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모나스커스 색소 아미노산 유도체를 이용한 신규 지방분해효소 저해제 탐색 및 저해 특성 분석
Reports NRF is supported by Research Projects( 모나스커스 색소 아미노산 유도체를 이용한 신규 지방분해효소 저해제 탐색 및 저해 특성 분석 | 2004 Year 신청요강 다운로드 PDF다운로드 | 김종훈(연세대학교) ) data is submitted to the NRF Project Results
Researcher who has been awarded a research grant by Humanities and Social Studies Support Program of NRF has to submit an end product within 6 months(* depend on the form of business)
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  • Researchers have entered the information directly to the NRF of Korea research support system
Project Number F00004
Year(selected) 2004 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2007년 03월 04일
Year type 결과보고
Year(final report) 2007년
Research Summary
  • Korean
  • 식품소재로 응용 가능한 지방분해효소 저해제를 모나스커스 색소 생산균을 이용하여 생합성하고, 생산된 각 유도체의 지방분해효소 저해특성을 분석하였다. L, D-아미노산과 비 천연 아미노산을 모나스커스 발효 시 첨가하여 여러 종류의 아미노산 색소 유도체를 제조하였고, 이중 지방분해효소를 효과적으로 저해하는 색소 유도체를 선별하였다. L, D-아미노산의 색소 유도체 중에서는 아미노산의 구조 중에 링구조를 포함하거나 비극성의 사슬구조를 지니는 색소 유도체가 비교적 우수한 지방분해효소 저해활성을 나타내었다. 이중 L-Tryptophan과 D-Tyrosine의 색소 유도체가 가장 우수한 저해활성을 나타내었고, 각각 61.2와 103.0 μM의 IC50 값을 나타내었다. 비 천연 아미노산의 색소 유도체의 경우에도 L, D-아미노산의 색소 유도체와 마찬가지로 링구조를 포함하거나 비극성의 사슬구조를 지니는 비 천연 아미노산의 색소 유도체가 비교적 우수한 지방분해효소 저해활성을 나타내었다. 이중 H-Penicillamine의 색소 유도체가 가장 높은 저해활성을 나타내었으며, 24.0 μM의 IC50 값을 나타내었다.
    색소유도체의 구조와 저해활성의 상관관계를 파악하고, 추가적인 색소유도체의 구조변화를 통해 저해활성 증진을 이루고자 하였다. 이를 위해 비교적 저해활성이 우수한 L-Tryptophan, L-Tyrosine, L-Leucine, L-Phenylalanine의 여러 아미노산 ester와 이들로 구성된 dipeptide를 이용하여 새로운 색소를 제조하였다. 새로이 제조한 색소 유도체 중 아미노산의 ester의 유도체가 dipeptide 유도체보다 높은 저해활성을 나타내었다. 이중 L-Leucine ethyl ester와 L-Tyrosine ethyl ester의 색소 유도체가 가장 높은 저해활성을 나타내었고, 각각 12.2와 13.8 μM의 IC50 값을 나타내었다.
    지방분해효소 저해활성이 우수한 색소 유도체를 선택하여 저해특성을 분석한 결과, 색소 유도체는 비교적 지방분해효소만 특이적인 저해 경향을 나타내었고, 다른 소화효소에는 저해 활성을 나타내지 않았다. 색소유도체는 지방분해효소를 비경쟁적으로 저해하는 것으로 나타났고, L-Tryptophan, H-Penicillamine, L-Leucine ethyl ester의 색소 유도체는 각각 34.6, 20.7, 9.4의 저해상수 (Ki)를 나타내었다. 각각의 색소 유도체가 지방분해효소에 결합하는 구조를 파악하기 위해 원평광 2색성 분광 분석을 수행하였고, L-Tryptophan, H-Penicillamine, L-Leucine ethyl ester 의 색소 유도체가 각각 지방분해효소의 β-sheet, β-turn, α-helix 부위에 특이적으로 결합하는 것을 밝혀내었다.
    색소 유도체의 산업적 이용을 위해 유도체 생산 시 필수적인 오렌지 색소의 생산 조건을 5 l 와 30 l 발효조에서 확립하였다. 5 l 발효조에서는 500 rpm에서 최대 24.8 g/l의 색소 생산량을 얻을 수 있었다. Impeller tip speed를 기준으로 30 l 발효조에서 색소 생산을 수행하여 5 l 발효조의 75% 이상의 색소를 획득할 수 있었다.
    쥐를 이용한 동물 실험을 통해 L-Leucine ethyl ester와 L-Tryptophan 색소유도체의 급성독성테스트를 수행하여 색소유도체의 안정성 확보하였다. L-Leucine ethyl ester와 L-Tryptophan 색소유도체의 항비만 활성을 테스트한 결과, 처리군의 전체 중량이 9%이상 감소하였고, 복강과 피하비만세포의 양이 각각 27-31%와 16-36% 정도 감소하였다. 혈중 콜레스테롤과 지질의 양도 각각 10%와 12% 정도 감소하였고, 간 콜레스테롤과 지질의 양도 30% 이상 감소하였다. 또한 L-Tryptophan 색소유도체 처리 군에서는 HDL-콜레스테롤의 양도 60% 이상 증가하였다. 모나스커스 색소 생산균을 이용해 생산한 색소유도체는 효과적인 지방분해효소 저해활성을 나타내었고, 생체 중에서도 비교적 우수한 항비만 작용을 나타내었다.
  • English
  • Derivatives of monascus pigment having high lipase-inhibitory activities were developed and characterized. Various derivatives of monascus pigment were produced during Monascus fermentation with added L, D- and unnatural amino acids. From them, compounds having high inhibitory activities against a porcine pancreatic lipase were selected. Monascus pigments produced with aromatic and non-polar aliphatic L, D- amino acids revealed strong inhibitory activities against the lipase. L-Trp and D-Tyr derivatives showed especially low IC50 values of 61.2 and 102.8 μM, respectively. In the case of unnatural amino acids derivatives, pigment derivatives produced with aromatic and non-polar aliphatic unnatural amino acids revealed strong inhibitory activities against the lipase. H-Pen derivative showed especially low IC50 values of 24.0 μM.
    Further structure modifications of pigment derivatives were made with amino acid esters and dipeptides for enhanced inhibitory activity. Secondary modifications to the derivatives of aromatic and nonpolar aliphatic amino acids were attempted by incorporation of the methyl, ethyl, t-butyl, and benzyl esters of L-Trp, L-Tyr, L-Leu, and L-Phe and dipeptide. The pigment derivatives of amino acid ester had high inhibitory activity more than dipeptide derivatives. L-Leu-OEt and L-Tyr-OEt derivatives made via further structure modifications showed high lipase-inhibitory activities with IC50 values of 12.2 and 13.8 μM, respectively.
    The pigment derivative exhibited some specificity against porcine pancreatic lipase but did not exhibit high activities against other digestive enzymes. All of the pigment derivatives appeared to be noncompetitive inhibitors against the lipase. The inhibition constants (Ki) of the L-Trp, H-Pen, and L-Leu-OEt derivatives against the lipase were estimated to be 34.6, 20.7, and 9.4 μM, respectively. The effects of the pigment derivative on the secondary structures of the lipase were examined using the CD spectropolarimeter. The L-Trp, H-Pen, and L-Leu-OEt derivatives influence dose dependently on β-sheet, β-turn, and α-helix of lipase structure, respectively.
    For the use of pigment derivative in large scale, the orange pigment that is essential for production of pigment derivative was produced in 5 and 30 l fermenter. At rotation speed 500 rpm, the maximum yield of orange pigment was obtained as 24.8 g/l. Scale-up of pigment fermentations from a 5 l jar fermenter to a 30 l fermeneter was conducted based on the impeller tip speed. The pigment yield in 30 l jar fermenter was obtained approximately 75% compared to the 5 l jar fermenter.
    The safety of pigment derivatives was confirmed using acute toxicity test. The L-Trp and L-Leu-OEt derivatives of monascus pigment, which previously exhibited high in vitro inhibitory activity against lipase, were tested for in vivo anti-obesity activity using mice. The average gained body weight of mice decreased 50% or less after feeding of the derivatives, likewise the weight of intra-peritoneal adipose tissue and subcutaneous adipose tissue were reduced by 23-31% and 16-36%, respectively. Derivative feeding reduced the total cholesterol level in sera by 10% or more and reduced the total triglyceride level by 12% or more. The HDL cholesterol level increased 60% or more due to feeding of the L-Trp derivative, whereas the total cholesterol level in the liver was reduced by more than 30%. Pigment derivatives having the inhibitory activities against porcine pancreatic lipase and cholesterol relating enzymes were found to have relatively high anti-obesity activities. The body fat, cholesterol, and triglyceride levels were significantly reduced by the feeding of both derivatives.
Research result report
  • Abstract
  • Derivatives of monascus pigment having high lipase-inhibitory activities were developed and characterized. Various derivatives of monascus pigment were produced during Monascus fermentation with added L, D- and unnatural amino acids. From them, compounds having high inhibitory activities against a porcine pancreatic lipase were selected. Monascus pigments produced with aromatic and non-polar aliphatic L, D- amino acids revealed strong inhibitory activities against the lipase. L-Trp and D-Tyr derivatives showed especially low IC50 values of 61.2 and 102.8 μM, respectively. In the case of unnatural amino acids derivatives, pigment derivatives produced with aromatic and non-polar aliphatic unnatural amino acids revealed strong inhibitory activities against the lipase. H-Pen derivative showed especially low IC50 values of 24.0 μM.
    Further structure modifications of pigment derivatives were made with amino acid esters and dipeptides for enhanced inhibitory activity. Secondary modifications to the derivatives of aromatic and nonpolar aliphatic amino acids were attempted by incorporation of the methyl, ethyl, t-butyl, and benzyl esters of L-Trp, L-Tyr, L-Leu, and L-Phe and dipeptide. The pigment derivatives of amino acid ester had high inhibitory activity more than dipeptide derivatives. L-Leu-OEt and L-Tyr-OEt derivatives made via further structure modifications showed high lipase-inhibitory activities with IC50 values of 12.2 and 13.8 μM, respectively.
    The pigment derivative exhibited some specificity against porcine pancreatic lipase but did not exhibit high activities against other digestive enzymes. All of the pigment derivatives appeared to be noncompetitive inhibitors against the lipase. The inhibition constants (Ki) of the L-Trp, H-Pen, and L-Leu-OEt derivatives against the lipase were estimated to be 34.6, 20.7, and 9.4 μM, respectively. The effects of the pigment derivative on the secondary structures of the lipase were examined using the CD spectropolarimeter. The L-Trp, H-Pen, and L-Leu-OEt derivatives influence dose dependently on β-sheet, β-turn, and α-helix of lipase structure, respectively.
    For the use of pigment derivative in large scale, the orange pigment that is essential for production of pigment derivative was produced in 5 and 30 l fermenter. At rotation speed 500 rpm, the maximum yield of orange pigment was obtained as 24.8 g/l. Scale-up of pigment fermentations from a 5 l jar fermenter to a 30 l fermeneter was conducted based on the impeller tip speed. The pigment yield in 30 l jar fermenter was obtained approximately 75% compared to the 5 l jar fermenter.
    The safety of pigment derivatives was confirmed using acute toxicity test. The L-Trp and L-Leu-OEt derivatives of monascus pigment, which previously exhibited high in vitro inhibitory activity against lipase, were tested for in vivo anti-obesity activity using mice. The average gained body weight of mice decreased 50% or less after feeding of the derivatives, likewise the weight of intra-peritoneal adipose tissue and subcutaneous adipose tissue were reduced by 23-31% and 16-36%, respectively. Derivative feeding reduced the total cholesterol level in sera by 10% or more and reduced the total triglyceride level by 12% or more. The HDL cholesterol level increased 60% or more due to feeding of the L-Trp derivative, whereas the total cholesterol level in the liver was reduced by more than 30%. Pigment derivatives having the inhibitory activities against porcine pancreatic lipase and cholesterol relating enzymes were found to have relatively high anti-obesity activities. The body fat, cholesterol, and triglyceride levels were significantly reduced by the feeding of both derivatives.
  • Research result and Utilization method
  • Pigment derivative were synthesized and the activities of lipase-inhibitory and anti-obesity were analyzed for using dietary additives. The original pigment had no activity to lipase but the pigment derivatives had highly inhibitory activity to lipase. The lipase-inhibitory activity of pigment derivatives is thought to be closely related to the structure of the amino acid moiety. Derivatives with incorporated nonpolar-aliphatic, aromatic or polar-uncharged amino acids showed a good activity and others had little or no activity to lipase. L-Trp, D-Tyr, and H-Pen derivatives of monascus pigment exhibited high activities. After further structure modifications of pigment derivatives, the ethyl ester moieties of pigment derivatives apparently enhance inhibitory activities against a pancreatic lipase. The L-Leu-OEt and L-Tyr-OEt derivatives showed the highest activities. The pigment derivatives all functioned as noncompetitive inhibitors against lipase, regardless of the derivative structures. The inhibitory activity of the pigment derivative was specific to the pancreatic lipase but not to other microbial sources of lipase and other digestive enzymes. Among pigment derivatives, the hydrophobic pigment had the relatively high inhibitory activity to lipase and did not inhibit such as α-amylase and α-glucosidase. Lipase is consisted with hydrophobic amino acid comparing to α-amylase and α-glucosidase. The interaction between lipase and pigment derivative is considered by hydrophobic interaction. The CD data showed that secondary structures of lipase were shifted by treatment of pigment derivatives.
    The feeding of pigment derivatives decreased the body fat contents and the cholesterol and triglyceride levels, but increased the HDL and HTR levels. The anti-obesity effect of the pigment derivatives is apparently related to their lipase-inhibitory activity and chemical stability in body. In this respect, the amino acid derivatives of monascus pigment are candidates for use as dietary supplements, especially for high fatty foods such as meats and dairy products.
  • Index terms
  • Monascus pigment, amino acid derivatives, amino acid ester derivatives, lipase inhibitor, anti-obesity activity
  • List of digital content of this reports
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