉진할 뿐만 아니라 T 세포의 분열을 촉진하는 효과가 있음을 확인하였다. 따라서 상기 혼합 추출물은 이 같은 면역증강 작용을 통해 면역기능 저하로 인해 유발되는 다양한 질환들을 예방 또는 치료하는 효과를 나타낼 수 있다. 본 발명에서 상기 면역기능 저하로 인한 질환은, 감기, 알러지 질환, 만성피로, 암으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다. 본 발명에서 사용되는 용어 "예방"은, 본 발명의 혼합물을 포함하는 조성물의 투여로 질환을 억제 또는 지연시키는 모든 행위를 의미한다. 또한, 본 발명에서 사용되는 용어 "치료"는, 본 발명의 혼합물을 포함하는 조성물의 투여로 질환의 증세가 호전되거나 완치되는 모든 행위를 의미한다.
The present inventors have found that a mixed extract of broiler and silkworm promotes the secretion of IL-2, IL-4, IL-10, IL-12 and IFN-γ in splenocytes as well as promotes the division of T cells Respectively. Therefore, the mixed extract can exhibit the effect of preventing or treating various diseases caused by immune function deterioration through the immunity enhancing action. In the present invention, the disease caused by decreased immune function may be any one selected from the group consisting of cold, allergic diseases, chronic fatigue, and cancer. The term "prophylactic, " as used herein, refers to any act that inhibits or delays disease by administration of a composition comprising a mixture of the present invention. The term "treatment ", as used herein, refers to any action that improves or alleviates a symptom of a disease upon administration of a composition comprising a mixture of the present invention.
상기 조성물이 약학적 조성물의 형태고 제공이 되는 경우에는, 본 발명의 상기 혼합물 이외에 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 함유할 수 있다. 상기에서 "약학적으로 허용되는" 이란 생리학적으로 허용되고 인간에게 투여될 때, 활성성분의 작용을 저해하지 않으며 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 비독성의 조성물을 말한다.
When the composition is in the form of a pharmaceutical composition, it may further contain one or more pharmaceutically acceptable carriers, excipients or diluents in addition to the mixture of the present invention. The term "pharmaceutically acceptable" as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, .
약학적으로 허용되는 담체로는 예컨대, 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 아울러, 펩티드 제제에 대한 경구투여용으로 사용되는 다양한 약물전달물질을 포함할 수 있다. 또한, 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코오스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 본 발명의 약학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현택제 등을 추가로 포함할 수 있다. 그 밖의 약학적으로 허용되는 담체 및 제제는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, it may contain various drug delivery materials used for oral administration to peptide preparations. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, etc. in addition to the above components. Other pharmaceutically acceptable carriers and preparations can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).
본 발명의 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들면, 경구 또는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다.
The composition of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI >
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다.The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.
경구 투여용 제제의 경우에 본 발명의 조성물은 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 등으로 당업계에 공지된 방법을 이용하여 제형화될 수 있다. 예를 들어, 경구용 제제는 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈,메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.
비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌(Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA,1995)에 기재되어 있다.
In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, Pa., 1995, which is a commonly known formulary for all pharmaceutical chemistries.
본 발명의 조성물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 바람직하게 본 발명의 약학적 조성물의 바람직한 전체 용량은 1일당 환자 체중 1㎏ 당 약 0.01㎍ 내지 10,000mg, 가장 바람직하게는 0.1㎍ 내지 1,000mg일 수 있다. 그러나 상기 약학적 조성물의 용량은 제제화 방법, 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율등 다양한 요인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 본 발명의 조성물의 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다.
The total effective amount of the composition of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the total preferred dose of the pharmaceutical composition of the present invention may be from about 0.01 μg to 10,000 mg, and most preferably from 0.1 μg to 1,000 mg per kg body weight of the patient per day. However, the dosage of the pharmaceutical composition may be determined depending on various factors such as the formulation method, administration route and frequency of treatment, as well as the patient's age, body weight, health condition, sex, severity of disease, diet and excretion rate, It will be possible to determine the appropriate effective dose of the composition of the present invention by those of ordinary skill in the art in view of this point. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
본 발명은 또한 육계와 누에의 혼합 추출물을 유효성분으로 포함하는 면역 증강용 식품 조성물을 제공한다.
The present invention also provides a food composition for enhancing immunity comprising a mixed extract of broiler chicks and silkworm as an active ingredient.
본 발명의 상기 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food)및 식품 첨가제(food additives)등의 모든 형태를 포함한다. 상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.
The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.
예를 들면, 건강 식품으로는 상기 조성물 자체를 차, 주스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마말레이드 등), 어류, 육류, 및 그 가공식품(예: 햄, 소시지 콘비프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등)등에 추출물을 첨가하여 제조할 수 있다. 또한, 본 발명의 조성물을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.
For example, as the health food, the composition itself may be prepared in the form of tea, juice, and drink and then taken for drinking, granulated, encapsulated and powdered. Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (such as canned fruits, bottled, jam, marmalade, etc.), fish, meats and their processed foods (eg, ham, sausage, Breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), juice, various drinks, cookies, sugar, dairy products such as butter, cheese, edible vegetable oil, margarine, vegetable protein, Frozen foods, various seasonings (eg soybean paste, soy sauce, sauces, etc.), and the like. In order to use the composition of the present invention in the form of a food additive, it may be prepared in the form of a powder or a concentrated liquid.
본 발명의 식품용 조성물 중 상기 추출물의 바람직한 함량은 식품용 조성물 총 중량에 대하여 0.001 내지 50% 일 수 있으며, 바람직하게는 0.1 내지 50% 범위로 함유될 수 있다.
The preferred content of the extract in the food composition of the present invention may be 0.001 to 50%, preferably 0.1 to 50%, based on the total weight of the food composition.
본 발명에 따른 조성물의 효과는 하기 실시예에 잘 나타나 있다.
The effect of the composition according to the invention is illustrated in the following examples.
본 발명의 조성물은 비장세포에서 IL-2, IL-4, IL-10, IL-12 및 IFN-γ의 분비를 촉진할 뿐만 아니라 T 세포의 분열을 촉진하여, 선천면역과 후천면역을 모두 증강시키는 효과를 나타낸다. 따라서, 본 발명의 조성물은 면역증강용 약학적 조성물 또는 식품 조성물의 개발에 매우 유용하게 활용될 수 있다.The composition of the present invention not only promotes the secretion of IL-2, IL-4, IL-10, IL-12 and IFN-y in splenocytes but also promotes cleavage of T cells to enhance both innate and acquired immunity . Therefore, the composition of the present invention can be very usefully utilized in the development of a pharmaceutical composition or a food composition for immunostaining.
도 1은 각각의 추출물 처리에 따른 T cell 분화 촉진여부를 확인한 결과이다.
도 2는 각각의 추출물 처리에 따라 비장세포에서 IL-2의 생성량 증가여부를 확인한 결과이다.
도 3은 각각의 추출물 처리에 따라 비장세포에서 IL-4의 생성량 증가여부를 확인한 결과이다.
도 4는 각각의 추출물 처리에 따라 비장세포에서 IL-10의 생성량 증가여부를 확인한 결과이다.
도 5는 각각의 추출물 처리에 따라 비장세포에서 IL-12의 생성량 증가여부를 확인한 결과이다.
도 6는 각각의 추출물 처리에 따라 비장세포에서 IFN-γ의 생성량 증가여부를 확인한 결과이다. FIG. 1 shows the result of confirming whether T cell differentiation is promoted according to each extract treatment.
FIG. 2 shows the results of confirming the increase in the amount of IL-2 produced in the splenocytes according to each of the extract treatments.
FIG. 3 shows the results of confirming the increase in IL-4 production in spleen cells according to each extract treatment.
FIG. 4 shows the results of confirming the increase in IL-10 production in splenocytes according to each extract treatment.
FIG. 5 shows the results of confirming the increase in the amount of IL-12 produced in the splenocytes according to the respective extract treatments.
FIG. 6 shows the results of confirming the increase in the amount of IFN-? Produced in the splenocytes according to each extract treatment.
이하 바람직한 실시예를 통하여 본 발명을 상세히 설명하기로 한다. 이에 앞서, 본 명세서 및 청구범위에 사용된 용어나 단어는 통상적이거나 사전적인 의미로 한정해서 해석되어서는 아니 되며, 발명자는 그 자신의 발명을 가장 최선의 방법으로 설명하기 위해 용어의 개념을 적절하게 정의할 수 있다는 원칙에 입각하여, 본 발명의 기술적 사상에 부합하는 의미와 개념으로 해석되어야만 한다. 따라서, 본 명세서에 기재된 실시예의 구성은 본 발명의 가장 바람직한 일실시예에 불과할 뿐이고 본 발명의 기술적 사상을 모두 대변하는 것은 아니므로, 본 출원시점에 있어서 이들을 대체할 수 있는 다양한 균등물과 변형예들이 있을 수 있음을 이해하여야 한다. 또한, 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한, 다른 구성요소를 제외하는 것이 아니라 다른 구성요소를 더 포함할 수 있음을 의미한다.
Hereinafter, the present invention will be described in detail with reference to preferred embodiments. Prior to this, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms, and the inventor should appropriately interpret the concepts of the terms appropriately The present invention should be construed in accordance with the meaning and concept consistent with the technical idea of the present invention. Accordingly, it is to be understood that the constituent features of the embodiments described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the inventive concepts of the present invention, so that various equivalents, And the like. Also, throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements, not excluding other elements, unless specifically stated otherwise.
이하 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
<실험방법> <Experimental Method>
1. 실험물질1. Experimental material
홍삼은 면역증진에 도움을 줄 수 있는 기능성소재를 시중에서 구입하였으며, Rg3는 순도 95%의 HPLC급 시약을 시그마알드리치에서 구입하였으며, 누에는 영천양잠협동조합에서 구입하였고, 육계는 옴니허브 식품용 소재를 구매하여 사용하였다.
Red ginseng was purchased from Sigma Aldrich with a purity of 95% and Rg3 was purchased from Sigma Aldrich. The silkworm was purchased from Yeongcheon Cooperative Federation and broiler was purchased from Omni Herb Foods Materials were purchased and used.
2. 추출물의 제조방법 및 2. Method of manufacturing extract and 실험군Experimental group
(1) 누에추출물 제조방법(1) Method of producing silkworm extract
1. 누에 무게를 달아 혼합한다.1. Weigh silkworm and mix.
2. 총중량에 10배수의 증류수를 넣어 2시간 동안 열수추출한다.2. Add 10 times of distilled water to the total weight, and heat-extract for 2 hours.
3. 추출액은 여과하여 불순물을 제거하고, 감압 농축한다.3. The extract is filtered to remove impurities and concentrated under reduced pressure.
4. 농축액은 동결건조기에 넣어 최종적으로 분말로 제조한다.4. The concentrate is put into a freeze-dryer and finally made into powder.
5. 분말은 냉동고에 보관하였으며, 이를 실험에 적당량 사용하였다.
5. The powder was stored in a freezer and used in an appropriate amount for the experiment.
(2) 육계추출물 제조방법(2) Method of manufacturing broiler chick extract
1. 육계 무게를 달아 혼합한다.1. Weigh the broiler weight and mix.
2. 총중량비 10배수의 증류수를 넣어 2시간 동안 열수추출한다.2. Distilled water with a total weight ratio of 10 is added and hot water is extracted for 2 hours.
3. 추출액은 여과하여 불순물을 제거하고, 감압 농축한다.3. The extract is filtered to remove impurities and concentrated under reduced pressure.
4. 농축액은 동결건조기에 넣어 최종적으로 분말로 제조한다.4. The concentrate is put into a freeze-dryer and finally made into powder.
5. 분말은 냉동고에 보관하였으며, 이를 실험에 적당량 사용하였다.
5. The powder was stored in a freezer and used in an appropriate amount for the experiment.
(3) 부추추출물 제조방법(3) Production method of leek extract
1. 부추 무게를 달아 혼합한다.1. Weigh the leek and mix.
2. 총중량비에 10배수의 증류수를 넣어 2시간 동안 열수추출한다.2. Add 10 times of distilled water to the total weight ratio, and extract hot water for 2 hours.
3. 추출액은 여과하여 불순물을 제거하고, 감압 농축한다.3. The extract is filtered to remove impurities and concentrated under reduced pressure.
4. 농축액은 동결건조기에 넣어 최종적으로 분말로 제조한다.4. The concentrate is put into a freeze-dryer and finally made into powder.
5. 분말은 냉동고에 보관하였으며, 이를 실험에 적당량 사용하였다.
5. The powder was stored in a freezer and used in an appropriate amount for the experiment.
(4) 누에와 부추 혼합추출물 제조방법(4) Manufacturing method of mixed extract of silkworm and leek
1. 누에와 부추를 1:1의 중량비로 무게를 달아 혼합한다.1. Weigh silkworm and leek at a weight ratio of 1: 1 and mix.
2. 총중량비에 10배수의 증류수를 넣어 2시간 동안 열수추출한다.2. Add 10 times of distilled water to the total weight ratio, and extract hot water for 2 hours.
3. 추출액은 여과하여 불순물을 제거하고, 감압 농축한다.3. The extract is filtered to remove impurities and concentrated under reduced pressure.
4. 농축액은 동결건조기에 넣어 최종적으로 분말로 제조한다.4. The concentrate is put into a freeze-dryer and finally made into powder.
5. 분말은 냉동고에 보관하였으며, 이를 실험에 적당량 사용하였다.
5. The powder was stored in a freezer and used in an appropriate amount for the experiment.
(5) 누에와 육계 혼합추출물 제조방법(5) Manufacturing method of silkworm and broiler mixed extract
1. 누에와 육계를 1:1의 중량비로 무게를 달아 혼합한다.1. Weigh silkworm and broiler at a weight ratio of 1: 1 and mix.
2. 총중량비에 10배수의 증류수를 넣어 2시간 동안 열수추출한다.2. Add 10 times of distilled water to the total weight ratio, and extract hot water for 2 hours.
3. 추출액은 여과하여 불순물을 제거하고, 감압 농축한다.3. The extract is filtered to remove impurities and concentrated under reduced pressure.
4. 농축액은 동결건조기에 넣어 최종적으로 분말로 제조한다.4. The concentrate is put into a freeze-dryer and finally made into powder.
5. 분말은 냉동고에 보관하였으며, 이를 실험에 적당량 사용하였다.
5. The powder was stored in a freezer and used in an appropriate amount for the experiment.
본 발명에서의 실험군은 다음과 같다. The test group in the present invention is as follows.
(1) 정상군: 시험물질을 투여하지 않고 용매만을 투여함.(1) Normal group: Only the solvent is administered without administering the test substance.
(2) 홍삼 추출물 투여군(200, 100, 50 ug/ml)(2) Red ginseng extract administration group (200, 100, 50 ug / ml)
(3) Rg3 투여군(200, 100, 50 ug/ml)(3) Rg3 administration group (200, 100, 50 ug / ml)
(4) 누에 추출물 단독 투여군(200, 100, 50 ug/ml)(4) Silkworm extract alone (200, 100, 50 ug / ml)
(5) 육계 추출물 단독 투여군(200, 100, 50 ug/ml)(5) Broiler extract alone (200, 100, 50 ug / ml)
(6) 부추 추출물 단독 투여군(200, 100, 50 ug/ml)(6) Leek extract alone (200, 100, 50 ug / ml)
(7) 누에 + 육계 혼합 추출물 투여군(200, 100, 50 ug/ml)(7) Silkworm + broiler mixed extract administration group (200, 100, 50 ug / ml)
(8) 누에 + 부추 혼합 추출물 투여군(200, 100, 50 ug/ml)
(8) Administration of silkworm + leek mixture extract (200, 100, 50 ug / ml)
3. 추출물의 림프구 3. Lymphocyte of the extract 증식능Proliferative ability 평가 evaluation
8주령의 Balb/c계 생쥐 (male)의 비장을 취하여 배양한 후, 누에가루 및 단미소재, 누에가루 복합소재 시료를 처리한 후 비장세포 증식능을 측정하였다.
8-week-old Balb / c mice were cultured in the spleen, and then spleen cell proliferative capacity was measured after treatment with silkworm powder, short-term material, and silkworm composite material.
(1) 세포독성 평가(MTT법)(1) Cytotoxicity evaluation (MTT method)
SPF에서 사육한 8주령의 Balb/c계 생쥐 (male)의 비장을 취하여 적당량의 무균 HBSS 용액이 담긴 용기에 넣고, 핀셋으로 잘게 절단하여 단일 비장세포 현탁액으로 만들었다. Filter로 여과하여 HBSS액에 세 번 세척하였다. 매 세척 시 1,000 rpm에서 원심분리하였다. 세포를 완전배양액 2 ㎖에 띄워 염색한 후 생존 세포 수를 계산하였다 (95%이상 생존도). 세포 농도를 2 × 106/㎖로 조절하고, 필요 시 순수 T 림프구 또는 B 림프구 등을 분리하였다. 비장세포 현탁액을 24 well plate에 넣고 각 well마다 1 ㎖씩 넣고 상기 방법에 따라 제조된 각각의 추출물을 400, 200, 100, 50 μg/ml 농도로 처리하였다.
Spleens of 8-week-old Balb / c mice (SPF) -milled spleens were taken and placed into a container containing an appropriate amount of sterile HBSS solution and finely cut into tweezers to prepare a single spleen cell suspension. Filtered and washed three times with HBSS solution. Each wash was centrifuged at 1,000 rpm. Cells were stained with 2 ml of complete culture medium and viable cells counted (95% viability). The cell concentration was adjusted to 2 × 10 6 / ml and, if necessary, pure T lymphocytes or B lymphocytes were isolated. The spleen cell suspension was placed in a 24-well plate, and 1 ml of each suspension was added to each well. Each of the extracts prepared according to the above method was treated at a concentration of 400, 200, 100, and 50 μg / ml.
대조군과 함께 CO2 5%, 37℃ 배양기에서 24시간 배양하였다. 배양 종료 4시간 전에 각 well마다 상등액 0.7 ㎖를 빼고 FBS를 함유하지 않은 RPMI 1640 배양액 0.7 ㎖를 넣는 동시에 MTT액 (5 ㎎/㎖)을 50 ㎕/well의 양으로 넣고 4시간 동안 계속 배양하였다. 배양 종료 후, well마다 1 ㎖의 산성 아이소프로페놀을 넣어 보라색 결정체가 완전히 용해될 때까지 혼합하였다. 그런 후 96 well plate에 나눠 담았다. 각 well은 3 well에 나눠 570 nm 파장에서 ELISA(enzyme-linked immunosorbent assay) reader로 측정한다. 또는 용해액을 바로 1 ㎖ 비색 비이커에 담아 분광 광도계로 570 nm에서 측정하였다.
And cultured in a CO 2 incubator at 37 ° C for 24 hours. Four hours before the end of the culture, 0.7 ml of the supernatant was subtracted from each well, 0.7 ml of RPMI 1640 culture medium without FBS was added, and MTT solution (5 mg / ml) was added at 50 μl / well. After completion of the incubation, 1 ml of isopropanol was added to each well and mixed until the purple crystals were completely dissolved. It was then divided into 96-well plates. Each well is divided into 3 wells and measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm. Or the dissolution solution was directly placed in a 1 ml colorimetric beaker and measured at 570 nm with a spectrophotometer.
(2) Cell proliferation 측정(EZ-CyTox법)(2) Cell proliferation measurement (EZ-CyTox method)
SPF에서 사육한 8주령의 Balb/c계 생쥐 (male)의 비장을 취하여 적당량의 무균 HBSS 용액이 담긴 용기에 넣고, 핀셋으로 잘게 절단하여 단일 비장세포 현탁액을 200 ㎕/well의 양으로 96 well plate에 넣었다. The spleen of 8-week-old Balb / c mice (SPF) fed into a container containing an appropriate amount of sterile HBSS solution was finely cut with a tweezers, and a single spleen cell suspension was added to a 96 well plate .
3개의 실험군은 Con A (0.5 μg/㎖)를 넣고 나머지 3개 실험군은 Con A를 넣지 않은 군, 그리고 3개 군은 상기 제조된 각각의 추출물을 400, 200, 100, 50, 0 μg/ml 농도로 처리한 실험군으로 하였다. 5% CO2, 37℃ 배양기에서 24시간, 72시간 동안 배양하였다. 배양 종료 6시간 전에 각 well에 EZ-CyTox assay reagent 10 ㎕을 넣어 30분에서 6시간 동안 계속 배양하였다. 배양 종료 후 420~480 nm 파장에서 ELISA (enzyme-linked immunosorbent assay) reader로 비장세포 증식능을 측정하였다.
In the three experimental groups, Con A (0.5 μg / ㎖) was added, the remaining three experimental groups were not in Con A, and the three groups were in the 400, 200, 100, 50 and 0 μg / ml , Respectively. And cultured in a 5% CO 2 , 37 ° C incubator for 24 hours and 72 hours. EZ-CyTox assay reagent (10 μl) was added to each well 6 hours before the end of incubation and continued for 30 minutes to 6 hours. After incubation, the splenocyte proliferative capacity was measured with an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 420 to 480 nm.
4. 세포활성물질 수준 측정4. Measurement of cell active substance level
SPF에서 사육한 8주령의 Balb/c계 생쥐 (male)의 비장을 취하여 적당량의 무균 HBSS 용액이 담긴 용기에 넣고, 핀셋으로 잘게 절단하여 단일 비장세포 현탁액을 200 ㎕/well의 양으로 96 well plate에 넣었다. 3개의 실험군은 Con A (0.5 μg/㎖)를 넣고 나머지 3개 실험군은 Con A를 넣지 않은 군, 그리고 3개 군은 상기 제조된 각각의 추출물을 400, 200, 100, 50, 0 μg/ml 농도로 처리한 실험군으로 하였다. 각 용기에는 10% FBS와 1% penicillin/streptomycin이 첨가된 RPMI 1640 배지에서 37℃, 5% CO2배양기에서 48시간 동안 배양하였다. The spleen of 8-week-old Balb / c mice (SPF) fed into a container containing an appropriate amount of sterile HBSS solution was finely cut with a tweezers, and a single spleen cell suspension was added to a 96 well plate . In the three experimental groups, Con A (0.5 μg / ㎖) was added, the remaining three experimental groups were not in Con A, and the three groups were in the 400, 200, 100, 50 and 0 μg / ml , Respectively. Each vessel was incubated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin / streptomycin at 37 ° C in a 5% CO 2 incubator for 48 hours.
48시간 배양 종료후, IFN-γ, IL-2, IL-4, IL-10, IL-12 등 세포활성물질의 측정은 ELISA방법으로 수행하였다. 즉 anti-mouse IFN-γ, IL-2, IL-4, IL-10 와 anti-mouse IL-12 capture 단클론 항체 등을 96-well plate에 1 ㎍/㎖농도로 코팅하여 4℃에서 12시간 방치하였다. 코팅 후, 비 특이적 결합부위를 막기 위해 2% BSA를 함유한 PBS에 blocking buffer를 첨가하여 37℃에서 2시간 동안 방치하였다. 0.05% tween-20 을 함유한 PBS로 4회 세척 후 재조합 사람 IFN-γ, IL-2, IL-4와 재조합 생쥐 IL-12 표준액과 상기 제조된 각각의 추출물(200, 100, 50, 0 μg/ml)를 처리한 검체의 배양 상등액을 각 well 에 100 ㎕씩 부가하여 37℃에서 2시간 동안 방치하였다. 0.05% tween-20 을 함유한 PBS로 4회 세척 후 biotinylated anti-mouse IFN-γ, IL-2, IL-4 와 anti-mouse IL-12는 1% BSA를 함유한 PBS를 이용하여 0.05 ㎍/ml 농도로 희석한 후 well에 처리하여 37℃에서 2시간 동안 방치한다. 세정용 완충용액으로 7회 세척한 후 avidin-conjugated enzyme을 2.5 ㎍/ml 농도로 각 well에 처리한 다음 37℃에서 30분 방치한 후 7회 세척한다. ABTS 기질액을 각 well 에 100 ㎕씩 가하여 10 분간 발색을 유도한 다음 ELISA reader를 이용하여 405 nm 파장에서 IFN-γ, IL-2, IL-4, IL-12 등을 측정하였다.
After 48 hours of culture, cell active substances such as IFN-y, IL-2, IL-4, IL-10 and IL-12 were assayed by ELISA. The anti-mouse IFN-γ, IL-2, IL-4, IL-10 and anti-mouse IL-12 capture monoclonal antibodies were coated on a 96-well plate at 1 μg / Respectively. After coating, blocking buffer was added to PBS containing 2% BSA to prevent non-specific binding sites, and the mixture was allowed to stand at 37 ° C for 2 hours. After washing four times with PBS containing 0.05% tween-20, the recombinant human IFN-γ, IL-2, IL-4 and recombinant mouse IL-12 standard solutions and the respective extracts (200, 100, / ml) was added to each well and incubated at 37 ° C for 2 hours. After washing four times with PBS containing 0.05% tween-20, the biotinylated anti-mouse IFN-γ, IL-2, IL-4 and anti- mouse IL- ml and then treated at 37 ° C for 2 hours. After washing 7 times with buffer solution for washing, avidin-conjugated enzyme was treated at 2.5 ㎍ / ml in each well, followed by incubation at 37 ℃ for 30 minutes and washing 7 times. IL-2, IL-4, and IL-12 were measured at 405 nm wavelength using an ELISA reader after 100 μl of ABTS substrate solution was added to each well.
<실험결과><Experimental Results>
1. 각 시험물질의 세포독성 평가 1. Assessment of cytotoxicity of each test substance
본 발명에서 실험에 사용된 각각의 시험물질들은 각각 200, 100, 50 ug/ml의 농도에서 비장세포에 대한 독성을 나타내지 않는 것으로 확인되어 이후 실험은 상기 농도에 따라 진행하였다(결과 미도시).
Each of the test substances used in the experiment of the present invention was confirmed to show no toxicity to spleen cells at the concentrations of 200, 100, and 50 ug / ml, respectively, and the experiment was then carried out according to the concentration (results not shown).
2. 2. 비장세포의Splenocyte T cell 증식 상승효과 T cell proliferation synergistic effect
본 발명에 따른 각각의 추출물이 비장세포 T cell의 증식에 어떠한 영향을 미치는지를 확인해 보았으며, 이에 대한 결과를 도 1에 나타내었다. The effect of each extract according to the present invention on the proliferation of splenic T cells was examined, and the results are shown in FIG.
도 1에서 볼 수 있는 바와 같이 세포만 존재하는 실험군(Only cells)과 비교하여 Con A를 처리한 대조군(Con-A_CTL)에서 T cell의 증식이 상승하는 것을 확인할 수 있었다. 한편, 누에와 육계의 혼합추출물군은 200 및 100 ug/ml의 농도에서 누에 단독 추출물 및 육계 단독 추출물과 비교해 현저히 우수한 상승효과를 나타내는 것을 확인할 수 있었다. 누에와 육계 혼합추출물의 T cell 증식 촉진효과는 양성대조군으로 사용된 Rg3보다 200 ug/ml의 농도에서 더 높게 나타났으며, 홍삼 투여군과 비교해도 거의 동등한 것으로 확인되었다.
As can be seen in FIG. 1, the increase in T cell proliferation was observed in the control group (Con-A_CTL) treated with Con A compared to the only cells having only cells. On the other hand, it was confirmed that the mixed extract group of silkworm and broiler chickens showed remarkably excellent synergistic effect at a concentration of 200 and 100 ug / ml as compared with the silkworm extract alone and the broiler chick extract alone. The T cell proliferation promoting effect of silkworm and broiler mixed extracts was higher than that of Rg3 used as positive control at 200 ug / ml and it was found to be almost equal to that of red ginseng group.
하지만, 누에와 부추의 혼합추출물군의 경우에는 아무런 상승효과가 관찰되지 않았으며, 심지어 누에 단독 추출물과 비교해 오히려 효과가 감소하는 경향을 나타내는 것으로 확인되었다.
However, no synergistic effect was observed in the mixed extract group of silkworm and leek, and it was found that the effect of the silkworm extract was lower than that of the silkworm extract alone.
3. 3. 비장세포에서In splenocytes IL-2 생산량의 상승효과 The synergistic effect of IL-2 production
IL-2는 T 림프구, B 림프구 성장 촉진 및 NK세포 활성화 기능을 하는 것으로 알려져 있다. 이에, 상기 제조된 각각의 추출물들이 비장세포에서 IL-2의 생산량에 미치는 영향을 확인해 보고자 하였다. 이에 대한 결과를 도 2에 나타내었다. IL-2 is known to promote T lymphocyte, B lymphocyte growth and NK cell activation. The effect of each of the extracts on the production of IL-2 in splenocytes was examined. The results are shown in Fig.
도 2에서 확인할 수 있는 바와 같이, 세포만 존재하는 실험군(Only cells)과 비교하여 Con A를 처리한 대조군(Con-A_CTL)에서 IL-2의 생산량이 상승하는 것을 확인할 수 있었다. 한편, 누에와 육계의 혼합추출물군은 모든 처리 농도에서 누에 단독 추출물 및 육계 단독 추출물과 비교해 현저히 우수한 상승효과를 나타내는 것을 확인할 수 있었다. 누에와 육계 혼합추출물의 IL-2 생성 촉진효과는 양성대조군으로 사용된 홍삼 및 Rg3보다도 현저히 우수하게 나타났다.
As shown in FIG. 2, it was confirmed that the production of IL-2 was increased in the control group (Con-A_CTL) treated with Con A compared with the only cells in which only cells were present. On the other hand, it was confirmed that the mixed extract group of silkworm and broiler chicks exhibited remarkably excellent synergistic effect in comparison with the single extract of silkworm and broiler alone extract at all treatment concentrations. IL-2 production promoting effect of silkworm and broiler mixed extracts was significantly better than red ginseng and Rg3 used as positive control.
4. 4. 비장세포에서In splenocytes IL-4 생산량의 상승효과 Increased effect of IL-4 production
IL-4는 B 림프구 성장 및 분화 항체 생성 유도, TH2 반응 유도하여 체액성면역증진을 나타내는 것으로 알려져 있다. 이에, 상기 제조된 각각의 추출물들이 비장세포에서 IL-4의 생산량에 미치는 영향을 확인해 보고자 하였다. 이에 대한 결과를 도 3에 나타내었다. IL-4 is known to induce B lymphocyte growth and differentiation, induce TH2 response, and enhance humoral immunity. The effect of each of the extracts on IL-4 production in spleen cells was examined. The results are shown in Fig.
도 3에서 확인할 수 있는 바와 같이, 세포만 존재하는 실험군(Only cells)과 비교하여 Con A를 처리한 대조군(Con-A_CTL)에서 IL-4의 생산량이 상승하는 것을 확인할 수 있었다. 한편, 누에와 육계의 혼합추출물군은 모든 처리 농도에서 누에 단독 추출물 및 육계 단독 추출물과 비교해 현저히 우수한 상승효과를 나타내는 것을 확인할 수 있었다. 누에와 육계 혼합추출물의 IL-4 생성 촉진효과는 100 및 50 ug/ml의 농도에서 양성대조군으로 사용된 홍삼 및 Rg3보다도 현저히 우수한 것으로 확인되었다.
As can be seen from FIG. 3, it was confirmed that the production of IL-4 was increased in the control group (Con-A_CTL) treated with Con A compared with the cells in which only cells were present. On the other hand, it was confirmed that the mixed extract group of silkworm and broiler chicks exhibited remarkably excellent synergistic effect in comparison with the single extract of silkworm and broiler alone extract at all treatment concentrations. The stimulating effect of IL-4 production in silkworm and broiler mixed extracts was significantly better than red ginseng and Rg3 used as positive control at 100 and 50 ug / ml.
5. 5. 비장세포에서In splenocytes IL-10 생산량의 상승효과 Increased effect of IL-10 production
IL-10은 B 림프구 성장 및 TH1 반응 억제하는 것으로 면역반응을 자극하기 보다는 조절하는 기능을 가진 항염증성 사이토카인으로, 대식세포, 수지상세포, 중성구, B세포 및 T세포, NK 세포 등이 IL-10의 수용체를 가지고 있으며, IL-10의 수용체 중 IL-10과 결합하는 체인의 구조는 IFN-a/ß나 IFN-r의 것과 동질성을 나타내어 IL-10도 Jak-ST AT 전달경로를 이용하여 신호를 전달하는 것으로 알려져 있다. 이에, 상기 제조된 각각의 추출물들이 비장세포에서 IL-10의 생산량에 미치는 영향을 확인해 보고자 하였다. 이에 대한 결과를 도 4에 나타내었다. IL-10 is an anti-inflammatory cytokine that inhibits B lymphocyte growth and TH1 response, rather than stimulates the immune response. IL-10 is an anti-inflammatory cytokine that controls IL- 10 receptor, and the structure of the chain that binds to IL-10 among the receptors of IL-10 is similar to that of IFN-a / ß or IFN-r. Signal. ≪ / RTI > Thus, the effect of each of the extracts prepared on the production of IL-10 in splenocytes was examined. The results are shown in Fig.
도 4에서 확인할 수 있는 바와 같이, 세포만 존재하는 실험군(Only cells)과 비교하여 Con A를 처리한 대조군(Con-A_CTL)에서 IL-10의 생산량이 상승하는 것을 확인할 수 있었다. 한편, 누에와 육계의 혼합추출물군은 200 ug/ml의 농도에서 누에 단독 추출물 및 육계 단독 추출물과 비교해 현저히 우수한 상승효과를 나타내는 것을 확인할 수 있었다. 누에와 육계 혼합추출물의 IL-10 생성 촉진효과는 200 ug/ml의 농도에서 양성대조군으로 사용된 홍삼 및 Rg3보다도 현저히 우수한 것으로 확인되었다.
As shown in FIG. 4, it was confirmed that the production of IL-10 was increased in the control group (Con-A_CTL) treated with Con A compared with the only cells containing only cells. On the other hand, it was confirmed that the mixed extract group of silkworm and broiler chicks showed a remarkably excellent synergistic effect at a concentration of 200 ug / ml as compared with the silkworm extract alone and the broiler chick extract alone. The promoting effect of IL-10 production in silkworm and broiler mixed extracts was significantly better than that of red ginseng and Rg3 as a positive control at a concentration of 200 ug / ml.
6. 6. 비장세포에서In splenocytes IL-12 생산량의 상승효과 Increased effect of IL-12 production
IL-12은 TH1 반응을 유도하는 것으로 생물활성으로는 정지기의 T세포와 자연살생(NK)세포에서 인터페론γ생산의 유도, NK세포의 활성유도, 정지기 T세포의 렉틴 자극에 의한 세포증식능의 항진, 나이브세포에서 TH1세포로의 분화 촉진, 인터류킨3 존재 하에서 조혈간세포의 증식 촉진, 이식종양에 대한 생체 내 항종양효과 등이 있는 것으로 알려져 있다. 이에, 상기 제조된 각각의 추출물들이 비장세포에서 IL-12의 생산량에 미치는 영향을 확인해 보고자 하였다. 이에 대한 결과를 도 5에 나타내었다. IL-12 induces the TH1 response. In terms of biological activity, IL-12 induces interferon gamma production in stably T cell and natural killer (NK) cells, induction of NK cell activation and cell proliferation by lectin stimulation , Promotion of differentiation from naive cells into TH1 cells, promotion of proliferation of hematopoietic stem cells in the presence of interleukin 3, and in vivo antitumor effect on transplant tumors. Thus, the effect of each of the extracts prepared on the production of IL-12 in splenocytes was examined. The results are shown in Fig.
도 5에 나타낸 바와 같이, 세포만 존재하는 실험군(Only cells)과 비교하여 Con A를 처리한 대조군(Con-A_CTL)에서 IL-12의 생산량이 상승하는 것을 확인할 수 있었다. 한편, 누에와 육계의 혼합추출물군은 모든 투여 농도에서 누에 단독 추출물 및 육계 단독 추출물과 비교해 현저히 우수한 상승효과를 나타내는 것을 확인할 수 있었다. 누에와 육계 혼합추출물의 IL-12 생성 촉진효과는 모든 농도에서 양성대조군으로 사용된 홍삼 및 Rg3보다도 현저히 우수한 것으로 확인되었다.
As shown in FIG. 5, it was confirmed that the production of IL-12 was increased in the control group (Con-A_CTL) treated with Con A compared to the cells only the cells containing only the cells. On the other hand, it was confirmed that the mixed extract group of silkworm and broiler chicks showed remarkably excellent synergistic effect compared to the silkworm extract alone and the broiler chick extract alone at all dose levels. The IL-12 production promoting effect of silkworm and broiler mixed extracts was found to be significantly better than red ginseng and Rg3 used as positive control at all concentrations.
7. 7. 비장세포에서In splenocytes IFNIFN -γ 생산량의 상승효과 -γ increase in production
IFN-γ는 TH1세포에서 생산되는 세포분열촉진물질로 그 기능이 아주 다양하다. 예를 들면, 바이러스로부터의 세포보호, 조직배양에서나 골수에서의 세포분열 억제, T세포의 작용 조절, 자연면역세포(NK세포)의 기능 항진을 유도하여 식균작용을 상승시키고, 또 특수 암세포의 분열 억제 등 이루 헤아릴 수 없이 많이 알려져 있다. 이에, 상기 제조된 각각의 추출물들이 비장세포에서 IFN-γ의 생산량에 미치는 영향을 확인해 보고자 하였다. 이에 대한 결과를 도 6에 나타내었다. IFN-γ is a cell division-promoting substance produced in TH1 cells. For example, cell protection from viruses, suppression of cell division in bone marrow, suppression of T cell function, and hyperactivation of naturally-occurring immune cells (NK cells) are enhanced to enhance phagocytosis, Suppression, etc. are known to be unthinkable. Thus, the effect of each of the extracts prepared on the production of IFN-y in spleen cells was examined. The results are shown in Fig.
도 6에 나타낸 바와 같이, 세포만 존재하는 실험군(Only cells)과 비교하여 Con A를 처리한 대조군(Con-A_CTL)에서 IFN-γ의 생산량이 상승하는 것을 확인할 수 있었다. 한편, 누에와 육계의 혼합추출물군은 100 및 50 ug/ml의 농도에서 누에 단독 추출물 및 육계 단독 추출물과 비교해 현저히 우수한 상승효과를 나타내는 것을 확인할 수 있었다. 누에와 육계 혼합추출물의 IFN-γ 생성 촉진효과는 100 및 50 ug/ml의 농도에서 양성대조군으로 사용된 홍삼 및 Rg3보다도 현저히 우수한 것으로 확인되었다.
As shown in FIG. 6, it was confirmed that IFN-y production was increased in the control group (Con-A_CTL) treated with Con A compared to the experimental cells (only cells) containing only cells. On the other hand, it was confirmed that the mixed extract group of silkworm and broiler chicks showed remarkably excellent synergistic effect at a concentration of 100 and 50 ug / ml as compared with the silkworm extract alone and the broiler chick extract alone. The promoting effect of IFN-γ production in silkworm and broiler mixed extracts was significantly better than red ginseng and Rg3 used as positive control at 100 and 50 ug / ml.
본 발명의 조성물은 비장세포에서 IL-2, IL-4, IL-10, IL-12 및 IFN-γ의 분비를 촉진할 뿐만 아니라 T 세포의 분열을 촉진하여, 선천면역과 후천면역을 모두 증강시키는 효과를 나타낸다. 따라서, 본 발명의 조성물은 면역증강용 약학적 조성물 또는 식품 조성물의 개발에 매우 유용하게 활용될 수 있어 산업상 이용가능성이 매우 우수하다. The composition of the present invention not only promotes the secretion of IL-2, IL-4, IL-10, IL-12 and IFN-y in splenocytes but also promotes cleavage of T cells to enhance both innate and acquired immunity . Accordingly, the composition of the present invention can be very usefully used in the development of a pharmaceutical composition or food composition for immunomodulation, and thus is highly industrially applicable.
Claims (5)
A mixture of broilers and silkworm extract.
The pharmaceutical composition according to claim 1, wherein the extract is one selected from the group consisting of water, an alcohol having 1 to 6 carbon atoms, hexane, ethyl acetate, and a mixed solvent thereof.
3. The pharmaceutical composition according to claim 2, wherein the alcohol is ethanol.
[Claim 2] The pharmaceutical composition according to claim 1, wherein the weight ratio of the broiler to the silkworm is 1: 0.5-3.
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