KR102472952B1 - A composition for immune enhancement comprising narrow-leaf erecta fig extract - Google Patents
A composition for immune enhancement comprising narrow-leaf erecta fig extract Download PDFInfo
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Abstract
본 발명은 좁은잎천선과 추출물을 유효성분으로 포함하는 면역 증진용 조성물에 관한 것이다.The present invention relates to a composition for enhancing immunity, comprising an extract of Astragalus nigra as an active ingredient.
Description
본 발명은 좁은잎천선과 추출물을 유효성분으로 포함하는 면역 증진용 조성물에 관한 것으로, 의약품 및 식품 등 다양한 용도로서 활용 가능하다.The present invention relates to a composition for enhancing immunity comprising an extract of Narrowleaf Astragalus as an active ingredient, and can be used for various purposes such as pharmaceuticals and foods.
모든 생물체는 생명을 영위하기 위해 끊임없이 많은 영양소를 필요로 하며, 이러한 영양소는 식품의 형태로 항상 외부로부터 공급되거나 일부는 체내에서 합성된다.All living organisms constantly need a lot of nutrients to sustain life, and these nutrients are always supplied from the outside in the form of food or some are synthesized in the body.
음식물 속에는 인간의 생명현상을 유지하는데 필요한 물질들이 포함되어 있으며, 인간은 음식물을 섭취하여 소화기관에서 소화시키고 흡수하여 체내의 조직세포에 공급함으로써 생명을 유지하고 있다.Food contains substances necessary for maintaining human life, and humans maintain life by ingesting food, digesting it in the digestive system, absorbing it, and supplying it to tissue cells in the body.
따라서 좋은 영양은 건강한 신체를 유지시켜주며, 건강한 신체는 건강한 정신을 보장한다.Therefore, good nutrition maintains a healthy body, and a healthy body guarantees a healthy mind.
영양은 직접, 간접으로 질병의 원인이 되기도 하며, 질병의 예방 및 치료에도 관여하는 것으로 알려져 있는데, 인체에 필요한 영양소의 균형섭취 제대로 이루어지지 않아 생기는 영양분의 부족도 문제이고, 과잉섭취 또한 심각한 문제이며, 이로 인해 각종 질병이 유발될 수 있다.Nutrition is known to directly or indirectly cause diseases, and is also involved in the prevention and treatment of diseases. , which can cause various diseases.
최근 생활수준이 높아지고 식생활이 서구화되면서 영양과잉, 운동부족 등으로 인한 비만, 당뇨, 고혈압 및 심장병 등과 같은 성인병 발병률이 증가하고 있다. BACKGROUND ART Recently, as living standards have increased and dietary habits have become westernized, the incidence of adult diseases such as obesity, diabetes, high blood pressure, and heart disease due to overnutrition and lack of exercise is increasing.
또한 평균수명 연장으로 인하여 인구가 점차 노령화되면서 건강에 대한 관심이 점차 높아지고 있다.In addition, as the population is gradually aging due to the extension of life expectancy, interest in health is gradually increasing.
건강증진(health promotion)은 질병, 특히 만성 퇴행성질환이나 감염증, 계속되는 스트레스에 대한 저항력의 증가, 일상의 활동력의 증대를 꾀하는 것으로 이를 위해 영양의 개선, 면역기능 강화, 적절한 운동과 휴식, 정신적 활동의 지속 등 생활양식 전반에 미치는 관리가 요구된다. Health promotion is to increase resistance to diseases, especially chronic degenerative diseases or infections, and to increase daily activity. Sustainability and overall lifestyle management are required.
면역은 체내에 존재하는 자기방어체계로서 인체가 외부에서 침입하는 각종 물질이나 생명체를 자기 자신에 대한 이물질로 인식하여 제거하고 대사시키는 과정이다.Immunity is a self-defense system that exists in the body, and is a process in which the human body recognizes various substances or organisms invading from the outside as foreign substances to itself, removes them, and metabolizes them.
외부 자극에 의한 손상이나 병원 미생물의 침입으로부터 자신을 방어하기도 하지만 염증반응 등과 같이 자기 자신의 조직에 손상을 줄 수도 있다.It defends itself from damage caused by external stimuli or the invasion of pathogenic microorganisms, but it can also damage its own tissue, such as an inflammatory response.
면역기능의 변화를 조절하여 정상으로 회복시키거나 변화의 폭을 줄여 주는 작용으로 면역기능 억제나 면역기능 증강으로 구분된다.It is an action that regulates changes in immune function to restore them to normal or reduce the width of changes, and is divided into suppression of immune function or enhancement of immune function.
면역 기능에 영향을 미치는 요인에는 유전적, 환경적 요인, 나이, 성별, 심리적 스트레스, 영양상태, 식이습관, 질병 등이 있다.Factors that affect immune function include genetic and environmental factors, age, gender, psychological stress, nutritional status, dietary habits, and diseases.
면역 기능의 조절 기능을 갖는 건강기능식품은 현재까지, 저하된 면역기능을 증진 시키는 기능성과 과도한 면역기능을 조절하는 기능성, 자기 성분에 대한 면역반응을 조절하는 자가면역기능 조절 기능성으로 구분된다.Functional health foods with the function of regulating immune function are classified into functionalities that promote reduced immune function, functions that control excessive immune function, and functions that regulate autoimmune function that regulate immune responses to self-components.
면역기능이 저하되면 과민반응을 일으키는 경우가 있는데 이러한 과민반응은 천식, 계절성 또는 동년성 비염, 알러지성 부비강염, 결막염, 식품과 약품에 의한 알러지, 아토피성 피부염, 두드러기, 벌침에 의한 알러지 등과 같이 다양하게 나타난다.When the immune function is weakened, hypersensitivity reactions may occur, and these hypersensitivity reactions are diverse, such as asthma, seasonal or winter rhinitis, allergic sinusitis, conjunctivitis, food and drug allergy, atopic dermatitis, hives, and allergy due to bee stings. appear
여러 원인으로 인한 면역기능 장애를 극복하기 위하여, 다양한 면역 증강제가 사용되고 있으나, 부작용 및 생체 안전성의 문제가 있으므로 장기 복용이 가능하고 예방용으로도 적합한 면역 증진 조성물이 요구되는 실정이다.In order to overcome immune dysfunction due to various causes, various immune enhancers are used, but there is a need for an immune enhancing composition that can be taken for a long time and is suitable for prevention because of side effects and biosafety problems.
본 발명은 전술한 종래 기술의 문제점을 해결하기 위한 것으로, 본 발명의 목적은 생체 안전성이 우수한 천연 식물 유래의 기능성 식의약품을 제공하는 것이다.The present invention is to solve the problems of the prior art described above, and an object of the present invention is to provide a functional food or drug derived from natural plants with excellent biosafety.
본 발명의 일 측면에 따르면, 좁은잎천선과 추출물을 유효성분으로 포함하는 면역 증진용 조성물이 제공된다.According to one aspect of the present invention, there is provided a composition for enhancing immunity comprising an extract of Astragalus nigra as an active ingredient.
일 실시예에 있어서, 상기 좁은잎천선과는 증자된 것일 수 있다.In one embodiment, the narrow leaf cloth may be increased.
일 실시예에 있어서, 상기 좁은잎천선과는 100 내지 200℃에서 증자한 것일 수 있다.In one embodiment, the narrow leaf cloth may be steamed at 100 to 200 ° C.
일 실시예에 있어서, 상기 좁은잎천선과는 100℃ 이상의 온도에서 30 내지 120분간 증자한 것일 수 있다.In one embodiment, the narrow leaf cloth may be steamed for 30 to 120 minutes at a temperature of 100 ° C or higher.
본 발명의 다른 측면에 따르면, 좁은잎천선과 추출물을 유효성분으로 포함하는 면역 증진용 건강기능식품이 제공된다.According to another aspect of the present invention, there is provided a health functional food for enhancing immunity comprising an extract of Narrowleaf Astragalus as an active ingredient.
일 실시예에 있어서, 상기 좁은잎천선과는 100℃ 이상의 온도에서 30 내지 120분간 증자할 것일 수 있다.In one embodiment, the narrow leaf cloth may be steamed for 30 to 120 minutes at a temperature of 100 ° C or higher.
본 발명의 다른 측면에 따르면, 좁은잎천선과 추출물을 유효성분으로 포함하는 면역 증진용 약학 조성물이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for enhancing immunity comprising an extract of Astragalus nigra as an active ingredient.
일 실시예에 있어서, 상기 좁은잎천선과는 100℃ 이상의 온도에서 30 내지 120분간 증자한 것일 수 있다.In one embodiment, the narrow leaf cloth may be steamed for 30 to 120 minutes at a temperature of 100 ° C or higher.
본 발명의 일 측면에 따른 조성물은 천연 추출물을 유효성분으로 포함하여 생체 안전성이 우수할 뿐만 아니라 면역 증진 활성이 우수하므로 건강기능식품뿐만 아니라 면역 관련 질환의 치료 용도로서 유용하게 활용할 수 있다.Since the composition according to one aspect of the present invention contains natural extracts as an active ingredient and has excellent biosafety and immune enhancing activity, it can be usefully used as a health functional food as well as for the treatment of immune-related diseases.
본 발명의 효과는 상기한 효과로 한정되는 것은 아니며, 본 발명의 상세한 설명 또는 청구범위에 기재된 발명의 구성으로부터 추론 가능한 모든 효과를 포함하는 것으로 이해되어야 한다.The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the configuration of the invention described in the claims.
도 1은 BALB/c mouse에서 NK cell cytotoxicity를 평가한 결과이다.
도 2는 ELISA를 통해 BALB/c 동물 모델의 T 세포의 활성 cytokine을 측정한 결과이다. (A)는 IL-2, (B)는 IL-4, (C)는 IL-10, (D)는 IL-12, (E)는 IFN-γ을 측정한 결과이다.
도 3은 복강침출 세포로부터 NO 생성량을 측정한 결과이다.
도 4는 마우스의 주요 면역관련 조직들의 총 세포수를 분석한 결과이다. (A)는 지라, (B)는 흉선, (C)는 페이어판, (D)는 DLN, (E)는 PEC에서 총 세포수를 측정한 결과이다.
도 5는 BALB/c mouse 모델의 PBMC에서 형광 유세포를 분석한 결과이다.
도 6은 마우스 비장세포에서 NK 세포의 기능적 활성을 측정한 결과이다.
도 7은 BALB/c 복강세포군 중 지라에서 절대세포수를 측정한 결과이다.
도 8은 BALB/c 흉선에서 절대세포수를 측정한 결과이다.
도 9는 BALB/c DLN에서 절대세포수를 측정한 결과이다.
도 10은 페이어(Peyer's patch)에서 절대세포수를 측정한 결과이다.
도 11은 BALB/c 마우스 PEC에서 절대세포수를 측정한 결과이다.1 is a result of evaluating NK cell cytotoxicity in BALB/c mice.
Figure 2 is the result of measuring the active cytokine of T cells of the BALB/c animal model through ELISA. (A) is IL-2, (B) is IL-4, (C) is IL-10, (D) is IL-12, and (E) is the result of measuring IFN-γ.
Figure 3 is the result of measuring the amount of NO production from the peritoneal leach cells.
Figure 4 is the result of analyzing the total number of cells in major immune-related tissues of mice. (A) is the spleen, (B) is the thymus, (C) is the Peyer's patch, (D) is the DLN, and (E) is the result of measuring the total number of cells in the PEC.
5 is a result of fluorescence flow cytometry analysis in PBMC of the BALB/c mouse model.
Figure 6 is a result of measuring the functional activity of NK cells in mouse splenocytes.
7 is a result of measuring the absolute cell number in the spleen among the BALB/c peritoneal cell population.
8 is a result of measuring the absolute cell number in BALB/c thymus.
9 is a result of measuring the absolute cell number in BALB/c DLN.
10 is a result of measuring the absolute cell number in Peyer's patch.
11 is a result of measuring the absolute cell number in BALB/c mouse PEC.
본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. The terms used in this specification have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but these may vary depending on the intention of a person skilled in the art, precedent, or the emergence of new technologies.
또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다. Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with the meaning in the context of the related art, and unless explicitly defined in this application, it should not be interpreted in an ideal or excessively formal meaning. don't
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여져 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.Numerical ranges are inclusive of the values defined therein. Every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written. Every minimum numerical limitation given throughout this specification includes every higher numerical limitation, as if such higher numerical limitations were expressly written. Every numerical limitation given throughout this specification will include every better numerical range within the broader numerical range, as if the narrower numerical limitations were expressly written.
이하, 본 발명의 실시예를 상세히 기술하나, 하기 실시예에 의해 본 발명이 한정되지 아니함은 자명하다.Hereinafter, embodiments of the present invention will be described in detail, but it is obvious that the present invention is not limited by the following examples.
본 발명의 일 측면에 따르면, 좁은잎천선과 추출물을 유효성분으로 포함하는 면역 증진용 조성물이 제공된다.According to one aspect of the present invention, there is provided a composition for enhancing immunity comprising an extract of Astragalus nigra as an active ingredient.
상기 “좁은잎천선과(Ficus erecta var. sieboldii)”는 무화과과에 속하는 식물로서 가는잎천선과나무라고도 하며 우리나라에서도 제주도와 남해안 지방, 특히, 제주도에 주로 자라는 나무이다.The "narrow-leaved Astragalus ( Ficus erecta var. sieboldii )" is a plant belonging to the fig family and is also known as a thin-leaved Astragalus tree, and is a tree that grows mainly in Jeju Island and the southern coastal region, especially Jeju Island, in Korea.
상기 좁은잎천선과는 바닷가 산기슭에서 자라며 높이 2 내지 4m이고, 나무껍질은 잿빛이 섞인 흰색이며 어두운 갈색 피목이 있고 털이 없다. 잎은 어긋나고 천선과 보다 좁은 바소꼴이며 길이 10 내지 20cm이다.The narrow-leaved celestial family grows at the foot of the seashore and is 2 to 4 m high, and the bark is grayish white, has dark brown fur, and has no hair. Leaves are alternate, lanceolate and narrower, 10 to 20 cm long.
상기 좁은잎천선과의 곁맥은 5 내지 6쌍이고, 잎자루는 길이가 1 내지 4cm이다. 상기 좁은잎천선과는 민간에서는 오래전부터 보중, 익기, 건비, 화습, 강근장골, 소종, 활혈, 해독의 효능으로 알려져 있다. There are 5 to 6 pairs of side veins with the narrow leaf trifolium, and the petiole is 1 to 4 cm long. The narrow-leaved Cheonseongwa has been known in the private sector for a long time for its efficacy of bojung, ripeness, gunbi, hwaseup, strong muscle long bone, small species, hwalhyeol, and detoxification.
또한, 상기 좁은잎천선과는 류마티스 관절염, 중기허약, 기혈쇠미, 사디산언, 근골분리, 타박상, 경폐, 산후유즙결핍 등의 치료에 이용되어 왔다.In addition, the narrow leaf aponeurosis has been used in the treatment of rheumatoid arthritis, mid-term weakness, qi and blood loss, sadisan-eon, musculoskeletal separation, bruises, cervical lungs, and postpartum milk deficiency.
상기 “면역(immunity)”은 생물체 내에서 병원체와 종양 세포 등을 탐지한 후 무력화하여 질병으로부터 생명체를 보호하는 기작을 의미하는 것으로, 상기 “면역 증진(Immunostimulation)”은 암 및 염증 등과 같은 다양한 질환에 대한 신체 방어 기전을 보강하는 일련의 치료학적 또는 예방학적 전략을 모두 포함할 수 있다.The “immunity” refers to a mechanism that detects pathogens and tumor cells in an organism and then neutralizes them to protect the organism from disease. The “immunostimulation” refers to various diseases such as cancer and inflammation. It can include any of a series of therapeutic or prophylactic strategies that reinforce the body's defense mechanisms against
상기 “유효성분으로 포함하는”은 투여하고자 하는 개체에 대한 치료적 또는 예방적 효과를 제공하기에 충분한 양을 포함하는 것을 의미하며, 상기 좁은잎천선과 추출물은 생체 안전성이 우수하므로 당업자가 양적 상한을 적절히 조정할 수 있다.The "contained as an active ingredient" means that it includes an amount sufficient to provide a therapeutic or preventive effect on the subject to be administered, and since the Narrowleaf Astragalus extract has excellent biosafety, those skilled in the art can limit the quantitative upper limit can be adjusted appropriately.
상기 “추출물”은 용매와 추출 원료를 특정 조건 하에서 접촉시킴으로써 추출 원료에 함유된 유효성분이 전이된 용매를 지칭하는 것으로, 천연물로부터 원료에 함유된 성분을 분리해낸 물질이라면, 추출 방법이나 성분의 종류와 무관하게 모두 포함할 수 있다. 예컨대, 물이나 유기용매를 이용하여 천연물로부터 용매에 용해되는 성분을 추출한 것, 천연물의 특정 성분, 예컨대 오일과 같은 특정 성분만을 추출하여 얻어진 것 등을 모두 포함할 수 있다.The "extract" refers to a solvent in which the active ingredient contained in the extraction material is transferred by contacting the solvent and the extraction material under specific conditions. Anything can be included regardless. For example, those obtained by extracting a component soluble in a solvent from a natural product using water or an organic solvent, and those obtained by extracting a specific component of a natural product, for example, only a specific component such as oil, may be included.
상기 추출물은 좁은잎천선과를 건조 후 분말화하거나, 열수추출법, 에탄올 추출법 등 종래 알려진 다양한 추출법에 의해 수득할 수 있다.The extract can be obtained by drying and pulverizing narrow-leaved Astragalus fruit, or by various extraction methods known in the art, such as hot water extraction and ethanol extraction.
상기 추출물은 물, 알코올, 에틸아세테이트, 아세톤, 핵산, 디클로로메탄 또는 이들의 혼합 용매로 추출될 수 있고, 바람직하게는 정제수를 이용하여 열수 추출될 수 있다.The extract may be extracted with water, alcohol, ethyl acetate, acetone, nucleic acid, dichloromethane, or a mixture thereof, preferably hot water extraction using purified water.
상기 원료에 포함된 유효성분은 용매의 극성에 따라 추출 비율이 상이해질 수 있으므로, 용매에 따른 생리 활성 물질의 선택성을 고려하여 달리할 수 있다.Since the active ingredient included in the raw material may have a different extraction ratio depending on the polarity of the solvent, it may be different in consideration of the selectivity of the physiologically active substance according to the solvent.
상기 추출물은 추출 원료를 물로 수세한 후 건조 및 분쇄하여, 원료 중량의 8 내지 12배에 달하는 부피의 용매로 약 1 내지 24시간 동안 환류 순환 추출, 가압 추출, 초음파 추출 등 통상적인 방법으로 추출 및 여과하여 제조할 수 있다.The extract is obtained by washing the extraction raw material with water, then drying and pulverizing, extracting by a conventional method such as reflux circulation extraction, pressure extraction, ultrasonic extraction, etc. for about 1 to 24 hours with a solvent in a volume of 8 to 12 times the weight of the raw material. It can be prepared by filtration.
상기 추출물은 감압 증류 또는 동결 건조 등과 같은 추가적인 공정에 의해 분말 상태로 수득할 수 있다.The extract may be obtained in powder form by additional processes such as distillation under reduced pressure or freeze drying.
상기 추출물은 통상적인 정제 과정을 거친 추출물도 포함할 수 있다. 예컨대, 상기 추출물은 일정한 분자량 컷-오프 값을 갖는 한외 여과막을 이용한 분리, 다양한 크로마토그래피(크기, 전하, 소수성 또는 친화성에 따른 분리를 위해 제작된 것)에 의한 분리 등 추가적으로 실시된 다양한 정제 방법을 통해 얻어진 분획물을 포함할 수 있다.The extract may also include an extract that has undergone a conventional purification process. For example, the extract is subjected to various additional purification methods such as separation using an ultrafiltration membrane having a certain molecular weight cut-off value and separation by various chromatography (made for separation according to size, charge, hydrophobicity or affinity). It may include fractions obtained through
일 실시예에서, 상기 좁은잎천선과는 증자(蒸煮)된 것일 수 있다.In one embodiment, the narrow leaf cheonseon line may be increased (蒸煮).
상기 “증자(蒸煮)”는 살청(殺靑)의 방법의 하나로 수증기를 통해 가열하여 산화효소의 활성을 정지시키는 공정을 의미한다. 상기 증자는 당해 기술분야에 알려진 통상의 장치를 통해 수행될 수 있다.The "steaming" means a process of stopping the activity of an oxidizing enzyme by heating with water vapor as one of the methods of killing. The steaming may be performed through a conventional device known in the art.
상기 증자에 의해 좁은잎천선과에 함유된 다양한 유효성분의 함량이 증감할 수 있으며, 본 발명자들은 상기 증자에 의해 좁은잎천선과의 면역 증진 활성이 현저히 증대될 수 있음을 확인하였다.By the above steaming, the content of various active ingredients contained in Astragalus trifolium can be increased or decreased, and the present inventors confirmed that the immunity enhancement activity of Astralanaria trifolium can be remarkably increased by the steaming.
상기 좁은잎천선과는 100 내지 200℃에서 증자할 수 있고, 상기 좁은잎천선과는 100℃ 이상의 온도에서 30 내지 120분간 증자할 수 있다.The narrow leaf Cheonseon can be steamed at 100 to 200 ° C, and the narrow leaf Cheonseon can be steamed for 30 to 120 minutes at a temperature of 100 ° C or higher.
상기 증자 온도가 과도하게 낮거나 증자 시간이 과도하게 짧으면 충분히 증자되지 아니하여 증자로 인한 면역 증진 효과가 충분히 구현되지 않을 수 있고, 과도하게 증자되는 경우 고열로 인해 일부 유효성분이 소실될 수 있다.If the temperature of the steaming is excessively low or the steaming time is excessively short, the immunity enhancing effect may not be sufficiently realized due to insufficient steaming, and in the case of excessive steaming, some active ingredients may be lost due to high heat.
상기 조성물은 식품 조성물, 예컨대 면역 증진용 건강기능식품으로서 사용될 수 있다.The composition can be used as a food composition, such as a health functional food for enhancing immunity.
상기 건강기능식품은 건강기능식품에 관한 법률에 따른 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, 상기 기능성은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건 용도에 유용한 효과를 얻을 목적으로 섭취하는 것을 의미할 수 있다.The health functional food refers to food manufactured and processed using raw materials or ingredients having functional properties useful for the human body in accordance with the Health Functional Food Act, and the functional food regulates nutrients with respect to the structure and function of the human body or physiologically It may mean ingestion for the purpose of obtaining useful effects for health purposes such as action.
상기 조성물은 통상의 식품 첨가물을 포함할 수 있으며, 상기 식품 첨가물은 다른 규정이 없는 한 식품의약품안전처에 승인된 식품 첨가물 공전의 총칙 및 일반시험법 등에 따라 해당 품목에 관한 규격 및 기준에 의하여 적합성 여부를 판단할 수 있다.The composition may include conventional food additives, and the food additives conform to the standards and standards for the item in accordance with the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. can determine whether
상기 식품 첨가물 공전에 기재된 품목은 예컨대 케톤류, 글리신, 구연산칼륨, 니코틴산, 계피산 등의 화학적 합성물, 감색소, 감초추출물, 결정셀룰로오스, 고량색소, 구아검 등의 천연첨가물, L-글루타민산나트륨 제제, 면류첨가알칼리제, 보존료제제, 타르색소제제 등의 혼합제제류를 들 수 있다.Items described in the food additives code include, for example, ketones, glycine, potassium citrate, chemical compounds such as nicotinic acid and cinnamic acid, natural additives such as persimmon pigment, licorice extract, crystalline cellulose, goyang pigment, guar gum, sodium L-glutamate preparations, and noodles. and mixed preparations such as added alkali agent, preservative preparation, and tar color preparation.
상기 조성물은 면역 증진을 위한 식품 및 음료 등에 다양하게 이용될 수 있으며, 예컨대, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능성 보조 식품, 식품 첨가제 등에 사용될 수 있다.The composition can be used in a variety of foods and beverages for immunity enhancement, for example, various foods, beverages, gum, tea, vitamin complexes, health functional supplements, food additives, and the like.
또한, 상기 건강기능식품은 면역 증진을 목적으로 정제, 과립, 분말, 캅셀, 액상의 용액 및 환으로 이루어진 군에서 선택된 어느 하나의 제형으로 제조 및 가공될 수 있다.In addition, the health functional food may be prepared and processed into any one formulation selected from the group consisting of tablets, granules, powders, capsules, liquid solutions, and pills for the purpose of enhancing immunity.
구체적으로 상기 정제 형태의 건강기능식품은 좁은잎천선과 추출물, 부형제, 결합제, 붕해제 및 다른 첨가제와의 혼합물을 통상의 방법으로 과립화한 후, 활택제 등을 넣어 압축 성형하거나, 상기 혼합물을 직접 압축 성형하여 제조할 수 있다. 또한, 상기 정제 형태의 건강기능식품은 필요에 따라 교미제 등을 함유할 수 있으며, 필요에 따라 적당한 제피제로 제피할 수도 있다.Specifically, the health functional food in the form of a tablet is obtained by granulating a mixture of extract, excipient, binder, disintegrant and other additives in a conventional manner, and then compression molding by adding a lubricant or the like, or the mixture It can be produced by direct compression molding. In addition, the health functional food in the form of a tablet may contain a corrigent, etc., if necessary, and may be coated with an appropriate coating agent, if necessary.
상기 캅셀 형태의 건강기능식품 중 경질캅셀제는 통상의 경질캅셀에 좁은잎천선과 추출물 및 부형제 등의 첨가제와의 혼합물 또는 그의 입상물 또는 제피한 입상물을 충진하여 제조할 수 있으며, 연질캅셀제는 좁은잎천선과 추출물 및 부형제 등의 첨가제와의 혼합물을 젤라틴 등 캅셀기제에 충진하여 제조할 수 있다. 상기 연질캅셀제는 필요에 따라 글리세린 또는 솔비톨 등의 가소제, 착색제, 보존제 등을 함유할 수 있다.Among the health functional foods in the form of capsules, hard capsules can be prepared by filling ordinary hard capsules with a mixture of narrow leaf astragalus and additives such as extracts and excipients, granular materials thereof, or coated granular materials. It can be prepared by filling a mixture of leaf chunseon with extracts and additives such as excipients in a capsule base such as gelatin. The soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, and a preservative, if necessary.
상기 환 형태의 건강기능식품은 좁은잎천선과 추출물, 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 성형하여 조제할 수 있으며, 필요에 따라 백당이나 다른 적당한 제피제로 제피를, 또는 전분, 탈크 또는 적당한 물질로 환의를 입힐 수도 있다.The health functional food in the form of a pill can be prepared by molding a mixture of narrow leaf celery extract, excipients, binders, disintegrants, etc. in an appropriate way, and if necessary, cover with white sugar or other suitable coating agent, or starch, talc Alternatively, it may be worn with a suitable material.
상기 과립형태의 건강기능식품은 좁은잎천선과 추출물, 부형제, 결합제, 붕해제 등의 혼합물을 적당한 방법으로 입상으로 제조할 수 있으며, 필요에 따라 착향제, 교미제 등을 함유할 수 있다. The health functional food in the form of a granule can be prepared in a granular form by a mixture of extracts, excipients, binders, disintegrants, etc. of Narrow Leaf Cheonseonseon, and may contain flavoring agents, flavoring agents, etc., if necessary.
또한, 상기 부형제, 결합제, 붕해제, 활택제, 교미제, 착향제 등에 대한 용어 정의는 당업계에 공지된 문헌에 기재된 것으로 그 기능 등이 동일 내지 유사한 것들을 포함할 수 있다.In addition, definitions of terms for excipients, binders, disintegrants, lubricants, corrigents, flavoring agents, etc. are described in literature known in the art, and may include those having the same or similar functions.
본 발명의 다른 측면에 따르면, 좁은잎천선과 추출물을 유효성분으로 포함하는 면역 증진용 약학적 조성물이 제공된다.According to another aspect of the present invention, there is provided a pharmaceutical composition for enhancing immunity comprising an extract of Astragalus nigra as an active ingredient.
또한, 상기 조성물은 면역활성을 증진시킴으로써, 면역결핍, 면역저하 또는 면역계 손상으로 인한 질환의 예방, 개선 또는 치료에 유용하게 이용될 수 있다In addition, the composition can be usefully used for preventing, improving or treating diseases caused by immunodeficiency, immunosuppression or immune system damage by enhancing immune activity.
상기 “예방”은 병리학적 현상의 발생 빈도 또는 정도를 감소시키는 모든 행위를 의미한다. 예방은 완전할 수 있으며 또는 부분적일 수도 있다. 이 경우에는 개체 내의 면역력 저하로 인해 발생되는 증상이 상기 조성물을 사용 하지 않은 경우와 비교하여 감소하는 현상을 의미할 수 있다.The "prevention" means any action that reduces the frequency or severity of pathological phenomena. Prevention can be complete or partial. In this case, it may mean a phenomenon in which symptoms caused by a decrease in immunity in the subject are reduced compared to the case where the composition is not used.
상기 “치료”는 치료하고자 하는 대상 또는 세포의 천연 과정을 변경시키기 위하여 임상적으로 개입하는 모든 행위를 의미하며, 임상 병리 상태가 진행되는 동안 또는 이를 예방하기 위하여 수행할 수 있다. The "treatment" refers to any action that clinically intervenes to change the natural process of a target or cell to be treated, and may be performed while a clinical pathology is progressing or to prevent it.
목적하는 치료 효과는 질병의 발생 또는 재발을 예방하거나, 증상을 완화시키거나, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키거나, 전이를 예방하거나, 질병 진행 속도를 감소시키거나, 질병 상태를 경감 또는 일시적 완화시키거나, 예후를 개선시키는 것을 포함할 수 있다.The desired therapeutic effect is to prevent the occurrence or recurrence of the disease, alleviate the symptoms, reduce any direct or indirect pathological consequences of the disease, prevent metastasis, reduce the rate of disease progression, or reduce the rate of disease progression. alleviating or palliating the condition, or improving the prognosis.
상기 조성물은 경구적 전달, 비경구적 전달의 형태로 투여될 수 있다. 상기 조성물은 전신 또는 국소 투여될 수 있으며, 상기 투여는 경구 투여 및 비경구 투여를 포함할 수 있다. 상기 조성물은 적절한 투여 형태를 제공하도록 적합한 양의 약학적으로 허용되는 비히클 또는 담체와 함께 제형화될 수 있다.The composition may be administered in the form of oral delivery or parenteral delivery. The composition may be administered systemically or locally, and the administration may include oral administration and parenteral administration. The composition may be formulated with a pharmaceutically acceptable vehicle or carrier in suitable amounts to provide an appropriate dosage form.
상기 조성물은 약학 조성물의 제조에 사용되는 담체, 부형제 및 희석제를 더 포함할 수 있다. The composition may further include carriers, excipients and diluents used in the preparation of pharmaceutical compositions.
상기 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유를 들 수 있으나, 이에 제한되는 것은 아니다.The carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil.
또한, 상기 조성물은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제제화하여 사용할 수 있다.In addition, the composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions.
경구 투여를 위한 고형제제는 정제, 환제, 산제, 과립제, 캡슐제 등이 사용될 수 있고, 상기 고형제제는 상기 좁은잎천선과 추출물과 이의 분획물들에 적어도 하나 이상의 부형제, 예컨대, 전분, 칼슘카보네이트, 수크로스, 락토오스, 또는 젤라틴 등을 혼합하여 조제할 수 있다. 또한, 상기 부형제 이외에 마그네슘 스티레이트, 탈크 같은 윤활제가 사용될 수 있다.Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and the solid preparations include at least one excipient, such as starch, calcium carbonate, It can be prepared by mixing sucrose, lactose, or gelatin. In addition to the above excipients, lubricants such as magnesium styrate and talc may be used.
경구 투여를 위한 액상 제제는 현탁제, 내용액제, 유제, 시럽제 등이 사용될 수 있고, 단순희석제인 물, 리퀴드 파라핀 외에 여러 가지 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 사용될 수 있다.Liquid preparations for oral administration may include suspensions, internal solutions, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives in addition to simple diluents such as water and liquid paraffin.
비경구 투여를 위한 제제는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 사용될 수 있다. 상기 비수성용제, 현탁제는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르가 사용될 수 있다. 상기 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴이 사용될 수 있다.Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used as the non-aqueous solvent or suspending agent. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, and glycerogeratin may be used.
상기 약학적 조성물은 약제학적으로 유효한 양이 대상체에 투여될 수 있다. 상기 “약제학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The pharmaceutical composition may be administered to a subject in a pharmaceutically effective amount. The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is based on the type and severity of the patient's disease, the activity of the drug, and the drug It may be determined according to factors including sensitivity, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical arts.
상기 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 바람직하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is preferable to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
이하 실시예를 통해, 본 발명을 더욱 상술하나 하기 실시예에 의해 본 발명이 제한되지 아니함은 자명하다.Through the following examples, the present invention is further detailed, but it is obvious that the present invention is not limited by the following examples.
제조예 : 열수 추출물(HR1902-W) 제조Preparation Example: Preparation of hot water extract (HR1902-W)
세척한 좁은잎천선과를 100℃의 온도에서 60분간 증자한 후 건조 및 분쇄하는 공정을 수행하였다. After steaming the washed narrow-leaved perianth fruit at a temperature of 100 ° C. for 60 minutes, a process of drying and crushing was performed.
상기 증자된 좁은잎천선과를 건조 및 분쇄한 후 20배수(v/v)의 증류수를 넣고 환류 추출장치에서 95°C의 온도로 4시간 동안 2회 추출하였다.After drying and pulverizing the steamed narrow-leaved Astragalus fruit, 20 times (v/v) distilled water was added and extracted twice for 4 hours at a temperature of 95°C in a reflux extractor.
1차 및 2차에서 얻어진 추출물을 혼합하여 여과지(ADVANTEC, Tokyo, Japan)로 여과하고, 일정 농도까지 농축(N-1300, Eyela, Tokyo, Japan)한 후 동결건조(7751041, Labconco, Kansas, MO, USA)하여 분말화하였다. The extracts obtained from the first and second steps are mixed, filtered through filter paper (ADVANTEC, Tokyo, Japan), concentrated to a certain concentration (N-1300, Eyela, Tokyo, Japan), and then freeze-dried (7751041, Labconco, Kansas, MO). , USA) and powdered.
실험예 1 : 동물실험 및 시료 제조Experimental Example 1: Animal testing and sample preparation
8주령의 수컷 BALB/c 마우스를 대한바이오링크(eumseong, Korea)에서 분양 받아, 2주간 실험실 환경(실온 22±2℃, 습도 55±15%, 12 시간 light-dark cycle)에 적응시킨 후 실험에 사용하였다. 8-week-old male BALB/c mice were acquired from Daehan Biolink (eumseong, Korea), adapted to the laboratory environment (room temperature 22±2°C,
실험동물군(5군, 무작위 배정)은 BALB/c 정상군, 양성대조군 홍삼 투여군(KRG 100, 200 mg/kg), HR02-W 투여군 및 단일성분 루틴 투여군(Rutin 10 mg/kg)으로 구성하여 수행하였다. Experimental animal groups (
실험 진행 및 투여 방법은 군당 5마리씩 5그룹으로 분리하여 사육하였으며, 식이 및 식수는 자유롭게 섭취하도록 하였다. 본 실험은 2주간 수행되었으며, 경구 투여로 진행되었다.Experiment progress and administration method was divided into 5 groups of 5 animals per group, and they were allowed to freely consume food and water. This experiment was conducted for 2 weeks and was conducted by oral administration.
실험군은 양성대조군(홍삼, KRG_100, 200 mg/kg), HR02-W, 단일성분(Rutin_10 mg/kg) 용량으로 각각 생리식염수에 녹여서 0.2mL 용량으로 투여하였다. For the experimental group, the positive control group (red ginseng, KRG_100, 200 mg/kg), HR02-W, and single component (Rutin_10 mg/kg) were dissolved in physiological saline and administered at a dose of 0.2mL.
약물투여는 경구투여용 금속제 존대(Zonde)를 이용하여 위 내로 강제 경구 투여하였다.Drug administration was forcibly orally administered into the stomach using a metal Zonde for oral administration.
실험예 2 : NK 세포 독성 시험Experimental Example 2: NK cytotoxicity test
2주간 시료의 경구 투여가 끝난 마우스에서 분리한 비장세포 현탁액을 YAC-1 cell과 1:50의 비율로 96-well plate에 공동 배양하여 CO2 incubator(37℃, 5% CO2)에서 4시간 동안 반응시켰다. The splenocyte suspension isolated from the mouse after oral administration of the sample for 2 weeks was co-cultured with YAC-1 cells at a ratio of 1:50 in a 96-well plate and incubated in a CO 2 incubator (37℃, 5% CO 2 ) for 4 hours. reacted during
배양 상층액을 lactate dehydrogenase 용액과 혼합하여 490 nm에서 흡광도를 측정하고, NK cell cytotoxicity를 다음 수식을 이용하여 계산하였다.The culture supernatant was mixed with a lactate dehydrogenase solution, the absorbance was measured at 490 nm, and the NK cell cytotoxicity was calculated using the following formula.
[수학식 1][Equation 1]
NK cell cytotoxicity (%) = [experimental release-spontaneous release)/maximum release-spontaneous release)] × 100.NK cell cytotoxicity (%) = [experimental release-spontaneous release)/maximum release-spontaneous release)] × 100.
NK 세포는 자연살해세포로, 선천면역반응 및 적응면역 모두에서 작용하는 림프구로서 선천면역반응으로 표적세포에 작용하여 세포살해를 담당하고, 사이토카인을 분비하여 세포독성 T 세포의 반응을 보조하는 백혈구로 알려져 있다.NK cells are natural killer cells, which are lymphocytes that act in both the innate immune response and adaptive immunity. They are white blood cells that are responsible for killing cells by acting on target cells with the innate immune response and secrete cytokines to assist the response of cytotoxic T cells. is known as
도 1을 참조하면, HR1902-W 투여시 100 mg/kg에서 각각의 cytotoxicity는 29.17%로 농도 대조군에서 19.01%로 측정된 것에 비하여 유의적으로 세포사멸능력이 증가하였으며, 홍삼처리군과 비슷한 수준까지 NK세포의 활성을 증가시켜 면역력 증진에 도움이 되는 것으로 해석할 수 있다.Referring to Figure 1, when HR1902-W was administered, each cytotoxicity at 100 mg/kg was 29.17%, compared to 19.01% in the concentration control group, and the apoptotic ability significantly increased, to a level similar to that of the red ginseng treatment group. It can be interpreted as helping to improve immunity by increasing the activity of NK cells.
실험예 3 : T 세포 활성 물질 측정 시험(ELISA)Experimental Example 3: T cell active substance measurement test (ELISA)
시료를 2주간 경구 투여한 후, mouse의 비장세포 현탁액을 96-well plate에 1×105의 밀도로 48시간동안 배양하였다. After oral administration of the sample for 2 weeks, the mouse splenocyte suspension was cultured in a 96-well plate at a density of 1×10 5 for 48 hours.
배양 후 상층액을 수집하여 ELISA kit를 이용하여 IL-2, IL-4, IL-1O, IL-12 및 IFN-γ의 발현양을 측정하였다.After culture, the supernatant was collected and the expression levels of IL-2, IL-4, IL-10, IL-12 and IFN-γ were measured using an ELISA kit.
HR1902-W 소재의 투여가 세포매개 면역반응의 중요한 역할을 하는 T 세포의 활성 cytokine에 미치는 영향을 ELISA 방법을 이용하여 측정하였다(도 2).The effect of administration of the HR1902-W material on active cytokines of T cells, which play an important role in cell-mediated immune response, was measured using an ELISA method (FIG. 2).
Interleukin-2(IL-2)는 HR1902-W 투여시 100 mg/kg에서 246.62 pg/mL로 측정되어 대조군의 160.27 pg/mL에 비해 높은 IL-2의 발현량을 보였으며, 홍삼처리군과 대등한 수준의 면역활성 효과를 보였다(도 2A). Interleukin-2 (IL-2) was measured at 246.62 pg/mL at 100 mg/kg when HR1902-W was administered, showing a higher IL-2 expression level compared to 160.27 pg/mL in the control group, comparable to the red ginseng treatment group. showed a level of immunostimulating effect (FIG. 2A).
Interleukin-4(IL-4)는 HR1902-W 투여시 100 mg/kg에서 96.30 pg/mL로 측정되어 대조군의 70.36 pg/mL보다 유의적으로 높은 IL-4의 발현량을 보였다(도 2B). Interleukin-4 (IL-4) was measured at 96.30 pg/mL at 100 mg/kg when HR1902-W was administered, showing a significantly higher IL-4 expression level than 70.36 pg/mL in the control group (FIG. 2B).
Interleukin-10(IL-10)는 HR1902-W 투여시 100 mg/kg에서 1939.21 pg/mL으로 측정되어 대조군의 824.65 pg/mL보다 2배이상 높은 IL-10의 발현량을 보였으며, 동일 농도의 홍삼투여군과 대등한 수준의 면역활성을 보였다(도 2C). Interleukin-10 (IL-10) was measured at 1939.21 pg/mL at 100 mg/kg when HR1902-W was administered, showing an expression level of IL-10 more than twice as high as 824.65 pg/mL in the control group. The red ginseng administration group showed an equivalent level of immune activity (FIG. 2C).
Interleukin-12(IL-12)는 HR1902-W 투여시 100 mg/kg에서 95.71 pg/mL로 측정되어 대조군의 67.69 pg/mL에 비해 유의적으로 높은 IL-12의 발현량을 보였으며, 동일 농도의 홍삼투여군과 대등한 수준의 면역활성을 보였다(도 2D). Interleukin-12 (IL-12) was measured at 95.71 pg/mL at 100 mg/kg when HR1902-W was administered, showing a significantly higher IL-12 expression level compared to 67.69 pg/mL in the control group. showed a level of immune activity equivalent to that of the red ginseng-administered group (FIG. 2D).
Interferon-γγ는 HR1902-W 투여시 100 mg/kg에서 1444.19 pg/mL로 측정되어 대조군의 606.94 pg/mL에 비해 유의적으로 높은 발현량을 보였으며, 동일 농도의 홍삼투여군보다 우수한 면역활성을 보였다(도 2E). Interferon-γγ was measured at 1444.19 pg/mL at 100 mg/kg when HR1902-W was administered, showing a significantly higher expression level compared to 606.94 pg/mL in the control group, and showed better immune activity than the red ginseng administration group at the same concentration. (Fig. 2E).
상기 결과는 HR1902-W가 대식세포, T 림프구, B 림프구, NK 세포의 활성에 관여하는 cytokine들의 발현량을 효과적으로 증가시킴으로써 선천면역 및 적응면역을 증진시킬 수 있음을 시사한다.The above results suggest that HR1902-W can enhance innate and adaptive immunity by effectively increasing the expression level of cytokines involved in the activation of macrophages, T lymphocytes, B lymphocytes, and NK cells.
실험예 4 : NO 생성량 측정 시험Experimental Example 4: NO production measurement test
경구 투여 종료 3일전, 1.5 mL의 10% proteose peptone 수용액을 마우스의 복강 내 투여한 후, 복부를 멸균시켰다. 3 days before the end of oral administration, 1.5 mL of 10% proteose peptone aqueous solution was intraperitoneally administered to mice, and then the abdomen was sterilized.
복막을 노출시킨 후, 4.5 mL의 HBSS(Hank's balanced salt solution)를 주사기로 복강 내 주사하여 복부를 30초 동안 마사지 한 후, 주사기를 사용하여 복강 내 세포를 회수하였다. After exposing the peritoneum, 4.5 mL of Hank's balanced salt solution (HBSS) was intraperitoneally injected with a syringe, the abdomen was massaged for 30 seconds, and cells were collected using a syringe.
회수된 세포를 HBSS 용액으로 2회 세척하고, RPMI 1640 배지에 현탁시켜 96-well plate에 2×106의 농도가 되도록 seeding 하였다. The recovered cells were washed twice with an HBSS solution, suspended in RPMI 1640 medium, and seeded in a 96-well plate at a concentration of 2×10 6 .
대식세포의 부착을 위하여 CO2 incubator에서 2시간 동안 배양하고, HBSS 용액으로 3회 세척하여 부유 세포를 제거하고, RPMI 1640 배지 100 μL를 각 well에 첨가하였다. For attachment of macrophages, they were cultured for 2 hours in a CO 2 incubator, washed three times with HBSS solution to remove floating cells, and 100 μL of RPMI 1640 medium was added to each well.
배양 72시간 후, 배양액 100 μL를 수집하여 Griess 시약과 10분간 반응시킨 후 540 nm에서 흡광도를 측정하여, 시료(HR1902-W)를 2주간 경구 투여한 마우스의 복강침출 세포로부터 NO 생성량을 측정하였다.After 72 hours of culture, 100 μL of the culture was collected, reacted with Griess reagent for 10 minutes, and the absorbance was measured at 540 nm to measure the amount of NO production from the peritoneal leaching cells of mice orally administered with the sample (HR1902-W) for 2 weeks. .
도 3을 참조하면, 면역증진 양성대조군(홍삼) 100, 200 mg/kg에서 각각의 O.D값이 3.60±0.53, 5.64±0.34으로 농도 의존적으로 NO 생성량이 증가하였으며, HR1902-W 100 mg/kg 농도로 투여하였을 4.37±0.54 값을 나타내어 NO 생성량이 유의적으로 증가하였다.Referring to Figure 3, the amount of NO production increased in a concentration-dependent manner, with the respective OD values of 3.60 ± 0.53 and 5.64 ± 0.34 in the immune enhancement positive control group (red ginseng) 100 and 200 mg/kg, respectively, and HR1902-
실험예 5 : 총 세포수 측정Experimental Example 5: Total cell count measurement
2주간 시료의 경구 투여 종료 2일 전, 2% 전분 수용액 0.5mL을 마우스 복강 내 투여하였다.2 days before the end of oral administration of the sample for 2 weeks, 0.5 mL of 2% aqueous starch solution was intraperitoneally administered to mice.
마우스 내의 지라(spleen), 흉선(thymus), 페이어판(peyer's patch), DLN(draining lymph node), PEC(peritoneal exudate cell)를 채취하여 HBSS에서 100 μm nylon 세포 여과기를 통해 여과하였다.The spleen, thymus, peyer's patch, DLN (draining lymph node), and PEC (peritoneal exudate cell) were collected and filtered through a 100 μm nylon cell strainer in HBSS.
ACK lysis buffer에 5분간 반응시킨 후, HBSS로 세척하여 FACS 완충용액 100 μL에 재현탁 시켰다. After reacting in ACK lysis buffer for 5 minutes, washed with HBSS and resuspended in 100 μL of FACS buffer.
분리된 세포를 FACS 완충액으로 2회 세척한 후, 4℃에서 면역 형광 염색을 수행하였으며, Hemocytometer를 이용하여 총 세포 수를 산출하였다. After washing the separated cells twice with FACS buffer, immunofluorescence staining was performed at 4° C., and the total number of cells was calculated using a hemocytometer.
FACS 데이터의 게이팅 퍼센트를 곱해 각 조직별 면역 세포의 절대수를 산출하였다.The absolute number of immune cells for each tissue was calculated by multiplying the FACS data by the gating percentage.
HR1902-W 소재와 대조군에 대한 마우스의 주요 면역관련 조직들의 총 세포수를 분석하였다(도 4).Total cell counts of major immune-related tissues of mice for the HR1902-W material and the control group were analyzed (FIG. 4).
DLN에서 총 세포수를 측정한 결과, HR1902-W 추출물 투여에 따라 1000 mg/kg 농도에서 118.5×104 cells/mL으로 대조군의 16.5×104 cells/mL에 비해 유의적으로 림프 세포의 수를 상승시키는 것을 확인하였다(도 4A). As a result of measuring the total number of cells in the DLN, the number of lymphocytes significantly increased to 118.5×10 4 cells/mL at a concentration of 1000 mg/kg according to the administration of the HR1902-W extract compared to 16.5×10 4 cells/mL in the control group. It was confirmed that it increased (FIG. 4A).
지라(Spleen)에서 총 세포수를 측정한 결과, HR1902-W 추출물 투여에 따라 100 mg/kg 농도에서 77.0×104×cells/mL로 분석되어, 대조군의 50.0×104 cells/mL에 비해 유의적으로 비장세포의 수를 증가시킨 것을 확인하였다(도 4B). As a result of measuring the total number of cells in the spleen, it was analyzed to be 77.0×10 4 ×cells/mL at a concentration of 100 mg/kg according to the administration of the HR1902-W extract, compared to 50.0 × 10 4 cells/mL in the control group. It was confirmed that the number of splenocytes was gradually increased (FIG. 4B).
흉선(thymus)에서 총 세포수를 측정한 결과, HR1902-W 추출물 투여에 따라 100 mg/kg 농도에서 127.6×104 cells/mL로 나타나 추출물 투여 농도가 증가할수록 흉선의 총 세포수 또한 증가하는 경향을 보였으나, 대조군과의 유의적인 차이는 확인하지 못하였다(도 4C). As a result of measuring the total number of cells in the thymus, it was found to be 127.6×10 4 cells/mL at a concentration of 100 mg/kg according to the administration of the HR1902-W extract, and the total number of cells in the thymus tended to increase as the concentration of the extract increased. , but no significant difference from the control group was confirmed (Fig. 4C).
페이어판(peyer's patch)에서 총 세포수를 측정한 결과, HR1902-W 투여에 따라 100 mg/kg 농도에서 75.5×104 cells/mL로, 투여농도가 증가할수록 복막 삼출 세포의 총 세포수가 유의적으로 증가하는 경향을 보였다(도 4D). As a result of measuring the total number of cells in Peyer's patch, HR1902-W was administered at 100 mg/kg concentration to 75.5×10 4 cells/mL, and as the concentration increased, the total number of peritoneal exudate cells was significant showed a tendency to increase (Fig. 4D).
PEC에서 총 세포수를 측정한 결과, HR1902-W 추출물 투여에 따라 100 mg/kg 농도에서 151.5×104 cells/mL로, 대조군의 총 세포수 99.5×104 cells/mL과 비교하여 유의적인 차이를 보였다(도 4E).As a result of measuring the total cell number in PEC, it was 151.5 × 10 4 cells / mL at 100 mg / kg concentration according to the administration of the HR1902-W extract, a significant difference compared to the total cell number of 99.5 × 10 4 cells / mL in the control group showed (Fig. 4E).
실험예 6 : PBMC에서의 형광 유세포 분석Experimental Example 6: Fluorescence flow cytometry analysis in PBMC
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
BALB/c mouse 모델의 PBMC에서 HR1902-W 소재의 형광 유세포를 분석하였다(도 5). The fluorescence flow cytometry of the HR1902-W material was analyzed in PBMC of the BALB/c mouse model (FIG. 5).
CD3e+ T cell & CD49b+ NK cell의 비율은 대조군에서 14.9%로 측정되었고, HR1902-W 투여에 의하여 100 mg/kg 농도에서 NK T 세포의 수가 증가하는 경향을 나타내었다(도 5A).The ratio of CD3e+ T cells & CD49b+ NK cells was measured to be 14.9% in the control group, and the number of NK T cells tended to increase at a concentration of 100 mg/kg by HR1902-W administration (FIG. 5A).
CD4+ helper T cell/CD8+ T cell의 비율은 대조군에서 17.5/6.6으로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여하였을 때, 18.1/10.2 비율이 증가하였다(도 5B).The ratio of CD4+ helper T cells/CD8+ T cells was measured as 17.5/6.6 in the control group, and when HR1902-W was administered at a concentration of 100 mg/kg, the ratio increased to 18.1/10.2 (FIG. 5B).
CD4+ & CD25+ regulatory T-cells의 비율은 대조군에서 2.6%로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여하였을 때, 상기 비율이 3.2%로 증가하는 결과값을 나타내었다(도 5C).The ratio of CD4+ & CD25+ regulatory T-cells was measured at 2.6% in the control group, and when HR1902-W was administered at a concentration of 100 mg/kg, the ratio increased to 3.2% (Fig. 5C).
CD8+ & CD25+ regulatory T-cells의 비율은 대조군에서 1.4%로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여하였을 때, 상기 비율이 3.0%로 증가하여 대조군 대비 2배이상 증가하는 결과값을 나타내었다(도 5D).The ratio of CD8+ & CD25+ regulatory T-cells was measured at 1.4% in the control group, and when HR1902-W was administered at a concentration of 100 mg/kg, the ratio increased to 3.0%, resulting in a more than two-fold increase compared to the control group. shown (Fig. 5D).
상기 결과는 세포성면역뿐만 아니라 체액성 면역에서도 우수한 면역증진 효능을 갖는 것을 시사한다.The above results suggest that it has excellent immune enhancing efficacy not only in cellular immunity but also in humoral immunity.
CD4+, CD62L, CD107a 표지자를 이용하여 마우스 비장세포에서 NK 세포 분석을 진행하였다(도 6).NK cell analysis was performed in mouse splenocytes using CD4+, CD62L, and CD107a markers (FIG. 6).
대조군에서 NK cell의 비율은 0.4%로 측정되었고, HR1902-W 투여시 100 mg/kg에서 6.3%로 농도 유의적으로 증가하였으며, 상기 값은 동일 농도의 홍삼 투여군보다 높게 측정되었다. The ratio of NK cells in the control group was measured at 0.4%, and when HR1902-W was administered, the concentration significantly increased from 100 mg/kg to 6.3%, and the value was higher than that of the red ginseng administration group at the same concentration.
상기 결과는 비장세포 배양을 통한 NK 세포의 cytotoxicity 측정 실험에서 HR1902-W 투여시, YAC-1 세포사멸능력이 증가한 결과를 뒷받침할 수 있다.The above result can support the result of YAC-1 apoptosis increased when HR1902-W was administered in the NK cell cytotoxicity measurement experiment through splenocyte culture.
실험예 7 : 지라(Spleen)에서 절대세포수 측정Experimental Example 7: Absolute cell count measurement in spleen
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
BALB/c 복강세포군 중 지라에서 HR1902-W 소재 투여에 따른 절대세포수를 측정하였다(도 7).Absolute cell counts were measured in the spleen of the BALB/c peritoneal cell population following the administration of the HR1902-W material (FIG. 7).
CD3e+ T cell/CD19+ B cell의 절대세포수는 대조군에서 각각 25.5×105 cells, 20.6×105 cells로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 45.1×105 cells, 22.8×105 cells로 T cell의 양이 유의적으로 증가하였으며, 동일 농도의 홍삼 처리군보다 그 증가량이 높은 수준이었다(도 7A). The absolute cell counts of CD3e+ T cell/CD19+ B cell were measured as 25.5×10 5 cells and 20.6×10 5 cells in the control group, respectively, and 45.1×10 5 cells and 22.8×10 when HR1902-W was administered at a concentration of 100 mg/kg. The amount of T cells increased significantly with 5 cells, and the increase was higher than that of the red ginseng treatment group at the same concentration (Fig. 7A).
CD4+ helper T cell/CD8+ T cell의 절대세포수는 대조군에서 각각 9.8×105 cells, 6.4×105 cells로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 18.2×105 cells, 11.0×105 cells로 T cell의 양이 유의적으로 상승하였다. 상기 결과는 동일 농도의 홍삼 처리군보다 증가량이 높은 수준이었으며, 대조군 대비 helper T cell과 T cell 마커에 있어서 모두 그 양이 증가하였다(도 7B). Absolute cell counts of CD4+ helper T cell/CD8+ T cell were measured as 9.8×10 5 cells and 6.4×10 5 cells in the control group, respectively, and 18.2×10 5 cells and 11.0×10 when HR1902-W was administered at a concentration of 100 mg/kg. The amount of T cells increased significantly with 5 cells. The above results showed a higher level of increase than the red ginseng treatment group at the same concentration, and both helper T cell and T cell markers increased in amount compared to the control group (FIG. 7B).
CD3e+ T cell & CD49b+ NK cell의 절대세포수는 대조군에서 1.7×105 cells로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 2.0×105 cells로 측정되어 대조군보다 일부 증가하였다(도 7C).The absolute cell count of CD3e+ T cell & CD49b+ NK cell was measured as 1.7×10 5 cells in the control group, and was measured as 2.0×10 5 cells when HR1902-W was administered at a concentration of 100 mg/kg, which slightly increased compared to the control group (FIG. 7C ).
CD4+ & CD25+ regulatory T-cells의 절대세포수는 대조군에서 1.2×105 cells로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 각각 2.6×105 cells로 측정되어 대조군보다 증가하였으며, 동일 농도의 홍삼 투여군과 대등한 수준까지 CD4+ & CD25+ regulatory T-cell의 양을 증가시켰다(도 7D).The absolute cell number of CD4+ & CD25+ regulatory T-cells was measured as 1.2×10 5 cells in the control group, and was measured as 2.6×10 5 cells respectively when HR1902-W was administered at a concentration of 100 mg/kg, which increased compared to the control group. The amount of CD4+ & CD25+ regulatory T-cells was increased to a level equivalent to that of the red ginseng-administered group (FIG. 7D).
CD8+ & CD25+ regulatory T cells의 절대세포수는 대조군에서 0.4×105 cells으로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 0.5×105 cells로 측정되어 대조군과 유의한 차이는 나타나지 않았다(도 7E).The absolute cell number of CD8+ & CD25+ regulatory T cells was measured as 0.4×10 5 cells in the control group, and was measured as 0.5×10 5 cells when HR1902-W was administered at a concentration of 100 mg/kg, showing no significant difference from the control group ( Figure 7E).
CD23+ B cell & B220+ B cell의 절대세포수는 대조군에서 12.5×105 cells으로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 18.8×105 cells로 측정되어 대조군보다 유의적으로 B cell의 양이 증가하였다(도 7F).The absolute cell number of CD23+ B cell & B220+ B cell was measured as 12.5×10 5 cells in the control group, and was measured as 18.8×10 5 cells when HR1902-W was administered at a concentration of 100 mg/kg, which significantly increased the number of B cells compared to the control group. amount increased (FIG. 7F).
GR-1 & CD11b+ Myeloid-Derived Suppressor cell의 절대세포수는 대조군에서 1.3×105 cells으로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 2.9×105 cells로 측정되어 대조군보다 두 배 이상 유의적으로 골수유래 과립구의 양이 증가하였다(도 7G).The absolute cell number of GR-1 & CD11b+ Myeloid-Derived Suppressor cells was measured as 1.3×10 5 cells in the control group, and was measured as 2.9×10 5 cells when HR1902-W was administered at a concentration of 100 mg/kg, more than twice that of the control group. The amount of bone marrow-derived granulocytes was significantly increased (FIG. 7G).
실험예 8 : 흉선(thymus)에서 절대세포수 측정Experimental Example 8: Measurement of absolute cell count in the thymus
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
BALB/c 흉선에서 HR1902-W 소재 투여에 따른 절대세포수를 측정하였다(도 8).In BALB/c thymus, the absolute cell number according to the administration of HR1902-W material was measured (FIG. 8).
도 8을 참조하면, CD4+ & CD25+ regulatory T cells의 절대세포수는 대조군에서 6.3×105 cells로 측정되었고, 100 mg/kg 농도의 HR1902-W 투여시 7.4×105 cells로 측정되어 대조군보다 유의적으로 그 양이 증가하였다.Referring to FIG. 8, the absolute cell number of CD4+ & CD25+ regulatory T cells was measured as 6.3×10 5 cells in the control group and 7.4×10 5 cells when HR1902-W was administered at a concentration of 100 mg/kg, which was significantly higher than that of the control group. consequently the amount increased.
실험예 9 : DLN에서의 절대세포수 측정Experimental Example 9: Absolute cell count measurement in DLN
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
BALB/c DLN에서 HR1902-W 소재 투여에 따른 절대세포수를 측정하였다(도 9).In BALB/c DLN, the absolute cell number according to the administration of the HR1902-W material was measured (FIG. 9).
CD3e+ T cell/CD19+ B cell의 절대세포수는 대조군에서 각각 9.3×105 cells, 5.6×105 cells로 측정되었고, HR1902-W 100 mg/kg 투여군에서 76.8×105 cells, 15.1×105 cells로 측정되어 T 세포군에서 대조군보다 7배 이상 증가하였다. 특히, 동일 농도의 홍삼 투여군과 비교하여 유의적으로 높은 수준까지 T cell의 양을 증가시켰다(도 9A). The absolute cell counts of CD3e+ T cell/CD19+ B cell were 9.3×10 5 cells and 5.6×10 5 cells in the control group, respectively, and 76.8×10 5 cells and 15.1×10 5 cells in the HR1902-
CD4+ helper T cell/CD8+ T cell의 절대세포수는 대조군에서 각각 7.3×105 cells, 3.6×105 cells로 측정되었고, HR1902-W 100 mg/kg 농도의 투여군에서 53.4×105 cells, 21.3×105 cells로 측정되어 CD4+와 CD8+ 표지자에 대하여 대조군 대비 모두 6배 이상 증가하였으며, 동일 농도의 홍삼 투여군과 비교하여 유의적으로 T cell의 양을 더 높은 수준까지 증가시켰다(도 9B). Absolute cell counts of CD4+ helper T cells/CD8+ T cells were measured as 7.3×10 5 cells and 3.6×10 5 cells in the control group, respectively, and 53.4×10 5 cells and 21.3× in the HR1902-
CD3e+ T cell & CD49b+ NK cell의 절대세포수는 대조군에서 0.3×105 cells로 측정되었고, HR1902-W 100 mg/kg 농도의 투여군에서 0.8×105 cells로 측정되어 대조군보다 유의적으로 증가한 결과를 보였으며, 동일 농도의 홍삼 투여군보다 높은 수준으로 NK T cell의 양을 증가시켰다(도 9C).The absolute cell count of CD3e+ T cell & CD49b+ NK cell was measured as 0.3×10 5 cells in the control group and 0.8×10 5 cells in the group administered with HR1902-
CD4+ & CD25+ regulatory T cells의 절대세포수는 대조군에서 1.1×105 cells로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여했을 때 8.6×105 cells로 측정되어 대조군보다 8배 이상 증가하였으며, 동일 농도의 홍삼 투여군과 비교하여 더 높은 수준까지 CD4+ & CD25+ regulatory T-cell의 양을 증가시켰다(도 9D).The absolute cell number of CD4+ & CD25+ regulatory T cells was measured as 1.1×10 5 cells in the control group, and when HR1902-W was administered at a concentration of 100 mg/kg, it was measured as 8.6×10 5 cells, which increased more than 8-fold than the control group. , increased the amount of CD4+ & CD25+ regulatory T-cells to a higher level compared to the red ginseng administration group at the same concentration (FIG. 9D).
CD23+ B cell & B220+ B cell의 절대세포수는 대조군에서 0.7×105 cells로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여했을 때 6.3×105 cells로 측정되어 대조군보다 9배 이상 증가하였으며, B cell 생성 또한 효과적으로 조절할 수 있을 것으로 확인되었다(도 9E).Absolute cell counts of CD23+ B cell & B220+ B cell were measured as 0.7×10 5 cells in the control group, and 6.3×10 5 cells when HR1902-W was administered at a concentration of 100 mg/kg, which is more than 9 times higher than that of the control group. And it was confirmed that B cell generation could also be effectively controlled (FIG. 9E).
실험예 10 : 페이어판에서 절대세포수 측정Experimental Example 10: Measurement of absolute cell count in Peyer's plate
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
페이어(Peyer's patch)에서 HR1902-W 소재 투여에 따른 절대세포수를 측정하였다(도 10).Absolute cell counts were measured according to the administration of the HR1902-W material in Peyer's patch (FIG. 10).
CD3e+ T cell & CD49b+ NK cell의 절대세포수는 대조군에서 7.4×105 cells로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여했을 때 10.4×105 cells로 측정되어 대조군 대비 유의적으로 증가하였으며, 동일 농도의 홍삼 투여군보다 우수하게 NK T cell의 생성을 촉진하였다(도 10A). The absolute cell count of CD3e+ T cell & CD49b+ NK cell was measured as 7.4×10 5 cells in the control group, and when HR1902-W was administered at a concentration of 100 mg/kg, it was measured as 10.4×10 5 cells, a significant increase compared to the control group. and promoted the generation of NK T cells better than the red ginseng administration group at the same concentration (FIG. 10A).
CD4+ helper T cell/CD8+ T cell의 절대세포수는 대조군에서 각각 2.2×105 cells, 2.5×105 cells로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여하였을 때 3.0×105 cells, 3.8×105 cells로 측정되어 CD4+와 CD8+ 표지자에 대하여 대조군대비 모두 유의적으로 증가하였다. 특히, 동일 농도의 홍삼 투여군과 비교하여 유의적으로 T cell의 양을 더 높은 수준까지 증가시키는 것으로 확인되었다(도 10B).Absolute cell counts of CD4+ helper T cell/CD8+ T cell were measured as 2.2×10 5 cells and 2.5×10 5 cells in the control group, respectively, and when HR1902-W was administered at a concentration of 100 mg/kg, it was 3.0×10 5 cells, As measured by 3.8×10 5 cells, both CD4+ and CD8+ markers were significantly increased compared to the control group. In particular, it was confirmed that the amount of T cells was significantly increased to a higher level compared to the red ginseng administration group at the same concentration (FIG. 10B).
실험예 11 : PEC에서 절대세포수 측정Experimental Example 11: Absolute cell count measurement in PEC
살아있는 세포 내 미토콘드리아의 탈수소효소에 의해 Tetrazolium salt(WST-1)에서 formazan으로 변화하는 것을 이용하여, 생성된 formazan의 농도를 spectrophotometer로 측정함으로써 세포 생존률을 측정하였다.Using the change from Tetrazolium salt (WST-1) to formazan by mitochondrial dehydrogenase in living cells, cell viability was measured by measuring the concentration of formazan produced using a spectrophotometer.
BALB/c 마우스 PEC에서 HR1902-W 소재 투여에 따른 절대세포수를 측정하였다(도 11).In BALB/c mouse PEC, the absolute cell number according to the administration of HR1902-W material was measured (FIG. 11).
CD4+ helper T cell/CD8+ T cell의 절대세포수는 대조군에서 각각 7.9×105 cells, 5.3×105 cells로 측정되었고, HR1902-W를 100 mg/kg 농도로 투여하였을 때 12.0×105 cells, 5.9×105 cells로 측정되어 CD4+와 CD8+ 표지자에 대하여 대조군 대비 모두 유의적으로 증가하였다(도 11A). The absolute cell counts of CD4+ helper T cell/CD8+ T cell were measured as 7.9×10 5 cells and 5.3×105 cells in the control group, respectively, and when HR1902-W was administered at a concentration of 100 mg/kg, it was 12.0×10 5 cells, 5.9 As measured by ×10 5 cells, both CD4+ and CD8+ markers were significantly increased compared to the control group (FIG. 11A).
CD69+ T cell & B220+ B cell의 절대세포수는 7.9×105 cells로 측정되었고, HR1902-W를 처리하였을 때 100 mg/kg 농도에서 12.2×105 cells로 측정되어 대조군보다 유의적으로 증가하였다(도 11B). The absolute cell number of CD69+ T cell & B220+ B cell was measured as 7.9×10 5 cells, and when treated with HR1902-W, it was measured as 12.2×10 5 cells at 100 mg/kg concentration, which was significantly increased compared to the control group ( Figure 11B).
CD23+ B cell & B220+ B cell의 절대세포수는 대조군에서 7.3×105 cells로 측정되었고, 1902를 100 mg/kg 농도로 투여하였을 때 13.6×105 cells로 측정되어 대조군 대비 유의적으로 증가하였으며, B cell 생성 또한 효과적으로 조절할 수 있을 것으로 확인되었다(도 11C).The absolute cell number of CD23+ B cell & B220+ B cell was measured as 7.3×10 5 cells in the control group, and was measured as 13.6×10 5 cells when 1902 was administered at a concentration of 100 mg/kg, which significantly increased compared to the control group. It was confirmed that B cell generation could also be effectively regulated (FIG. 11C).
상기 결과는 증자된 좁은잎천선과 추출물의 면역 증진 효과가 우수하며, 의약품 및 식품 등 다양한 용도로 활용될 수 있음을 시사한다.The above results suggest that the immunity-enhancing effect of the steamed Narrowleaf Astragalus extract is excellent, and that it can be used for various purposes such as medicines and foods.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다. 예를 들어, 단일형으로 설명되어 있는 각 구성 요소는 분산되어 실시될 수도 있으며, 마찬가지로 분산된 것으로 설명되어 있는 구성 요소들도 결합된 형태로 실시될 수 있다.The above description of the present invention is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined form.
본 발명의 범위는 후술하는 청구범위에 의하여 나타내어지며, 청구범위의 의미 및 범위 그리고 그 균등 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and equivalent concepts should be interpreted as being included in the scope of the present invention.
Claims (8)
상기 좁은잎천선과는 100 내지 200℃의 수증기를 통해 증자 처리된 것인, 면역 증진용 조성물.Containing the extract of Narrow Leaf Astragalus as an active ingredient,
The narrow leaf astragalus and the steaming treatment through steam at 100 to 200 ° C., the composition for enhancing immunity.
상기 좁은잎천선과는 100℃ 이상의 온도에서 30 내지 120분간 증자한 것인 면역 증진용 조성물.According to claim 1,
The composition for enhancing immunity with the narrow leaf astragalus and steamed for 30 to 120 minutes at a temperature of 100 ° C or higher.
상기 좁은잎천선과는 100 내지 200℃의 수증기를 통해 증자 처리된 것인, 면역 증진용 건강기능식품.Containing the extract of Narrow Leaf Astragalus as an active ingredient,
A health functional food for enhancing immunity with the narrow leaf fern and steamed through steam at 100 to 200 ° C.
상기 좁은잎천선과는 100℃ 이상의 온도에서 30 내지 120분간 증자한 것인 건강기능식품.According to claim 5,
A health functional food that is steamed for 30 to 120 minutes at a temperature of 100 ° C. or higher with the narrow leaf fern.
상기 좁은잎천선과는 100 내지 200℃의 수증기를 통해 증자 처리된 것인, 면역 증진용 약학 조성물.Containing the extract of Narrow Leaf Astragalus as an active ingredient,
The narrow leaf astragalus and the steamed treatment through steam at 100 to 200 ° C., a pharmaceutical composition for enhancing immunity.
상기 좁은잎천선과는 100℃ 이상의 온도에서 30 내지 120분간 증자한 것인 면역 증진용 약학 조성물.
According to claim 7,
The pharmaceutical composition for enhancing immunity with the narrow leaf astragalus and steamed for 30 to 120 minutes at a temperature of 100 ° C or higher.
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| JP2010150207A (en) * | 2008-12-26 | 2010-07-08 | Noevir Co Ltd | Moisturizer, anti-aging agent, antioxidant, neutral fat-accumulation inhibitor, skin whitening agent, anti-inflammatory agent, immunostimulator, skin care preparation, oral medication |
| KR101807605B1 (en) | 2016-10-25 | 2017-12-11 | 한국 한의학 연구원 | Composition for prevention, improvement or treatment of cognitive dysfunction comprising Ficus erecta extract as effective component |
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| KR102100553B1 (en) * | 2018-03-19 | 2020-04-14 | 재단법인 제주테크노파크 | Composition for Anti-Arthritis Using a Leaf Extract of Ficus erecta Thunb. var. sieboldii(Miq.)King |
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| JP2010150207A (en) * | 2008-12-26 | 2010-07-08 | Noevir Co Ltd | Moisturizer, anti-aging agent, antioxidant, neutral fat-accumulation inhibitor, skin whitening agent, anti-inflammatory agent, immunostimulator, skin care preparation, oral medication |
| KR101807605B1 (en) | 2016-10-25 | 2017-12-11 | 한국 한의학 연구원 | Composition for prevention, improvement or treatment of cognitive dysfunction comprising Ficus erecta extract as effective component |
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