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, compou ...
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.