Activity Quaternary Ammonium Benzophenanthridine Alkaloids And Derivatives Of Tryptophan Decarboxylase Inhibition

Tryptophan decarboxylase (AAD) is also called the dopa decarboxylase(DDC).Through catalytic the decarboxylation of L-3,4-dihydroxy phenylalanine (L-DOPA) and L-5-hydroxy tryptophan to synthesis important physiological activity of the neurotransmitter dopamine and serotonin.At the same time it can also produce some aromatic amino acid decarboxylation catalyzed by corresponding biogenic amines.The brain serotonin levels decrease will lead to a common mood disorders such as depression and anxiety.Lesions in the substantia nigra and the striatum fewer dopamine synthesis can also affect the occurrence of Parkinson’s disease.Due to the presence of the blood-brain barrier, the serotonin and dopamine in the blood can hardly through into the brain.Because the AAD activity is higher in the nervous system and kidney, peripheral AAD inhibitors such as carbidopa can effectively reduce the tryptophan, L-dopa decarboxylation degradation rate among the tissues which has been clinically used to treat Parkinson’s disease and alleviate adverse symptoms caused by higher amine content in peripheral biogenicis, but the price is expensive and the adverse reactions are obvious.In terms of economic animals, the free tryptophan in the blood get through the blood brain barrier then metabolizing to5-hydroxy tryptamine and melatonin, which can adjust the animal feed intake and circadian rhythms.Lack of tryptophan will led to the decrease of the animal feed intake and symptom such as growth retardation and rough coat.As protein resources increasingly tense and diet worldwide high protein or amino acid imbalance results in fecal nitrogen and urinary nitrogen discharge which cause serious environmental pollution and many other problems and considering the pig amino acid low protein diet nutrition balance has been increasingly used in the process of production of live pigs, tryptophan as the restrictive amino acid in the basic diet, its importance start to clearly reflected, become the key factor of the cost of pig feed intake, production performance and feed. Small intestin is the main place for metabolism of amino acid in food, as a special community in the intestinal,microorganism colonize and participate in metabolic activity with the intestinal mucosa cells,effect the use ratio of the amino acids significant.In part of the tryptophan in diet through anaerobic metabolism of microbes to generate tryptamine, amine intermediates in the intestinal metabolism of anaerobic environment further to produce skatole, reduces the utilization rate of tryptophan and increased fecal nitrogen emissions, caused the environment pressure.AAD secreted by microbes is a key enzyme which catalysis tryptophan in the gut metabolism, it can effective catalytic tryptophan, phenylalanine, tyrosine and aromatic amino acid in the body by decarboxylation to generate the corresponding biogenic amine.Inhibition of AAD could reduce tryptophan metabolism in the pig intestines, and this is the effective way to improve the utilization rate of tryptophan and other aromatic amino acid and will also help to improve the feed conversion rate at the same time,promote animal growth and reduce fecal nitrogen emissions and other environmental problems.Our team found the main secondary metabolitesin in medicinal plants macleaya cordata:sanguinarine(SG)、chelerythrine(CHE)(figure2,P8) and other quaternary benzophenathridine alkaloids(QBA) have significant anti-inflammatory and antimicrobial activity.Animal experiments showed that QBA has high security and also can improvet the feed intake of pigle and promote its growth.Currently, extractive of macleaya cordata has been developed for the country’s first secondary categories of veterinary drugs, the mechanism could be related to that QBA can inhibit the activity of AAD, reduce tryptophan degradation of aromatic amino acids in the intestine and promote the level of transformation of protein.At present, foreign literature reports SG and CHE has inhibitory effect on AAD, the enzyme be used was extracted and purified from the mice kidney, the purity is not high and only the two alkaloids were tested.This topic is based on the existing experimental results, using the human recombinant AAD (AAD structure is slightly different from different sources, but the conformation of active site are basically the same), after validating the effect of SG and CHE, different structural modification to the two alkaloids are performed, the variety of derivatives were test for its inhibitory activity of AAD.After analyzing alkaloids are performed, the variety of derivatives and test its inhibitory activity of AAD.After analyzing the change of the inhibitory activity before and after the modification, provides a new train of thought for the inhibition mechanism of SG and CHE to AAD.1. Determinate the experimental conditions of the reaction of AAD catalytic L-DopaBy single factor and orthogonal experiment, finally determine the experimental conditions of AAD catalytic L-Dopa, namely, the molar ratio of AAD,L-Dopa and PLP is9.6354×10-5:5:2.5×10-2, the PH value of buffer solution (NaH2PO4-NaH2PO4) is6.8, the reaction temperature is37℃, the reaction time is30min. Under this condition, L-Dopa is catalytic of90%or more.2. Test the inhibitory activities of5alkaloids to AADAccording to the different dissolution properties of alkaloids to configure it into a solution of different concentrations as a inhibitor, set up the control response.Testing by HPLC, the group which containing inhibitors obviously increase the amount of unreacted substrate and decreased the amount of dopamine, and the changes over concentration of inhibitor in reaction system. This showed that QBA have the inhibition activity to AAD and this effect is influenced by the structure of inhibitor. The synthesis of QBA derivativesIn this study, several kinds of natural QBA alkaloid are tested for the first time as AAD inhibitors.We have synthesized a series of5-methyl phenanthridine ramifications to test their inhibitory activity of AAD.In synthetic compounds,2,8-dimethoxy-5-methyl phenanthridine hydrochloride showed good inhibitory activity (IC50=0.19mM). A preliminary conclusion of structure-activity relationship showed that the inhibition of5-methyl phenanthridine derivatives to AAD is influenced by the electron cloud density of C=N double bond of the imine cations.4. Analysis the inhibitory activities of synthetic compounds to AADBy comparing IC50of synthetic compounds of AAD, we come to the conclusion that QBAs and phenanthridine salt have the same inhibition mechanism to AAD, namely by destroying the thiol of enzyme active site to inhibit the enzyme activity. The inhibitory activity of5-methyl phenanthrene derivatives to AAD primarily related to the electron cloud density of the imine cations on the C=N.