KR20030091101A - Composition comprising of soluble dietary fiber from corn hull - Google Patents
Composition comprising of soluble dietary fiber from corn hull Download PDFInfo
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- KR20030091101A KR20030091101A KR1020020028514A KR20020028514A KR20030091101A KR 20030091101 A KR20030091101 A KR 20030091101A KR 1020020028514 A KR1020020028514 A KR 1020020028514A KR 20020028514 A KR20020028514 A KR 20020028514A KR 20030091101 A KR20030091101 A KR 20030091101A
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- South Korea
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- composition
- dietary fiber
- water
- soluble dietary
- iii
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Abstract
Description
본 발명은 옥피 유래의 수용성 식이섬유를 포함하는 조성물에 관한 것으로, 더욱 상세하게는 비만예방 효과가 우수할 뿐만 아니라, 항암활성 및 면역조절능력이 우수한 옥피 유래의 수용성 식이섬유를 포함하는 항암용, 또는 면역조절용 조성물에 관한 것이다.The present invention relates to a composition comprising a water-soluble dietary fiber derived from oxime, and more particularly, for anticancer comprising a water-soluble dietary fiber derived from oxy, which is excellent in anti-obesity effect and excellent in anticancer activity and immunomodulatory ability. Or to a composition for immunomodulation.
식이섬유(dietary fiber)는 인체에 존재하는 소화효소로 소화되지 않는 식물 중의 난소화성 성분으로, 검(gum) 및 점액질(mucilage) 이외에도 셀룰로오스, 헤미셀룰로오즈, 리그닌, 및 팩틴과 같은 세포벽 물질을 포함한다.Dietary fiber is an indigestible component in plants that is indigestible by the digestive enzymes present in the human body. In addition to gum and mucilage, dietary fiber includes cell wall materials such as cellulose, hemicellulose, lignin, and pectin. .
식이섬유는 수용성과 불용성으로 구분되며, 수용성 식이섬유는 혈중 콜레스테롤 농도의 저하, 식후 혈당치 상승 억제 등의 생리활성을 가지고, 불용성 식이섬유는 저작시간 및 장내체류시간 지연, 변량증가 등의 효과와 대장암 예방, 다이옥신류 등 환경오염물질의 체외 배설작용을 가진다. 특히, 수용성 식이섬유의 경우 혈청 콜레스테롤 상승억제 효과가 우수한 것으로 알려지고 있는데, 지질대사 개선 작용의 메커니즘은 소화관에서의 지질 흡수의 저해, 담즙산의 배설 촉진, 대장에서섬유소의 발효로 발생되는 단쇄 지방산, 특히 프로피온산(propionic acid)이 간장에서의 콜레스테롤 생합성을 억제하는 점 등을 들 수 있다.Dietary fiber is divided into water-soluble and insoluble, and water-soluble fiber has physiological activities such as lowering blood cholesterol level and suppressing post-prandial blood sugar level increase.Insoluble fiber has effects such as delay in chewing time, intestinal stay time, and increase in variance. It has the effect of excretion of environmental pollutants such as cancer prevention and dioxins. In particular, water-soluble dietary fiber is known to have an excellent effect on inhibiting serum cholesterol elevation. The mechanism of lipid metabolism improvement is inhibition of lipid absorption in the digestive tract, promotion of excretion of bile acids, and short-chain fatty acids produced by fermentation of fiber in the large intestine, In particular, propionic acid (propionic acid) inhibits cholesterol biosynthesis in the liver and the like.
한편, 옥수수전분 제조시 부산물로 발생되는 옥피는 식이섬유, 특히 헤미셀룰로오즈가 다른 곡류의 외피보다 풍부하게 함유되어 있어 우수한 식이섬유 소재로서 이용될 수 있다.On the other hand, the oxy produced as a by-product in the production of corn starch is rich in dietary fiber, especially hemicellulose than the shell of other grains can be used as an excellent dietary fiber material.
헤미셀룰로오즈는 셀룰로오즈 및 펙틴질을 제외한 식물 세포벽을 구성하는 다당류의 총칭으로, 단당류가 중합되어 복잡하게 결합되어 있는 형태이다. 이는 불용성의 헤미셀룰로오즈 A와 수용성인 헤미셀룰로오즈 B로 구분되며, 수용성의 헤미셀룰로오즈 B는 5탄당인 자일로스와 아라비노스로 이루어진 다당류인 아라비노자일란(arabinoxylan)을 주성분으로 한다. 특히, 상기 아라비노자일란은 사람의 소화 효소로 분해되지 않는 베타-1,4-결합을 가지기 때문에 우수한 식이섬유로 사용될 수 있다.Hemicellulose is a generic term for polysaccharides constituting the plant cell wall excluding cellulose and pectin, and is a form in which monosaccharides are polymerized and complexly bonded. It is classified into insoluble hemicellulose A and water-soluble hemicellulose B, and the water-soluble hemicellulose B is mainly composed of arabinoxyllan (arabinoxylan), a polysaccharide composed of pentose xylose and arabinose. In particular, the arabinoxylan can be used as an excellent dietary fiber because it has a beta-1,4-linkage that is not degraded by human digestive enzymes.
이와 같은 이유로 곡류의 껍질로부터 제조된 식이섬유를 이용하는 연구는 계속되어 왔다. 일본공개특허공보 평3-209331 및 평4-5649는 쌀겨, 소맥겨, 옥수수껍질 등으로부터 제조된 헤미셀룰로오즈를 비피더스균 등 장내 유익균의 증식을 촉진시키는 정장제로 이용하는 방법에 대하여 개시하고 있으며, 일본공개특허공보 소59-1689 및 제2814172호는 식용식물로부터 제조된 셀룰로오즈 및 리그닌 주체의 식이섬유를 혈청 콜레스테롤 상승억제물질 및 혈중지질조절제로 이용하는 방법에 대하여 개시하고 있다.For this reason, research on using dietary fiber made from grain husks has continued. Japanese Patent Application Laid-Open Nos. Hei 3-209331 and Hei 4-5649 disclose a method of using hemicellulose prepared from rice bran, wheat bran, corn husk, and the like as a formal agent for promoting the growth of enteric beneficial bacteria such as bifidus. Patent Publications Nos. 59-1689 and 2814172 disclose a method of using cellulose and lignin-derived dietary fiber prepared from edible plants as serum cholesterol elevation inhibitors and blood lipid control agents.
또한 일본공개특허공보 소62-201820 및 평1-242530은 옥수수, 소맥겨 등 곡류의 껍질로부터 얻어진 헤미셀룰로오즈를 지방간 억제물질로 이용하는 방법에 대하여 개시하고 있으며, 일본공개특허공보 제2787252호는 곡류의 껍질에서 추출한 수용성 다당류를 대장암 억제제로 이용하는 방법에 대하여 개시하고 있으며, 일본공개특허공보 평7-89886 및 미국특허 제 5,198,363호는 옥수수 껍질로부터 수득한 셀룰로오즈와 헤미셀룰로오즈 주체의 중성세제추출 식이섬유가 돌연변이원성 물질인 디니트로피렌(dinitropyrene)을 불활성화시키는 방법에 대하여 개시하고 있다.In addition, Japanese Patent Laid-Open Nos. 62-201820 and 1-242530 disclose a method of using hemicellulose obtained from the husks of cereals such as corn and wheat bran as fatty liver inhibitors, and Japanese Patent Laid-Open No. 2787252 discloses grains. A method of using water-soluble polysaccharides extracted from husks as a colorectal cancer inhibitor is disclosed. Disclosed is a method of inactivating a mutagenic substance, dinitropyrene.
그러나, 상기 종래기술들은 곡류의 외피로부터 제조된 식이섬유를 혈중 지질대사개선, 간장보호, 대장암 억제제로 이용하거나, 장내 유익균의 증식 효과 등 일반적으로 식이섬유가 가지고 있는 기능성의 범주를 크게 벗어나지 않으며, 옥피로부터 제조된 헤미셀룰로오즈가 주성분을 이루는 수용성 식이섬유의 생리활성에 대하여는 연구된 바 없다.However, the conventional techniques do not deviate significantly from the range of functionalities that dietary fiber generally has, such as using dietary fiber prepared from the outer shell of cereals as blood lipid metabolism improvement, hepatic protection, colon cancer inhibitor, or proliferation effect of enteric beneficial bacteria. The physiological activity of water-soluble dietary fiber composed mainly of hemicellulose prepared from oxime has not been studied.
상기와 같은 문제점을 해결하고자, 본 발명은 옥피로부터 추출한 수용성 식이섬유인 헤미셀룰로오즈를 포함하는 항암용 조성물을 제공하는 것을 목적으로 한다.In order to solve the problems as described above, an object of the present invention is to provide an anticancer composition comprising hemicellulose, which is a water-soluble dietary fiber extracted from oxy.
본 발명의 다른 목적은 옥피로부터 추출한 수용성 식이섬유인 헤미셀룰로오즈를 포함하는 면역활성 조절용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for modulating immune activity comprising hemicellulose, which is a water-soluble dietary fiber extracted from octape.
상기 목적을 달성하기 위하여, 본 발명은 옥피 유래의 수용성 식이섬유를 포함하는 항암용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for anticancer comprising a water-soluble dietary fiber derived from oxidized.
또한 본 발명은 옥피 유래의 수용성 식이섬유를 포함하는 면역활성 조절용 조성물을 제공한다.In another aspect, the present invention provides a composition for modulating immune activity comprising a water-soluble dietary fiber derived from oxime.
이하 본 발명을 상세하게 설명한다.Hereinafter, the present invention will be described in detail.
본 발명자들은 옥피로부터 추출한 수용성 식이섬유의 생리활성에 대하여 연구하던 중, 상기 수용성 식이섬유가 비만예방효과를 가질 뿐만 아니라, 항암활성 및 면역조절능력이 우수하고, 특히 항암제, 면역억제제, 또는 면역촉진제와 함께 처리할 경우 그 활성을 조절할 수 있음을 확인하고, 이를 토대로 본 발명을 완성하게 되었다.The inventors of the present invention, while studying the physiological activity of the water-soluble dietary fiber extracted from the octaphyte, the water-soluble dietary fiber not only has an anti-obesity effect, but also has excellent anti-cancer activity and immunomodulatory ability, in particular an anticancer agent, an immunosuppressive agent, or immunity When the treatment with the promoter was confirmed that the activity can be adjusted, based on this, the present invention was completed.
본 발명의 조성물은 옥수수 껍질(옥피) 유래 수용성 식이섬유를 유효성분으로 포함하며, 바람직하게는 하기 방법으로 제조된 옥피 유래 수용성 식이섬유를 포함한다. 하기 방법으로 제조된 옥피 유래 수용성 식이섬유는 헤미셀룰로오즈를 주성분으로 포함한다.The composition of the present invention comprises a corn husk (okpi) -derived water-soluble fiber as an active ingredient, preferably comprises an ox-derived water-soluble fiber prepared by the following method. The oxime-derived water soluble fiber prepared by the following method contains hemicellulose as a main component.
본 발명의 바람직한 옥피 유래 수용성 식이섬유의 제조방법은 옥피에서 전분 및 단백질을 제거하고, 알칼리 추출하여 수득한 알칼리 추출액에 효소를 처리하여 반응시킨 후, 여과, 정제, 및 건조하여 제조하는 것이다. 본 발명에서 사용되는 옥피는 상업적으로 용이하게 입수할 수 있으며, 품질 수준은 시중에 유통되고 있는 것이면 특별한 제한 없이 사용할 수 있다. 구체적인 옥피 유래 수용성 식이섬유의 제조방법은 본 출원인의 대한민국 공개특허공보 제 2002-32310호에 자세히 기재되어 있다. 본 발명의 구체적인 일례에서, 본 발명에서 사용되는 옥피 유래의 수용성 식이섬유는Preferred method for preparing oxime-derived water-soluble fiber of the present invention is to remove starch and protein from oxime, react with an alkaline extract obtained by alkali extraction, and then treat the enzyme, and then filter, purify and dry. The oxy used in the present invention can be easily obtained commercially, the quality level can be used without particular limitation as long as it is commercially available. The method for producing a specific oxime-derived water-soluble dietary fiber is described in detail in Korean Patent Application Publication No. 2002-32310. In a specific example of the present invention, the water-soluble dietary fiber derived from oxime used in the present invention
ⅰ) 옥피로부터 전분 및 단백질을 제거하는 단계;Iii) removing starch and protein from the oxid;
ⅱ) 상기 ⅰ)단계에서 전분 및 단백질이 제거된 옥피를 알칼리 용액으로 추Ii) adding the oxyphyte from which starch and protein have been removed in step
출하고 여과하는 단계;Extracting and filtering;
ⅲ) 상기 ⅱ)단계에서 수득한 알칼리 추출액에 셀룰라제와 셀로비아제를 처Iii) Cellulase and cellobiase were applied to the alkaline extract obtained in step ii).
리하여 반응시키는 단계;Reaction to react;
ⅳ) 상기 ⅲ)단계에서 수득한 효소 반응액에 흡착제를 처리하고, 막여과를Iii) treating the enzyme reaction solution obtained in step iii) with adsorbent, and membrane filtration
통하여 여과액을 수득하는 단계; 및Obtaining a filtrate through; And
ⅴ) 상기 ⅳ)단계의 여과액을 정제하는 단계를 포함하는 방법으로 제조된다.Iii) purifying the filtrate of step iii).
또한 본 발명은 투명도 및 여과성을 더욱 향상시키기 위하여 상기 ⅲ)단계에서 자일라나제를 추가로 처리할 수 있으며, ⅲ)단계의 효소반응 전에 알칼리 추출액에 대해 탈염 및 탈색처리를 위하여 이온교환수지 처리를 수행하는 공정을 추가로 포함할 수 있다.In addition, the present invention can be further treated with xylanase in the step iii) to further improve the transparency and filterability, the ion exchange resin treatment for desalting and decolorizing the alkaline extract before the enzymatic reaction of step iii). The process may further comprise.
상기 ⅰ)단계의 전분 및 단백질 제거는 일반적으로 알려진 방법을 사용할 수 있으며, 예를 들어 아밀라제, 글루코아밀라제 등의 전분분해효소와 단백질분해효소를 처리하는 효소처리 방법을 사용할 수 있다. 상기 효소처리 방법은 1 종의 전분분해효소와 단백질분해효소를 동시에 처리하거나, 하나씩 처리할 수 있다. 상기 ⅰ)단계에서 효소를 이용하여 옥피 중의 전분 및 단백질을 제거하고, 여과포를 이용하여 여과함으로써 옥피의 회수율을 향상시킬 수 있다.Starch and protein removal of step iii) can be generally used a known method, for example, an enzyme treatment method for treating starch and protease, such as amylase, glucoamylase. In the enzyme treatment method, one starch enzyme and protease can be treated simultaneously, or one by one. In step iii), starch and protein in oxy are removed using an enzyme, and filtration can be improved by filtration using a filter cloth.
상기 ⅱ)단계는 옥피 중 70 % 이상을 차지하는 헤미셀룰로오즈를 추출하기 위해 알칼리 용액을 사용하여 고온에서 교반하는 단계이다. 상기 알칼리 용액은수산화나트륨 단독 또는 수산화나트륨과 수산화칼슘을 병용해서 사용할 수 있으며, 더 높은 회수율, 식이섬유함량, 및 작업공정상의 잇점을 위해서는 수산화나트륨 단독으로 사용하는 것이 바람직하다. 또한, 헤미셀룰로오즈를 효과적으로 추출하기 위해서는 저농도의 수산화나트륨을 사용하는 것이 바람직하며, 알칼리 용액으로 추출한 후 여과포를 이용하여 여과함으로써 추출액의 회수율을 최대로 할 수 있다.Step ii) is a step of stirring at high temperature using an alkaline solution to extract hemicellulose, which occupies 70% or more of octane. The alkaline solution may be used alone or in combination with sodium hydroxide and calcium hydroxide, and sodium hydroxide alone is preferred for higher recovery, dietary fiber content, and work process advantages. In addition, in order to effectively extract hemicellulose, it is preferable to use a low concentration of sodium hydroxide, and the extraction rate can be maximized by extracting with an alkaline solution and then filtering with a filter cloth.
상기 ⅲ)단계는 상기 ⅱ)단계의 알칼리 추출액을 산으로 중화한 후, 셀룰라제와 셀로비아제를 처리하여 반응시키는 단계로, 상기 셀룰라제와 셀로비아제에 자일라나제를 추가로 첨가하여 반응시킬 수도 있다. 특히, 수용성 식이섬유의 수율 향상, 추출액의 점도 저하, 투명도 향상, 및 여과성 향상을 위하여 셀룰라제, 셀로비아제, 및 자일라나제를 모두 처리하는 것이 바람직하다.In step iii), the alkaline extract of step ii) is neutralized with an acid, followed by reaction with a cellulase and a cellobiase. The reaction is further performed by adding xylanase to the cellulase and the cellobiase. You can also In particular, in order to improve the yield of the water-soluble dietary fiber, lower the viscosity of the extract, improve the transparency, and improve the filterability, it is preferable to treat all the cellulase, the cellobia agent, and the xylanase.
또한, 상기 ⅲ)단계에서 알칼리 추출액을 효소로 처리하기 전에 양이온과 음이온 교환수지로 탈염 및 탈색할 경우, 이후 첨가되는 효소의 양을 감소시키면서 동질의 수용성 식이섬유를 얻을 수 있는 잇점이 있다.In addition, when desalting and decolorizing the cation and anion exchange resin before treating the alkaline extract with an enzyme in step (iii), there is an advantage in that the same water-soluble dietary fiber can be obtained while reducing the amount of the added enzyme.
상기 ⅳ)단계는 상기 ⅲ)단계의 효소반응액을 활성탄 등의 흡착체로 처리한 후 최종 막여과를 수행하고, 이온교환수지를 처리하여 최종 정제한 후, 건조함으로써 높은 수율로 수용성 식이섬유를 제조할 수 있다.In step iii), the enzyme reaction solution of step iii) is treated with an adsorbent such as activated carbon, followed by final membrane filtration, final purification by treatment with ion exchange resin, and drying to prepare water-soluble dietary fiber with high yield. can do.
상기와 같이 제조된 본 발명의 옥피 유래의 수용성 식이섬유는 셀룰로오즈에 비하여 비만예방효과가 우수하고, 세포에 대해 독성을 나타내지 않는다. 또한, 적정 농도의 옥피 유래의 수용성 식이섬유는 우수한 항암활성을 나타내며, 인체 면역세포의 림프구와 과립구의 면역 활성화를 향상시키는 작용을 한다.The water-soluble dietary fiber derived from oxime of the present invention prepared as described above is superior to cellulose in preventing obesity and exhibits no toxicity to cells. In addition, the water-soluble dietary fiber derived from the appropriate concentration of oxime shows excellent anticancer activity, and serves to improve the immune activation of lymphocytes and granulocytes of human immune cells.
본 발명은 상기 옥피 유래의 수용성 식이섬유과 항암제를 포함하는 항암용 조성물에 관한 것이다. 상기 조성물에 포함되는 옥피 유래의 수용성 식이섬유는 상기 설명한 방법으로 제조되는 헤미셀룰로오즈인 것이 바람직하다. 본 발명의 항암용 조성물에 사용가능한 항암제는 항암제로 알려진 모든 항암제를 포함하는 의도이며, 예를 들면, 시스플라틴(cisplatin), 아드리아마이신(adriamycin) 등을 사용할 수 있다. 상기 항암제와 옥피 유래 수용성 식이섬유를 함께 사용할 경우에 항암활성을 더욱 향상시켜 그 항암 효과를 상승시키는 작용을 한다.The present invention relates to a composition for anticancer comprising the water-soluble fiber and the anticancer agent derived from the above-mentioned oxy. It is preferable that the water-soluble dietary fiber derived from oxime contained in the said composition is hemicellulose manufactured by the method mentioned above. The anticancer agent usable in the anticancer composition of the present invention is intended to include all anticancer agents known as anticancer agents. For example, cisplatin, adriamycin, and the like can be used. When the anticancer agent and oxime-derived water-soluble dietary fiber are used together, the anticancer activity is further enhanced to increase the anticancer effect.
본 발명의 항암용 조성물은 조성물 내에 옥피 유래의 수용성 식이섬유를 총 조성물중 0.002중량% 이상, 예를 들면, 0.005%를 포함하나 식이섬유는 유해한 성분이 아니므로 100중량%까지 포함되어도 무방하다. 또한, 항암제는 총 조성물중 0.001중량% 이상, 예를 들면 0.0025%를 포함할 수 있으며, 환자의 상태, 병의 위중도, 및 항암제의 종류에 따라 적절하게 함량을 선택할 수 있다. 예컨대, 본 발명의 조성물은 50∼500 ㎍/mL, 및 항암제 25 ㎍/mL의 농도로 포함할 수 있다. 암세포에 상기 농도로 옥피 유래의 수용성 식이섬유와 항암제를 동시에 포함하는 조성물을 처리할 경우 항암제를 단독으로 처리한 경우와 비교하여 유의성 있는 암세포 사멸율을 나타내었다.The anticancer composition of the present invention comprises at least 0.002% by weight, for example, 0.005% of the total water-soluble dietary fiber from the composition in the composition, but dietary fiber may contain up to 100% by weight since it is not a harmful ingredient. In addition, the anticancer agent may comprise at least 0.001% by weight of the total composition, for example, 0.0025%, and may be appropriately selected depending on the condition of the patient, the severity of the disease, and the type of the anticancer agent. For example, the composition of the present invention may comprise at a concentration of 50-500 μg / mL, and 25 μg / mL anticancer agent. Treatment of cancer cells with a composition containing both water-soluble dietary fiber and an anticancer agent at the same time showed a significant cancer cell death rate compared with the treatment with an anticancer agent alone.
본 발명은 상기 옥피 유래의 수용성 식이섬유을 포함하는 면역활성 조절용 조성물에 관한 것이다. 상기 조성물에 포함되는 옥피 유래의 수용성 식이섬유는 상기 제조 방법으로 제조되는 수용성 식이섬유가 바람직하고, 더욱 바람직하게는 헤미셀룰로오즈이다.The present invention relates to a composition for modulating immunological activity comprising the water-soluble dietary fiber derived from the above-mentioned oxy. The water-soluble dietary fiber derived from oxime contained in the composition is preferably a water-soluble dietary fiber produced by the above-mentioned production method, more preferably hemicellulose.
본 발명의 면역활성 조절용 조성물은 면역활성을 촉진할 수 있다. 장내 면역세포에서 주요 조직적합성 복합체(major histocompatibility complex, MHC)의 발현은 병원체 제거에 중요한 기능을 나타내는데, 본 발명의 조성물이 혈액 중 림프구와 과립구에서 MHC Ⅰ과 MHC Ⅱ의 발현 양상에 미치는 영향을 조사한 결과, 이들을 발현하는 세포수 및 단위 세포당 MHC 분자의 막발현이 증가함을 알 수 있다. 따라서, 본 발명에 따른 옥피 유래 수용성 식이섬유의 면역활성 조절용 조성물은 면역활성을 향상시킨다.The composition for modulating immune activity of the present invention can promote immune activity. The expression of major histocompatibility complex (MHC) in intestinal immune cells has an important function in pathogen elimination. The effects of the composition of the present invention on the expression patterns of MHC I and MHC II in lymphocytes and granulocytes in the blood were investigated. As a result, it can be seen that the number of cells expressing them and the expression of MHC molecules per unit cell increases. Therefore, the composition for regulating the immunological activity of oxime-derived soluble fiber according to the present invention improves the immunological activity.
본 발명의 일례에서, 본 발명의 면역활성 조절용 조성물이 임파구와 과립구에서의 면역활성화를 위해서는 5 ㎍/mL의 농도로 포함할 수 있으며, 상기와 같은 농도로 포함된 옥피 유래의 수용성 식이섬유를 사람 말초혈액세포에 처리할 경우, 임파구에서는 단위세포당 MHC Ⅰ 분자의 발현량이 유의하게 증가하고, 과립구에서는 MHC 분자가 발현된 세포수와 단위세포당 MHC 분자의 발현량이 모두 유의하게 증가하였다.In one example of the present invention, the composition for regulating immunological activity of the present invention may include a concentration of 5 μg / mL for immunoactivation in lymphocytes and granulocytes, and the water-soluble dietary fiber derived from oxime contained in the above concentrations. When treated with peripheral blood cells, the expression level of MHC I molecules per unit cell was significantly increased in lymphocytes, and in granulocytes, both the number of cells expressing MHC molecules and the amount of MHC molecules per unit cell were significantly increased.
또한, 본 발명의 면역활성 조절용 조성물은 면역촉진제와 함께 사용할 경우에 면역촉진효과를 경감시켜, 과민성 면역촉진효과로 인한 부작용을 최소화할 수 있다. 특히, 조성물 내에 옥피 유래의 수용성 식이섬유를 0.5 ㎍/mL 이상의 농도로 포함할 경우 피부발진, 피부발적, 피부염, 천식, 알러지, 동종이식 거절, 자가면역반응 등의 과민성 면역촉진효과로 인한 부작용을 최소화할 수 있으며, 이는 상기 조성물이 세균성 또는 케미칼에 의한 과민성 면역촉진 효과를 완화시킴으로써 나타나는 효과인 것이다.In addition, the composition for modulating the immunological activity of the present invention may reduce the immunostimulating effect when used in combination with an immunostimulating agent, thereby minimizing side effects due to an overactive immune promoting effect. Particularly, when the composition contains water-soluble dietary fiber derived from oxime at a concentration of 0.5 ㎍ / mL or more, side effects due to irritable immune promoting effects such as skin rash, skin redness, dermatitis, asthma, allergy, allograft rejection, autoimmune reaction, etc. It can be minimized, which is an effect exhibited by the composition to alleviate the hypersensitive immune promoting effect by bacterial or chemical.
본 발명에 사용 가능한 면역촉진제는 본 기술분야에서 통상의 지식을 가진 자가 적절히 선택할 수 있으며, 예를 들면 PHA-L(phaseolus vulgaris agglutinin-L type), PHA-P(phaseolus vulgaris agglutinin-P type), 또는 PMA(phorbol 12-myristate 13-acetate)/Ionomycin 등이다. 본 발명의 옥피 유래의 수용성 식이섬유를 면역촉진제인 PHA-L 또는 PHA-P와 병용처리할 경우 옥피 유래의 수용성 식이섬유 50 ㎍/mL 이하의 농도에서 면역세포 활성화를 유의하게 억제시켰으며, PMA/Ionomycin와 병용처리할 경우에는 0.5 ㎍/mL 이상의 농도에서 면역 세포 활성화를 유의하게 억제시킴을 확인할 수 있었다. 반면, 셀룰로오즈는 면역촉진제인 PMA/Ionomycin와 병용처리시에만 면역 세포 활성 억제 효과를 나타내었다. 이로부터 본 발명의 옥피 유래의 수용성 식이섬유의 과민성 면역반응 억제 작용 즉, 과민성 대장염 완화 작용을 기대할 수 있다.Immunostimulators usable in the present invention may be appropriately selected by one of ordinary skill in the art, for example, phaseolus vulgaris agglutinin-L type (PHA-L), phaseolus vulgaris agglutinin-P type (PHA-P), Or PMA (phorbol 12-myristate 13-acetate) / Ionomycin. When the water-soluble dietary fiber derived from oxime of the present invention was co-treated with PHA-L or PHA-P as an immunostimulator, immune cell activation was significantly inhibited at a concentration of 50 μg / mL or less of water-soluble dietary fiber derived from oxime. When combined with / Ionomycin it was confirmed that significantly inhibit the immune cell activation at a concentration of 0.5 ㎍ / mL or more. Cellulose, on the other hand, showed an inhibitory effect on immune cell activity only in combination with the immunostimulant PMA / Ionomycin. From this, it is possible to expect the action of inhibiting the hypersensitive immune response of the soluble fiber derived from oxime of the present invention, that is, the alleviating of irritable colitis.
본 발명의 면역활성 조절용 조성물은 면역억제제와 함께 사용할 경우에 상기 면역억제제로 인한 과민성 면역억제효과를 경감시키고, 면역세포활성을 회복시킴으로써 과민성 면역억제효과로 인한 부작용을 최소화할 수 있다. 특히, 조성물 내에 옥피 유래의 수용성 식이섬유를 500 ㎍/mL 이상의 농도로 포함할 경우, 혈액 면역계 질환, 바이러스 감염, 면역부전증 등의 과민성 면역억제효과로 인한 부작용을 경감시킬 수 있으며, 림프구 수가 1000/㎕ 이하인 성인에게서 세포성 면역기능 부전이 강하게 나타날 수 있다. 본 발명에 사용 가능한 면역억제제는 본 기술분야에서 통상의 지식을 가진 자가 적절히 선택할 수 있으며, 예를 들면 덱사메타손(dexamethasone) 등이 있다. 즉, 상기 면역조절용 조성물은 옥피 유래의 수용성 식이섬유와 면역억제제를 추가로 포함함으로써 면역조절을 할 수 있는 효과가 있다.When used in combination with an immunosuppressive agent, the composition for adjusting immunological activity of the present invention may reduce the hypersensitive immunosuppressive effect caused by the immunosuppressive agent and restore immune cell activity, thereby minimizing side effects due to the hypersensitive immunosuppressive effect. Particularly, when the composition includes oxime-derived water-soluble fiber at a concentration of 500 μg / mL or more, side effects due to overactive immunosuppressive effects such as blood immune system disease, viral infection, immunodeficiency, etc. may be reduced, and lymphocyte count is 1000 /. Cellular immune dysfunction may be strongly manifested in adults below μl. Immunosuppressants usable in the present invention may be appropriately selected by those of ordinary skill in the art, for example dexamethasone. In other words, the immunomodulatory composition has an effect capable of immunomodulation by additionally including a water-soluble dietary fiber and an immunosuppressive agent derived from oxy.
또한 본 발명의 면역활성 조절용 조성물은 상기와 같은 면역촉진제 또는 면역억제제와 병용하여 사람 말초혈액세포에 처리할 경우, 병용한 면역촉진제 또는 면역억제제의 효과를 완화시켜주는 효과가 있다.In addition, the composition for modulating the immunological activity of the present invention has an effect of alleviating the effects of the combined immunostimulator or immunosuppressant when treated in human peripheral blood cells in combination with the above-described immunostimulator or immunosuppressant.
본 발명에 따른 조성물은 약제 조성물 또는 식품 조성물일 수 있으며, 본 발명세서 식품 조성물이라 함은 식품, 식품첨가제, 음료, 및 음료첨가제 등을 포함하는 의도이다.The composition according to the present invention may be a pharmaceutical composition or a food composition, the food composition of the present invention is intended to include food, food additives, beverages, beverage additives and the like.
본 발명의 옥피 유래의 수용성 식이섬유를 포함하는 조성물은 항암활성 및 면역조절능력을 증가시키기 위하여 약제학적으로 허용되는 담체와 혼합하여 항암활성 및 면역조절용 약제 조성물을 제조할 수 있다. 예를 들어, 유효량의 옥피 유래 식이섬유를 약제학적 분야에서 통상적으로 사용하는 담체, 부형제 등과 혼합하여 제조할 수 있다.The composition comprising the water-soluble dietary fiber derived from the octaphytes of the present invention can be mixed with a pharmaceutically acceptable carrier in order to increase the anticancer activity and immunomodulatory ability to prepare a pharmaceutical composition for anticancer activity and immunomodulation. For example, it may be prepared by mixing an effective amount of dietary fiber derived from oxime, carriers, excipients and the like commonly used in the pharmaceutical field.
상기 옥피 유래의 수용성 식이섬유는 항암용 및 면역조절용 약제 조성물은 1 회에 500 ㎎ 또는 1,000 ㎎ 씩 1일 3회 투여하는 것이 바람직하다.The water-soluble dietary fiber derived from the octaphyte is preferably administered once a day 500 mg or 1,000 mg of the anticancer and immunomodulatory pharmaceutical composition at a time.
상기 담체로는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산체, 안정화제, 현탁화제, 색소, 또는 향료 등을 사용할 수 있으며, 이들을 혼합하여 정제, 트포키, 캡슐, 산제 과립제 등의 형태로 사용할 수 있다. 상기 조성물은 경구 투여하는 것이 바람직하며, 이외에 각종의 형태로 통용되는 기법에 따라 제조할 수 있다. 투여량은 체내에서 활성 성분의 흡수도, 불활성율 및 배설속도, 환자의 연령,성별, 상태, 및 치료할 질병의 중증 정도에 따라 적절히 선택할 수 있다.As the carrier, a binder, a suspending agent, a disintegrating agent, an excipient, a solubilizer, a dispersion, a stabilizer, a suspending agent, a dye, or a flavoring agent can be used, and the like can be mixed with tablets, tablets, capsules, powder granules, and the like. Available in form. The composition is preferably administered orally, and can be prepared according to techniques commonly used in various forms. The dosage may be appropriately selected depending on the absorbency, inactivation rate and rate of excretion of the active ingredient in the body, the age, sex, condition of the patient, and the severity of the disease to be treated.
또한 본 발명의 옥피 유래의 수용성 식이섬유는 식품 총 중량부에 대하여 1 내지 100 중량부로 첨가하여 과자류, 라면류, 음료수, 마요네즈, 드레싱, 아이스크림, 비타민 복합제 등의 건강기능성 식품으로 제조할 수 있다.In addition, the water-soluble dietary fiber derived from oxime of the present invention may be added to 1 to 100 parts by weight based on the total weight of food, it can be prepared as a health functional food such as confectionery, ramen, beverages, mayonnaise, dressings, ice cream, vitamin complexes.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시하나, 하기 실시예는 본 발명을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다.Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.
[실시예]EXAMPLE
실시예 1: 옥피 유래의 수용성 식이섬유 제조Example 1 Preparation of Water-Soluble Dietary Fiber from Oxygen
건조 옥피 200 g을 8 % 농도가 되도록 증류수와 혼합하고, 정속 교반기로 교반하면서 1N 수산화나트륨 용액을 가하여 pH 5.8로 조절하였다. 이를 미리 95 ℃로 조절된 항온수조에 옮기고, 내열성 알파-아밀라제(Novo Nordisk Ltd., Termaryl 120LS, Denmark)를 건조 옥피의 고형분 대비 1.0 중량%로 첨가 후, 2 시간 동안 반응한 다음 여과포로 탈수하였다. 상기 전분이 제거된 옥피에 3.0 L의 증류수를 첨가하고 교반하면서 1N 수산화나트륨 용액을 가하여 pH 7.0으로 조절하였다. 여기에 단백질분해효소(Novo Nordisk Ltd., Flavourzyme, Denmark)를 건조 옥피의 고형분 대비 1.0 중량%로 첨가하여 50 ℃로 조절된 항온수조에서 3 시간 동안 반응시켰다. 이어서, 여과포를 이용하여 탈수하고 오븐에서 건조하였다. 여기에 0.5 % 농도의 수산화나트륨 용액 3 L를 혼합하고 80 ℃로 조절된 항온수조에서 3 시간 동안 충분히 교반한 다음, 여과포로 여과하여 1차 알칼리 추출액을 수득하였다. 그 다음, 10 % 염산 용액으로 추출액의 pH를 4.8로 조절하여 50 ℃로 조절된 항온수조에 옮긴 다음, 셀룰라제(Novo Nordisk LTD., Celluclast, Denmark)와 셀로비아제(Novo Nordisk Ltd., Novozyme 188, Denmark)를 건조 옥피 대비 각각 1.0 중량%씩 동시에 투입하여 5 시간 동안 반응시켰다. 반응 종료 후 분말 활성탄을 옥피 대비 10 중량%로 첨가하여 95 ℃에서 30 초간 가열하고, 0.45 ㎛의 막여과(Gelman Co., Metricel, USA)를 최종적으로 실시하여 옥피 유래의 수용성 식이섬유를 제조하였다.200 g of dry oxy was mixed with distilled water to a concentration of 8%, and adjusted to pH 5.8 by adding 1N sodium hydroxide solution while stirring with a constant speed stirrer. This was transferred to a constant temperature water bath adjusted to 95 ° C., and heat-resistant alpha-amylase (Novo Nordisk Ltd., Termaryl 120LS, Denmark) was added at 1.0% by weight relative to the solid content of dry octaves, and then reacted for 2 hours, followed by dehydration with a filter cloth. . 3.0 L of distilled water was added to the octane from which the starch was removed, and then adjusted to pH 7.0 by adding 1N sodium hydroxide solution while stirring. Proteolytic enzyme (Novo Nordisk Ltd., Flavorzyme, Denmark) was added to 1.0% by weight relative to the solid content of dry octape and reacted for 3 hours in a constant temperature water bath adjusted to 50 ℃. Subsequently, the filter cloth was dehydrated and dried in an oven. 3 L of sodium hydroxide solution at a concentration of 0.5% was mixed thereto, stirred sufficiently for 3 hours in a constant temperature water bath adjusted to 80 ° C, and filtered through a filter cloth to obtain a primary alkali extract. Then, the pH of the extract was adjusted to 4.8 with 10% hydrochloric acid solution and transferred to a constant temperature water bath adjusted to 50 ° C., followed by cellulase (Novo Nordisk LTD., Celluclast, Denmark) and cellovia (Novo Nordisk Ltd., Novozyme). 188, Denmark) was added at 1.0% by weight, respectively, relative to the dry octagon and reacted for 5 hours. After the completion of the reaction, powdered activated carbon was added at 10% by weight relative to octane, heated at 95 ° C. for 30 seconds, and finally subjected to 0.45 μm membrane filtration (Gelman Co., Metricel, USA) to prepare a water-soluble dietary fiber derived from oxime. .
실시예 2: 비만 예방 효과Example 2: Obesity Prevention Effect
스프라그-돌리(Sprague-Dawley)계 흰쥐를 한 군당 8 마리씩 준비하여 1 주일간 예비사육하였다. 그 후, 상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유(CF)를 4 주간 경구투여하였다. 반면, 대조군으로는 동일량의 셀룰로오즈(CD)를 경구투여시켰다. 사육환경은 온도 23 ±1 ℃, 습도 50 % 전후로 유지하였다. 사육기간 중 각 실험동물은 식이와 음료를 자유로이 섭취토록 하였으며, 체중은 매주 1 회, 식이섭취량은 매일 오전 중에 측정하였다. 실험식이는 AIN-93G를 기준으로 하였으며, 식이의 조성은 하기 표 1과 같고, 순수 섬유소의 중량으로 환산하여 식이 총 무게의 5 %가 되도록 첨가하였다.Sprague-Dawley rats were prepared for 8 rats per group and preliminarily bred for 1 week. Thereafter, the water-soluble dietary fiber (CF) derived from oxime prepared in Example 1 was orally administered for 4 weeks. On the other hand, the same amount of cellulose (CD) was orally administered as a control. Breeding environment was maintained at a temperature of 23 ± 1 ℃, humidity around 50%. During the breeding period, each experimental animal was free to eat food and drink, weight was measured once a week, and dietary intake was measured every morning. The experimental diet was based on AIN-93G, and the composition of the diet was as shown in Table 1 below, and added to be 5% of the total weight of the diet in terms of the weight of pure fiber.
또한, 혈당치는 전자동 혈액분석기(Bayer Co., Chiron Diagnostics)를 이용하여 측정하였으며, 모든 실험 데이터는 평균치표준편차로 나타내었고, 실험식이에 따른 유의성은 아노바(ANOVA)법에 의해 분석하여 각 군의 평균치 차이를 포스트-혹(Post-hoc) 테스트에 의해 P<0.05수준에서 검증하여, 그 결과를 하기 표 2에 나타내었다.In addition, blood glucose levels were measured using a fully automated blood analyzer (Bayer Co., Chiron Diagnostics), and all experimental data were expressed as mean standard deviation, and the significance according to the diet was analyzed by ANOVA. The average difference of was verified at the level of P <0.05 by a post-hoc test, and the results are shown in Table 2 below.
상기 표 2를 통하여, 본 발명의 옥피 유래의 수용성 식이섬유를 섭취한 옥피 식이섬유 그룹(CF)이 셀룰로오즈 그룹(CD)에 비하여 사육 1 주일 후부터의 체중증가량이 낮았으며, 특히 2 주째에 유의하게 낮게 나타났다. 4 주간의 실험기간 중 평균 1 일 식이섭취량을 보면 옥피 유래의 수용성 식이섬유를 섭취한 그룹이 셀룰로오즈를 섭취한 그룹과 비교하여 유의하게 낮았으며, 4 주간의 체중증가량을 총 식이섭취량으로 나누어 식이효율을 계산한 결과, 수용성인 옥피 식이섬유 그룹이 불용성인 셀룰로오즈 그룹보다 다소 낮은 경향을 보였다.Through the above Table 2, the weight gain from oxy dietary fiber group (CF) ingestion of the oxidized fiber derived from the oxidized fiber of the present invention (CF) after 1 week of breeding compared to the cellulose group (CD) was low, especially in the second week significantly Appeared low. The average daily dietary intake during the four-week period was significantly lower in the group that consumed water-soluble fiber from octane compared to the group ingesting cellulose.The dietary efficiency was divided by the total diet intake for four weeks. As a result, the water-soluble octaphyte fiber group tended to be somewhat lower than the insoluble cellulose group.
실시예 3. 항암제와 병용 배양시 항암활성에 대한 영향Example 3. Effect on anticancer activity in co-culture with anticancer agent
한국인의 위암세포인 SNU-1에 본 발명에 따른 상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유와 항암제인 시스플라틴과 병용처리하였을 경우 항암활성에 미치는 영향을 측정하였다.The effect of anti-cancer activity was measured when SNU-1, a gastric cancer cell of Koreans, was used in combination with the phytic-soluble fiber produced in Example 1 according to the present invention and cisplatin, an anticancer agent.
먼저, 위암세포를 10% fetal calf serum(FCS)이 함유된 RPMI 1640 배지를 사용하여 배양한 후, 트리판 블루 배제법으로 세포를 카운팅하고, 96-웰 플렛에 2.0104 개씩 분주하였다. 각 웰에 옥피 식이섬유와 시스플라틴을 0:2.5, 0.5:2.5, 5:2.5, 50:2.5, 500:2.5의 농도로 혼합하여 처리하였다. 반면, 대조군으로는 옥피 유래의 수용성 식이섬유 대신에, 셀룰로오즈와 시스플라틴을 각각 혼합하여 사용하였다. 이를 CO2인큐베이터에서 72 시간 동안 배양시킨 다음, MTT 분석법으로 암세포의 생존도(viability)를 측정하고, 그 결과를 하기 표 3에 나타내었다.First, gastric cancer cells were cultured using RPMI 1640 medium containing 10% fetal calf serum (FCS), and the cells were counted by trypan blue exclusion, and 2.0104 cells were divided into 96-well plates. Oxygen fiber and cisplatin were mixed and treated in each well at a concentration of 0: 2.5, 0.5: 2.5, 5: 2.5, 50: 2.5, and 500: 2.5. On the other hand, cellulose and cisplatin were mixed and used instead of soluble fiber derived from octaphy. This was incubated for 72 hours in a CO 2 incubator, and then measured the viability of cancer cells by MTT assay, the results are shown in Table 3 below.
상기 표 3를 통하여, 위암세포주에 항암제와 함께 상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유를 처리하였을 경우 항암제 단독 처리하였을 경우(암세포 활성 : 100 %)보다 옥피 유래의 수용성 식이섬유는 500 ㎍/mL의 농도에서 암세포 활성을 24.4 %의 수준으로 낮추었으며, 같은 농도의 셀룰로오즈 처리군에서는 18.9 %의 수준까지 낮춤으로써 시스플라틴의 활성을 크게 증가시켜 암세포의 사멸을 촉진함을 알 수 있었다.Through the Table 3, when treated with the anti-cancer soluble fiber fiber prepared in Example 1 to the gastric cancer cell line with the anticancer agent alone (cancer cell activity: 100%) water-soluble dietary fiber derived from oxime 500 It was found that the cancer cell activity was lowered to 24.4% at the concentration of ㎍ / mL, and in the same cellulose treatment group, the activity of cisplatin was greatly increased by lowering the level to 18.9%, thereby promoting the death of cancer cells.
실시예 4: 세포독성 실험Example 4: Cytotoxicity Experiment
상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유와 셀룰로오즈를 한국인 위암세포(SNU-1)에 0, 0.5, 1, 5, 10, 100, 1000 ㎍/mL의 농도별로 처리하고, 72 시간 후 세포 사멸 양상을 현미경으로 관찰하여 세포 독성 유무를 측정하였다. 양성대조군으로 항암제인 시스플라틴과 에토포사이드를 사용하였으며, 암세포는 한국세포주은행에서 제공받아 사용하였다.Oxygen-soluble soluble fiber and cellulose prepared in Example 1 were treated to Korean gastric cancer cells (SNU-1) at concentrations of 0, 0.5, 1, 5, 10, 100, and 1000 μg / mL, and after 72 hours. Apoptosis was observed under a microscope to determine the cytotoxicity. Anti-cancer drugs, cisplatin and etoposide, were used as positive controls, and cancer cells were supplied from Korea Cell Line Bank.
먼저, SNU-1을 10 % 페털 캘프 시럼(fetal calf serum)이 함유된 RPMI 1640 배지를 사용하여 배양한 후, 트립판 블루 배제(trypan blue exclusion)법으로 세포를 카운팅하고 96-웰 플렛(flat bottom)에 2.0104 개씩 분주하였다. 각 웰에 옥피 식이섬유를 0, 1, 10, 100, 1000 ㎍/mL의 농도로 처리하였고, 양성대조군인 시스플라틴과 에토포사이드를 0, 0.05, 1.0, 5.0, 10.0 ㎍/mL의 농도로 처리하였다. 이를 CO2인큐베이터(5% CO2humidified)에서 72 시간 동안 배양한 다음, 위상차 현미경을 이용하여 세포사멸여부를 육안으로 확인하고, 그 결과를 하기 표 4에 나타내었다.First, SNU-1 was incubated using RPMI 1640 medium containing 10% fetal calf serum, followed by counting cells by trypan blue exclusion and 96-well flat. 2.0104 were dispensed at the bottom). Each well was treated with oxidized fiber at concentrations of 0, 1, 10, 100, 1000 μg / mL and positive controls cisplatin and etoposide at concentrations of 0, 0.05, 1.0, 5.0 and 10.0 μg / mL. . This was incubated for 72 hours in a CO 2 incubator (5% CO 2 humidified), and then visually confirmed apoptosis using a phase contrast microscope, the results are shown in Table 4 below.
상기 표 4를 통하여, 상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유와 대조군인 셀룰로오즈를 농도별로 위암세포주에 직접 처리하였을 경우, 1000 ㎍/mL의 고농도에서도 세포의 성장 및 증식에 거의 영향을 미치지 않음을 통하여, 실험에 사용된 식이가 세포독성을 나타내지 않으며, 인체에 안전함을 확인할 수 있었다. 또한, 항암실험이 제대로 진행되었는지를 비교하기 위해 시중에서 임상으로 사용되고 있는 시스플라틴은 1 ㎍/mL에서 항암작용을 나타내기 시작하였으며, 시스플라틴과 에토포사이드는 5 ㎍/mL에서 50 % 이상의 암세포가 사멸하였다.Through Table 4, when the water-soluble dietary fiber derived from oxime prepared in Example 1 and cellulose, a control group, were directly treated to gastric cancer cell lines by concentration, the growth and proliferation of cells were hardly affected even at high concentrations of 1000 ㎍ / mL. It was confirmed that the diet used in the experiment does not exhibit cytotoxicity and is safe for humans. In addition, cisplatin, which is clinically used in the market, began to exhibit anticancer activity at 1 μg / mL, and cisplatin and etoposide were killed at 50 μg / mL by 50% or more. .
실시예 5: 활성화 면역세포의 MHC Ⅰ과 MHC Ⅱ 분자 발현에 미치는 영향Example 5 Effect on MHC I and MHC II Molecular Expression of Activated Immune Cells
장내 면역세포에서 주요 조직적합성 복합체(major histocompatibility complex, MHC)의 발현은 병원체 제거에 중요한 기능을 나타내는데, 우선 하기의 방법으로 혈액 중 림프구와 과립구를 분리하였다. 즉, 사용 직전 헤파린을 처리한 혈액 20 mL를 50 mL의 코니칼 튜브에 넣고, 같은 양의 RMPI(-) 배지를 넣어 희석하였다. 15 mL의 코니칼 원심분리용 튜브에 4 mL Histopaque-1077를 넣은 후, 그 위에 10 mL의 희석액을 넣었다. 이를 실온에서 1500 rpm으로 30 분간 원심분리하고, 상층액(혈장+혈소판)을 파스퇴르 피펫으로 제거한 다음, histopaque층 위에 생성된 사람말초혈액단핵세포(PBMC)층을 새로운 튜브에 옮겼다. 그 다음, PBMC층 부피의 3 배에 해당하는 PBS를 넣어 주고, 1500 rpm으로 10 분간 원심분리하여 상층액을 제거하였다. 이를 2 회 반복하여 세포를 세척한 후, 세포 펠렛을 RPMI 배지에 현탁시켰다. 트리판 블루 배제법으로 세포수를 측정하여 8.0 ×105cells/well, 1.2 ×105cells/well의 세포를 각각 96 웰 플렛(flat bottom)에 분주하였다. 각 웰에 상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유, IFN-γ, TNF-α를 각각 하기 표 5, 및 표 6에 나타낸 조성으로 처리하여 36 시간 동안 배양하였다(37 ℃, 5 % CO2hmidified). 배양 3 일 경과 후, 세포들을 각각의 웰 별로 서로 다른 에펜도르프 튜브로 옮기고, 800 ㎕의 PBS를 첨가하여 2100 rpm으로 10 분간 원심분리하여 상층액을 제거하고, 펠렛을 현탁시킨 후, 200 ㎕의 PBS(0.2 % NaN3)를 넣고 얼음위에서 30 분간 배양하였다. 이를 다시 2100 rpm으로 10 분간 원심분리하여 상층액을 제거하고, 여기에 50 ㎕의 항체 희석액을 첨가하였다. 각각의 튜브를 교반기(rotator)에 장착하여 4 ℃의 암실에서 1 시간 동안 반응시킨 다음, PBS(0.2 % NaN3) 1 mL로 3 회 세척하고, 최종적으로 100 ㎕의 PBS(0.2 % NaN3)로 세포를 현탁시켰다. 상기 준비된 세포를 5 mL의 튜브에 옮기고 PBS로 희석시킨 후, FACScan의 시료주입부(sample injection port)에 장착하여 MHC Ⅰ과 MHC Ⅱ의 발현 양상을 임파구(lymphocyte)와 과립구(granulocyte)로 각각 분석하고, 그 결과를 각각 하기 표 5와 6에 나타내었다.The expression of major histocompatibility complex (MHC) in intestinal immune cells is an important function for pathogen removal. Lymphocytes and granulocytes were isolated from blood by the following method. That is, 20 mL of heparinized blood immediately before use was placed in a 50 mL conical tube, and the same amount of RMPI (-) medium was added and diluted. 4 mL Histopaque-1077 was placed in a 15 mL conical centrifuge tube, followed by 10 mL dilution. It was centrifuged at 1500 rpm for 30 minutes at room temperature, the supernatant (plasma + platelet) was removed with a Pasteur pipette, and then the human peripheral blood mononuclear cell (PBMC) layer formed on histopaque layer was transferred to a new tube. Then, PBS corresponding to three times the volume of the PBMC layer was added thereto, and the supernatant was removed by centrifugation at 1500 rpm for 10 minutes. This was repeated twice to wash the cells and then the cell pellets were suspended in RPMI medium. Cell number was measured by trypan blue exclusion method and the cells of 8.0 × 10 5 cells / well, 1.2 × 10 5 cells / well were dispensed into 96 well flat (flat bottom), respectively. Oxygen-derived water-soluble fiber, IFN-γ, and TNF-α prepared in Example 1 were treated in each well with the compositions shown in Tables 5 and 6, respectively, and incubated for 36 hours (37 ° C., 5%). CO 2 hmidified). After 3 days of culture, cells were transferred to different Eppendorf tubes for each well, centrifuged at 800 rpm for 10 minutes at 2100 rpm to remove supernatant, and pellets were suspended. PBS (0.2% NaN 3 ) was added and incubated for 30 minutes on ice. This was again centrifuged at 2100 rpm for 10 minutes to remove the supernatant, to which 50 μl of antibody dilution was added. Each of mounting the tubing to the agitator (rotator) to perform reaction for 1 hour at 4 ℃ dark room, and then, PBS (0.2% NaN 3) 1 mL to 3 times wash, and finally a 100 ㎕ PBS (0.2% NaN 3 ) Cells were suspended. The prepared cells were transferred to 5 mL tubes, diluted with PBS, mounted on a sample injection port of FACScan, and analyzed for expression of MHC I and MHC II by lymphocytes and granulocytes, respectively. And the results are shown in Tables 5 and 6, respectively.
일반적으로 MHC Ⅰ의 증가는 바이러스의 침입 억제능의 증가와 관련있으며, MHC Ⅱ의 증가는 세균과 장내 독소의 침입 억제 및 제거능의 증가와 관련있다고 알려져 있다.In general, an increase in MHC I is associated with an increase in the ability to inhibit the invasion of viruses, and an increase in MHC II is known to be associated with an increase in the inhibition and clearance of bacterial and enteric toxins.
상기 표에서 막발현 세포수의 증가는 MHC 분자의 막발현 세포수가 증가한 것을 의미하며, 형광세기의 증가는 단위 세포당 MHC 분자의 막발현 개수가 증가한 것을 의미한다. 상기 표 5에 나타낸 바와 같이, 임파구에서는 MHC Ⅰ의 발현이 뚜렷히 나타났으며, 본 발명의 5 ㎍/mL 농도의 옥피 유래의 수용성 식이섬유 처리시 약 10 배 이상의 형광세기(fluorescent intensity) 증가를 나타내었다. 또한, 본 발명의 옥피 유래의 수용성 식이섬유를 IFN-γ 및 TNF-α와 병용처리한 경우, IFN-γ 및 TNF-α만을 처리한 경우와 비교하여 막발현 세포수가 식이섬유의 처리 농도에 비례하여 증가함을 확인할 수 있었다.In the above table, the increase in the number of membrane expressing cells means that the number of membrane expressing cells of MHC molecules is increased, and the increase in fluorescence intensity means that the number of membrane expressions of MHC molecules per unit cell is increased. As shown in Table 5, the expression of MHC I was clearly observed in lymphocytes, and an increase in fluorescence intensity of about 10 times or more was observed upon treatment with water-soluble dietary fiber derived from the oxidized 5 ㎍ / mL concentration of the present invention. It was. In addition, when the water-soluble dietary fiber derived from the oxime of the present invention was treated in combination with IFN-γ and TNF-α, the number of membrane-expressing cells was proportional to the treatment concentration of the dietary fiber compared to the case where only IFN-γ and TNF-α were treated. It was confirmed that the increase.
또한, 상기 표 6에 나타낸 바와 같이, 과립구에서는 MHC Ⅰ과 Ⅱ가 모두 발현하였으며, 상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유와 IFN-γ및 TNF-α를 병용처리할 경우, MHC Ⅰ의 형광세기가 5 ㎍/mL 이하 농도에서는 증가를, 50 ㎍/mL 이상의 농도에서는 감소를 보였다. 반면, MHC Ⅱ의 형광세기는 50 ㎍/mL 이하의 농도에서는 증가를, 500 ㎍/mL의 농도에서는 감소를 보였다. 셀룰로오즈의 경우, 옥피 유래의 수용성 식이섬유에 비해 효과가 미약하게 나타났다.In addition, as shown in Table 6, in the granulocytes, both MHC I and II were expressed, and MHC I was treated in combination with IFN-? And TNF-? The fluorescence intensity of was increased at the concentration of 5 µg / mL or less and decreased at the concentration of 50 µg / mL or more. On the other hand, the fluorescence intensity of MHC II increased at concentrations below 50 μg / mL and decreased at concentrations of 500 μg / mL. In the case of cellulose, there was a slight effect compared to soluble fiber derived from oxime.
이상의 결과를 통하여, 적정농도의 옥피 유래의 수용성 식이섬유는 임파구와 과립구의 면역 활성화를 향상시키는 작용이 있음을 확인할 수 있었다.Through the above results, it was confirmed that the water-soluble dietary fiber derived from the appropriate concentration of oxime has the effect of improving the immune activation of lymphocytes and granulocytes.
실시예 6: 면역활성 조절에 대한 옥피 유래 식이섬유의 영향Example 6 Effect of Oxy-derived Dietary Fiber on Immune Activity Regulation
사용 직전 헤파린을 처리한 혈액(fresh heparized blood) 20 mL을 50 mL의 코티칼 튜브(conical tube)에 넣고, 같은 양의 RMPI(-) 배지를 넣어 희석였다. 15 mL의 코티칼 원심분리용 튜브(conical centrifuge tube)에 Histopaque-1119 4 mL를 넣은 후, 그 위에 10 mL 희석액을 넣었다. 이를 실온에서 2700 rpm으로 30 분간 원심분리하고, 상층액(혈장)을 파스퇴르 피펫으로 제거한 다음, histopaque-1119층 위에 생성된 상층(PBMC + granulocyte)을 새로운 튜브에 옮겼다. 튜브당 총 부피가 10 mL가 되도록 PBS를 넣고, 1600 rpm으로 10 분간 원심분리하여 상층액을 제거하였다. 이를 2 회 반복하여 세척한 후, 세포 펠렛(cell pellet)을 RPMI(without phenol red) 배지에 현탁시켰다. 트립판 블루 배제법으로 세포농도와 생존도를 측정한 결과 95 % 이상의 생존도를 나타냈다. 이를 1.5 ×107세포/mL의 농도로 100 ㎕씩 96 웰 플렛에 분주하였다. 이에 상기 실시예 1에서 제조한 옥피 유래의 수용성 식이섬유와 면역촉진제인 PHA-L, PHA-P, 또는 PMA/Ionomycin, 면역억제제인 덱사메타손(dexamethasone)을 각각 0:2.5, 0.5:2.5, 5:2.5, 50:2.5, 500:2.5 ㎍/mL의 농도로 혼합하여 세포에 처리하였다. 이를 3 일간 배양한 후(37 ℃, 5 % CO2hmidified), MTT 분석법을 실시하여 사람말초혈액단핵세포(PBMA)의 세포 생존도를 측정하였다. 각각의 실험은 3 회 반복실시하고, 그 평균값을 하기 표 7 내지 표 10에 나타내었다.Immediately before use, 20 mL of fresh heparized blood was placed in 50 mL of conical tubes and diluted in the same amount of RMPI (-) medium. 4 mL of Histopaque-1119 was added to a 15 mL conical centrifuge tube, followed by a 10 mL dilution. It was centrifuged at 2700 rpm for 30 minutes at room temperature, the supernatant (plasma) was removed with a Pasteur pipette and the supernatant (PBMC + granulocyte) produced on histopaque-1119 layer was transferred to a new tube. PBS was added so that the total volume per tube was 10 mL, and the supernatant was removed by centrifugation at 1600 rpm for 10 minutes. After washing twice, the cell pellet was suspended in RPMI (without phenol red) medium. Cell concentration and viability were measured by trypan blue exclusion and showed a survival rate of more than 95%. This was dispensed into 96 well plates at 100 μl at a concentration of 1.5 × 10 7 cells / mL. Thus, the water-soluble fiber derived from the oxime prepared in Example 1 and PHA-L, PHA-P, or PMA / Ionomycin, an immunostimulant, dexamethasone (dexamethasone), respectively, were 0: 2.5, 0.5: 2.5, and 5: The cells were mixed and mixed at concentrations of 2.5, 50: 2.5, and 500: 2.5 μg / mL. After culturing for 3 days (37 ° C., 5% CO 2 hmidified), MTT assay was performed to measure cell viability of human peripheral blood mononuclear cells (PBMA). Each experiment was repeated three times, and the average value is shown in Tables 7 to 10 below.
상기 표 7, 표 8, 및 표 9에 나타난 바와 같이, 본 발명의 옥피 유래의 수용성 식이섬유를 면역촉진제인 PHA-L 또는 PHA-P와 병용처리할 경우 옥피 유래의 수용성 식이섬유 50 ㎍/mL 이하의 농도에서 면역세포에서의 과민성 면역촉진 효과를 유의하게 경감시켰으며, PMA/Ionomycin와 병용처리할 경우에는 0.5 ㎍/mL 이상의 농도에서 면역세포에서의 과민성 면역촉진 효과를 유의하게 경감시킴을 확인할 수 있었다. 반면, 셀룰로오즈는 면역촉진제인 PMA/Ionomycin와 병용처리시에만 과민성 면역촉진 효과를 경감시키는 효과를 나타내었다. 이로부터 본 발명의 옥피 유래의 수용성 식이섬유의 과민성 면역반응 억제 작용 즉, 과민성 대장염 완화 작용을 기대할 수 있었다.As shown in Table 7, Table 8, and Table 9, 50 μg / mL of the water-soluble dietary fiber derived from the oxime when the water-soluble dietary fiber derived from the oxime of the present invention is used in combination with an immunostimulating agent, PHA-L or PHA-P. In the following concentrations, the immune-stimulating effect on immune cells was significantly reduced, and when combined with PMA / Ionomycin, it significantly reduced the immune-stimulating effect on immune cells at concentrations of 0.5 ㎍ / mL or more. Could. Cellulose, on the other hand, showed an effect of reducing the hypersensitive immunostimulating effect only in combination with the immunostimulant PMA / Ionomycin. From this, it was anticipated that the sensitizing immune response inhibitory action, that is, the sensitizing colitis relieving action, of the water-soluble dietary fiber derived from oxime of the present invention.
또한, 상기 표 10을 통하여 본 발명의 옥피 유래의 수용성 식이섬유를 면역억제제인 덱사메타손과 병용처리할 경우 면역세포활성을 회복시킴을 확인할 수 있었다.In addition, it can be seen from Table 10 that the treatment with oxime-derived fiber of the present invention in combination with dexamethasone, an immunosuppressive agent, restores immune cell activity.
상기에서 살펴본 바와 같이, 본 발명의 옥피 유래의 수용성 식이섬유를 포함하는 조성물은 비만예방 효과가 우수할 뿐만 아니라, 항암활성 및 면역조절능력이 우수한 효과가 있다. 특히, 항암제나 면역촉진제와 병용처리시 상승작용 나타내므로 약제, 가공식품, 기능성 식품, 식품첨가제, 기능성 음료, 또는 음료첨가제 등에 유용하게 사용될 수 있다.As described above, the composition comprising the water-soluble dietary fiber derived from oxime of the present invention is not only excellent in preventing obesity, but also has an excellent anti-cancer activity and immunomodulatory ability. In particular, since it shows synergistic effect in combination with anticancer agents or immunostimulants, it can be usefully used in drugs, processed foods, functional foods, food additives, functional beverages, or beverage additives.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6277418B1 (en) * | 1998-06-02 | 2001-08-21 | Baylor College Of Medicine | Corn extract contraceptive |
| US20020010151A1 (en) * | 2000-05-15 | 2002-01-24 | Nihon Shokuhin Kako Co., Ltd. | Immuno-active agent, method of its use, and method of immunity activation |
| JP2002338475A (en) * | 2001-05-14 | 2002-11-27 | Nippon Shokuhin Kako Co Ltd | Antitumor composition |
| KR100413384B1 (en) * | 2000-10-24 | 2004-01-03 | 주식회사 삼양제넥스 | Method for preparing soluble dietary fiber from corn hull |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6277418B1 (en) * | 1998-06-02 | 2001-08-21 | Baylor College Of Medicine | Corn extract contraceptive |
| US20020010151A1 (en) * | 2000-05-15 | 2002-01-24 | Nihon Shokuhin Kako Co., Ltd. | Immuno-active agent, method of its use, and method of immunity activation |
| KR100413384B1 (en) * | 2000-10-24 | 2004-01-03 | 주식회사 삼양제넥스 | Method for preparing soluble dietary fiber from corn hull |
| JP2002338475A (en) * | 2001-05-14 | 2002-11-27 | Nippon Shokuhin Kako Co Ltd | Antitumor composition |
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| Title |
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| Carcinogenesis, 1984 Feb, 5(2), 261-4 * |
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