| Sanguinarine (SA) is a quaternary benzo[c]phenanthridine alkaloid and has been extensively studied because of its antimicrobial, antiproliferative and antiplatelet activities, which are worth applying it to veterinary medicine. SA has a little toxin, but it could be metabolized to less toxic dihydrosanguinarine (DHSA). This experiment was conducted to study the metabolic enzymes involved in the reduction metabolism of SA to DHSA by liver microsome, cytosol, intestinal mucosa and microbiota, and compare distribution of reduction enzymes and metabolic characteristics of rat, pig and chicken. Metabolic pathways of SA in pig liver microsomes and structures of metabolites were also aimed to be proposed.1. The study on reductive metabolism of SA by rat, pig and chicken liver preparationsThe results showed that when SA was incubated with riboflavin(RIB), flavin mononucleotide(FMN), reduced form of nicotinamide-adenine dinucleotid I (NADH), reduced form of nicotinamide-adenine dinucleotid II (NADPH), respectively, DHSA, the iminium bond reductive metabolite was formed by NAD(P)H. The reductase activity of the liver microsomes or cytosol of rat, pig or chicken was enhanced significantly in the presence of NADPH or NADH.When SA was incubated in different tissue microsomes and cytosol in the absence of NAD(P)H, DHSA could be formed in some tissues microsomes or cytosol which are as following, rat liver and kidney microsomes, pig heart, liver and kidney microsomes, rat heart, liver and kidney cytosol, pig heart, liver, kidney and lung cytosol, chicken kidney cytosol. In the presence of NADPH, the order of reduction activity of rat tissue microsomes was kidney> lung> heart> spleen>liver>brain>testicle, and the order of pig tissue microsomes was kidney>liver> heart> spleen> lung. In the presence of NADH, the order of reduction activity of rat tissue cytosol was heart>kidney> lung>liver> spleen>brain>testicle, and the order of pig tissue cytosol was kidney> spleen> lung>liver>heart. There was no significant difference in reduction activity among chicken tissue microsomes and cytosol.Inhibition studies indicated that NADPH-CYP450reductase was responsible for DHSA formation by chicken liver microsomes. Quinone oxidoreductase1(NQO1), quinone oxidoreductase2(NQO2) and/or carbonyl reductases(CBR) were responsible for DHSA formation by rat or pig liver microsomes. NQO2or CBR played the major role by rat, pig or chicken tissue cytosol. And there was still NQO1in rat spleen cytosol, chicken heart, lung, brain cytosol, and pig all tissue cytosol.2. The reductive metabolism of SA by intestinal metabolism systemDHSA couldn’t be formed when SA was incubated in intestinal microsomes, cytosol and mucosa in the absence of NAD(P)H, but DHSA could be formed in the presence of NAD(P)H. The reduction activity of pig intestinal cytosol was highest. Inhibition studies indicated that there was NQO2or CBR in rat, pig and chicken intestinal cytosol, and there was still NQO1in rat colon, pig and chicken intestinal cytosol.DHSA could be formed when SA was incubated in intestinal microbiota. The order of DHSA amount by rat intestinal microbiota was colon>jejunum> ileum> duodenum. The order by chicken intestinal microbiota was ileum>jejunum> duodenum. The order by pig intestinal microbiota was ileum> duodenum>jejunum> colon. The reduction activity of pig intestinal microbiota was highest among these animals intestinal microbiota.3. Identification of sanguinarine metabolites in pig liver preparationsDHSA was the main metabolite formed in liver microsomes and the only one in cytosol. One oxidative metabolite and two O-demethylene metabolites of SA were found in the TCA-treated microsomal samples. SA pseudobase and two additional O-demethylene metabolites of DHSA were only found in the acetonitrile-treated microsomal samples.These results indicated that the SA reduction proceeds via two routes in the rat, pig and chicken cytosol. One route is direct non-enzymatic reduction by NAD(P)H, and the other is enzymatic reduction by possible carbonyl and/or quinone reductases. NADPH-CYP450reductase was involved in rat, pig and chicken microsomes additionally. DHSA could be formed when SA was incubated in intestinal microbiota. And the amount of DHSA was different obviously among different intestines of rat, pig and chicken. A total of seven metabolites in pig liver preparations were identified. The metabolic pathway was proposed to be reduction of iminium bond and O-demethylenation. These results laid a good foundation for making use of SA in the future. |
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