KR102581324B1 - Composition for preventing and treating immunodeficiency of Glycogen storage disease type Ib or Glycogen storage disease type Ib comprising Oenothera odorata extract or its fraction as effective component - Google Patents
Composition for preventing and treating immunodeficiency of Glycogen storage disease type Ib or Glycogen storage disease type Ib comprising Oenothera odorata extract or its fraction as effective component Download PDFInfo
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- KR102581324B1 KR102581324B1 KR1020210053262A KR20210053262A KR102581324B1 KR 102581324 B1 KR102581324 B1 KR 102581324B1 KR 1020210053262 A KR1020210053262 A KR 1020210053262A KR 20210053262 A KR20210053262 A KR 20210053262A KR 102581324 B1 KR102581324 B1 KR 102581324B1
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- evening primrose
- extract
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Abstract
본 발명은 달맞이꽃 추출물 또는 이의 분획물을 유효성분으로 함유하는 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 치료용 조성물에 관한 것으로, 구체적으로 달맞이꽃 추출물 또는 이의 분획물은 세포독성 없이, 당원병 제Ib형에서의 단핵구 생장조절 및 대식세포로의 분화을 유도하여, 지속적인 박테리아 감염등의 면역결핍증에 대한 예방 및 치료용 조성물의 유효성분으로 유용하게 사용될 수 있다.The present invention relates to a composition for preventing and treating immunodeficiency of glycogenopathy type Ib or glycogenopathy type Ib, which contains evening primrose extract or fractions thereof as an active ingredient. Specifically, the evening primrose extract or fractions thereof is non-cytotoxic and contains glycogen disease type Ib. By regulating the growth of monocytes and inducing differentiation into macrophages in type Ib disease, it can be usefully used as an active ingredient in compositions for preventing and treating immunodeficiency disorders such as persistent bacterial infections.
Description
본 발명은 달맞이꽃 추출물 또는 이의 분획물을 유효성분으로 함유하는 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 치료용 조성물에 관한 것이다.The present invention relates to a composition for the prevention and treatment of glycogenic disease type Ib or glycogenic disease type Ib immunodeficiency, containing evening primrose extract or a fraction thereof as an active ingredient.
우리 몸의 세포는 기능을 수행하기 위해 포도당을 주 에너지원으로 사용한다. 포도당은 음식을 통해 쉽게 얻을 수 있기에 필요 이상으로 섭취하게 되면 포도당은 글리코겐의 형태로 체내에 저장이 된다. 저장된 글리코겐은 필요시 다시 포도당으로 분해되어 에너지원으로 사용된다. 그러나 일부 사람들은 태어나면서부터 특정 유전자에 돌연변이가 발생하여 글리코겐을 합성하거나 분해할 수 없게 된다. Cells in our body use glucose as their main energy source to perform their functions. Glucose can be easily obtained through food, so if you consume more than necessary, glucose is stored in the body in the form of glycogen. When needed, stored glycogen is broken down into glucose and used as an energy source. However, some people are born with mutations in certain genes, making them unable to synthesize or break down glycogen.
당원병 제Ib형은 SLC37A4 유전자의 돌연변이에 의해 간, 신장, 소장의 글리코겐을 분해할 수 없는 희귀성 유전질환이다. SLC37A4 유전자에 의해 발현되는 glucose-6-phosphate transporter(G6PT) 단백질은 세포의 소포체막에 존재한다. 글리코겐 분해 과정의 마지막 단계에서 G6P는 G6PT를 통해 세포질에서 소포체로 이동하고 간 또는 신장에서는 glucose-6-phosphatase-α에 의해, 포도당을 스스로 만들어 낼 수 없는 세포에서는 glucose-6-phosphatase-β에 의해 포도당으로 분해되어 혈당을 유지하거나 세포의 생장과 기능을 유지하는데 각각 이용된다. 그러나 SLC37A4 유전자의 돌연변이로 인해 G6PT가 발현되지 않으면 G6P가 소포체막으로 들어가지 못해 포도당으로 전환하지 못하고 글리코겐이 간과 신장에 축적되거나 세포의 생장 및 기능에 문제가 생긴다. 결과적으로 당원병 제Ib형은 간 또는 신장에 글리코겐 축적으로 인한 간비대와 신장비대, 저혈당 경련, 젖산혈증, 고요산혈증, 고지혈증 등이 나타나게 되며, 호중구 감소증, 호중구 기능저하, 단핵구 기능저하로 인한 면역결핍을 유발하여 박테리아 감염에 취약하게 된다.Glycogen type Ib is a rare genetic disease in which glycogen in the liver, kidneys, and small intestine cannot be broken down due to a mutation in the SLC37A4 gene. The glucose-6-phosphate transporter (G6PT) protein expressed by the SLC37A4 gene exists in the endoplasmic reticulum membrane of cells. In the final step of the glycogenolysis process, G6P moves from the cytoplasm to the endoplasmic reticulum through G6PT and is then transported by glucose-6-phosphatase-α in the liver or kidney and by glucose-6-phosphatase-β in cells that cannot produce glucose on their own. It is broken down into glucose and used to maintain blood sugar levels or to maintain cell growth and function. However, if G6PT is not expressed due to a mutation in the SLC37A4 gene, G6P cannot enter the endoplasmic reticulum membrane and cannot be converted to glucose, glycogen accumulates in the liver and kidneys, or problems with cell growth and function occur. As a result, glycogen disease type Ib causes hepatomegaly and renal enlargement, hypoglycemic convulsions, lactic acidemia, hyperuricemia, and hyperlipidemia due to glycogen accumulation in the liver or kidneys, and immunity due to neutropenia, decreased neutrophil function, and decreased monocyte function. Deficiency causes susceptibility to bacterial infections.
현재 당원병 제Ib형의 호중구 세포에 대한 연구가 가장 활발하게 진행되고 있으며, 호중구와 유사한 기능을 하는 단핵구 세포에 대한 연구도 보고되고 있다. 단핵구 세포는 감염부위에서 대식세포로 분화되며 호중구와 함께 병원균을 포식하는 중요한 기능을 한다. 또한, 대식세포는 항원 제시 기능을 통해 T세포 등을 활성화시키고, 염증반응이 끝나고 난 후, 조직 재생과 같은 역할을 하기 때문에 단핵구로부터 대식세포로의 분화가 적절하게 이루어지는 것은 중요하다. Currently, research on neutrophil cells of type Ib glycemic disease is being conducted most actively, and research on monocyte cells that function similar to neutrophils has also been reported. Monocyte cells differentiate into macrophages at the site of infection and play an important role in phagocytosing pathogens together with neutrophils. In addition, macrophages activate T cells and the like through their antigen presentation function and play a role such as tissue regeneration after the inflammatory response is over, so it is important to properly differentiate from monocytes to macrophages.
당원병 제Ib형 환자의 면역결핍에 대한 치료는 호중구 수치를 정상화시키기 위해 granulocyte colony-stimulating factor(G-CSF)를 투여하는 것이다. 그러나 호중구의 기능 개선에는 영향을 미치지 않으며 장기간 투여시 급성 골수성 백혈병을 야기한다고 보고된다. 아울러, 현재까지 호중구 또는 단핵구의 기능을 개선시키는 치료제는 보고된 바 없다. Treatment for immunodeficiency in patients with type Ib diabetes is the administration of granulocyte colony-stimulating factor (G-CSF) to normalize neutrophil levels. However, it has no effect on improving neutrophil function and is reported to cause acute myeloid leukemia when administered for a long period of time. In addition, no therapeutic agent that improves the function of neutrophils or monocytes has been reported to date.
한편, 달맞이꽃(Oenothera odorata)은 바늘꽃과에 속하는 한해살이풀로서, 오래전 우리나라에 들어와 자생하는 귀화식물이다. 우리나라 각지의 길가나 빈터 등 전국에서 쉽게 볼 수 있는 달맞이꽃은 긴잎달맞이꽃, 금달맞이꽃, 큰달맞이꽃, 애기달맞이꽃 등 다양한 종이 자라고 있으며, 높이가 50 센티미터에서 1미터 이하로 자란다. 또한, 달맞이꽃의 종류는 145가지 정도로 다양하지만, 성분과 효능은 모두 비슷한 것으로 알려져 있고, 이들에게는 공통적으로 플라보노이드와 점액질 및 탄닌 그리고 식물스테롤이 풍부한 것으로 알려져 있다. Meanwhile, evening primrose ( Oenothera odorata ) is an annual herb belonging to the Needle family and is a naturalized plant that came to Korea a long time ago and grows naturally. Evening primrose, which can be easily seen all over the country, including roadsides and vacant lots, grows in various species such as long-leaf evening primrose, golden evening primrose, large evening primrose, and baby evening primrose, and grows from 50 centimeters to less than 1 meter in height. In addition, there are about 145 different types of evening primrose, but they are all known to have similar ingredients and efficacy, and they are known to be rich in flavonoids, mucilage, tannin, and plant sterols in common.
한편, 달맞이꽃을 이용한 선행연구로, 달맞이꽃 종자유는 피 순환을 원활하게 하여 심장병 예방 및 류마티스성 관절염, 생리통, 당뇨병 등에 효과가 있는 것으로 알려져 있다. 또한, 동물실험에서 달맞이꽃 종자유를 피하주사하였더니 혈중 콜레스테롤이나 중성지방이 낮아지고 고밀도단백은 증가하며 지방세포가 작아지면서 체내 지방의 축적이 감소된다는 보고되어 있으나, 당원병에 대한 효과에 대해서는 전혀 알려진 바 없다.Meanwhile, according to previous research using evening primrose, evening primrose seed oil is known to be effective in preventing heart disease, rheumatoid arthritis, menstrual pain, and diabetes by improving blood circulation. In addition, in animal experiments, it has been reported that subcutaneous injection of evening primrose oil lowers blood cholesterol and neutral fat, increases high-density protein, and reduces fat accumulation in the body as fat cells become smaller. However, the effect on glycogenosis is not known at all. There is no bar.
이에, 본 발명자들은 부작용이 없는 천연물 유래 당원병 제Ib형의 면역결핍증 예방 및 치료제를 개발하기 위해 노력한 결과, 달맞이꽃 추출물의 분획물이 G6PT 결여로 인해 나타나는 비정상적으로 증가하는 단핵구 세포의 생장과 저하된 대식세포로의 분화를 정상화시키고, 특히, 단핵구의 세포사멸을 유도하지 않아 세포독성이 없으므로, 희귀성 난치질환인 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 치료용 조성물로 사용될 수 있음을 밝힘으로써, 본 발명을 완성하였다. Accordingly, the present inventors made efforts to develop a preventive and therapeutic agent for immune deficiency disease type Ib derived from natural products without side effects, and as a result, a fraction of evening primrose extract was used to reduce the growth of abnormally increased mononuclear cells caused by the lack of G6PT and the decreased immune response. It normalizes differentiation into phagocytes and, in particular, does not induce apoptosis of monocytes and is therefore non-cytotoxic, so it can be used as a composition for preventing and treating immunodeficiency of glycogen type Ib or glycogen type Ib, a rare incurable disease. By revealing that there is, the present invention has been completed.
본 발명의 목적은 달맞이꽃 추출물 또는 이의 분획물을 유효성분으로 함유하는 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 치료용 조성물을 제공하기 위한 것이다.The purpose of the present invention is to provide a composition for preventing and treating glycogenopathies type Ib or immunodeficiency of glycogenopathies type Ib, containing evening primrose extract or fractions thereof as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 달맞이꽃 추출물 또는 이의 분획물을 유효성분으로 함유하는 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of glycogen type Ib or glycogen type Ib immunodeficiency, containing evening primrose extract or a fraction thereof as an active ingredient.
아울러, 본 발명은 달맞이꽃 추출물 또는 이의 분획물을 유효성분으로 함유하는 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 개선용 건강식품용 조성물을 제공한다.In addition, the present invention provides a composition for health food for preventing and improving immunodeficiency of glycogenic disease type Ib or glycogenic disease type Ib, containing evening primrose extract or a fraction thereof as an active ingredient.
본 발명의 달맞이꽃 추출물의 분획물은 세포독성 없이, 당원병 제Ib형에서의 단핵구 생장조절 및 대식세포로의 분화를 유도하여, 지속적인 박테리아 감염 등의 면역결핍증에 대한 예방 및 치료용 조성물의 유효성분으로 유용하게 사용될 수 있다.The fraction of the evening primrose extract of the present invention induces monocyte growth regulation and differentiation into macrophages in glycogenopathies type Ib without cytotoxicity, and is used as an active ingredient in compositions for the prevention and treatment of immunodeficiency disorders such as persistent bacterial infections. It can be useful.
도 1은 달맞이꽃 추출물의 물 분획물의 단핵구세포와 G6PT가 낙아웃(knockout)된 단핵구세포에 대한 세포독성을 조사한 결과로서, 사람의 말초혈액에서 얻은 단핵구 세포주인 THP-1(+/+)과 이를 CRISPR-Cas9으로 G6PT 낙아웃한 THP-1(G6PT가 결핍된 THP-1)(-/-)에 달맞이꽃 추출물의 물 분획물(Odorata)을 각각 10 ug/ml, 25 ug/ml, 50 ug/ml, 100 ug/ml, 200 ug/ml 농도로 24, 48, 72, 96시간 처리한 후 유세포분석기를 통해 세포사멸을 측정한 도이다.
도 2는 THP-1 대조군과 G6PT가 결핍된 THP-1 실험군에 달맞이꽃 추출물의 물 분획물(Odorata)을 각각 10 ug/ml, 25 ug/ml, 50 ug/ml, 100 ug/ml, 200 ug/ml 농도로 24, 48, 72, 96시간 동안 처리하여 세포 성장의 변화를 정량적으로 측정한 도이다.
도 3은 THP-1 대조군(WT)과 G6PT가 결핍된 THP-1 실험군(G6PT-/-)에 달맞이꽃 추출물의 물 분획물(O.Odorata) 50 ug/ml 농도로 72시간 동안 전처리하고 PMA(phorbol 12-myristate 13-acetate) 100 nM을 후처리한 후, 세포 분화를 현미경 상에서 관찰한 도이다.
도 4는 THP-1 대조군(+/+)과 G6PT가 결핍된 THP-1 실험군(-/-)에 달맞이꽃 추출물의 물 분획물(O.Odorata) 50 ug/ml 농도로 72시간 동안 전처리하고 PMA 100 nM을 후처리한 후 유세포분석기를 통해 FSC(forward scatter)와 SSC(side scatter), 그리고 CD86의 형광을 측정한 도이다.
도 5는 THP-1 대조군(+/+)과 G6PT가 결핍된 THP-1 실험군(-/-)에 달맞이꽃 추출물의 물 분획물 50 ug/ml 농도로 72시간 동안 전처리하고 PMA 100 nM을 후처리한 후 실시간 중합효소연쇄반응을 통해 염증성 사이토카인(inflammatory cytokine)의 mRNA의 발현량을 측정한 도이다.
Figure 1 shows the results of examining the cytotoxicity of the water fraction of the evening primrose extract against monocyte cells and monocyte cells in which G6PT has been knocked out. The results were obtained from THP-1(+/+), a monocyte cell line obtained from human peripheral blood, and its The aqueous fraction of evening primrose extract (Odorata) was administered to THP-1 (G6PT-deficient THP-1)(-/-) in which G6PT was knocked out using CRISPR-Cas9 at 10 ug/ml, 25 ug/ml, and 50 ug/ml, respectively. , 100 ug/ml, and 200 ug/ml concentrations were used to measure cell death using flow cytometry after treatment for 24, 48, 72, and 96 hours.
Figure 2 shows the water fraction (Odorata) of evening primrose extract in the THP-1 control group and G6PT-deficient THP-1 experimental group at doses of 10 ug/ml, 25 ug/ml, 50 ug/ml, 100 ug/ml, and 200 ug/ml, respectively. This is a diagram quantitatively measuring changes in cell growth after treatment at ml concentration for 24, 48, 72, and 96 hours.
Figure 3 shows the THP-1 control group (WT) and the G6PT-deficient THP-1 experimental group (G6PT -/- ) pretreated with the water fraction of evening primrose extract (O.Odorata) at a concentration of 50 ug/ml for 72 hours and treated with PMA (phorbol). This is a diagram showing cell differentiation observed under a microscope after post-treatment with 100 nM (12-myristate 13-acetate).
Figure 4 shows the THP-1 control group (+/+) and the G6PT-deficient THP-1 experimental group (-/-) pretreated with the water fraction of evening primrose extract (O.Odorata) at a concentration of 50 ug/ml for 72 hours and treated with PMA 100. This is a diagram measuring FSC (forward scatter), SSC (side scatter), and fluorescence of CD86 using a flow cytometer after nM post-processing.
Figure 5 shows the THP-1 control group (+/+) and the G6PT-deficient THP-1 experimental group (-/-) pretreated with the water fraction of evening primrose extract at a concentration of 50 ug/ml for 72 hours and post-treated with 100 nM PMA. This is a diagram measuring the mRNA expression level of inflammatory cytokines through real-time polymerase chain reaction.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 달맞이꽃 추출물 또는 이의 분획물을 유효성분으로 함유하는 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for the prevention and treatment of glycogenopathies type Ib or immunodeficiency of glycogenopathies type Ib, containing evening primrose extract or a fraction thereof as an active ingredient.
상기 달맞이꽃 추출물 또는 이의 분획물은 하기의 단계들을 포함하는 제조방법에 의해 제조되는 것이 바람직하나 이에 한정되지 않는다:The evening primrose extract or fraction thereof is preferably, but not limited to, prepared by a production method comprising the following steps:
1) 달맞이꽃에 추출용매를 가하여 추출하는 단계;1) Extracting evening primrose by adding an extraction solvent;
2) 단계 1)의 추출물을 여과하는 단계;2) filtering the extract of step 1);
3) 단계 2)의 여과한 추출물을 감압 농축한 후 건조하여 달맞이꽃의 추출물을 제조하는 단계; 및3) preparing an extract of evening primrose by concentrating the filtered extract of step 2) under reduced pressure and then drying it; and
4) 단계 3)의 달맞이꽃 추출물을 추가적으로 유기용매로 추출하여 달맞이꽃 분획물을 제조하는 단계.4) Preparing an evening primrose fraction by additionally extracting the evening primrose extract of step 3) with an organic solvent.
상기 방법에 있어서, 단계 1)의 달맞이꽃는 재배한 것 또는 시판되는 것 등 제한 없이 사용할 수 있다. 상기 달맞이꽃은 꽃, 잎, 가지, 뿌리, 열매, 껍질, 전초 및 이들의 혼합물로 구성된 군으로부터 선택되는 어느 하나 이상을 이용할 수 있으나, 전초인 것이 보다 바람직하다. In the above method, the evening primrose of step 1) can be used without limitation, whether cultivated or commercially available. The evening primrose can be any one or more selected from the group consisting of flowers, leaves, branches, roots, fruits, bark, whole plants, and mixtures thereof, but it is more preferable that the evening primroses are whole plants.
상기 방법에 있어서, 상기 단계 1)의 추출용매는 물, 알코올 또는 이들의 혼합물을 사용하는 것이 바람직하다. 상기 알코올로는 C1 내지 C2 저급 알코올을 이용하는 것이 바람직하며, 저급 알코올로는 에탄올 또는 메탄올을 이용하는 것이 바람직하다. 추출방법으로는 진탕추출, Soxhlet 추출 또는 환류 추출을 이용하는 것이 바람직하나 이에 한정되지 않는다. 상기 추출용매를 건조된 달맞이꽃 분량에 1 내지 10배 첨가하여 추출하는 것이 바람직하고, 2 내지 3배 첨가하여 추출하는 것이 더욱 바람직하다. 추출온도는 20℃ 내지 100℃인 것이 바람직하고, 20℃ 내지 40℃인 것이 더욱 바람직하고, 실온인 것이 가장 바람직하나, 이에 한정하지 않는다. 또한, 추출시간은 10 내지 48시간인 것이 바람직하며, 15 내지 30시간인 것이 더욱 바람직하고, 24시간인 것이 가장 바람직하나, 이에 한정하지 않는다. 아울러, 추출 횟수는 1 내지 5회인 것이 바람직하며, 3 내지 4회 반복 추출하는 것이 더욱 바람직하고, 3회인 것이 가장 바람직하나, 이에 한정되는 것은 아니다. In the above method, it is preferable to use water, alcohol, or a mixture thereof as the extraction solvent in step 1). It is preferable to use C1 to C2 lower alcohol as the alcohol, and it is preferable to use ethanol or methanol as the lower alcohol. The extraction method is preferably shaken extraction, Soxhlet extraction, or reflux extraction, but is not limited thereto. It is preferable to extract by adding 1 to 10 times the amount of the extraction solvent to the amount of dried evening primrose, and more preferably to extract by adding 2 to 3 times the amount of the extraction solvent. The extraction temperature is preferably 20°C to 100°C, more preferably 20°C to 40°C, and most preferably room temperature, but is not limited thereto. In addition, the extraction time is preferably 10 to 48 hours, more preferably 15 to 30 hours, and most preferably 24 hours, but is not limited thereto. In addition, the number of extractions is preferably 1 to 5 times, more preferably 3 to 4 times, and most preferably 3 times, but is not limited thereto.
상기 방법에 있어서, 단계 3)의 감압농축은 진공감압농축기 또는 진공회전증발기를 이용하는 것이 바람직하나 이에 한정하지 않는다. 또한, 건조는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the vacuum concentration in step 3) is preferably performed using a vacuum vacuum concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, drying is preferably performed by reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, but is not limited thereto.
상기 방법에 있어서, 단계 4)의 유기용매는 n-헥산, 클로로포름, 에틸아세테이트, n-부탄올 또는 물인 것이 바람직하나, 이에 한정하지 않는다. 상기 분획물은 달맞이꽃 추출물을 물에 현탁시킨 후 n-헥산, 클로로포름, 에틸아세테이트, n-부탄올 및 물로 순차적으로 계통 분획하여 수득한 n-헥산 분획물, 클로로포름 분획물, 에틸아세테이트 분획물, n-부탄올 분획물 또는 물 분획물 중 어느 하나인 것이 바람직하며, 물 분획물임이 더욱 바람직하나, 이에 한정하지 않는다. 상기 분획물은 상기 달맞이꽃 추출물로부터 분획 과정을 1 내지 5회, 바람직하게는 3회 반복하여 수득할 수 있고, 분획 후 감압 농축하는 것이 바람직하나 이에 한정하지 않는다.In the above method, the organic solvent in step 4) is preferably n-hexane, chloroform, ethyl acetate, n-butanol, or water, but is not limited thereto. The fraction is an n-hexane fraction, chloroform fraction, ethyl acetate fraction, n-butanol fraction, or water obtained by suspending the evening primrose extract in water and sequentially fractionating it with n-hexane, chloroform, ethyl acetate, n-butanol, and water. Any one of the fractions is preferred, and the water fraction is more preferred, but is not limited thereto. The fraction can be obtained from the evening primrose extract by repeating the fractionation process 1 to 5 times, preferably 3 times, and concentration under reduced pressure after fractionation is preferred, but is not limited to this.
또한, 상기 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증은 간비대증, 신장비대증, 저혈당, 젖산혈증, 고요산혈증, 고지혈증, 호중구 감소증 및 호중구와 단핵구 기능저하로 이루어진 군으로부터 선택되는 어느 하나 이상인 것이 바람직하나 이에 한정되지 않는다.In addition, the glycogenic disease type Ib or glycogenic immunodeficiency type Ib is any one selected from the group consisting of hepatomegaly, renal hypertrophy, hypoglycemia, lactic acidemia, hyperuricemia, hyperlipidemia, neutropenia, and decreased neutrophil and monocyte function. It is preferable to be above this, but it is not limited to this.
본 발명의 구체적인 실시예에서, 본 발명자들은 본 발명의 달맞이꽃 추출물의 물 분획물의 세포독성을 확인하기 위하여, 단핵구 세포주인 THP-1 세포 및, G6PT가 결핍된 THP-1 세포주에 달맞이꽃 추출물의 물 분획물을 처리한 결과, 본 발명의 달맞이꽃 추출물의 물 분획물은 높은 농도에서도 세포 독성이 나타나지 않았음을 확인하였다(도 1 참조).In a specific embodiment of the present invention, in order to confirm the cytotoxicity of the water fraction of the evening primrose extract of the present invention, the present inventors applied the water fraction of the evening primrose extract to THP-1 cells, a mononuclear cell line, and a G6PT-deficient THP-1 cell line. As a result of treatment, it was confirmed that the water fraction of the evening primrose extract of the present invention did not exhibit cytotoxicity even at high concentrations (see Figure 1).
또한, 본 발명자들은 달맞이꽃 추출물의 물 분획물처리에 의한 단핵구 세포 성장 변화를 확인하기 위하여, THP-1 세포 및, G6PT가 결핍된 THP-1 세포주에 달맞이꽃 추출물의 물 분획물을 농도별로 처리한 결과, 대조군 세포와 비교하여 G6PT가 결핍된 THP-1 실험군 세포의 세포 성장 변화는 달맞이꽃 추출물의 물 분획물의 농도가 높을수록 세포 성장이 저해되는 것을 확인하였다(도 2 참조).In addition, in order to confirm changes in monocyte cell growth caused by treatment with the water fraction of the evening primrose extract, the present inventors treated THP-1 cells and the G6PT-deficient THP-1 cell line with the water fraction of the evening primrose extract at different concentrations, and as a result, the control group Compared to cells in the G6PT-deficient THP-1 experimental group, changes in cell growth confirmed that cell growth was inhibited as the concentration of the water fraction of evening primrose extract increased (see Figure 2).
또한, 본 발명자들은 달맞이꽃 추출물의 물 분획물처리에 의한 단핵구의 대식세포로의 분화능 확인하기 위하여, THP-1 세포 및, G6PT가 결핍된 THP-1 세포주에 달맞이꽃 추출물의 물 분획물 및 대식세포로의 분화를 위해 PMA를 처리한 결과, 달맞이꽃 추출물의 물 분획물을 전처리하고 분화를 유도한 G6PT가 결핍된 THP-1에서는 대조군 세포와 비슷하게 대식세포로의 분화가 유도되는 것을 확인하였다(도 3 및 도 4참조).In addition, in order to confirm the ability of monocytes to differentiate into macrophages by treatment with the water fraction of the evening primrose extract, the present inventors used the water fraction of the evening primrose extract and differentiation into macrophages in THP-1 cells and a G6PT-deficient THP-1 cell line. As a result of treatment with PMA, it was confirmed that differentiation into macrophages was induced similarly to control cells in G6PT-deficient THP-1, which was pretreated with the water fraction of evening primrose extract and induced differentiation (see Figures 3 and 4 ).
아울러, 본 발명자들은 달맞이꽃 추출물의 물 분획물처리에 의해 분화된 세포의 사이토카인 발현량을 확인한 결과, 달맞이꽃 추출물의 물 분획물을 전처리하고 분화를 유도한 G6PT가 결핍된 THP-1 세포에서 대식세포 특성인 염증성 사이토카인(IL-1β, IL-8)의 발현량이 증가하는 것을 확인하였다(도 5 참조).In addition, the present inventors confirmed the cytokine expression level of cells differentiated by treatment with the water fraction of evening primrose extract and found that G6PT-deficient THP-1 cells, which were pretreated with the water fraction of evening primrose extract and induced differentiation, exhibited macrophage characteristics. It was confirmed that the expression level of inflammatory cytokines (IL-1β, IL-8) increased (see Figure 5).
따라서, 본 발명의 달맞이꽃 추출물 또는 이의 분획물은 세포독성 없이, 당원병 제Ib에서의 단핵구의 생장조절 및 대식세포로의 분화촉진을 유도하여 지속적인 박테리아 감염 등의 면역결핍증에 대한 예방 및 치료용 약학적 조성물의 유효성분으로 유용하게 사용될 수 있다.Therefore, the evening primrose extract or fraction thereof of the present invention induces growth regulation of monocytes and promotion of differentiation into macrophages in glycogenopathies type Ib without cytotoxicity, and is used as a pharmaceutical for the prevention and treatment of immunodeficiency disorders such as persistent bacterial infections. It can be usefully used as an active ingredient in a composition.
상기 약학적 조성물은 성인의 경우, 1일 1회 내지 수회 투여 시, 0.01 내지 500 mg/kg의 용량으로 투여될 수 있으며, 구체적으로는 0.1 내지 200 mg/kg, 보다 구체적으로 0.1 내지 100 mg/kg, 보다 더 구체적으로 0.1 내지 20 mg/kg, 보다 더 구체적으로 1 내지 10 mg/kg의 용량으로 일일 1 내지 3회 투여될 수 있고 제형화 된 단위 투여형 제제는 필요에 따라 일정시간 간격으로 수회 투여될 수 있다. For adults, the pharmaceutical composition may be administered at a dose of 0.01 to 500 mg/kg, specifically 0.1 to 200 mg/kg, more specifically 0.1 to 100 mg/kg, when administered once or several times a day. kg, more specifically 0.1 to 20 mg/kg, and even more specifically 1 to 10 mg/kg, and can be administered 1 to 3 times daily, and the formulated unit dosage form is administered at regular time intervals as needed. It may be administered multiple times.
또한, 상기 약학적 조성물은 약학적으로 허용 가능한 담체 및 비히클을 추가로 포함할 수 있다. 구체적으로 이온 교환 수지, 알루미나, 알루미늄 스테아레이트, 레시틴, 혈청 단백질(예, 사람 혈청 알부민), 완충 물질(예, 각종 인산염, 글리신, 소르브산, 칼륨 소르베이트, 포화 식물성 지방산의 부분적인 글리세라이드 혼합물), 물, 염 또는 전해질(예, 프로타민 설페이트, 인산수소이나트륨, 인산수소칼륨, 염화나트륨 및 아연 염), 교질성 실리카, 마그네슘 트리실리케이트, 폴리비닐피롤리돈, 셀룰로즈계 기질, 폴리에틸렌 글리콜, 나트륨 카르복시메틸셀룰로즈, 폴리아릴레이트, 왁스 또는 양모지 등을 포함할 수 있으나 이에 제한되지 않는다.Additionally, the pharmaceutical composition may further include a pharmaceutically acceptable carrier and vehicle. Specifically, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), and buffering substances (e.g., various phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids). ), water, salts or electrolytes (e.g. protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic matrices, polyethylene glycol, sodium carboxylate. It may include, but is not limited to, methylcellulose, polyarylate, wax, or wool paper.
약학적으로 허용 가능한 담체 및 비히클을 포함하는 상기 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. The composition containing a pharmaceutically acceptable carrier and vehicle may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 약학적 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로즈(Sucrose)또는 락토오스(Lactose), 탄산칼슘, 젤라틴 등을 섞어 조제될 수 있다. 또한 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용될 수 있다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the pharmaceutical composition of the present invention, such as starch, calcium carbonate, It can be prepared by mixing sucrose or lactose, calcium carbonate, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used.
경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is.
비경구투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로젤라틴 등이 사용될 수 있다.Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.
상기 약학적 조성물은 정제, 환제, 산제, 과립제, 피복정, 캡슐제, 현탁제, 내용액제, 유제, 시럽제, 즙, 멸균된 수용액, 비수성용제, 주사제, 동결건조제제 및 좌제로 이루어진 군으로부터 선택되는 어느 하나의 제형을 가질 수 있다.The pharmaceutical composition is selected from the group consisting of tablets, pills, powders, granules, coated tablets, capsules, suspensions, oral solutions, emulsions, syrups, juices, sterilized aqueous solutions, non-aqueous solutions, injections, lyophilized preparations and suppositories. It can have any one formulation.
상기 약학적 조성물은 쥐, 생쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 또는 뇌혈관내 주사에 의해 투여될 수 있다.The pharmaceutical composition can be administered to mammals such as rats, mice, livestock, and humans through various routes. All modes of administration are contemplated, for example, oral, rectal or by intravenous, intramuscular, subcutaneous, or intracerebrovascular injection.
본 발명에 따른 약학적 조성물의 투여량은 투여 목적, 질환의 종류, 질환의 중증도, 조성물에 함유된 유효 성분 및 다른 성분의 종류 및 함량, 제형의 종류 및 환자의 연령, 체중, 일반 건강 상태, 성별 및 식이, 투여 시간, 투여 경로 및 조성물의 분비율, 치료 기간, 동시 사용되는 약물을 비롯한 다양한 인자에 따라 조절될 수 있다.The dosage of the pharmaceutical composition according to the present invention is determined by the purpose of administration, the type of disease, the severity of the disease, the type and content of the active ingredient and other ingredients contained in the composition, the type of dosage form, and the patient's age, weight, general health condition, It can be adjusted according to various factors, including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, and concurrently used drugs.
아울러, 본 발명은 달맞이꽃 추출물 또는 이의 분획물을 유효성분으로 함유하는 당원병 제Ib형 또는 당원병 제Ib형의 면역결핍증 예방 및 개선용 건강식품용 조성물을 제공한다.In addition, the present invention provides a composition for health food for preventing and improving immunodeficiency of glycogenic disease type Ib or glycogenic disease type Ib, containing evening primrose extract or a fraction thereof as an active ingredient.
본 발명의 달맞이꽃 추출물 또는 이의 분획물을 식품 첨가물로 사용할 경우, 상기 추출물 또는 이의 분획물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 상기 달맞이꽃 추출물은 열수 및 에탄올을 이용하여 추출하는 것이 바람직하며, 상기 에탄올의 농도는 50% 내지 70%인 것이 바람직하다. 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 조성물은 원료에 대하여 15 중량부 이하, 바람직하게는 10 중량부 이하의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다. When using the evening primrose extract or fraction thereof of the present invention as a food additive, the extract or fraction thereof can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The evening primrose extract is preferably extracted using hot water and ethanol, and the concentration of ethanol is preferably 50% to 70%. The mixing amount of the active ingredient can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when producing a food or beverage, the composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.
상기 식품의 종류에는 특별한 제한은 없다. 본 발명의 달맞이꽃 추출물 또는 이의 분획물을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There are no special restrictions on the types of foods above. Examples of foods to which the evening primrose extract or fractions thereof of the present invention can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, It includes beverages, teas, beverages, alcoholic beverages, and vitamin complexes, and includes all health foods in the conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 달맞이꽃 추출물 또는 이의 분획물 100 ㎖당 일반적으로 약0.01 ~ 0.04 g, 바람직하게는 약 0.02 ~ 0.03 g 이다.The health drink composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional drinks. Natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 ml of the evening primrose extract or fraction thereof of the present invention.
상기 외에 본 발명의 달맞이꽃 추출물 또는 이의 분획물은 여러가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 달맞이꽃 추출물 또는 이의 분획물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다.In addition to the above, the evening primrose extract or fraction thereof of the present invention may include various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, and glycerin. , alcohol, and carbonating agents used in carbonated beverages. In addition, the evening primrose extract or fraction thereof of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination.
이하, 본 발명을 실시예에 의하여 상세히 설명한다.Hereinafter, the present invention will be described in detail through examples.
단, 하기 실시예는 본 발명을 구체적으로 예시하는 것이며, 본 발명의 내용이 하기 실시예에 의해 한정되는 것은 아니다.However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.
<실시예 1> 달맞이꽃(<Example 1> Evening primrose ( Oenothera odorataOenothera odorata ) 추출물 및 이의 분획물의 제조) Preparation of extracts and fractions thereof
긴잎달맞이꽃의 전초에서 메틸알코올 99.9%(HPLC grade)를 추출용매로 사용하여 섭씨 45도에서 30분 초음파처리(sonication)와 2시간 정지를 10회 반복하여 획득한 달맞이꽃 추출물을 한국식물추출물은행에서 분양받아 본 실험의 시료로 사용하였다.Evening primrose extract obtained from the longleaf evening primrose plant using 99.9% methyl alcohol (HPLC grade) as an extraction solvent and repeating sonication for 30 minutes at 45 degrees Celsius and stopping for 2 hours 10 times was distributed by the Korea Plant Extract Bank. It was received and used as a sample for this experiment.
또한, 한국식물추출물은행에 의뢰하여 상기 달맞이꽃 추출물을 헥산(Hexane), 클로로포름(Chloroform), 에틸 아세테이트(ethyl acetate), 부탄올Butyl alcohol), 증류수(Distilled water)의 5가지 용매로 추출하여 분획물을 수득하고 이중, 물 분획물을 본 실험의 시료로 사용하였다.In addition, by requesting the Korea Plant Extract Bank, the evening primrose extract was extracted with five solvents: hexane, chloroform, ethyl acetate, butanol, and distilled water to obtain fractions. And of these, the water fraction was used as a sample in this experiment.
<실시예 2> 달맞이꽃 추출물의 물 분획물의 세포독성 확인<Example 2> Confirmation of cytotoxicity of water fraction of evening primrose extract
사람의 말초혈액에서 얻은 단핵구 세포주인 THP-1 세포는 RPMI1640 배지에 10% FBS(fetal bovine serum)과 1% Pen/Strep(penicillin/streptomycin)을 섞어 37℃, 5% CO2의 조건에서 배양하였다. THP-1 cells, a mononuclear cell line obtained from human peripheral blood, were cultured in RPMI1640 medium mixed with 10% FBS (fetal bovine serum) and 1% Pen/Strep (penicillin/streptomycin) at 37°C and 5% CO2 . .
서울대학교 박태섭 교수님 연구팀을 통해 G6PT가 결핍된 THP-1(CRISPR-Cas9으로 G6PT 낙아웃한 THP-1) 세포주를 확보하고 이를 THP-1 대조군 세포와 동일한 조건에서 배양하였다. 도 1에서처럼 배양한 THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포에 상기 <실시예 1>에서 제조한 달맞이꽃 추출물의 물 분획물을 각각 10 ug/ml, 25 ug/ml, 50 ug/ml, 100 ug/ml, 200 ug/ml의 농도별로 처리하고 48시간 배양하였다. 48시간에 배지를 교환하고 달맞이꽃 추출물의 물 분획물을 농도별로 다시 처리하여 96시간까지 배양하였다. 24, 48, 72, 96시간마다 세포를 얻어 아넥신 V(Annexin V) 와 프로피디움 아이오다이드(Propidium Iodide; PI)로 염색하고 유세포 분석기를 통해 세포사멸을 관찰하였다. Through Professor Tae-seop Park's research team at Seoul National University, a G6PT-deficient THP-1 (THP-1 with G6PT knocked out using CRISPR-Cas9) cell line was obtained and cultured under the same conditions as THP-1 control cells. 10 ug/ml, 25 ug/ml, and 50 ug/ml of the water fraction of the evening primrose extract prepared in <Example 1> was administered to the THP-1 control cells and G6PT-deficient THP-1 test group cells cultured as in Figure 1, respectively. Treated at different concentrations of ml, 100 ug/ml, and 200 ug/ml and cultured for 48 hours. At 48 hours, the medium was changed, and the water fraction of evening primrose extract was treated again at different concentrations and cultured for up to 96 hours. Cells were obtained every 24, 48, 72, and 96 hours, stained with Annexin V and propidium iodide (PI), and apoptosis was observed using flow cytometry.
그 결과, 도 1에 나타낸 바와 같이, 본 발명의 달맞이꽃 추출물의 물 분획물을 처리한 대조군과 실험군 세포는 달맞이꽃 추출물의 물 분획물을 처리하지 않은 대조군과 실험군 세포와 비교했을 때 유의미한 세포사멸이 관찰되지 않았다. As a result, as shown in Figure 1, no significant cell death was observed in the control and experimental group cells treated with the water fraction of the evening primrose extract of the present invention compared to the control and experimental group cells not treated with the water fraction of the evening primrose extract. .
따라서, 본 발명의 달맞이꽃 추출물의 물 분획물은 높은 농도에서도 세포 독성이 나타나지 않았음을 확인하였다(도 1).Therefore, it was confirmed that the water fraction of the evening primrose extract of the present invention did not exhibit cytotoxicity even at high concentrations (Figure 1).
<실시예 3> 달맞이꽃 추출물의 물 분획물처리에 의한 세포 성장 변화 확인<Example 3> Confirmation of cell growth changes by treatment of water fraction of evening primrose extract
THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포는 RPMI1640 배지에 10% FBS과 1% Pen/Strep을 섞어 37℃, 5% CO2의 조건에서 배양하였다. THP-1 control cells and G6PT-deficient THP-1 experimental group cells were cultured in RPMI1640 medium mixed with 10% FBS and 1% Pen/Strep at 37°C and 5% CO 2 .
도 2에서처럼 배양한 THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포에 달맞이꽃 추출물의 물 분획물을 각각 10 ug/ml, 25 ug/ml, 50 ug/ml, 100 ug/ml, 200 ug/ml의 농도별로 처리하고 48시간 배양하였다. 48시간에 배지를 교환하고 달맞이꽃 추출물의 물 분획물을 농도별로 다시 처리하여 96시간까지 배양하였다. As shown in Figure 2, 10 ug/ml, 25 ug/ml, 50 ug/ml, 100 ug/ml, and 200 ug of the water fraction of evening primrose extract was added to THP-1 control cells and G6PT-deficient THP-1 experimental group cells, respectively. They were treated at different concentrations of /ml and cultured for 48 hours. At 48 hours, the medium was changed, and the water fraction of evening primrose extract was treated again at different concentrations and cultured for up to 96 hours.
그 결과, 도 2에 나타낸 바와 같이, 24, 48, 72, 96시간마다 세포수를 측정한 결과, THP-1 대조군 세포의 세포 성장 변화는 큰 차이를 보이지 않는 반면에 G6PT가 결핍된 THP-1 실험군 세포의 세포 성장 변화는 달맞이꽃 추출물의 물 분획물의 농도가 높을수록 세포 성장이 저해되는 것을 확인하였다(도 2). As a result, as shown in Figure 2, as a result of measuring cell numbers every 24, 48, 72, and 96 hours, there was no significant difference in cell growth changes in THP-1 control cells, whereas THP-1 lacking G6PT showed no significant difference. Changes in cell growth of cells in the experimental group confirmed that cell growth was inhibited as the concentration of the water fraction of evening primrose extract increased (Figure 2).
<실시예 4> 달맞이꽃 추출물의 물 분획물처리에 의한 세포분화능 확인<Example 4> Confirmation of cell differentiation ability by treatment of water fraction of evening primrose extract
THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포는 RPMI1640 배지에 10% FBS과 1% Pen/Strep을 섞어 37℃, 5% CO2의 조건에서 배양하였다. THP-1 control cells and G6PT-deficient THP-1 experimental group cells were cultured in RPMI1640 medium mixed with 10% FBS and 1% Pen/Strep at 37°C and 5% CO 2 .
도 3에서처럼 THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포에 달맞이꽃 추출물의 물 분획물을 50 ug/ml의 농도로 처리하고 48시간 배양하였다. 48시간에 배지를 교환하고 달맞이꽃 추출물의 물 분획물을 50 ug/ml의 농도로 다시 처리하여 72시간까지 배양하였다. 72시간 후 달맞이꽃 추출물의 물 분획물이 없는 배지로 교환하고 대식세포로의 분화를 위해 PMA(phorbol 12-myristate 13-acetate) 100 nM을 처리하고 120시간까지 배양하였다. 120시간에 PMA가 없는 배지로 교환하고 달맞이꽃 추출물의 물 분획물을 농도별로 다시 처리하여 144시간까지 배양하였다. As shown in Figure 3, THP-1 control cells and G6PT-deficient THP-1 experimental group cells were treated with the water fraction of evening primrose extract at a concentration of 50 ug/ml and cultured for 48 hours. At 48 hours, the medium was changed, and the water fraction of evening primrose extract was treated again at a concentration of 50 ug/ml and cultured for up to 72 hours. After 72 hours, the medium was changed to one without the water fraction of evening primrose extract, and for differentiation into macrophages, 100 nM of PMA (phorbol 12-myristate 13-acetate) was added and cultured for up to 120 hours. At 120 hours, the medium was replaced with PMA-free medium, and the water fraction of evening primrose extract was treated again at different concentrations and cultured for up to 144 hours.
그 결과, 도 3에 나타낸 바와 같이, PMA만 처리한 대조군 THP-1 단핵구 세포는 플라스크 바닥에 붙는 부착능력과 시간이 지남에 따라 핵과 세포질의 비율이 감소하는 분화된 대식세포의 특징이 관찰되었으나, G6PT가 결핍된 THP-1은 부착능력을 보이는 세포가 거의 없음을 확인할 수 있었다. 한편, 달맞이꽃 추출물의 물 분획물을 전처리하고 분화를 유도한 G6PT가 결핍된 THP-1에서는 대조군 세포와 비슷하게 부착능력이 나타나는 것을 확인하였다(도 3).As a result, as shown in Figure 3, the control THP-1 monocyte cells treated only with PMA were observed to have characteristics of differentiated macrophages, such as the adhesion ability to stick to the bottom of the flask and the decrease in the ratio of nucleus and cytoplasm over time. It was confirmed that THP-1 lacking G6PT had almost no cells showing adhesion ability. Meanwhile, it was confirmed that G6PT-deficient THP-1, which was pretreated with the water fraction of evening primrose extract and induced differentiation, showed adhesion ability similar to that of control cells (Figure 3).
또한, 도 4에서처럼 THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포에 도 3과 동일하게 달맞이꽃 추출물의 물 분획물을 전처리하고 PMA로 후처리하여 배양하였다. 대식세포로 분화되어 플라스크 바닥에 붙은 세포를 1 ml PBS(Phosphate-Buffered Saline) 버퍼로 3번 세척하고 0.25% 트립신(trypsin)을 이용해 세포를 얻어 대식세포 마커인 CD86으로 염색하고 유세포분석기를 통해 형광을 측정하였다. In addition, as shown in Figure 4, THP-1 control cells and G6PT-deficient THP-1 experimental group cells were pretreated with the water fraction of evening primrose extract in the same manner as in Figure 3 and then post-treated with PMA and cultured. Cells differentiated into macrophages and attached to the bottom of the flask were washed three times with 1 ml Phosphate-Buffered Saline (PBS) buffer, obtained using 0.25% trypsin, stained with CD86, a macrophage marker, and fluorescently analyzed using a flow cytometer. was measured.
그 결과, 도 4에 나타낸 바와 같이 도 3과 동일하게 달맞이꽃 추출물 물 분획물을 전처리하고 분화를 유도한 G6PT가 결핍된 THP-1 세포에서 대식세포로의 분화능력과 세포 내 과립의 수가 증가하는 것을 확인하였다(도 4).As a result, as shown in Figure 4, it was confirmed that the differentiation ability into macrophages and the number of intracellular granules increased in G6PT-deficient THP-1 cells, which were induced to differentiate by pretreatment with the evening primrose extract water fraction in the same manner as in Figure 3. (Figure 4).
<실시예 5> 달맞이꽃 추출물의 물 분획물처리에 의해 분화된 세포의 사이토카인 발현량 확인<Example 5> Confirmation of cytokine expression level in differentiated cells by treatment with water fraction of evening primrose extract
THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포는 RPMI1640 배지에 10% FBS과 1% Pen/Strep을 섞어 37℃, 5% CO2의 조건에서 배양하였다. 도 3에서 처럼 THP-1 대조군 세포와 G6PT가 결핍된 THP-1 실험군 세포에 달맞이꽃 추출물의 물 분획물을 50 ug/ml의 농도로 처리하고 48시간 배양하였다. 48시간에 배지를 교환하고 달맞이꽃 추출물의 물 분획물을 50 ug/ml의 농도로 다시 처리하여 72시간까지 배양하였다. 72시간 후 달맞이꽃 추출물의 물 분획물이 없는 배지로 교환하고 대식세포로의 분화를 위해 PMA 100 nM을 처리하고 120시간까지 배양하였다. 120시간에 PMA가 없는 배지로 교환하고 달맞이꽃 추출물의 물 분획물을 농도별로 다시 처리하여 144시간까지 배양하였다. 대식세포로 분화되어 플라스크 바닥에 붙은 세포를 1 ml PBS 버퍼로 3번 세척하고 트리졸(TRIzol)을 이용해 세포 내 RNA를 추출하였다. 그런 다음, 실시간 중합효소연쇄반응을 통해 염증성 사이토카인(inflammtory cytokine)의 mRNA이 발현량을 측정하였다. THP-1 control cells and G6PT-deficient THP-1 experimental group cells were cultured in RPMI1640 medium mixed with 10% FBS and 1% Pen/Strep at 37°C and 5% CO 2 . As shown in Figure 3, THP-1 control cells and G6PT-deficient THP-1 experimental group cells were treated with the water fraction of evening primrose extract at a concentration of 50 ug/ml and cultured for 48 hours. At 48 hours, the medium was changed, and the water fraction of evening primrose extract was treated again at a concentration of 50 ug/ml and cultured for up to 72 hours. After 72 hours, the medium was replaced with a medium without the water fraction of evening primrose extract, treated with 100 nM of PMA for differentiation into macrophages, and cultured for up to 120 hours. At 120 hours, the medium was replaced with PMA-free medium, and the water fraction of evening primrose extract was treated again at different concentrations and cultured for up to 144 hours. Cells differentiated into macrophages and attached to the bottom of the flask were washed three times with 1 ml PBS buffer, and intracellular RNA was extracted using TRIzol. Then, the mRNA expression level of inflammatory cytokines was measured through real-time polymerase chain reaction.
그 결과, 도 5에 나타낸 바와 같이 달맞이꽃 추출물 물 분획물을 전처리하고 분화를 유도한 G6PT가 결핍된 THP-1 세포에서 대식세포 특성인 염증성 사이토카인(IL-1β, IL-8)의 발현량이 증가하는 것을 확인하였다(도 5). As a result, as shown in Figure 5, the expression level of inflammatory cytokines (IL-1β, IL-8), which are characteristic of macrophages, increased in G6PT-deficient THP-1 cells pretreated with the evening primrose extract water fraction and induced differentiation. This was confirmed (Figure 5).
Claims (8)
Glycopathies type Ib containing evening primrose extract or a fraction thereof as an active ingredient, or any one selected from the group consisting of hepatomegaly, nephromegaly, hypoglycemia, lactic acidemia, hyperuricemia, neutropenia, neutrophil dysfunction, and monocyte hypofunction. Composition for health food for preventing and improving immunodeficiency in diabetes mellitus type Ib.
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"선천성 대사질환모델을 활용한 호중구의 기능, 분화 및 골수성 백혈병 발병 기전에 대한 연구" 이공학개인기초연구지원사업 최종보고서, 주관연구기관:고려대학교 (2019.11)* |
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