DFT Study On The Photochemical Features And The Cholesterol-Lowering Activities Of Monascus Pigments

Monascus pigments are the secondary metabolites of monascus,more than 50 monascus pigments have been found and identified up to now.There are six major well-known original water-insoluble polyketide monascus pigments,including two red pigments（R5,R7）,two orange pigments（O5,O7）and two yellow pigments（Y5,Y7）.As a kind of natural food colorant,monascus pigments have been widely used in the coloration of food,such as processed meats,tomato ketchup and beverage and so on.Monascus pigments also have many good biological activities,and no adverse effect has been reported,which make them a kind of potential food supplements and drugs.However,the water-insolubility and the weak photostability have largely restricted their application span in food and pharmaceutical industries.Meanwhile,the photobleaching mechanism of the six original monascus pigments is still unclear yet.It has been reported that their fading was a photochemical process.Their saturated side chains(C₅H₁₁,C₇H₁₅)do not belong to the chromophore（conjugated π bonds）,so they are irrelative with the fading of monascus pigments.We optimized the molecular geometries of R5,O5,Y5 and their relevant molecules which possess the same side chains(C₅H₁₁)with DFT at B3LYP/6-311+G（d,p）level.Also,we calculated their UV-vis absorption spectra,and optimized the first singlet excited states（S1）of some relevant molecules with TDDFT at the same level.On the basis of the calculated data,we speculated the possible photobleaching mechanism of the six original monascus pigments.It is the break of the conjugated π bonds which has induced their decoloration,the photodissociation of these pigments involves three steps,excitation of the large conjugated π system,water addition reaction and Norrish type I photochemical cleavage reactions of the side chains.Excitation of the conjugated π system include two steps,excitation from the ground state（S0）to the S1,an intersystem crossing（ISC）from the S1 to the first triplet excited state（T1）.T1 is much more stable than S1 and have much longer lifetime,which could have an addition reaction with water.This addition reaction has destroyed the conjugated π bond,and induced the fading of the six water-insoluble monascus pigments ultimately.The photodegradation mechanism we have speculated may help find some solutions to enhance the photostability of these monascus colorants during the food processing process and extend the shelf life of the foods added with monascus pigments.Because of their water-insolubility and weak photostability,many literatures have been focused on the enhancement of their photostability and water-soluble.A much more common,effective and convenient strategy is to add amino acids as the nitrogen source during the monascus fermentation process,the oxygen moieties of O5 and O7 could be replaced by the-NH₂ of the added amino acids,and the generated red pigments are water-soluble and more stable to light than R5 and R7,which are named the amino acid derivatives of monascus pigments.They also possess many beneficial biological activities.Popularity of the food possessing red color,water-solubility,stronger photostability and numerous biological effects have made these amino acid derivatives a kind of promising food supplements and medicines.Here,we try to theoretically elucidate the reason which has induced their larger photostability with DFT and TDDFT.R5 and the amino acid derivatives of O5 have been selected to exclude the influence of different side chains on our calculated results.At B3LYP/6-311+G（d,p）level,the molecular geometries of R5 and 17 amino acid derivatives of O5 have been optimized with DFT,their UV-vis absorption spectra have been calculated with TDDFT.According to the calculated data,the frontier orbital energy gaps of the amino acid derivatives have been increased,the oscillator strengths of their excitation from S0 to S1 are larger than that of R5,the molar absorptivity at the absorption wavelength corresponding to the excitation from S0 to S1 is also larger than that of R5.These all make the excitation from S0 to S1 more difficult,which has caused much larger photostability of the amino acid derivatives of monascus pigments.The photobleaching of monascus pigments involves three steps,excitation from S0 to S1,an ISC from S1 to T1 and a water addition reaction with T1.The latter two steps may also have an effect on their photostability.Adding amino sugars,nucleotides and nucleic acid which possess-NH2 and various biological activities during the monascus fermentation process,the generated red water-soluble derivatives of monascus pigments which are more stable to light could be a kind of potential and popular food supplements,this may be the future development direction of the water-soluble amino derivatives of monascus pigments.Y5 and Y7 could lower the total cholesterol levels in serum and liver significantly by blocking its biosynthesis in liver,but how can they achieve this capability? 3-hydroxy-3-methylglutaryl-coenzyme A（HMG-CoA）reductase is the committed enzyme in the liver cholesterol biosynthetic pathway,which could catalyze HMG-CoA to mevalonate.It has been reported that R5,R7,O5,O7 and some amino acid derivatives of monascus pigments possess inhibitory activities against the HMG-CoA reductase.Also,O5,O7 and the threonine,tryptophan and leucine-OEt derivatives of monascus pigments could decrease the total cholesterol levels.Due to the extremely similar molecular geometries between the six original water-insoluble monascus pigments and the water-soluble amino acid derivatives of monascus pigments,Y5 and Y7 may also be able to inhibit the catalysis of HMG-CoA reductase,thus blocking the biosynthesis of cholesterol in liver.According to the reported experimental data,the inhibitory activities of Y5 and Y7 against the HMG-CoA reductase should be much larger than that of the other four water-insoluble monascus pigments and the water-soluble amino acid derivatives.Statins,also called HMG-CoA reductase inhibitors,only their hydroxy-acid forms obtained through the catalyzed hydrolysis of their δ-lactone rings could competitively inhibit the catalysis of HMG-CoA reductase because of their characteristic HMG-like moieties.On the basis of the γ-lactone rings monascus pigments possess,we supposed they may have the same functional mechanism like statins.For R5,R7,O5,O7,Y5 and Y7,their hydroxy-acid forms have similar molecular geometries,which has induced their approximate linking modes and energies with the HMG-CoA reductase.Thus their hydrolytic activation free energies of the γ-lactone rings may also influence their inhibitory abilities against the HMG-CoA reductase,the smaller the hydrolytic activation free energies are,the larger inhibitory abilities they possess.The hydrolytic patterns of esters under the catalysis of carboxylesterases and water are similar,and the six water-insoluble monascus pigments have similar molecular geometries,indicating the order of their activation free energies calculated in water environment could be consistent with their practical sequence under the enzymatic catalysis.A Cluster-Continuum Model including up to six explicit water molecules at B3LYP-D3（BJ）level has been used to calculate the hydrolytic activation free energies of their γ-lactone rings.The order of the calculated activation free energies agree very well with their inhibitory activities against the HMG-CoA reductase,meaning that our hypothesis should be right,the hydroxy-acid forms of Y5 and Y7 could inhibit the catalysis of HMG-CoA reductase competitively.But their linking modes with HMG-CoA reductase also need further theoretical research.The obtained inhibitory mechanism against the HMG-CoA reductase may guide people to improve the cholesterol-lowering capabilities of Y5 and Y7,develop some their analogues which also possess this ability.