血根碱在大鼠、猪和鸡的体外代谢研究
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摘要
血根碱是一种天然的异喹啉类生物碱,具有抗菌、促进动物生长及杀虫等作用,在兽医领域具有广阔的应用前景。但血根碱具有一定的毒性,其主要代谢途径是被还原成毒性较低的二氢血根碱,而其中所参与的代谢酶和还原代谢机制尚未有研究报道,为此,本试验建立肝微粒体与胞液体外代谢系统、肠道代谢系统进行血根碱的体外代谢研究,旨在研究参与还原的代谢酶、肠道菌与代谢部位,以及种属间差异的比较,并鉴定血根碱在猪肝微粒体与胞液中的代谢产物与代谢路径的推导,为血根碱的临床合理、安全使用及残留代谢奠定基础。
1血根碱在大鼠、猪和鸡微粒体与胞液中的还原代谢研究
辅助因子核黄素(RIB)、黄素单核苷酸(FMN)、还原型烟酰胺腺嘌呤二核苷酸(NADH)、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)直接与血根碱孵育时,NADH、 NADPH能将血根碱还原为二氢血根碱；当与血根碱在大鼠、猪和鸡肝微粒体或胞液中孵育时,NADP、NADH能显著增强肝微粒体或胞液的还原能力。
没有NAD (P) H参与时,大鼠肝、肾微粒体与心、肝、肾胞液,猪心、肝、肾微粒体与心、肝、肺、肾胞液,鸡肾胞液能将血根碱还原为二氢血根碱；NAD (P) H参与时,微粒体还原强弱依次为大鼠肾、肺、心、脾、脑、睾丸、肝；猪肾、肝、心、脾、肺；胞液还原强弱为大鼠心、肾、肺、肝、脾、脑、睾丸；猪肾、脾、肝、肺、心；鸡微粒体与胞液在组织间没有差异。
还原酶抑制试验表明,鸡肝微粒体中是NADPH-细胞色素P450还原酶在发挥作用,在大鼠、猪肝微粒体中还有醌氧化还原酶1(NQO1)、醌氧化还原酶2(NQO2)或羰基还原酶发挥作用；在大鼠、猪和鸡各组织胞液中为NQO2或羰基还原酶发挥作用,而大鼠脾胞液、鸡心、肺、脑胞液、猪各组织胞液还含有(?)NQO1。
2血根碱在肠道系统中的还原代谢研究
NAD (P) H能让大鼠、猪和鸡肠微粒体、肠胞液与肠粘膜匀浆液生成二氢血根碱,猪各肠胞液还原酶活性最强,还原酶抑制试验表明大鼠、猪和鸡各个肠胞液中含有NQO2或羰基还原酶,而大鼠结肠胞液、猪、鸡各个肠胞液中还有NQO1.
血根碱在大鼠、猪和鸡肠道内容物中能被还原为二氢血根碱,生成量由多到少依次为大鼠结肠、空肠、回肠、十二指肠；鸡回肠、空肠、十二指肠；猪回肠、十二指肠、空肠、结肠；三种动物肠道内容物还原强弱为猪、鸡、大鼠。
3血根碱在猪肝微粒体与胞液中的代谢产物鉴定
二氢血根碱是肝微粒体中的主要代谢产物和肝胞液中的唯一代谢产物,经三氯乙酸处理的肝微粒体样品中还鉴定出一个氧化产物,两个O-去甲基化代谢物,经乙腈处理的样品中还鉴定出伪血根碱和另外两个O-去甲基化代谢产物。
综上所述,在大鼠、猪和鸡胞液中存在两条将血根碱转变为的二氢血根碱的代谢途径,一条是直接通过NAD(P)H进行非酶还原,另一条路径是通过胞液中的醌还原酶或羰基还原酶进行还原；在微粒体代谢途径中还有NADPH-细胞色素P450还原酶参与。在肠道内容物中能将血根碱还原成二氢血根碱降低其毒性,在不同种属动物肠道,同种动物不同肠道中,二氢血根碱的生成量有较大差异。从猪肝微粒体与胞液中总共鉴定出7种代谢产物,两条主要代谢路径是亚胺键还原与O-去甲基化,以上研究为血根碱的临床合理、安全使用,以及血根碱的残留代谢奠定试验研究基础。
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 preparations
The 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 system
DHSA 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 preparations
DHSA 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.
1血根碱在大鼠、猪和鸡微粒体与胞液中的还原代谢研究
辅助因子核黄素(RIB)、黄素单核苷酸(FMN)、还原型烟酰胺腺嘌呤二核苷酸(NADH)、还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)直接与血根碱孵育时,NADH、 NADPH能将血根碱还原为二氢血根碱；当与血根碱在大鼠、猪和鸡肝微粒体或胞液中孵育时,NADP、NADH能显著增强肝微粒体或胞液的还原能力。
没有NAD (P) H参与时,大鼠肝、肾微粒体与心、肝、肾胞液,猪心、肝、肾微粒体与心、肝、肺、肾胞液,鸡肾胞液能将血根碱还原为二氢血根碱；NAD (P) H参与时,微粒体还原强弱依次为大鼠肾、肺、心、脾、脑、睾丸、肝；猪肾、肝、心、脾、肺；胞液还原强弱为大鼠心、肾、肺、肝、脾、脑、睾丸；猪肾、脾、肝、肺、心；鸡微粒体与胞液在组织间没有差异。
还原酶抑制试验表明,鸡肝微粒体中是NADPH-细胞色素P450还原酶在发挥作用,在大鼠、猪肝微粒体中还有醌氧化还原酶1(NQO1)、醌氧化还原酶2(NQO2)或羰基还原酶发挥作用；在大鼠、猪和鸡各组织胞液中为NQO2或羰基还原酶发挥作用,而大鼠脾胞液、鸡心、肺、脑胞液、猪各组织胞液还含有(?)NQO1。
2血根碱在肠道系统中的还原代谢研究
NAD (P) H能让大鼠、猪和鸡肠微粒体、肠胞液与肠粘膜匀浆液生成二氢血根碱,猪各肠胞液还原酶活性最强,还原酶抑制试验表明大鼠、猪和鸡各个肠胞液中含有NQO2或羰基还原酶,而大鼠结肠胞液、猪、鸡各个肠胞液中还有NQO1.
血根碱在大鼠、猪和鸡肠道内容物中能被还原为二氢血根碱,生成量由多到少依次为大鼠结肠、空肠、回肠、十二指肠；鸡回肠、空肠、十二指肠；猪回肠、十二指肠、空肠、结肠；三种动物肠道内容物还原强弱为猪、鸡、大鼠。
3血根碱在猪肝微粒体与胞液中的代谢产物鉴定
二氢血根碱是肝微粒体中的主要代谢产物和肝胞液中的唯一代谢产物,经三氯乙酸处理的肝微粒体样品中还鉴定出一个氧化产物,两个O-去甲基化代谢物,经乙腈处理的样品中还鉴定出伪血根碱和另外两个O-去甲基化代谢产物。
综上所述,在大鼠、猪和鸡胞液中存在两条将血根碱转变为的二氢血根碱的代谢途径,一条是直接通过NAD(P)H进行非酶还原,另一条路径是通过胞液中的醌还原酶或羰基还原酶进行还原；在微粒体代谢途径中还有NADPH-细胞色素P450还原酶参与。在肠道内容物中能将血根碱还原成二氢血根碱降低其毒性,在不同种属动物肠道,同种动物不同肠道中,二氢血根碱的生成量有较大差异。从猪肝微粒体与胞液中总共鉴定出7种代谢产物,两条主要代谢路径是亚胺键还原与O-去甲基化,以上研究为血根碱的临床合理、安全使用,以及血根碱的残留代谢奠定试验研究基础。
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 preparations
The 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 system
DHSA 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 preparations
DHSA 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.
引文
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