KR100963511B1 - Exopolysaccharides for type 2 diabetes therapy from Phellinus baumii mycellium and a preparation method thereof - Google Patents
Exopolysaccharides for type 2 diabetes therapy from Phellinus baumii mycellium and a preparation method thereof Download PDFInfo
- Publication number
- KR100963511B1 KR100963511B1 KR1020070036778A KR20070036778A KR100963511B1 KR 100963511 B1 KR100963511 B1 KR 100963511B1 KR 1020070036778 A KR1020070036778 A KR 1020070036778A KR 20070036778 A KR20070036778 A KR 20070036778A KR 100963511 B1 KR100963511 B1 KR 100963511B1
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- South Korea
- Prior art keywords
- phellinus
- extracellular polysaccharide
- baumii
- mycelium
- type
- Prior art date
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Abstract
본 발명은 상황 버섯(Phellinus baumii)의 균사체로부터 얻은 항당뇨 활성이 있는 세포외다당체 및 그 제조방법에 관한 것으로, 상황 버섯(Phellinus baumii) 균사체로부터 생산되는 세포외다당체를 2형 당뇨 마우스(ob/ob)에 투여할 경우, 혈장 내 글루코우즈 함량을 현저하게 감소시킬 수 있을 뿐만 아니라 혈중의 지질을 감소시킴으로써 뛰어난 고지혈증 치료 효과도 동시에 얻을 수 있어 당뇨환자의 혈당을 조절하거나 비만 등으로 인한 고지혈증을 개선시킬 수 있는 매우 뛰어난 효과가 있다.The present invention is a situation mushroom ( Phellinus Cells that have antidiabetic activity obtained from mycelia of baumii) other polysaccharides, and relates to a method of manufacturing the same, circumstances mushroom (Phellinus When the extracellular polysaccharide produced from the mycelium is administered to type 2 diabetic mice ( ob / ob ), not only can the glucose content in the plasma be significantly reduced, but also the excellent hyperlipidemic effect can be achieved by reducing the blood lipids. It is possible to obtain a very excellent effect to control the blood sugar of diabetic patients or improve hyperlipidemia due to obesity.
상황 버섯, 당뇨병, 혈당강하, 세포외다당체, 고지혈증 Situation Mushroom, diabetes, hypoglycemic, extracellular polysaccharide, hyperlipidemia
Description
도 1은 2형 당뇨 마우스(ob/ob)에서 혈장 내 글루코우즈 농도에 대한 상황 버섯(Phellinus baumii) 균사체 유래 세포외다당체의 혈당저하효과를 나타낸 그래프이다.1 is a situation mushroom ( Phellinus) for the concentration of glucose in the plasma in type 2 diabetic mice ( ob / ob ) baumii ) is a graph showing the hypoglycemic effect of mycelia derived extracellular polysaccharides.
도 2는 2형 당뇨 마우스(ob/ob)에서 혈장 내 글루코우즈 농도에 대한 상황 버섯(Phellinus baumii) 균사체 유래 세포외다당체의 당부하실험 결과를 나타낸 그래프이다.2 is a situation mushroom ( Phellinus ) for the glucose concentration in plasma in type 2 diabetic mice ( ob / ob ) baumii ) is a graph showing the results of a glucose tolerance test on mycelia derived extracellular polysaccharides.
도 3은 2형 당뇨 마우스(ob/ob)에서 분리해 낸 지방세포에서 PPARγ유전자 발현량을 조사하여 혈당강하 효과를 분자 생물학적으로 증명한 그래프이다.FIG. 3 is a graph showing molecular biological effects of hypoglycemic effect by examining the expression level of PPARγ gene in adipocytes isolated from type 2 diabetic mice ( ob / ob ).
도 4는 2형 당뇨 마우스(ob/ob)에서 분리해 낸 혈액에서 PPARγ유전자 발현량을 조사하여 혈당강하 효과를 분자 생물학적으로 증명한 그래프이다.4 is a graph showing molecular biological effects of hypoglycemic effect by investigating the expression of PPARγ gene in blood isolated from type 2 diabetic mice ( ob / ob ).
본 발명은 상황 버섯 균사체 유래의 항당뇨 활성 세포외다당체 및 그 제조방 법에 관한 것으로, 더욱 상세하게는 상황 버섯(Phellinus baumii) 균사체의 액침배양액으로부터 세포외다당체를 분리하고 상기 다당체의 혈당저하효과를 조사함으로써 제공할 수 있는 상황버섯 균사체 유래의 항당뇨 활성 세포외다당체 및 그 제조방법에 관한 것이다.The present invention relates to an anti-diabetic active extracellular polysaccharide derived from S. mushroom mycelium and a method for preparing the same, and more particularly, to separate the extracellular polysaccharide from the immersion medium of Phellinus baumii mycelium and to lower the glycemic effect of the polysaccharide. The present invention relates to an antidiabetic active extracellular polysaccharide derived from a situation mushroom mycelium and a method for producing the same.
제 2형 당뇨병은 현재 전 세계인구의 5~7%인 1억 3500만 명에 이환되어 있으며 향후 20년 내에 3억 명으로 폭발적인 증가가 예측된다. 이러한 2형 당뇨를 치료하기 위해 다양한 유형의 구강 당뇨병치료제들이 이용되어 왔으나, 부작용이 없는 천연물 유래의 항당뇨 물질을 이용하고자 하는 환자들이 늘어가고 있다. 합성 항당뇨제를 계속적으로 이용할 경우 부작용과 독성이 유발되기 때문이다. 따라서 효율, 부작용의 제한, 비교적 낮은 비용 때문에 생물학적 활성 성분이 밝혀지지 않음에도 불구하고 한약재 등의 천연물질들이 널리 처방되고 있다. 오랜 치료를 통해, 한방에서는 당뇨병 치료에 대한 많은 유용한 경험이 축적되어 왔다. 오늘날, 당뇨병과 그것의 합병증을 임상적으로 치료하기 위해 민간 한약 처방으로 주로 사용되어온 한약재로는 대략 33종이 있다. 한약재에 있는 항당뇨 활성 물질로는 다당류, 테르페노이드, 플라보노이드, 스테롤 및 알카노이드가 있다. 많은 나라들에서, 다양한 약용식물에서 추출한 천연 항당뇨제를 발견하기 위한 노력을 기울여 왔다.Type 2 diabetes is currently affected by 135 million people, 5-7% of the world's population, and an explosive increase is projected to 300 million in the next 20 years. Various types of oral diabetes treatments have been used to treat type 2 diabetes, but more and more patients are trying to use anti-diabetic substances derived from natural products without side effects. Continuous use of synthetic antidiabetics can cause side effects and toxicity. Therefore, even though biologically active ingredients are not revealed due to efficiency, limitation of side effects, and relatively low cost, natural substances such as herbal medicines are widely prescribed. Through long treatments, many useful experiences for treating diabetes have been accumulated in oriental medicine. Today, there are approximately 33 types of herbal medicines that have been used mainly in folk medicine prescriptions to clinically treat diabetes and its complications. Antidiabetic active substances in herbal medicines include polysaccharides, terpenoids, flavonoids, sterols and alkanoids. In many countries, efforts have been made to find natural antidiabetics derived from various medicinal plants.
버섯과 동충하초는 항당뇨 활성을 갖는 대표적인 천연 약재원이다. 과학적 증거가 크게 부족함에도 불구하고, 많은 연구자가 트리멜라 아우란티아(Tremella aurantia), 시넨시스 동충하초(Cordyceps sinensis), 표고버섯(Lentinus edodes)을 포함하여 다양한 식용 및 약용 곰팡이의 자실체 또는 균사체로부터 항당뇨 효과를 연구하기 위해 노력해 왔다. 로 등(Lo, H.C. et al., Life Sciences 74, 2897-2908, 2004)은 동충하초 자실체가 당뇨병 치료를 위한 기능성 식품으로 이용가능성이 있음을 보고하였다. 키호 등(Kiho, T. et al., Carbohydrate research 251, 81-87, 1994)은 버들볏짚버섯(Agrocybe cylindracea) 자실체의 열수추출물이 항당뇨 효과를 갖는다고 주장하였다. 유안 등(Yuan, Z. et al., Bioscience, Biotechnology, and Biochemistry 62, 1898-1903, 1998)은 목이버섯(Auricularia auricula - judae Que)의 자실체로부터 수용성 다당체의 항당뇨 활성을 조사하였다. 그럼에도 불구하고, 이러한 목적을 위해, 약용버섯의 자실체가 아닌 균사체 배양액으로부터 세포외다당체를 얻으려는 노력은 지금까지 크게 부족한 실정이다.Mushrooms and Cordyceps sinensis are representative natural medicinal sources with antidiabetic activity. Despite the large lack of scientific evidence, many researchers have found Tremella aurantia ), Cordyceps sinensis ) and Lentinus edodes have been working to study antidiabetic effects from fruiting bodies or mycelium of various edible and medicinal fungi. (Lo, HC et al., Life Sciences 74, 2897-2908, 2004) reported that Cordyceps Sinensis may be used as a functional food for the treatment of diabetes. Kiho, T. et al., Carbohydrate research 251, 81-87, 1994, described Agrocybe cylindracea ) fruit water extracts were claimed to have antidiabetic effects. Yuan, Z. et al., Bioscience, Biotechnology, and Biochemistry 62, 1898-1903, 1998, reported that Auricularia The antidiabetic activity of water-soluble polysaccharides from fruiting bodies of auricula - judae Que) was investigated. Nevertheless, for this purpose, efforts to obtain extracellular polysaccharides from mycelium culture medium, which are not fruiting bodies of medicinal mushrooms, have been greatly lacked until now.
한편, 상황버섯(Phellinus baumii)은 몇몇 아시아 국가들에서 사람의 다양한 질병을 위한 민간 약재로써 이용되어온 버섯이다. 장 등(Jang et al., Biotechnology Letters 26, 31-33, 2004)은 상황버섯(Phellinus baumii) 추출물이 사람의 급성폐렴을 억제하는데 유용할 것이라고 보고하였다. 또한, 손 등(Shon et al., Food Chemistry 82, 593-597, 2003)은 상황버섯(Phellinus baumii) 추출물의 항산화활성 및 자유 라디칼 소거능을 규명하였다. 생물학적 활성능을 조사하기 위해, 대부분의 연구자들이 균사체 액침배양액에서 얻은 세포외다당체보다는 상황버섯(Phellinus baumii)의 자실체를 주로 이용하였으나, 균사체배양에 의해 생산되는 세포외다당체에 관한 항당뇨 효과에 관한 보고는 없다.Meanwhile, the situation mushroom ( Phellinus baumii ) is a mushroom that has been used as a medicinal herb for various human diseases in some Asian countries. Zhang et al., Biotechnology Letters 26, 31-33, 2004, describe Phellinus baumii ) extract may be useful for suppressing acute pneumonia in humans. In addition, Shon et al., Food Chemistry 82, 593-597, 2003, described Phellinus baumii ) extracts were characterized for their antioxidant activity and free radical scavenging ability. In order to investigate the biological activity, most researchers used Phellinus rather than the extracellular polysaccharide obtained from the mycelial immersion medium. baumii fruiting body was mainly used, but there are no reports of antidiabetic effects on extracellular polysaccharides produced by mycelial culture.
다만, 본 발명자가 특허출원하여 등록된 대한민국 특허 제10-0663712호에 상황버섯 균사체의 액침배양액을 과당, 효모추출물, 펩톤 및 염화칼슘이 함유된 배지 에서 배양온도를 30℃로 하여 4일 동안 진탕배양한 후 원심분리한 다음 여과하여 얻은 배양여과액에 에탄올을 첨가하여 교반한 뒤 방치하고 원심분리함으로써 제조한 항당뇨 활성 세포외다당체가 개시되어 있으나 상기 세포외다당체는 그 구성 단당류가 주로 만노우즈 48.7%와 아라비노우즈 38.4%로 이루어져 있으며 단백질 부분은 주로 아르기닌 14.1%와 글리신 12.0%로 구성된 다당체에 관한 것이다.However, in Korean Patent No. 10-0663712, which the inventors have applied for a patent, the immersion culture liquid of the situation mushroom mycelium was cultured in a culture medium containing fructose, yeast extract, peptone and calcium chloride at 30 ° C. for 4 days for shaking culture. Thereafter, antidiabetic active extracellular polysaccharides prepared by centrifugation, filtration and filtration of the culture filtrate obtained by adding ethanol, left to stand, and centrifugation are disclosed, but the extracellular polysaccharide is mainly composed of mannose 48.7. % And arabinose 38.4%, and the protein portion mainly relates to polysaccharides consisting of 14.1% arginine and 12.0% glycine.
본 발명자들은 상기와 같은 점을 감안하여 연구하던 중 상황 버섯(Phellinus baumii)의 균사체 액침배양액으로부터 강력한 항당뇨 활성을 가지는, 탄수화물(78.9%)과 단백질(21.1%)로 구성되며, 단백질부분은 글루타민산을 비롯한 17개의 아미노산으로 이루어져 있고, 탄수화물부분은 만노우즈와 갈락토우즈로 이루어져 있는 세포외다당체를 분리할 수 있음을 확인함으로써 본 발명을 완성하였다.In view of the above, the inventors of the present invention consisted of carbohydrate (78.9%) and protein (21.1%), which have strong antidiabetic activity from the mycelium immersion medium of Phellinus baumii , and the protein part is glutamic acid. It consists of 17 amino acids, including the carbohydrate portion was completed by confirming that the extracellular polysaccharide consisting of mannose and galactose can be separated.
따라서, 본 발명의 목적은 상황 버섯(Phellinus baumii)의 균사체 액침배양액으로부터 생산되는 항당뇨 활성을 갖는 세포외다당체를 제공하는 것이다.Therefore, an object of the present invention is a situation mushroom ( Phellinus It is to provide an extracellular polysaccharide having antidiabetic activity produced from the mycelium liquid immersion medium of baumii ).
본 발명의 또 다른 목적은 상황 버섯(Phellinus baumii)의 균사체 액침배양액으로부터 세포외다당체를 제조하는 방법을 제공하는 것이다.Another object of the present invention is a situation mushroom ( Phellinus It is to provide a method for producing an extracellular polysaccharide from mycelium immersion culture solution of baumii ).
본 발명의 상기 목적은 상황 버섯(Phellinus baumii) 균사체를 액침배양하여 균사체배양액을 얻고 상기 균사체배양액을 여과하여 배양여과액을 얻은 다음 여기에 에탄올을 첨가하여 추출함으로써 세포외다당체를 얻은 다음 2형 당뇨 마우스(ob/ob)에 구강투여하여 혈장 내 글루코우즈, 콜레스테롤 및 트리글리세라이드 농도를 측정하여 상기 세포외다당체의 항당뇨 활성을 확인하고 마우스의 지방세포 를 분리하여 혈당강하에 핵심적인 요소인 PPARγ 유전자를 분석하여 분자적 메카니즘을 규명함으로써 달성하였다.The object of the present invention is to immerse the mycelium ( Phellinus baumii ) mycelium culture to obtain a mycelium culture broth, and to obtain the culture filtrate by filtering the mycelium culture broth and then to obtain the extracellular polysaccharide by extraction with ethanol added to type 2 diabetes Plasma glucose, cholesterol, and triglyceride levels in mice ( ob / ob ) were orally administered to confirm the antidiabetic activity of the extracellular polysaccharides and to separate the fat cells of the mouse PPARγ gene, a key factor in hypoglycemic activity By analyzing the molecular mechanism.
이하, 발명의 구성을 구체적으로 설명한다.EMBODIMENT OF THE INVENTION Hereinafter, the structure of this invention is demonstrated concretely.
본 발명은 상황 버섯(Phellinus baumii)의 균사체액침배양액으로부터 세포외다당체를 얻는 단계; 2형 당뇨 마우스(ob/ob)에 상기 세포외다당체를 구강투여하여 혈장 내 글루코우즈, 콜레스테롤 및 트리글리세라이드 농도 측정를 측정하여 상기 세포외다당체의 항당뇨 활성을 확인하는 단계; 및 마우스의 지방세포를 분리하여 혈당강하에 핵심적인 요소인 PPARγ 유전자를 분석하여 분자적 메카니즘을 규명하는 단계로 구성된다.The present invention is a situation mushroom ( Phellinus obtaining extracellular polysaccharide from the mycelium solution immersion medium of baumii ); Confirming antidiabetic activity of the extracellular polysaccharide by orally administering the extracellular polysaccharide to type 2 diabetic mice ( ob / ob ) to measure glucose, cholesterol and triglyceride concentrations in plasma; And isolating mouse fat cells to analyze the PPARγ gene, which is a key factor in hypoglycemic activity, to identify molecular mechanisms.
본 발명은 항당뇨 활성을 가지며, 탄수화물 75~85중량%와 단백질 15~25중량%로 구성되고, 단백질 부분은 아스파틴산을 비롯한 17개의 아미노산 즉, 트레오닌, 세린, 글루타민산, 프롤린, 글리신, 알라닌, 시스테인, 발린, 메티오닌, 이소루신, 루신, 티로신, 페닐알라닌, 히스티딘, 라이신, 아르기닌으로 이루어져 있고, 탄수화물 부분의 주요당은 만노우즈와 갈락토우즈을 특징으로 하는 상황 버섯(Phellinus baumii)의 균사체배양액 유래의 세포외다당체를 제공한다.The present invention has an anti-diabetic activity and consists of 75 to 85% by weight carbohydrate and 15 to 25% by weight protein, the protein portion of 17 amino acids including aspartic acid, that is, threonine, serine, glutamic acid, proline, glycine, alanine, It consists of cysteine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine and arginine, and the main sugars in the carbohydrate fraction are derived from the mycelium culture of Phellinus baumii characterized by mannose and galactose It provides an extracellular polysaccharide.
본 발명에서 상기 항당뇨 활성 상황 버섯(Phellinus baumii)의 균사체배양액 유래의 세포외다당체를 구성하는 단백질 부분은 아스파틴산 13~17중량%, 트레오닌 5~9중량%, 세린 6~10중량%, 글루타민산 12~16중량%, 프롤린 4~8중량%, 글리신 7~11중량%, 알라닌 3~7중량%, 시스테인 1~2중량%, 발린 2~6중량%, 메티오닌 1~2중량%, 이소루신 1~4중량%, 루신 1~4중량%, 티로신 1~2중량%, 페닐알라닌 1~4중량%, 히스티딘 1~3중량%, 라이신 8~12중량%, 아르기닌 2~6중량%로 이루어져 있고, 탄수화물 부분의 주요당은 만노우즈 88~94중량%와 갈락토우즈 5~10중량%임을 특징으로 한다.In the present invention, the protein portion constituting the extracellular polysaccharide derived from the mycelium culture medium of the anti-diabetic activity ( Phellinus baumii ) is 13 to 17% by weight, aspartic acid, 5 to 9% by weight, threonine, 6 to 10% by weight, glutamic acid 12-16 wt%, Proline 4-8 wt%, Glycine 7-11 wt%, Alanine 3-7 wt%, Cysteine 1-2 wt%, Valine 2-6 wt%, Methionine 1-2 wt%, Isoleucine 1-4% by weight, leucine 1-4%, tyrosine 1-2%, phenylalanine 1-4%, histidine 1-3%, lysine 8-12%, arginine 2-6% The main sugars in the carbohydrate portion are mannose 88-94% by weight and galactose 5-10% by weight.
본 발명 상황 버섯(Phellinus baumii) 균사체배양액 유래 항당뇨 활성 세포외다당체의 가장 바람직한 구성은 탄수화물 78.9중량%와 단백질 21.1중량%로 구성되며, 탄수화물은 만노우즈 92.18중량%와 갈락토우즈 6.24중량%로 이루어져 있는 것을 특징으로 한다.In the present invention, the most preferred composition of the mushroom ( Phellinus baumii ) mycelium culture-derived antidiabetic active extracellular polysaccharide is composed of 78.9% by weight carbohydrate and 21.1% by weight protein, carbohydrate is 92.18% by weight mannose and 6.24% by weight galactose It is characterized by consisting of.
본 발명은 또한 상기 세포외다당체를 유효성분으로 포함하는 항당뇨용 식품조성물을 제공함을 특징으로 한다.The present invention is also characterized by providing an antidiabetic food composition comprising the extracellular polysaccharide as an active ingredient.
본 발명은 또한 상기 세포외다당체를 유효성분으로 포함하는 고지혈증 개선용 식품조성물을 제공함을 특징으로 한다.The present invention is also characterized by providing a food composition for improving hyperlipidemia comprising the extracellular polysaccharide as an active ingredient.
본 발명 상황 버섯(Phellinus baumii)의 균사체배양액 유래의 세포외다당체는 상황 버섯(Phellinus baumii)의 균사체 액침배양여과액에 4배 부피의 에탄올을 첨가하고 4℃에서 방치하여 얻는다.Invention situation mushroom ( Phellinus Extracellular Polysaccharide of mycelial culture of origin baumii) is Phellinus (Phellinus baumii ) was obtained by adding 4 times the volume of ethanol to the mycelial immersion filtrate and standing at 4 ° C.
본 발명의 상황 버섯(Phellinus baumii) 균사체배양액에서 생산되는 세포외다당체는 1일 기준으로 마우스 체중 1kg 당 200 mg으로 구강 투여한다. 실험 마지막 날 마우스의 지방세포를 채취한 후 지방세포의 전체 RNA를 분리하여 분자생물학 실험을 수행한다.Situation mushroom of the present invention ( Phellinus baumii ) Extracellular polysaccharide produced in mycelial culture is administered orally at 200 mg per kg body weight of the mouse on a daily basis. On the last day of the experiment, mouse cells were harvested and the entire RNA of the adipocytes was isolated to perform molecular biology experiments.
이하, 본 발명의 구체적인 방법을 실시예를 들어 상세히 설명하고자 하지만 본 발명의 권리범위는 이들 실시예에만 한정되는 것은 아니다.Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.
실시예Example 1: 상황 버섯( 1: situation mushroom ( PhellinusPhellinus baumiibaumii )의 균사체배양액에서 In the mycelia culture medium 세포외다당체의Extracellular polysaccharide 제조 Produce
상황 버섯(Phellinus baumii)에서 항당뇨 효과가 있는 세포외다당체를 얻기 위해 사용된 상황 버섯(Phellinus baumii)의 균사체는 본 발명자들이 신선한 자실체로부터 직접 분리하였다(대구대학교 생명공학과 기능성소재연구실).Situation Mushroom ( Phellinus baumii ) is used to obtain extracellular polysaccharides with antidiabetic effect ( Phellinus baumii ) was directly isolated from fresh fruiting bodies by the present inventors (Daegu University Department of Biotechnology and Functional Materials Laboratory).
접종 미생물을 제조하기 위해, 처음에 페트리디쉬 내 PDA 배지(2.4% 포테이토 덱스트로우즈 브로스와 2% 아가)에서 상황 버섯(Phellinus baumii)의 균사체를 배양한 다음, 살균된 절단기로 아가 플레이트를 직경 5mm 정도 펀칭하여 이를 종배양배지로 옮겼다. 종배양액을 PMP 배지(2.4% 포테이토 덱스트로우즈 브로스, 1% 맥아추출물, 0.1% 펩톤) 50mL이 들어있는 250mL-플라스크에 넣고 28℃에서 150 rpm 으로 4일 동안 진탕배양하였다.To prepare the inoculated microorganism, Phellinus was firstly placed in Petri dish in PDA medium (2.4% potato dextrose broth and 2% agar). The mycelium of baumii ) was incubated, and then the agar plate was punched about 5 mm in diameter with a sterilized cutter and transferred to the seed culture medium. Seed cultures were placed in 250 mL-flasks containing 50 mL of PMP medium (2.4% potato dextrose broth, 1% malt extract, 0.1% peptone) and shaken for 4 days at 28 ° C. at 150 rpm.
세포외다당체(exopolysaccharides, EPS)를 생산하기 위해, 상황버섯(Phellinus baumii)의 액침배양액을 다음의 배양조건 하에서 5리터-교반식 발효탱크에 넣고 발효시켰다: 과당 30 g/L, 효모추출물 10 g/L, 펩톤 10g/l, 염화칼슘 0.55 g/L, 황산마그네슘 0.5 g/L; 배양온도: 30℃; 포기 속도: 2 vvm; 교반 속도: 200 rpm; 초기 pH: 6.0; 배양 부피: 3L.To produce exopolysaccharides ( EPs ), immersion cultures of Phellinus baumii were placed in a 5 liter-stirred fermentation tank under the following culture conditions and fermented: fructose 30 g / L, yeast extract 10 g / L, peptone 10g / l, calcium chloride 0.55 g / L, magnesium sulfate 0.5 g / L; Incubation temperature: 30 ° C .; Abandonment speed: 2 vvm; Stirring speed: 200 rpm; Initial pH: 6.0; Culture volume: 3 L.
세포외다당체를 제조하기 위해, 배양액을 10,000 × g에서 20분간 원심분리한 다음, 상등액을 와트만 여과지 제2번(Whatman filter paper No.2, 영국 메이드 스톤, 와트만 인터내셔널 주식회사)으로 여과하였다. 배양여과액에 4배의 에탄올을 첨가하여 격렬하게 교반한 다음 4℃에서 밤새도록 방치하였다. 침전된 세포외다당체를 10,000 × g에서 20분 동안 원심분리한 다음, 상등액은 버리고 동결건조함으로써 본 발명 세포외다당체를 제조하였다.To prepare the extracellular polysaccharide, the culture was centrifuged at 10,000 x g for 20 minutes, and then the supernatant was filtered with Whatman filter paper No. 2 (Watman International, UK-made Stone, Inc.). Four times of ethanol was added to the culture filtrate, the mixture was stirred vigorously, and left overnight at 4 ° C. The extracellular polysaccharide of the present invention was prepared by centrifugation of the precipitated extracellular polysaccharide at 10,000 x g for 20 minutes, and then the supernatant was discarded and lyophilized.
상기와 같이 상황 버섯(Phellinus baumii)에서 생산되는 세포외다당체의 탄수화물 총 함량은 글루코우즈를 표준물질로 하여 페놀-황산법으로 측정하였다. 총 단백질은 우태아혈청의 알부민을 표준물질로 하여 Lowry법에 따라 측정하였다. 세포외다당체에서 단백질 부분은 280nm에서 흡광도를 측정하고, 탄수화물 부분은 480nm에서 측정하였다.Situation mushrooms as above ( Phellinus The total carbohydrate content of the extracellular polysaccharide produced in baumii ) was measured by phenol-sulfuric acid method using glucose as a standard. Total protein was measured according to the Lowry method using albumin of fetal calf serum as a standard. The protein portion of the extracellular polysaccharide was measured at 280 nm and the carbohydrate portion was measured at 480 nm.
5리터-교반식 발효탱크에서 균사체의 생장과 세포외다당체를 생산할 경우, 최대 균사체 농도는 12일째에 약 15 g/L에 도달한 반면, 최대 세포외다당체 생산(약 3.5 g/L)은 13일째에 달성되었다(미도시됨). In mycelial growth and extracellular polysaccharide production in a 5 liter-stirred fermentation tank, the maximum mycelial concentration reached about 15 g / L on day 12, whereas the maximum extracellular polysaccharide production (about 3.5 g / L) was 13 Achieved on day 1 (not shown).
세포외다당체에서 탄수화물과 단백질 함량은 각각 78.9%와 21.1%였으며 평균분자량 (Mw)은 1.3~1.7×106 Dalton(g/mol)으로 결정되었다. 세포외다당체에서 단백질부분은 글루타민산을 비롯한 17개의 아미노산으로 구성되어 있고, 탄수화물부분은 주로 만노우즈 92.18%와 갈락토우즈 6.24%로 구성되어 있었다(표 1).The carbohydrate and protein contents of the extracellular polysaccharide were 78.9% and 21.1%, respectively, and the average molecular weight (Mw) was determined to be 1.3 ~ 1.7 × 10 6 Daltons (g / mol). In the extracellular polysaccharide, the protein part is composed of 17 amino acids including glutamic acid, and the carbohydrate part is mainly composed of 92.18% of mannose and 6.24% of galactose (Table 1).
실시예Example 2: 상황 버섯( 2: situation mushrooms ( PhellinusPhellinus baumiibaumii ) 균사체 유래의 Derived from mycelium 세포외다당체의Extracellular polysaccharide 항당Hangang 뇨 효과 조사Urine effect investigation
상기 실시예 1에서 얻은 상황 버섯(Phellinus baumii)의 세포외다당체의 항당뇨 효과를 조사하기 위해 2형 당뇨 마우스(ob/ob)를 사용하였다. ob/ob 마우스는 유전적으로 렙틴(leptin)이 결핍되어 인슐린 비의존성 당뇨병(제 2형 당뇨) 환자에서 나타나는 비만 및 고혈당 등의 유사한 대사장애가 나타나는 마우스로 2형당뇨 효능 검증을 위해 많은 연구자들이 사용하는 실험동물이다.Situation mushroom ( Phellinus) obtained in Example 1 Type 2 diabetic mice ( ob / ob ) were used to investigate the antidiabetic effect of extracellular polysaccharide of baumii ). ob / ob mice are genetically deficient in leptin and have similar metabolic disorders such as obesity and hyperglycemia in insulin-independent diabetes (type 2 diabetes) patients. It is an experimental animal.
체중이 25~35 g인 C57BL/6 J Ham SIc-ob/ob 수컷 쥐(일본, SLC)를 각각 마우스 전용 케이스에서 사육하고, 냉난방실(23± 2℃, 55± 5% 습도)에서 12:12시간 명암주기 하에서 적어도 2주일 동안 사료와 물을 자유롭게 식이 하도록 순화시켰다. 실험기간 동안, 삼양사에서 구입한 구형 사료를 쥐에게 급이시켰다.C57BL / 6 J Ham SI- ob / ob male rats (Japan, SLC) weighing 25-35 g each were housed in mouse-only cases, and 12: in an air-conditioned room (23 ± 2 ° C, 55 ± 5% humidity): The feed and water were allowed to freely diet for at least 2 weeks under a 12 hour light cycle. During the experiment, rats were fed with old food purchased from Samyang.
ob/ob 마우스는 유전자 변형마우스이기 때문에 생후 7주가 지나면 고혈당 및 비만현상이 나타난다. 7주령이 되는 날 절식시킨 마우스의 혈액 내 글루코우즈를 측정한 다음, 혈액 내 글루코우즈 함량이 >300 mg/dl인 쥐를 당뇨군에 포함시켰다. Because ob / ob mice are transgenic mice, hyperglycemia and obesity occur after 7 weeks of age. Glucose in the blood of mice fasted at 7 weeks of age was measured, and mice with a glucose content of> 300 mg / dl were included in the diabetic group.
모든 동물들을 1군당 6마리로 하여 2군으로 무작위로 나누었다: 대조군, 0.9% 염화나트륨 식염수를 투여한 2형 당뇨 마우스; 처리군, 세포외다당체(EPS)를 처리한 2형 당뇨 마우스, 상황 버섯(Phellinus baumii)의 세포외다당체를 200 mg/체중 1kg의 농도로 54일 동안 매일 구강 투여함. 54일 차에는 당부하검사(glucose tolerance test)를 수행하였다. 마우스를 15시간 절식시킨 후 글루코오스 2g/체중 1kg의 농도로 구강투여를 한 후 -30, 0, 30, 60, 120 분 시간 간격으로 혈당을 조사하였다.All animals were randomized into two groups, 6 per group: control group, type 2 diabetic mice administered 0.9% sodium chloride saline; Treatment group, type 2 diabetic mouse treated with extracellular polysaccharide (EPS), situation mushroom ( Phellinus) baumii ) extracellular polysaccharide is administered orally daily for 54 days at a concentration of 200 mg / 1 kg body weight. On day 54, the glucose tolerance test was performed. After fasting the mice for 15 hours, the mice were orally administered at a concentration of 2 g of glucose and 1 kg of body weight, and then blood glucose levels were examined at -30, 0, 30, 60, and 120 minute intervals.
당뇨병 치료를 위해 천연물질을 이용하는 몇몇 연구자들의 다른 실험 설계(정상 투여 농도: 100-300 mg/kg)를 기반으로 하여, 200 mg/kg을 본 실험의 적절한 투여농도로 선택하였다.Based on the different experimental designs (normal dose concentration: 100-300 mg / kg) of some researchers using natural substances for the treatment of diabetes, 200 mg / kg was chosen as the appropriate dose for this experiment.
다음으로, 획득체중과 사료섭취는 시간별로 측정하였다. 항응고제로서 0.1M EDTA가 처리된 튜브에 실험동물의 혈액 샘플을 채취한 다음, 3000 × g에서 10분 동안 원심분리하여 혈장을 분리하였다. 혈장 내 글루코우즈, 콜레스테롤 및 트리글리세라이드 농도는 효소비색법에 따라 측정하였다(대한민국, YD 진단회사). Next, gain weight and feed intake were measured by time. Blood samples of experimental animals were taken in tubes treated with 0.1 M EDTA as anticoagulant and then centrifuged at 3000 x g for 10 minutes to separate plasma. Plasma glucose, cholesterol and triglyceride concentrations were measured according to the enzyme colorimetric method (Korea, YD Diagnostic Company).
그 결과 당뇨 마우스에서 혈장 내 글루코우즈 농도에 대한 상황 버섯 유래 세포외다당체의 혈당저하효과를 도 1에 그래프로 도시하였으며 당부하실험결과를 도 2에 나타내었다.As a result, the hypoglycemic effect of the situation mushroom-derived extracellular polysaccharide on plasma glucose concentration in diabetic mice is shown graphically in FIG.
도 1은 2형 당뇨 마우스(ob/ob)에서 54일 동안 상황 버섯(Phellinus baumii) 세포외다당체의 혈장 내 글루코우즈 농도에 대한 효과를 나타내고 있다. 두 당뇨군에서, 혈장 내 초기 글루코우즈 농도는 오차범위 내에서 거의 같았다. 하지만 대조군에서, 혈장 내 글루코우즈 농도는 실험동안 계속적으로 증가하여 최종 442 mg/L에 도달하였다. 이에 반해, 대조군과 비교하여 상황 버섯(Phellinus baumii)의 세포외다당체 투여마우스는 52일째에 혈장 내 글루코우즈 농도를 34%로 유의하게 낮추었다. 1 is a situation mushroom ( Phellinus ) for 54 days in type 2 diabetic mice ( ob / ob ) baumii ) shows the effect of extracellular polysaccharide on plasma glucose concentration. In both diabetic groups, the initial glucose concentration in plasma was about the same within the margin of error. However, in the control group, plasma glucose concentration continued to increase during the experiment to reach a final 442 mg / L. In contrast, the extracellular polysaccharide mice of Phellinus baumii significantly lowered the plasma glucose concentration to 34% on day 52 compared with the control group.
도 2는 상황 버섯(Phellinus baumii)의 세포외다당체의 혈당 강하현상을 재검증하기 위해 당부하검사(glucose tolerance test)를 수행하였다. 그 결과 대조군 및 처리군 모두 다 글루코오스 투여 후 30분이 지나서 고혈당 현상이 나타났지만 처리군의 경우 60분부터 급격한 혈당 강하 현상이 나타났으며 글루코오스 투여 후 120분에는 초기 혈당으로 떨어짐을 확인할 수 있었다. 하지만 대조군의 경우 혈액 내 글루코우즈 함량이 500 mg/dl이상의 고혈당이 유지됨을 확인하였으며 이러한 결과를 토대로 상황 버섯(Phellinus baumii)의 세포외다당체의 혈당 강하 현상을 재확인할 수 있었다.Figure 2 is a situation mushroom Phellinus glumii ) glucose tolerance test (glucose tolerance test) was performed to re-examine the hypoglycemic effect of the extracellular polysaccharide. As a result, both control and treatment groups showed hyperglycemia after 30 minutes of glucose administration, but the treatment group showed a rapid drop in blood glucose from 60 minutes and dropped to
실시예Example 3: 3: ob/obob / ob 마우스 지방세포에서 In mouse fat cells PPARPPAR γ유전자 발현량을 조사Examine the amount of γ gene expression
동물실험 마지막 날 ob/ob 마우스의 지방세포를 분리하였다. 전체 RNA 분리를 분리하는 시약을 사용하여 지방세포의 RNA를 분리한 후 RT-PCR기법을 사용하여 지방세포의 PPARγ발현량을 조사하였다.Adipocytes from ob / ob mice were isolated on the last day of animal testing. After separating the RNA from the adipocytes using a reagent that separates the total RNA separation, the PPARγ expression of the adipocytes was examined using RT-PCR.
SPSS 프로그램을 이용하여 ANOVA 테스트의 one-way 분석을 통해 통계적 유의성을 분석하였다. 모든 데이터들은 평균 ± 표준편차(P<0.05)로 표시하였다. protective LSD의 기술에 따라 측정할 때, 평균치가 P<0.05에서 유의한 차이가 있는 것으로 간주하였다. Statistical significance was analyzed by one-way analysis of ANOVA test using SPSS program. All data are expressed as mean ± standard deviation (P <0.05). When measured according to the technique of protective LSD, the mean value was considered to be significant difference at P <0.05.
52일 동안 2형 당뇨 마우스(ob/ob)에서 획득체중과 사료섭취에 대한 상황 버섯(Phellinus baumii)의 세포외다당체(EPS)의 투여 효과는 하기 표 2에 나타내었다. The effects of the administration of extracellular polysaccharide (EPS) of Phellinus baumii on gain weight and feed intake in type 2 diabetic mice ( ob / ob ) for 52 days are shown in Table 2 below.
b: 획득체중/사료섭취
c: 평균±표준편차(n=6)를 나타냄.
*: 대조군과 비교하여 유의한 차이가 있음, P<0.05[Note] a: Classification
b: weight gain / feeding
c: The mean ± standard deviation (n = 6).
*: Significant difference compared to control, P <0.05
52일 동안 2형 당뇨 마우스(ob/ob)에서 혈장 내 총 콜레스테롤 및 트리글리세라이드에 대한 상황 버섯(Phellinus baumii)의 세포외다당체(EPS)의 효과는 하기 표 3에 나타내었다.Situation Mushroom ( Phellinus ) for Total Cholesterol and Triglycerides in Plasma in Type 2 Diabetic Mice ( ob / ob ) for 52 Days baumii ) effect of extracellular polysaccharide (EPS) is shown in Table 3 below.
b: 평균±표준편차(n=6)를 나타냄.
*: NC 군과 비교하여 유의한 차이가 있음, P<0.05[Note] a: Classification
b: The mean ± standard deviation (n = 6).
*: Significant difference compared to NC group, P <0.05
상기 표 3에 나타난 바와 같이, 실험기간 동안 실험군들 간의 혈장 내 총 콜레스테롤 농도는 정상범위에 속해 있었다. 하지만 혈장 내 트리글리세라이드 농도는 상황 버섯(Phellinus baumii)의 세포외다당체(EPS) 구강 투여된 쥐에서 현저하게 감소되어 정상범위에 들어와 있었지만 대조군은 정상수치보다 높은 수치를 기록하였다.As shown in Table 3, the total cholesterol concentration in plasma between the experimental groups during the experimental period was in the normal range. Plasma triglyceride levels, however, were significantly reduced in oral administration of Phellinus baumii oral extracellular polysaccharide (EPS) mice, but the control group recorded higher values than normal values.
도 3 및 도 4는 상황 버섯(Phellinus baumii)의 세포외다당체를 투여한 마우스 와 대조군 마우스에서 지방세포를 분리한 후 그 속의 PPARγ라는 유전자 발현량 및 혈액 중의 PPARγ을 각각 조사한 그래프이다. 2형 당뇨병 치료제인 TZD계열 약물의 경우 지방세포의 PPARγ에 리간드로 작용하여 PPARγ를 활성화 시켜 인슐린 민감도를 증가 시켜 혈당 강하현상을 일으킨다. 도 3 및 도 4에서 보는 바와 같이 상황 버섯(Phellinus baumii)의 세포외다당체도 대조군에 비해 PPARγ의 유전자 발현을 증가 시켰다. 따라서 앞선 혈당 강하 효과가 이러한 분자적 메카니즘에 의해 일어났음을 알 수 있었다.3 and 4 is a situation mushroom ( Phellinus Adipose cells were isolated from mice treated with the extracellular polysaccharide of baumii ) and control mice, and the gene expression levels of PPARγ in the blood and PPARγ in the blood were examined. TZD-based drugs, type 2 diabetes drugs, act as ligands on PPARγ of adipocytes, activate PPARγ, and increase insulin sensitivity, leading to hypoglycemic effects. As shown in Figures 3 and 4 the situation mushroom ( Phellinus baumii ) also increased PPARγ gene expression compared to the control. Therefore, it can be seen that the previous hypoglycemic effect was caused by this molecular mechanism.
상기 실험결과들로부터, 본 발명 상황 버섯(Phellinus baumii) 균사체 유래의 세포외다당체가 2형 당뇨 마우스(ob/ob)의 당뇨병 정도를 유의적으로 감소시킬 수 있음을 알 수 있었으며, 이러한 결과들을 통해 본 발명 상황 버섯(Phellinus baumii) 균사체 유래의 세포외다당체가 당뇨병을 억제하는데 유용할 것임을 확인할 수 있었다.From the above experimental results, the present invention situation mushroom ( Phellinus baumii ) mycelium-derived extracellular polysaccharides can significantly reduce the degree of diabetes in type 2 diabetic mice ( ob / ob ), and these results suggest that the cells of the present invention mushroom ( Phellinus baumii ) mycelium derived It was confirmed that the exogenous polysaccharide may be useful in suppressing diabetes.
이상, 상기 실시예를 통하여 설명한 바와 같이, 본 발명은 상황 버 섯(Phellinus baumii)의 균사체로부터 얻은 항당뇨 활성이 있는 세포외다당체 및 그 제조방법에 관한 것으로, 상황 버섯(Phellinus baumii) 균사체로부터 생산되는 세포외다당체를 2형 당뇨 마우스(ob/ob)에 투여할 경우, 혈장 내 글루코우즈 함량을 현저하게 감소시킬 수 있을 뿐만 아니라 혈중의 지질을 감소시킴으로써 뛰어난 고지혈증 치료 효과도 동시에 얻을 수 있어 당뇨환자의 혈당을 조절하거나 비만 등으로 인한 고지혈증을 개선시킬 수 있는 매우 뛰어난 효과가 있으므로 식품 및 의약산업상 매우 유용한 발명인 것이다.As described above, the present invention relates to an extracellular polysaccharide having an anti-diabetic activity obtained from the mycelium of Phellinus baumii and a method for preparing the same, Phellinus When the extracellular polysaccharide produced from the mycelium is administered to type 2 diabetic mice ( ob / ob ), not only can the glucose content in the plasma be significantly reduced, but also the excellent hyperlipidemic effect can be achieved by reducing the blood lipids. It can be obtained because it has a very excellent effect to control the blood sugar of diabetic patients or improve hyperlipidemia due to obesity, etc. It is a very useful invention in the food and pharmaceutical industry.
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