KR101467350B1 - Glycoprotein fraction isolated from wheat bran and method for producing the same - Google Patents
Glycoprotein fraction isolated from wheat bran and method for producing the same Download PDFInfo
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- KR101467350B1 KR101467350B1 KR1020120132909A KR20120132909A KR101467350B1 KR 101467350 B1 KR101467350 B1 KR 101467350B1 KR 1020120132909 A KR1020120132909 A KR 1020120132909A KR 20120132909 A KR20120132909 A KR 20120132909A KR 101467350 B1 KR101467350 B1 KR 101467350B1
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- South Korea
- Prior art keywords
- glycoprotein
- bran
- glycoprotein fraction
- present
- supernatant
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Abstract
본 발명은 밀기울 유래 당단백 분획 및 이의 제조방법에 관한 것으로, 좀 더 상세하게는 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70kDa인 것을 특징으로 하는 밀기울 유래 당단백 분획물 및 이를 유효성분으로 포함하는 면역기능 증진용 약학적, 식품 조성물에 관한 것이다.
본 발명의 당단백 분획물은 대식세포의 활성을 면역활성증진용 의약품인 크레스틴(PSK) 보다도 촉진하는 효능이 매우 뛰어나며 세포독성이 전혀 없어, 새로운 기능성 식품 및 면역기능 증강용 의약품 제조에 효과적이다.The present invention relates to a bran-derived glycoprotein fraction and a method for producing the same, and more particularly, to a bran-derived glycoprotein fraction comprising (a) (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater, wherein the glycoprotein fraction has a molecular weight of 16 to 70 kDa, Pharmaceutical compositions and food compositions.
The glycoprotein fraction of the present invention is highly effective in promoting the activity of macrophages than that of cresustine (PSK), which is a drug for promoting immunological activity, and has no cytotoxicity, and thus is effective for the production of new functional foods and immunomodulating drugs.
Description
본 발명은 밀기울 유래 당단백 분획 및 이의 제조방법에 관한 것으로, 좀 더 상세하게는 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70kDa인 것을 특징으로 하는 밀기울 유래 당단백 분획물 및 이를 유효성분으로 포함하는 면역기능 증진용 약학적, 식품 조성물에 관한 것이다.
The present invention relates to a bran-derived glycoprotein fraction and a method for producing the same, and more particularly, to a bran-derived glycoprotein fraction comprising (a) (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater, wherein the glycoprotein fraction has a molecular weight of 16 to 70 kDa, Pharmaceutical compositions and food compositions.
당단백질은 탄수화물이 단백질의 특정 아미노산에 복합체로 이루어진 생체분자로서 동식물의 세포조직에 널리 분포하고 있으며, 주로 세포외벽에 많이 존재하면서 수많은 세포표면의 반응에 관여한다. 또한 당단백질은 단백질 및 핵산과 달리 순수한 선형 및 가지형 구조와 같은 거대한 삼차원적 구조를 지니고 있어 구조적으로 다양할 뿐만 아니라 생물학적으로 잠재적인 기능들을 많이 갖고 있다.
Glycoprotein is a biomolecule composed of a complex of carbohydrates and specific amino acids of proteins. It is widely distributed in the cell tissues of plants and animals. Unlike proteins and nucleic acids, glycoproteins also have a large three-dimensional structure, such as pure linear and branched structures, which are not only structurally diverse, but also have many biological potential functions.
면역이란 인체 내에서 외부로부터 침입하는 미생물 동종의 조직 등의 다른 물질, 특이 세포가 발생하면 면역계가 관여하여 이들을 항원으로 인식하여 특이하게 반응하여 항체를 생산하는 능력을 나타냄으로서 개체의 항상성을 유지하려는 현상을 말하며, 면역 반응에 관여하는 세포는 임파구, macrophage, NK cell, 수지상세포 등이 있다. 최근 생체의 면역세포로부터 cytokine 방출을 촉진시켜 암세포를 괴사시키는 면역요법이 주목받고 있다. 면역요법은 면역반응의 증강 및 억제 유도를 통해 생체의 방어능력을 조절함으로써 암세포를 사멸하기 때문에 기존의 약물에 의한 항암치료법보다 더 큰 효과를 기대할 수 있다. 따라서 안전성이 보고된 천연물로부터 면역활성 증진 효능을 갖는 천연물을 탐색하고자 하는 시도가 많이 이루어지고 있다. Immunity refers to the ability of the immune system to participate in the generation of specific cells, such as microbial homologous tissues that invade from the outside in the body, and to produce antibodies by recognizing them as antigens and reacting singly. And the cells involved in the immune response are lymphocytes, macrophages, NK cells, and dendritic cells. In recent years, immune therapy has been attracting attention as a means of accelerating the release of cytokines from immune cells of living organisms and necrosing cancer cells. Immunotherapy regulates the defenses of the body through stimulation of the immune response and induces the suppression of cancer cells. Therefore, it can be expected to have a greater effect than the conventional chemotherapy. Therefore, attempts have been made to search for natural products having the immunostimulatory activity enhancing activity from natural substances reported to have safety.
일반적으로 밀기울이란, 밀에서 가루를 빼고 남은 찌꺼기를 말하는 것으로, 밀의 속껍질이 많이 섞인 12~15% 가량으로 밀기울은 밀에서 차지하는 용적이 크고 기호성이 높고 소화가 잘 되어 가축의 사료로 많이 쓰이는데, 열량은 적으나 무기물이 많이 들어 있으므로 젖소에는 농후사료의 25~30%, 육계에는 10%, 산란계에는 23%까지 사용할 수 있으며, 이 밖에 된장, 누룩 등의 원료로도 쓰인다. 그러나 밀기울 유래 당단백 분획물이 면역 기능 증진 활성이 있는지에 대해서는 알려진 바가 전혀 없다.
Generally, wheat bran refers to the residue left after removing the flour from the wheat. It is about 12 ~ 15% mixed with wheat bran. The bran is big in volume, high in palatability and digestible, There are few, but a lot of minerals in the dairy cow, 25 ~ 30% of the concentrated feed,
본 발명자들은 면역기능을 증진시킬 수 있는 새로운 방법에 관하여 연구하던 중 본 발명의 밀기울유래 당단백 분획물이 대식세포의 활성을 촉진하는 효능이 매우 뛰어난 것을 확인하여 본 발명을 완성하였다.
The inventors of the present invention have investigated a novel method for enhancing immune function, and found that the bran-derived glycoprotein fraction of the present invention has an excellent effect of promoting the activity of macrophages, thereby completing the present invention.
따라서 본 발명의 목적은 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70 kDa인 것을 특징으로 하는 밀기울 유래 당단백 분획물을 제공하는 것이다.
SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide (a) (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater, wherein the glycoprotein fraction has a molecular weight of 16 to 70 kDa.
본 발명의 다른 목적은 상기 당단백 분획물을 유효성분으로 포함하는 면역기능 증진용 식품 조성물을 제공하는 것이다.
Another object of the present invention is to provide a food composition for enhancing immune function comprising the glycoprotein fraction as an active ingredient.
본 발명의 다른 목적은 상기 당단백 분획물을 유효성분으로 포함하는 감기, 아토피, 만성피로 및 암으로 이루어진 군에서 선택되는 어느 하나 이상의 면역 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.
Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of immune diseases selected from the group consisting of cold, atopic, chronic fatigue and cancer comprising the glycoprotein fraction as an active ingredient.
본 발명의 다른 목적은Another object of the present invention is
(a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 밀기울 유래 당단백 분획물 제조방법을 제공하는 것이다.
(a) degreasing the bran; (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seaweed.
상기의 목적을 달성하기 위하여 본 발명은 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70 kDa인 것을 특징으로 하는 밀기울 유래 당단백 분획물을 제공한다.
In order to accomplish the above object, the present invention provides a method for producing a bran, comprising: (a) degreasing a bran; (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater, wherein the glycoprotein fraction has a molecular weight of 16 to 70 kDa.
본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 당단백 분획물을 유효성분으로 포함하는 면역기능 증진용 식품 조성물을 제공한다.
In order to accomplish another object of the present invention, there is provided a food composition for enhancing immune function comprising the glycoprotein fraction as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여 본 발명은 상기 당단백 분획물을 유효성분으로 포함하는 감기, 아토피, 만성피로 및 암으로 이루어진 군에서 선택되는 어느 하나 이상의 면역 질환 예방 또는 치료용 약학적 조성물을 제공한다.
In order to accomplish another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating immunological diseases selected from the group consisting of cold, atopic, chronic fatigue and cancer comprising the glycoprotein fraction as an active ingredient .
본 발명의 다른 목적을 달성하기 위하여 본 발명은According to another aspect of the present invention,
(a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 밀기울 유래 당단백 분획물 제조방법을 제공한다.(a) degreasing the bran; (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater.
이하 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70 kDa인 것을 특징으로 하는 밀기울 유래 당단백 분획물을 제공한다.
(A) degreasing the wheat bran; (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater, wherein the glycoprotein fraction has a molecular weight of 16 to 70 kDa.
상기 당단백 분획물을 Laemmli의 방법에 따라 분석한 결과, 분자량이 16 내지 70kDa 으로 확인되었다(실시예 3-4 및 도 2 참조). 또한, 당단백 분획물의 단백질과 당의 중량비 비율이 4:1 내지 6:1 인 것을 특징으로 하고, 더욱 상세하게는 4.5 : 1 내지 5.5 : 1 인 것을 특징으로 한다.
The glycoprotein fractions were analyzed according to the method of Laemmli and found to have a molecular weight of 16 to 70 kDa (see Example 3-4 and Fig. 2). Also, the glycoprotein fraction has a weight ratio of protein to sugar of from 4: 1 to 6: 1, more specifically from 4.5: 1 to 5.5: 1.
또한 상기 당단백 분획물은 전체 아미노산 대비 아스파르트산은 5 내지 15%(w/w), 트레오닌은 3 내지 7%(w/w), 세린은 3 내지 7%(w/w), 글루탐산은 10 내지 25%(w/w), 프롤린은 3 내지 10%(w/w), 글리신은 3 내지 10%(w/w), 알라닌은 3 내지 10%(w/w), 발린은 3 내지 10%(w/w), 메티오닌은 1 내지 5%(w/w), 이소루신은 3 내지 10%(w/w), 루신은 3 내지 10%(w/w), 티로신은 1 내지 5%(w/w), 페닐알라닌은 3 내지 10%(w/w), 리신은 3 내지 10%(w/w), 히스티딘은 1 내지 5%(w/w), 아르기닌은 5 내지 15%(w/w)의 비율로 포함되는 것을 특징으로 한다.
The glycoprotein fraction has 5 to 15% (w / w) of aspartic acid, 3 to 7% (w / w) of threonine, 3 to 7% (w / w), proline is 3 to 10% (w / w), glycine is 3 to 10% (w / w), alanine is 3 to 10% w / w), methionine is 1 to 5% w / w, isoleucine is 3 to 10% w / w, leucine is 3 to 10% (w / w) of arginine, 3 to 10% (w / w) of phenylalanine, 3 to 10% (w / w) of lysine, 1 to 5% As shown in FIG.
본 발명의 당단백 분획물을 아미노산 조성을 조사한 결과, 아스파르트산은 8.88%, 트레오닌은 4.23%, 세린은 5.21%, 글루탐산은 21.18%, 프롤린은 6.04%, 글리신은 6.05%, 알라닌은 6.18%, 발린은 5.32%, 메티오닌은 1.63%, 이소루신은 3.64%, 류신은 8.63%, 티로신은 1.61%, 페닐알라닌은 4.55%, 리신은 5.63%, 히스티딘은 2.96%, 아르기닌은 8.27%의 중량비로 나왔다.(실시예 3-2 및 표 2 참조)
The glycoprotein fraction of the present invention was found to have an aspartic acid content of 8.88%, threonine content of 4.23%, serine content of 5.21%, glutamic acid content of 21.18%, proline content of 6.04%, glycine content of 6.05%, alanine content of 6.18%, valine content of 5.32% , Methionine was 1.63%, isoleucine was 3.64%, leucine was 8.63%, tyrosine was 1.61%, phenylalanine was 4.55%, lysine was 5.63%, histidine was 2.96% and arginine was 8.27% -2 and Table 2)
본 발명의 당단백 분획물은 전체 당 대비 퓨코오스는 0.1 내지 3%(w/w), 람노오스는 0.1 내지 3%(w/w), 아라비노스는 20 내지 40%(w/w), 갈락토오스는 3 내지 10%(w/w), 글루코오스는 10 내지 20%(w/w), 만노오스는 1 내지 10%(w/w), 자일로스는 20 내지 50%(w/w), N-아세틸글루코사민(GlcNAc)은 0.5 내지 5%(w/w)의 비율로 포함되는 것을 특징으로 한다.
The glycoprotein fraction of the present invention may contain 0.1 to 3% (w / w) of fructose, 0.1 to 3% (w / w) of rhamnose, 20 to 40% (w / w) of arabinose, (W / w) of glucose, 10 to 20% (w / w) of glucose, 1 to 10% (w / w) of mannose, 20 to 50% Glucosamine (GlcNAc) is included in a ratio of 0.5 to 5% (w / w).
상기 당단백 분획물의 당 구성을 조사한 결과, 퓨코오스는 0.40%, 람노오스는 0.26%, 아라비노스는 32.39%, 갈락토오스는 5.89%, 글루코오스는 14.83%, 만노오스는 3.85%, 자일로스는 40.68%, N-아세틸글루코사민(GlcNAc)은 1.72%의 중량비로 나왔다. (실시예 3-3 및 표 3 참조)
The glycoprotein fractions were found to contain 0.40% of fucose, 0.26% of rhamnose, 32.39% of arabinose, 5.89% of galactose, 14.83% of glucose, 3.85% of mannose, 40.68% of xylose, -Acetylglucosamine (GlcNAc) was present in a weight ratio of 1.72%. (See Examples 3-3 and Table 3)
또한 본 발명의 당단백 분획물은 면역 기능 증진 활성이 있는 것을 특징으로 한다.
The glycoprotein fraction of the present invention is also characterized in that it has an immunomodulating activity.
면역기능이란 생물체 내에서 병원체와 종양 세포 등을 탐지한 다음 죽임으로써 질병으로부터 생명체를 보호하는 기능을 말한다.Immune function refers to the ability to protect living organisms from diseases by detecting pathogens and tumor cells in the organism and then killing them.
면역기능은 내재면역(Innate immune)과 적응면역(adaptive immune) 기능이 있으며, 내재면역은 미생물들과 독소들이 체내로 진입하는 데 성공하였을 때 곧바로 작용하는 면역 기능으로 패턴 인식 수용체가 미생물들 사이에 널리 보존되어 있는 부분을 인지하여 외부의 미생물을 감지하는 방식으로 작용하거나, 손상된 세포들이 방출하는 경고성 신호를 같은 종류의 수용체가 이를 인지하여 미생물의 존재 사실을 감지하는 방식으로 작용한다.The immune function is innate immune and adaptive immune function. Immunity is a function of immune function that acts immediately when microorganisms and toxins enter the body, It acts in such a way that it perceives the widely preserved parts and acts in a way to sense external microorganisms, or it senses the presence of microorganisms by recognizing the warning signals emitted by damaged cells by the same kind of receptors.
적응면역은 항원의 표식을 기억하는 기능을 의미하는 면역기억(immunological memory)을 비롯한 보다 강력한 면역 반응을 말하며 항원에 특이적이며 항원제시(antigen presentation) 과정을 통한 비자기항원의 인지를 필요로 한다.Adaptive immunity refers to a stronger immune response, including immunological memory, which refers to the function of memorizing an antigen, and is specific for an antigen and requires the recognition of non-magnetic antigens through an antigen presentation process .
본 발명의 당단백 분획물은 대식세포의 NO, TNF-α, IL-6의 생성을 촉진시켜 내재면역(Innate immune)과 적응면역(adaptive immune)을 모두 증진시키는 효능이 있다(실시예 4-2 및 4-3 참조).The glycoprotein fraction of the present invention has an effect of promoting the production of macrophages NO, TNF-a and IL-6, thereby enhancing both innate and adaptive immune (Examples 4-2 and 4- 4-3).
또한 본 발명의 당단백 분획물은 세포독성이 없어 식품 또는 의약품에 활용이 용이하다.Furthermore, the glycoprotein fraction of the present invention has no cytotoxicity and is thus easily applicable to foods or pharmaceuticals.
이와 같은 본 발명의 효과는 본 발명의 일실시예에 잘 나타나 있다.The effects of the present invention are well illustrated in an embodiment of the present invention.
본 발명의 일실시예에서는 본 발명의 당단백 분획물을 대식세포에 처리하여 세포 생존율을 측정하는 방법으로 세포독성이 있는지 시험하였다. 그 결과 본 발명의 당단백 분획물은 세포독성이 없는 것을 확인하였다(실시예 4-1 참조).In one embodiment of the present invention, the glycoprotein fraction of the present invention was treated with macrophages to determine cell viability, and then tested for cytotoxicity. As a result, it was confirmed that the glycoprotein fraction of the present invention was not cytotoxic (see Example 4-1).
본 발명의 다른 일실시예에서는 본 발명의 당단백 분획물을 대식세포에 처리하여 산화질소(NO) 생성에 미치는 영향을 측정하였다. 그 결과 본 발명의 당단백 분획물을 처리하는 경우 대식세포의 산화질소(NO) 생성량이 증가하는 것을 확인하였다. 상기 NO 생성량은 면역활성 증진 의약품인 PSK(Krestin) 보다도 훨씬 높은 수치를 보였다(실시예 4-2 및 도 4 참조).In another embodiment of the present invention, the glycoprotein fraction of the present invention was treated with macrophages to determine the effect on nitric oxide (NO) production. As a result, it was confirmed that when the glycoprotein fraction of the present invention was treated, the amount of nitric oxide (NO) produced in macrophages increased. The NO production amount was much higher than that of PSK (Krestin), which is an immunosuppressive drug (see Example 4-2 and Fig. 4).
본 발명의 다른 일실시예에서는 본 발명의 당단백 분획물을 대식세포에 처리하여 면역관련 cytokine의 생성에 미치는 영향을 실험하였다. 그 결과 본 발명의 당단백 분획물을 처리하는 경우 TNF-α,IL-6 등의 Cytokine의 생성량이 매우 증가하는 것을 확인하였다. 상기 싸이토킨 생성량 또한 PSK 보다 월등히 높았다(실시예 4-3 및 도 5, 도 6 참조)In another embodiment of the present invention, the glycoprotein fraction of the present invention was treated with macrophages to study the effect on the production of immune-related cytokines. As a result, it was confirmed that when the glycoprotein fraction of the present invention was treated, the amount of cytokines such as TNF-α and IL-6 was greatly increased. The amount of cytokine produced was much higher than that of PSK (see Example 4-3 and Figures 5 and 6)
IL-6는 B세포 자극인자2(BSF2) 또는 인터페론 ß2라고도 호칭된 사이토카인이다. IL-6는 B 림프구계 세포의 활성화에 관여하는 분화인자로서 발견되고(Hirano, T. et al., Nature, 324, 73-76, 1986), 그 후, 각종 세포의 기능에 영향을 미치는 다기능 사이토카인인 것이 명확해졌다(Akira, S. et al., Adv. in Immunology, 54, 1-78, 1993). 또한 IL-6는 T 림프구계 세포의 성숙화를 유도하는 것이 보고되어 있으며 (Lotz, M. et al., J. Exp. Med. 167, 1253-1258, 1988), T cell이나 thymocytes에 co-stimulator로 작용을 하여 항암효과를 나타낸다고 알려져 있다(Kuby J. In 'Immunology' 2nd ed. 1994, Chap. 15. W.H. Freemn company. USA.).IL-6 is a cytokine, also called B cell stimulating factor 2 (BSF2) or interferon beta2. IL-6 is found as a differentiation factor involved in the activation of B lymphocytic cells (Hirano, T. et al., Nature, 324, 73-76, 1986) Cytokine (Akira, S. et al., Adv. In Immunology, 54, 1-78, 1993). In addition, IL-6 has been reported to induce the maturation of T lymphocyte cells (Lotz, M. et al., J. Exp. Med. 167, 1253-1258, 1988) (Kuby J. In 'Immunology' 2nd ed., 1994,
TNF-α는 특정 암세포에 대한 세포독성과 항 바이러스성 작용이 있고, 급성 및 만성 염증질환에서 일어나는 여러 가지 생체반응에도 중요한 역할을 한다고 알려져 있다 (Kuby J. In 'Immunology' 2nd ed. 1994, Chap. 15. W.H. Freemn company. USA.)).TNF-α has cytotoxic and antiviral effects on specific cancer cells and is known to play an important role in various biological reactions occurring in acute and chronic inflammatory diseases (Kuby J. In 'Immunology' 2nd ed. 1994,
이로서 본 발명의 당단백 분획물의 면역 기능 증진 활성이 매우 뛰어난 것을 확인하였다.
Thus, it was confirmed that the glycoprotein fraction of the present invention has excellent immunosuppressive activity.
따라서 본 발명은 본 발명의 당단백 분획물을 유효성분으로 포함하는 면역기능 증진용 식품 조성물을 제공한다.
Accordingly, the present invention provides a food composition for enhancing immune function comprising the glycoprotein fraction of the present invention as an active ingredient.
본 발명의 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. 상기 유형의 식품 조성물은 당 업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다.
The food composition of the present invention includes all forms such as functional food, nutritional supplement, health food and food additives. Food compositions of this type may be prepared in a variety of forms according to conventional methods known in the art.
예를 들면, 건강식품으로는 본 발명의 밀기울 유래 당단백 분획물 자체를 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 하거나, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 본 발명의 밀기울유래 당단백 분획물과 면역기능 증진의 효과가 있다고 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.
For example, as a health food, the bran-derived glycoprotein fraction of the present invention itself can be prepared in the form of tea, juice, and drink for drinking, granulated, encapsulated, and powdered. In addition, the bran-derived glycoprotein fraction of the present invention may be mixed with a known substance or active ingredient known to have an effect of enhancing the immune function together with the bran-derived glycoprotein fraction in the form of a composition.
또한, 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 밀기울유래 당단백 분획물을 첨가하여 제조할 수 있다.Functional foods also include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruits, bottled, jam, maalmalade, etc.), fish, meat and processed foods such as ham, Etc.), breads and noodles (eg udon, buckwheat noodles, ramen noodles, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, , Retort food, frozen food, various seasonings (for example, soybean paste, soy sauce, sauce, etc.) by adding the bran-derived glycoprotein fraction of the present invention.
본 발명의 식품 조성물 중 상기 본 발명의 밀기울 유래 당단백 분획물의 바람직한 함유량으로는 이에 한정되지 않지만 바람직하게는 최종적으로 제조된 식품 중 0.01 내지 50 중량%이다.
The preferred content of the bran-derived glycoprotein fraction of the present invention in the food composition of the present invention is not particularly limited, but is preferably 0.01 to 50% by weight in the finally prepared food.
또한, 본 발명의 밀기울유래 당단백 분획물을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.In order to use the bran-derived glycoprotein fraction of the present invention in the form of a food additive, it may be prepared in the form of a powder or a concentrated liquid.
본 발명의 조성물은 면역기능 저하로 기인되는 질환의 예방 및 개선에 효과가 있으며, 상기 면역기능 저하로 기인되는 질환으로는 감기 등의 감염성 질환 및 염증성 질환, 아토피 등의 알러지 질환, 만성피로, 및 암으로 이루어진 군으로부터 선택되는 어느 하나인 것이 바람직하나 이에 한정되는 것은 아니며, 당업자에 알려진 면역기능 저하로 기인되는 질환은 모두 본 발명에 포함된다.
The composition of the present invention is effective for the prevention and improvement of diseases caused by immune dysfunction. Examples of the diseases caused by the immune dysfunction include infectious diseases such as cold, inflammatory diseases, allergic diseases such as atopy, chronic fatigue, Cancer, but the present invention is not limited thereto, and all diseases caused by immune function deterioration known to those skilled in the art are included in the present invention.
또한 본 발명은 본 발명의 당단백 분획물을 유효성분으로 포함하는 감기, 아토피, 만성피로 및 암으로 이루어진 군에서 선택되는 어느 하나 이상의 면역 질환 예방 또는 치료용 약학적 조성물을 제공한다.
The present invention also provides a pharmaceutical composition for preventing or treating immunological diseases selected from the group consisting of cold, atopic, chronic fatigue and cancer, which comprises the glycoprotein fraction of the present invention as an active ingredient.
본 발명에 따른 약학적 조성물은 본 발명의 당단백 분획물 또는 이들의 약학적으로 허용가능한 염을 단독으로 함유하거나 또는 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 추가로 함유할 수 있다.
The pharmaceutical composition according to the present invention may contain the glycoprotein fraction of the present invention or a pharmaceutically acceptable salt thereof, alone or in combination with one or more pharmaceutically acceptable carriers, excipients or diluents.
약학적으로 허용되는 담체로는 예컨대, 경구 투여용 담체 또는 비경구 투여용 담체를 추가로 포함할 수 있다. 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 또한, 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등을 포함할 수 있으며, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. 그 밖의 약학적으로 허용되는 담체로는 다음의 문헌에 기재되어 있는 것을 참고로 할 수 있다(Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
The pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may contain water, a suitable oil, a saline solution, an aqueous glucose and a glycol, and may further contain a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).
본 발명의 면역기능 증진용 약학적 조성물은 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들면, 경구 또는 비경구적으로 투여할 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다. 바람직하게는 본 발명의 약학적 조성물은 경피 투여될 수 있다. 상기에서 경피 투여'란 본 발명의 약학적 조성물을 세포 또는 피부에 투여하여 본 발명 조성물에 함유된 활성성분이 피부 내로 전달되도록 하는 것을 말하다. 예컨대, 본 발명의 약학적 조성물을 주사형 제형으로 제조하여 이를 30 게이지의 가는 주사 바늘로 피부를 가볍게 단자(prick)하는 방법, 또는 피부에 직접적으로 도포하는 방법으로 투여될 수 있다.
The pharmaceutical composition for enhancing immune function of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal administration Lt; / RTI > Preferably, the pharmaceutical composition of the present invention can be transdermally administered. Refers to administering the pharmaceutical composition of the present invention to cells or skin to allow the active ingredient contained in the composition of the present invention to be delivered into the skin. For example, the pharmaceutical composition of the present invention can be administered in a scanning type formulation, pricking the skin lightly with a 30 gauge needle, or directly applying it to the skin.
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다.
The pharmaceutical composition of the present invention can be formulated into oral preparations or parenteral administration preparations according to the administration route as described above.
경구 투여용 제제의 경우에 본 발명의 조성물은 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 등으로 당업계에 공지된 방법을 이용하여 제형화될 수 있다. 예를 들어, 경구용 제제는 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. 적합한 부형제의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈, 메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 본 발명의 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.
In the case of a preparation for oral administration, the composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a liquid, a gel, a syrup, a slurry, . For example, an oral preparation can be obtained by combining the active ingredient with a solid excipient, then milling it, adding suitable auxiliaries, and then processing the mixture into a granular mixture. Examples of suitable excipients include, but are not limited to, sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition of the present invention may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.
비경구 투여용 제제의 경우에는 주사제, 크림제, 로션제, 외용연고제, 오일제, 보습제, 겔제, 에어로졸 및 비강 흡입제의 형태로 당업계에 공지된 방법으로 제형화할 수 있다. 이들 제형은 모든 제약 화학에 일반적으로 공지된 처방서인 문헌(Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour)에 기재되어 있다.In the case of a preparation for parenteral administration, it can be formulated by a method known in the art in the form of injection, cream, lotion, external ointment, oil, moisturizer, gel, aerosol and nasal aspirate. These formulations are described in Remington's Pharmaceutical Science, 15th edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a commonly known formulary for all pharmaceutical chemistries.
본 발명의 당단백 분획물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 본 발명의 약학적 조성물은 질환의 정도에 따라 유효성분의 함량을 달리할 수 있다. 바람직하게는 본 발명의 당단백 분획물의 바람직한 전체 용량은 1일당 환자 체중 1 당 약 0.01ug 내지 1,000 mg, 가장 바람직하게는 0.1 ug 내지 100 mg일 수 있다. 그러나 상기 본 발명의 당단백 분획물의 용량은 약학적 조성물의 투여 경로 및 치료 횟수뿐만 아니라 환자의 연령, 체중, 건강 상태, 성별, 질환의 중증도, 식이 및 배설율 등 다양한 요인들을 고려하여 환자에 대한 유효 투여량이 결정되는 것이므로, 이러한 점을 고려할 때 당 분야의 통상적인 지식을 가진 자라면 상기 본 발명의 당단백 분획물을 면역기능 증진제로서의 특정한 용도에 따른 적절한 유효 투여량을 결정할 수 있을 것이다. 본 발명에 따른 약학적 조성물은 본 발명의 효과를 보이는 한 그 제형, 투여 경로 및 투여 방법에 특별히 제한되지 아니한다. The total effective amount of the glycoprotein fraction of the present invention may be administered to a patient in a single dose and may be administered by a fractionated treatment protocol administered over a prolonged period of time in multiple doses. In the pharmaceutical composition of the present invention, the content of the active ingredient may be varied depending on the degree of the disease. Preferably, the preferred total dose of the glycoprotein fraction of the present invention can be from about 0.01 μg to 1,000 mg per patient body weight per day, most preferably from 0.1 μg to 100 mg per patient body weight per day. However, the dose of the glycoprotein fraction of the present invention is effective for the patient in consideration of various factors such as the route of administration and the number of treatments of the pharmaceutical composition as well as the age, body weight, health condition, sex, severity of disease, It will be understood by those skilled in the art that the glycoprotein fraction of the present invention can be used to determine an appropriate effective dose according to the specific use as an immunological enhancer. The pharmaceutical composition according to the present invention is not particularly limited to its formulation, administration route and administration method as long as the effect of the present invention is exhibited.
본 발명의 조성물은 면역기능 저하로 기인되는 질환의 예방 및 치료에 효과가 있으며, 상기 면역기능 저하로 기인되는 질환으로는 감기 등의 감염성 질환 및 염증성 질환, 아토피 등의 알러지 질환, 만성피로, 및 암으로 이루어진 군으로부터 선택되는 어느 하나인 것이 바람직하나 이에 한정되는 것은 아니며, 당업자에 알려진 면역기능 저하로 기인되는 질환은 모두 본 발명에 포함된다.
The composition of the present invention is effective for the prevention and treatment of diseases caused by immune dysfunction. Examples of diseases caused by the immune dysfunction include infectious diseases such as cold, inflammatory diseases, allergic diseases such as atopy, chronic fatigue, Cancer, but the present invention is not limited thereto, and all diseases caused by immune function deterioration known to those skilled in the art are included in the present invention.
본 발명은 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 밀기울유래 당단백 분획물의 제조방법을 제공한다.
(A) degreasing the wheat bran; (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater.
상기 본 발명의 당단백 분획물을 제조하는 방법을 단계별로 설명하면 다음과 같다.
The method for producing the glycoprotein fraction of the present invention will be described step by step.
(a) 밀기울을 탈지하는 단계;(a) degreasing the bran;
(a) 단계에서는 본 발명의 분획물을 제조하기 위한 원료인 밀기울을 탈지(脫脂)한다.In step (a), bran, which is a raw material for producing the fractions of the present invention, is degreased.
탈지는 지방을 제거하는 것을 말하며, 밀기울은 탈지를 통하여 산패를 막고, 가공성을 높일 수 있다.Degreasing refers to removing fat, while bran can prevent rancidity through degreasing and improve processability.
탈지하는 방법은 특별히 제한되지 아니하며, 예를 들어 초임계 추출 또는 n-hexane을 이용하는 방법일 수 있다. 바람직하게는 n-hexane에 침지하는 방법일 수 있다.
The method of degreasing is not particularly limited, and may be, for example, a method using supercritical extraction or n-hexane. Preferably, it may be a method of immersing in n-hexane.
(b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계;(b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0;
(b) 단계에서는 상기 탈지된 밀기울을 완충용액에 혼합한다.In step (b), the degreased bran is mixed with a buffer solution.
상기 완충용액은 일반적인 완충용액이면 어떠한 것도 가능하며, 바람직하게는 Tris-HCl 용액 일 수 있다. 완충용액의 pH는 바람직하게는 pH 7.0 내지 pH 8.0일 수 있다.The buffer solution may be any ordinary buffer solution, preferably a Tris-HCl solution. The pH of the buffer solution may preferably be from pH 7.0 to pH 8.0.
혼합용액은 밀기울의 유효성분이 추출될 수 있도록 교반할 수 있으며, 교반 시간 및 온도는 특별히 제한이 없으나, 바람직하게는 10시간 내지 24시간동안 교반할 수 있으며, 교반시 혼합용액의 온도는 4℃ 내지 24℃ 일 수 있다.
The mixing solution can be stirred so that the effective component of the wheat bran can be extracted. The mixing time and temperature are not particularly limited, but it is preferable that the mixing solution is stirred for 10 hours to 24 hours. Lt; / RTI >
(c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계;(c) centrifuging the mixture to obtain a supernatant;
(c) 단계에서는 상기 혼합물을 원심분리하여 상등액을 수득한다.In step (c), the mixture is centrifuged to obtain a supernatant.
본 단계의 원심분리는 상등액과 침전물을 분리할 수 있는 정도이면 회전수 및 온도에 제한이 없다.Centrifugation in this step is not limited to the number of revolutions and temperature as long as it can separate the supernatant and the precipitate.
또한 원심 분리 후 여과지로 상등액을 여과하는 과정을 추가할 수 있다.
Also, a process of filtering the supernatant liquid by centrifugation and then filtering paper can be added.
(d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계;(d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000);
(d) 단계에서는 상기 수득한 상등액을 투석 반투막으로 투석한다.In step (d), the obtained supernatant is dialyzed against a dialysis semipermeable membrane.
투석에 사용되는 용액은 특별히 제한되지 아니하나 바람직하게는 Tris-HCl 완충용액 일 수 있으며, 용액의 pH는 바람직하게는 pH 7.0 내지 pH 8.0일 수 있다. 투석 시, 분자량 cuf-off는 6000 내지 8000이 바람직하다.The solution used for dialysis is not particularly limited, but may preferably be a Tris-HCl buffer solution, and the pH of the solution may preferably be from pH 7.0 to pH 8.0. When dialyzing, the molecular weight cuf-off is preferably 6000 to 8000.
투석 시 온도는 상기 (b)단계의 혼합용액과 동일한 온도에서 투석할 수 있으며, 투석 시간은 상기 (c) 단계의 상등액의 부피에 따라 다를 수 있으며, 바람직하게는 24시간 내지 48시간 일 수 있다.
The dialysis temperature may be dialyzed at the same temperature as the mixed solution of step (b), and the dialysis time may vary depending on the volume of the supernatant of step (c), preferably 24 to 48 hours .
(e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계;(e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant;
(e) 단계에서는 투석이 완료된 상등액으로부터 단백질을 분리한다.In step (e), the protein is separated from the dialyzed supernatant.
당단백질을 침전시키기 위하여, 단백질을 침전시키는 염을 사용하는데, 상기 침전 염은 단백질 함유 수용액의 이온강도(ionic strength)를 증가시켜 단백질 침전(salting-out)시킬 수 있는 염은 제한없이 사용될 수 있으며, 바람직하게는 양이온이 NH4 +, K+ 및 Na+로 구성된 군에서 선택된 어느 하나이고, 음이온이 씨트레이트(citrate), PO4 3 -, SO4 2 -, CH3COO-, Cl-, OH- 및 NO3 -로 구성된 군에서 선택된 어느 하나인 상기 양이온과 음이온이 결합된 화합물이고, 더욱 바람직하게는 NH4OH, (NH4)2SO4, KNO3, NaNO3, NaSO4 등이고, 가장 바람직하게는 황산 암모늄 일 수 있다. 단백질 침전에 사용되는 염의 농도는 분리하고자 하는 단백질의 종류에 따라 달라질 수 있으며, 바람직하게는 70%(w/v) 내지 80%(w/v)의 농도일 수 있다. 황산 암모늄의 첨가에 따라 당단백질이 침전되며, 반응 시간은 4시간 내지 10시간 일 수 있다. 침전된 당단백질의 분리는 통상의 침전물 분리 방법에 의할 수 있으며, 바람직하게는 원심분리에 의하여 분리 할 수 있다.
In order to precipitate the glycoprotein, a salt for precipitating a protein is used. The precipitated salt can be used without limitation, for example, by salting-out by increasing the ionic strength of a protein-containing aqueous solution , Preferably the cation is any one selected from the group consisting of NH 4 + , K + and Na + , and the anion is selected from the group consisting of citrate, PO 4 3 - , SO 4 2 - , CH 3 COO - , Cl - or the like, and any one of the cation and the anion binding compound selected from the group, more preferably NH 4 OH, consisting of a (NH 4) 2 SO 4, KNO 3, NaNO 3, NaSO 4, - OH - , and NO 3 And most preferably ammonium sulfate. The concentration of the salt used for protein precipitation may vary depending on the kind of the protein to be separated, and may be preferably 70% (w / v) to 80% (w / v). Upon addition of ammonium sulfate, the glycoprotein precipitates, and the reaction time can be from 4 hours to 10 hours. The precipitated glycoprotein can be separated by a conventional precipitate separation method, preferably by centrifugation.
(f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계;(f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000);
(f) 단계에서는 상기 회수된 침전물을 완충용액에 용해한 후 투석하여 염을 제거한다. 투석에 사용되는 용액은 특별히 제한되지 아니하나 바람직하게는 Tris-HCl 완충용액 일 수 있으며, 용액의 pH는 바람직하게는 pH 7.0 내지 pH 8.0일 수 있다.In step (f), the recovered precipitate is dissolved in a buffer solution, followed by dialysis to remove the salt. The solution used for dialysis is not particularly limited, but may preferably be a Tris-HCl buffer solution, and the pH of the solution may preferably be from pH 7.0 to pH 8.0.
투석 시 온도는 회수된 유효성분이 비가역적인 변형을 일으키지 않는 한 특별히 제한되지 아니하며, 투석 시간은 상기 (c) 단계의 상등액의 부피에 따라 다를 수 있으며, 바람직하게는 24시간 내지 72시간 일 수 있다.
The dialysis temperature is not particularly limited as long as the recovered effective ingredient does not cause irreversible deformation, and the dialysis time may vary depending on the volume of the supernatant of step (c), preferably 24 to 72 hours.
(g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계;(g) obtaining a glycoprotein fraction from the dialyzed precipitated seafood;
(g) 단계에서는 상기 침전 용해물에서 당단백 분획물을 수득한다. 당단백 분획물 수득 과정은 해당기술분야에서 통상적으로 쓰이는 건조 방법일 수 있고, 바람직하게는 분무건조, 열풍건조 또는 동결건조 방법일 수 있으며, 가장 바람직하게는 동결건조 방법에 의할 수 있다.
In step (g), the glycoprotein fraction is obtained from the above-mentioned precipitated seafood. The process for obtaining glycoprotein fractions may be a drying method commonly used in the technical field, and may be preferably spray drying, hot air drying or freeze drying, and most preferably, freeze-drying.
따라서 본 발명은 밀기울 유래 당단백 분획 및 이의 제조방법에 관한 것으로, 좀 더 상세하게는 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70 kDa인 것을 특징으로 하는 밀기울 유래 당단백 분획물, 이의 제조 방법 및 이를 유효성분으로 포함하는 면역기능 증진용 약학적, 식품 조성물을 제공한다. 본 발명의 당단백 분획물은 대식세포의 활성을 촉진하는 효능이 매우 뛰어나며 세포독성이 전혀 없어 새로운 기능성 식품 및 면역기능 증강용 의약품 제조에 효과적이다.
Accordingly, the present invention relates to a bran-derived glycoprotein fraction and a method for producing the same. More particularly, the present invention relates to a bran- (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) a step of obtaining a glycoprotein fraction from the dialyzed precipitated seaweed, and having a molecular weight of 16 to 70 kDa, a method for producing the same, and a method for producing the same A pharmaceutical composition for promoting immune function. The glycoprotein fraction of the present invention is highly effective in promoting the activity of macrophages and has no cytotoxicity, so that it is effective in the production of new functional foods and immunomodulating drugs.
도 1은 가시오가피, 달맞이, 들깨박 등 총 10종의 다양한 식물체(의 건조 분말)의 추출물의 macrophage NO 생성에 미치는 영향을 살펴본 결과이다. (con: 음성대조군, LPS: 리포폴리사카라이드, Nitric Oxide(M): 생성된 일산화질소의 농도)
도 2는 본 발명의 방법에 의해 제조된 밀기울 유래 당단백 분획물에 포함되는 당단백질의 분자량을 측정한 결과이다(Marker: 사이즈 마커, wheat bran: 본 발명 당단백 분획물의 전기영동 결과).
도 3은 본 발명 밀기울 유래 당단백 분획물의 세포독성을 실험한 결과 그래프이다(con: 음성대조군, LPS: 리포폴리사카라이드, 25, 50, 100 : 밀기울 유래 당단백 분획물 처리 농도(ug/mL), Cell viability(%): 세포 생존율(%)).
도 4는 밀기울 유래 당단백 분획물의 대식세포의 NO생성에 미치는 영향을 측정한 실험 결과 그래프이다(con: 음성대조군, LPS: 리포폴리사카라이드, 25, 50, 100 : 밀기울 유래 당단백 분획물 또는 PSK 처리 농도(ug/mL), Nitro oxide(uM): 산화질소 농도(uM)).
도 5는 밀기울 유래 당단백 분획물의 대식세포의 TNF-에 미치는 영향을 측정한 실험 결과 그래프이다(con: 음성대조군, LPS: 리포폴리사카라이드, 25, 50, 100 : 밀기울 유래 당단백 분획물 또는 PSK 처리 농도(ug/mL), TNF-alpha(pg/ml): TNF-alpha농도(pg/ml)).
도 6은 밀기울 유래 당단백 분획물의 대식세포의 IL-6에 미치는 영향을 측정한 실험 결과 그래프이다. (blank: 음성대조군, LPS: 리포폴리사카라이드, 25, 50, 100 : 밀기울 유래 당단백 분획물 또는 PSK 처리 농도(ug/mL), IL-6(pg/ml): IL-6농도(pg/ml)).FIG. 1 shows the results of examining the effect of extracts of 10 kinds of various plants (dried powder), such as caustic potato, evening primrose, and perilla seed, on macrophage NO production. (con: negative control, LPS: lipopolysaccharide, Nitric Oxide (M): concentration of produced nitric oxide)
FIG. 2 shows the result of measuring the molecular weight of the glycoprotein contained in the bran-derived glycoprotein fraction produced by the method of the present invention (Marker: size marker, wheat bran: electrophoresis result of glycoprotein of the present invention).
FIG. 3 is a graph showing cytotoxicity of the glycoprotein-derived bran protein fraction of the present invention (con: negative control, LPS: lipopolysaccharide, 25, 50, 100: bran- viability (%): cell viability (%)).
FIG. 4 is a graph showing the results of measurement of the effect of bran-derived glycoprotein fractions on the NO production of macrophages (con: negative control, LPS: lipopolysaccharide, 25, 50, 100: glycoprotein- (ug / mL), nitric oxide (uM): nitric oxide concentration (uM).
FIG. 5 is a graph showing the effect of the bran-derived glycoprotein fraction on the TNF- of macrophages (con: negative control, LPS: lipopolysaccharide, 25, 50, 100: glycoprotein fraction derived from bran or PSK- (pg / ml), TNF-alpha (pg / ml): TNF-alpha concentration (pg / ml)).
FIG. 6 is a graph showing the results of an experiment to measure the effect of bran-derived glycoprotein fractions on IL-6 of macrophages. (pg / ml): IL-6 concentration (pg / ml), and the concentration of IL-6 (pg / ml) )).
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail with reference to examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.
However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
<실시예 1> ≪ Example 1 >
면역 증진 활성이 있는 식물 소재 선별Selection of Plant Materials with Immune Enhancement Activity
가시오가피, 산사, 산약, 어성초 등의 생약재는 경동시장에서 구입하였고, 부산물 중 들깨박, 참깨박은 국산 들깨와 참깨를 시중에서 구입해서 볶고 기름을 짜고 남은 박이며, 밀기울은 전남 광주지역에서 수집하였다. 상기 확보된 다양한 식물체 시료를 n-Hexane으로 3시간 처리하여 탈지한 후 건조, 분쇄하여 실험에 사용하였으며, 탈지시료 100 g을 20 mM Tris-HCl (pH 8.0) 2000 mL에 용해하고, 4℃에서 16시간 교반 후 원심분리하였다. 상등액을 여과지 (Whatman, No. 4)를 이용하여 불순물을 제거하고, 여과 용액은 투석 반투막 (MWCO 6000 내지 8000)을 이용하여 4℃의 20 mM Tris-HCl (pH 7.4) 용액에서 48시간 투석한 후 동결건조하여 -18℃ 이하에 보관하면서 실험에 사용하였다. Korean herbs and sesame seeds were bought from the market, fried and oiled, and the bran was collected in Gwangju area, Jeonnam province. Korean herbs and sesame seeds were purchased from Kyungdong market. The obtained various plant samples were treated with n-hexane for 3 hours, followed by degreasing, drying and pulverization. 100 g of the degreasing sample was dissolved in 2000 mL of 20 mM Tris-HCl (pH 8.0) After stirring for 16 hours, the mixture was centrifuged. The supernatant was filtered using a filter paper (Whatman, No. 4), and the filtrate was dialyzed in a 20 mM Tris-HCl (pH 7.4) solution at 4 ° C for 48 hours using a dialysis semipermeable membrane (MWCO 6000 to 8000) Followed by lyophilization and storage at -18 ° C or below.
RAW 264.7 macrophage 세포를 2.0x105 cells/well의 농도로 96 well plate에 분주하고 24시간 후 시료를 2.0x105 cells/well의 농도로 처리하고 37℃, 5% CO2의 조건에서 24시간 배양하였다. 이때 양성 대조군으로는 LPS 1 ug/mL을 단독 처리하였고, 음성 대조군으로는 아무것도 처리 안한 RAW 264.7 macrophage 세포로 하여 동일 조건에서 배양하였다. Cell culture medium 100 uL와 Griess reagent (sigma-aldrich co.) 100uL 동량을 혼합하여 상온에서 15분 반응 후 ELISA reader를 이용하여 540 nm에서 흡광도를 측정하였다. Sodium nitrite로 표준곡선을 작성하여 NO 함량을 정량하였다.
RAW 264.7 macrophage cells were the cultured for 24 hours under the conditions of 2.0x10 5 cells / well with the concentration of the dispensed in 96 well plate and process the samples after 24 hours at a concentration of 2.0x10 5 cells / well and 37 ℃, 5% CO 2 . At this time, 1 ug / mL of LPS was treated as a positive control, and RAW 264.7 macrophage cells as a negative control were cultured under the same conditions. 100 uL of cell culture medium and 100 uL of Griess reagent (Sigma-aldrich co.) Were mixed and reacted at room temperature for 15 minutes. Absorbance was measured at 540 nm using an ELISA reader. The NO content was quantified by preparing a standard curve with sodium nitrite.
10종의 식물 추출물의 macrophage NO 생성에 미치는 영향을 살펴본 결과 [도 1]에서 보는 바와 같이, LPS로 활성화된 세포의 경우 NO 함량이 31.64±1.38 uM수준을 나타내었으며, 특히 밀기울 추출물의 NO 생성 활성이 높은 것으로 나타났다.
As shown in FIG. 1, the NO content of the LPS-activated cells was 31.64 ± 1.38 μM, and the NO production activity of bran extract Respectively.
<실시예 2> ≪ Example 2 >
밀기울 유래 당단백 분획물 제조Production of glycoprotein fractions derived from bran
전남 광주에서 수집한 밀(Triticum aestivum L.)에서 분리한 밀기울을 n-hexane에 3시간 동안 침지하여 탈지처리 한 후, 건조 분쇄 하였다.The wheat bran extracted from wheat ( Triticum aestivum L. ) collected in Gwangju, South Jeolla Province was dipped in n-hexane for 3 hours and then dried and pulverized.
탈지한 밀기울 100g을 20mM Tris-HCl(pH 8.0)용액 2L에 용해하고, 4℃에서 16시간동안 교반한 후 원심분리 하였다. 상등액을 여과지(Whatman, No4.)로 여과하고, 여과 용액을 투석 반투막(MWCO 6000~8000)으로 4℃의 20mM Tris-HCl(pH 7.4)용액으로 48시간 투석하여 밀기울 유래 당단백 추출물을 제조하였다.100 g of degreased bran was dissolved in 2 L of 20 mM Tris-HCl (pH 8.0) solution, stirred at 4 캜 for 16 hours, and centrifuged. The supernatant was filtered through a filter paper (Whatman, No. 4), and the filtrate was dialyzed against a dialysis semipermeable membrane (MWCO 6000-8000) for 48 hours with a 20 mM Tris-HCl (pH 7.4) solution at 4 ° C to prepare a bran-derived glycoprotein extract.
밀기울 유래 당단백 추출물에서 당단백 분획물을 분리하기 위하여, 상기 당단백 추출물에 황산암모늄(ammonium sulfate)를 80% 농도가 되도록 첨가하여 6시간 정치 후 형성된 침전물을 원심분리하여 회수하였다. 회수한 침전물을 20mM Tris-HCl(pH 7.4)용액으로 용해 후 투석 반투막(MWCO 6000~8000)으로 4℃의 증류수로 48시간 투석한 후 동결건조 하였다.In order to separate the glycoprotein fractions from the bran extract-derived glycoprotein extracts, ammonium sulfate was added to the glycoprotein extract at a concentration of 80%, and after standing for 6 hours, the precipitate formed was centrifuged and recovered. The recovered precipitate was dissolved in 20 mM Tris-HCl (pH 7.4) solution, dialyzed semi-permeable membrane (MWCO 6000 ~ 8000) was dialyzed with distilled water at 4 ° C for 48 hours and lyophilized.
그 결과 약 11.06g의 당단백 분획물을 수득하였으며, 본 발명의 방법에 의한 밀기울 유래 당단백 분획물의 수득율은 약 11.1% 임을 확인하였다.
As a result, about 11.06 g of glycoprotein fractions were obtained, and the yield of bran-derived glycoprotein fractions by the method of the present invention was found to be about 11.1%.
<실시예 3> ≪ Example 3 >
밀기울 유래 당단백 분획물의 분자구조적 특성 규명Identification of Molecular Structural Properties of Glycoprotein Derived from Bran
<3-1> 단백질 함량 및 당함량 측정<3-1> Measurement of protein content and sugar content
제조된 당단백 분획물의 단백질 함량은 BCA방법으로 측정하였다. 25uL의 시료에 BCA working reagent(Cat No. 23227, Thermo, 독일) 200ul을 가하여 37℃에서 30분간 반응하여 발색시킨 후 spectrophotometer(Biotek, 미국)를 이용하여 540nm에서 흡광도를 측정하였다. 표준곡선은 Bovine serum albumin을 이용하여 작성한 후 단백질 함량을 계산하였다.The protein content of the prepared glycoprotein fractions was measured by the BCA method. After incubation at 37 ° C for 30 min, 200 uL of BCA working reagent (Cat No. 23227, Thermo, Germany) was added to 25 uL of sample, and the absorbance was measured at 540 nm using a spectrophotometer (Biotek, USA). Standard curves were prepared using Bovine serum albumin and protein content was calculated.
제조된 당단백 분획물의 당 함량은 시료를 증류수에 희석한 희석액 200ul와 5% 페놀 용액 200ul, 황산 1ml을 혼합하고, 실온에서 20분간 방치한 후, spectrophotometer(Biotek, USA)를 이용하여 490nm에서 흡광도를 측정하는 방법으로 측정하였다. 표준곡선은 glucose를 이용하여 작성한 후 당함량을 계산하였다.
The glucose content of the prepared glycoprotein fractions was measured by measuring the absorbance at 490 nm using a spectrophotometer (Biotek, USA) after mixing 200 μl of the diluted sample diluted in distilled water, 200 μl of 5% phenol solution and 1 ml of sulfuric acid, . The standard curve was prepared using glucose and the sugar content was calculated.
그 결과 [표 1]에서 보는 바와 같이, 본 발명 당단백 분획물의 단백질 함량은 22.93% 이며, 당함량은 4.7%인 것을 확인하였다. 또한 본 발명의 당단백 분획물은 단백질과 당의 비율이 4.9:1 인 것을 확인하였다.As a result, as shown in Table 1, the protein content of the glycoprotein of the present invention was 22.93% and the sugar content was 4.7%. The glycoprotein fraction of the present invention was confirmed to have a protein to sugar ratio of 4.9: 1.
<3-2> 아미노산 조성 분석<3-2> Amino acid composition analysis
본 발명의 당단백 분획물을 약 10mg 함유하는 시료를 가수분해 시험관에 넣고 6N HCl 약 15mL을 가한 후 봉관하여 110℃에서 24시간 가수분해 하였다. 가수분해 종료 후 봉관을 절단하여 이 여액을 50ml 플라스크에 넣고 증류수로 정용한 다음 여과하였으며, 여과한 여액을 정당히 희석하여 아미노산 자동분석기(Hitachi AAA L-8900, 일본)를 이용하여 아미노산 조성을 분석하였다.
A sample containing about 10 mg of the glycoprotein fraction of the present invention was placed in a hydrolysis test tube, and about 15 mL of 6N HCl was added, followed by hydrolysis at a temperature of 110 ° C for 24 hours. After completion of the hydrolysis, the capillary was cut, and the filtrate was added to a 50 ml flask, which was then filtered with distilled water. The filtrate was diluted well and analyzed for amino acid composition using an amino acid automatic analyzer (Hitachi AAA L-8900, Japan).
그 결과 본 발명의 밀기울 유래 당단백 분획물은 [표 2]와 같은 아미노산 구성을 가지고 있으며, glutamic acid가 21.18%로 가장 많이 함유되어 있으며, 그 다음으로 aspartic acid 및 leucine이 각각 8.88%, 8.63%를 차지하는 것으로 나타났다. As a result, the bran-derived glycoprotein fractions of the present invention had an amino acid composition as shown in Table 2, with glutamic acid being the largest as 21.18%, followed by aspartic acid and leucine as 8.88% and 8.63%, respectively Respectively.
<3-3> 당 조성 분석<3-3> Analysis of sugar composition
미량원심분리 튜브에 시료를 1mg 넣고 건조시킨 후 4N HCl을 가하고, 100에서 4℃시간 동안 반응시킨다. 상온에서 식힌 시료는 Speed-vac으로 건조시킨 후, 정제수를 이용하여 2회 수세 건조하였다. 50ul의 정제수로 시료를 녹인 후, CarboPac PA1(Dionex, 미국) 컬럼을 이용하여 이동상 A: 200nm NaOH와 B: DW로 80분에 걸쳐 HPAEC-PAD(Dionex, ICS-3000, 미국)로 분석하였다.
Add 1 mg of sample to a microcentrifuge tube, dry it, add 4N HCl, and allow to react at 100 ℃ for 4 hours. The samples cooled at room temperature were dried by speed-vac and washed twice with purified water. (Dionex, ICS-3000, USA) over a period of 80 minutes with mobile phase A: 200 nm NaOH and B: DW using a CarboPac PA1 (Dionex, USA) column after dissolving the sample in 50 ul of purified water.
그 결과 본 발명의 밀기울 유래 당단백 분획물은 [표 3]과 같은 당구성을 가지고 있으며, xylose가 40.68%로 가장 많은 함량을 차지하고 있으며, 그 다음으로는 arabinose가 32.39% 함유되어 있었다. As a result, the bran-derived glycoprotein fraction of the present invention had the sugar structure as shown in Table 3, and xylose occupied the largest amount of 40.68%, followed by arabinose 32.39%.
<3-4> 전기영동<3-4> Electrophoresis
제조한 당단백 분획물에 포함된 당단백질의 분자량을 측정하기 위하여 Laemmli의 방법에 따라 15% polyacrylamide gel을 제작 후 당단백 분획물을 주입하고 전기영동 완충용액(0.025M Tris-HCl, 0.192M glycine, 0.1% SDS, pH 8.3)으로 전기영동(SDS-PAGE)을 실시하였으며, 이를 Coomassie Blue로 염색한 후 탈색하였다. 분자량 표준물질은 15~250 kDa Marker(Dokdo)를 이용하였다.To measure the molecular weight of the glycoprotein contained in the prepared glycoprotein fractions, 15% polyacrylamide gel was prepared according to the method of Laemmli, and the glycoprotein fractions were injected. The glycoprotein fractions were then added to the gelatin buffer solution (0.025M Tris-HCl, 0.192M glycine, , pH 8.3) and electrophoresed (SDS-PAGE), stained with Coomassie Blue and discolored. The molecular weight standard was 15 ~ 250 kDa Marker (Dokdo).
그 결과 [도 2]에서 보는 바와 같이 분자량 16, 18, 21, 24, 26, 35, 44, 46, 55, 65, 70 kDa 정도의 분자량을 가진 당단백질이 포함된 것을 확인하였다.
As a result, it was confirmed that a glycoprotein having a molecular weight of about 16, 18, 21, 24, 26, 35, 44, 46, 55, 65 and 70 kDa was contained as shown in FIG.
<실시예 4> <Example 4>
밀기울 유래 당단백 분획물의 면역활성 증진 효능 실험Effectiveness of immunoglobulin activity of bran-derived glycoprotein fractions
<4-1> 세포독성 시험<4-1> Cytotoxicity test
RAW 264.7 대식세포(macrophage)를 2.0X105 cells/well의 농도로 96well plate에 분주하고, 24시간 후 시료를 12, 25, 50, 100ug/ml의 농도로 처리하고, 5% CO2의 조건에서 24시간 동안 배양하였다. 이때 대조군으로 lipopolysaccharide(LPS) 1ug/ml을 단독 처리하여 동일 조건에서 배양하였다.RAW 264.7 macrophages (macrophage) to 2.0X10 the frequency division, and after 24 hours the sample in 96well plate at a concentration of 5 cells / well 12, 25, 50, treated with a concentration of 100ug / ml, and, under the conditions of 5% CO 2 And cultured for 24 hours. At this time, 1 ug / ml of lipopolysaccharide (LPS) was treated as a control and cultured under the same conditions.
세포 배양 후 0.5mg/ml농도의 MTT( (3-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide)를 넣고 4시간 동안 37℃ incubator에서 반응시킨 후 형성된 formazan을 540nm에서 흡광도를 측정하였다.
After incubation, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) was added at a concentration of 0.5 mg / ml and incubated for 4 hours at 37 ° C in an incubator. Absorbance was measured.
그 결과 [도 3]에서 보는 바와 같이, 본 발명의 당단백 분획물을 RAW 264.7 cell에 25, 50, 100 ug/mL 농도로 24시간 처리하였을 때 세포독성이 나타나지 않는 것을 확인하였다.
As a result, as shown in FIG. 3, when the glycoprotein fraction of the present invention was treated with RAW 264.7 cells at 25, 50, and 100 ug / mL for 24 hours, it was confirmed that no cytotoxicity was observed.
<4-2> 대식세포의 NO 생산에 미치는 영향 측정<4-2> Measurement of effect on macrophage NO production
RAW 264.7 macrophage 세포를 2.0x105 cells/well의 농도로 96 well plate에 분주하고 24시간 후 시료를 25, 50 ㎍/mL의 농도로 처리하고 37℃, 5% CO2의 조건에서 24시간 배양하였다. 이때 대조군은 LPS 1 ㎍/mL을 단독 처리하여 동일 조건에서 배양하였다. Cell culture medium 100 ㎕와 Griess reagent (sigma-aldrich co.) 100 ㎕ 동량을 혼합하여 상온에서 15분 반응 후 ELISA reader를 이용하여 540 nm에서 흡광도를 측정하였다. Sodium nitrite로 표준곡선을 작성하여 NO 함량을 정량하였다. RAW 264.7 macrophage cells were seeded in 96 well plates at a concentration of 2.0x10 5 cells / well. After 24 hours, the samples were treated with 25, 50 ㎍ / mL and cultured at 37 ° C and 5% CO 2 for 24 hours . At this time, 1 ㎍ / mL of LPS was treated alone in the control group and cultured under the same conditions. 100 μl of cell culture medium and 100 μl of Griess reagent (Sigma-aldrich co.) Were mixed and reacted at room temperature for 15 minutes. Absorbance was measured at 540 nm using an ELISA reader. The NO content was quantified by preparing a standard curve with sodium nitrite.
밀기울 당단백 분획물의 macrophage NO 생성에 미치는 영향을 농도별로 처리하여 조사하였으며, 면역활성 증진을 위한 의약품으로 사용되고 있는 polysaccharide K (PSK - 'kerastin' 이라고도 함)와 비교하였다. [도 4]에서 보는 바와 같이, LPS로 활성화된 세포의 경우 NO 함량이 37.08±0.91 uM수준을 나타내었고 밀기울 당단백 분획물을 25, 50, 100 ug/mL 처리시에는 각각 18.57±0.03 uM, 23.05±0.56 uM, 27.56±0.21 uM 으로 농도 의존적으로 증가하는 것으로 나타나 음성 대조군에 비해 월등히 높은 함량을 나타내는 것을 확인하였다.
The effects of bran glycoprotein fraction on macrophage NO production were investigated by concentration and compared with polysaccharide K (PSK - 'kerastin') which is used as a drug for enhancing immune activity. As shown in FIG. 4, the NO content of the LPS-activated cells was 37.08 ± 0.91 μM, and the bran glycoprotein fractions were 18.57 ± 0.03 μM, 23.05 ± 0.2 μM, 0.56 uM, and 27.56 ± 0.21 uM, respectively, which were significantly higher than the negative control.
<4-3> 대식세포의 Cytokine 분비에 미치는 영향 측정<4-3> Measurement of effect on macrophage Cytokine secretion
TNF-α 등의 cytokine은 ELISA kit(Enzo)을 이용하여 측정하였다. RAW 264.7 대식세포(macrophage)를 2.0X105 cells/well의 농도로 96well plate에 분주하고, 24시간 후 시료를 25, 50, 100 ug/ml의 농도로 처리하고, 37℃, 5% CO2의 조건에서 24시간 동안 배양하였다. 이때 양성 대조군으로는 LPS 1 ug/mL을 단독 처리하였고, 음성 대조군으로는 아무것도 처리 안한 RAW 264.7 macrophage 세포로 하여 동일 조건에서 배양하였다. 밀기울 당단백 분획물이 macrophage의 TNF-α 등의 cytokine의 발현에 미치는 영향을 조사하였으며, 면역 활성 증진을 위한 의약품으로 사용되고 있는 polysaccharide K (PSK -'kerastin' 이라고도 함)와 비교하였다. 배양 후 상등액을 취하여 ELISA kit(Enzo)를 이용하여 사이토카인(cytokine) 생성량을 측정하였다.
Cytokines such as TNF-α were measured by ELISA kit (Enzo). RAW 264.7 macrophages (macrophage) of 2.0X10 5 cells / well dispensed into 96well plate with a concentration of, treated with a concentration of the
그 결과 [도 5]에서 보는 바와 같이 TNF-α의 경우, LPS를 단독으로 처리 시 25,825 pg/mL로 나타났으며, 밀기울 당단백 분획물을 25, 50, 100 ug/mL 농도로 처리 시 각각 18,380±12.00, 27,195±59.50, 32,465±146.50 pg/mL으로 나타나 음성대조군은 물론 PSK를 동일 농도로 처리하였을 때보다 월등히 높은 수준으로 나타나 밀기울 당단백 분획물이 PSK(krestin)보다 TNF-α의 발현에 높은 영향을 주는 것으로 나타났다.
As shown in FIG. 5, when TNF-α was treated with LPS alone, it was found to be 25,825 pg / mL. When bran glycoprotein fractions were treated at 25, 50 and 100 μg / 12, 27,195 ± 59.50, and 32,465 ± 146.50 pg / mL, respectively, indicating that the bran glycoprotein fraction had a higher effect on TNF-α expression than PSK (krestin) .
또한 IL-6 발현을 관찰한 결과 [도 6]에서 보는 바와 같이, 밀기울 당단백 분획물을 25, 50, 100 ug/mL 농도로 처리시 각각 772.50±7.25, 2,562.50±13.75, 4,480±11.50 pg/mL의 IL-6 농도를 나타냈는데, 이는 음성 대조군에 비해 월등히 높은 수치이다. 반면 PSK 처리군의 경우에는 100 ug/mL로 처리하였을 때 1,335±1.0 pg/mL의 IL-6 함량을 나타내었다.
As shown in FIG. 6, when bran glycoprotein fractions were treated at 25, 50, and 100 ug / mL, 772.50 ± 7.25, 2,562.50 ± 13.75, and 4,480 ± 11.50 pg / mL of IL-6 IL-6, which is significantly higher than the negative control group. In contrast, the PSK-treated group showed a IL-6 content of 1,335 ± 1.0 pg / mL when treated with 100 μg / mL.
이상 살펴본 바와 같이, 따라서 본 발명은 밀기울 유래 당단백 분획 및 이의 제조방법에 관한 것으로, 좀 더 상세하게는 (a) 밀기울을 탈지하는 단계; (b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계; (c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계; (d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; (e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계; (f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및 (g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70kDa인 것을 특징으로 하는 밀기울 유래 당단백 분획물 및 이를 유효성분으로 포함하는 면역기능 증진용 약학적, 식품 조성물에 관한 것이다. As described above, the present invention relates to a bran-derived glycoprotein fraction and a method for producing the same. More particularly, the present invention relates to a bran- (b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0; (c) centrifuging the mixture to obtain a supernatant; (d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000); (e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant; (f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And (g) obtaining a glycoprotein fraction from the dialyzed precipitated seawater, wherein the glycoprotein fraction has a molecular weight of 16 to 70 kDa, Pharmaceutical compositions and food compositions.
본 발명의 당단백 분획물은 대식세포의 활성을 촉진하는 효능이 매우 뛰어나며 세포독성이 전혀 없어 새로운 기능성 식품 및 면역기능 증강용 의약품 제조에 효과적이므로 산업상 이용가능성이 높다.The glycoprotein fraction of the present invention is highly effective in promoting the activity of macrophages and has no cytotoxicity. Therefore, the glycoprotein fraction is highly industrially applicable because it is effective in the production of new functional foods and immunomodulating drugs.
Claims (7)
(b) 밀기울을 pH가 7.0 내지 8.0인 완충용액에 혼합하는 단계;
(c) 상기 혼합물을 원심분리하여 상등액을 수득하는 단계;
(d) 상기 상등액을 투석 반투막(MWCO 6000-8000)으로 투석하는 단계;
(e) 상기 투석된 상등액에 단백질을 침전시키는 염을 첨가하여 당단백질을 침전시키는 단계;
(f) 상기 침전물을 pH가 7.0 내지 8.0인 완충용액에 용해하여 투석 반투막(MWCO 6000-8000)으로 투석하는 단계; 및
(g) 상기 투석된 침전 용해물에서 당단백 분획물을 수득하는 단계;
를 포함하는 방법에 의하여 제조되고, 분자량이 16 내지 70 kDa이고, 면역 기능 증진 활성이 있는 것을 특징으로 하는 밀기울 유래 당단백 분획물.
(a) degreasing the bran;
(b) mixing wheat bran into a buffer solution having a pH of 7.0 to 8.0;
(c) centrifuging the mixture to obtain a supernatant;
(d) dialyzing the supernatant with a dialysis semipermeable membrane (MWCO 6000-8000);
(e) precipitating the glycoprotein by adding a salt to precipitate the protein in the dialyzed supernatant;
(f) dissolving the precipitate in a buffer solution having a pH of 7.0 to 8.0 and dialyzing with a dialysis semipermeable membrane (MWCO 6000-8000); And
(g) obtaining a glycoprotein fraction from the dialyzed precipitated seafood;
, Wherein the bran-derived glycoprotein fraction has a molecular weight of 16 to 70 kDa and has an immuno-enhancing activity.
The glycoprotein fraction according to claim 1, wherein the glycoprotein fraction comprises 5 to 15% (w / w) of aspartic acid, 3 to 7% (w / w) of threonine, 3 to 7% (W / w) of proline, 3 to 10% (w / w) of glycine, 3 to 10% (w / w) of alanine, 3 to 10% To 10% (w / w) of methionine, 1 to 5% (w / w) of methionine, 3 to 10% (w / w) of isoleucine, 3 to 10% 5% w / w phenylalanine, 3-10% w / w lysine, 1 to 5% w / w histidine, 5-15% arginine, (w / w) of the glycoprotein fraction.
The method of claim 1, wherein the glycoprotein fraction comprises 0.1 to 3% (w / w) of fucose, 0.1 to 3% (w / w) of rhamnose, 20 to 40% (W / w) of galactose, 10 to 20% (w / w) of glucose, 1 to 10% (w / w) of mannose, 20 to 50% , And N-acetylglucosamine (GlcNAc) are contained in a proportion of 0.5 to 5% (w / w).
A food composition for enhancing an immune function comprising the glycoprotein fraction of claim 1 as an active ingredient.
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| KR101170998B1 (en) * | 2011-12-26 | 2012-08-08 | 한국식품연구원 | Glycoprotein fraction isolated from rice bran and method for producing the same |
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| KR20140066826A (en) | 2014-06-02 |
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