不同抗生素对ICR小鼠大豆异黄酮转化菌株体内外转化能力影响与菌株定植
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摘要
大豆异黄酮是大豆在其生长过程中形成的次生代谢产物,主要包括：黄豆苷原(daidzein)和染料木素(genistein)。摄入动物体内的大豆异黄酮,在胃肠道微生物作用下被代谢为不同代谢产物,其中黄豆苷原最终被降解为雌马酚(equol)和去氧甲基安哥拉紫檀素(O-desmethylangolensin,简称O-Dma)。现阶段研究表明,在大豆异黄酮中雌马酚的生理活性最强。然而,由于受饮食习惯、年龄、疾病、抗生素使用等诸多因素影响,寄居在胃肠道内的大豆异黄酮转化菌株的正常生长与转化也随之受到影响。
本试验采用转化黄豆苷原稳定且重现性好的ICR小鼠为试验动物。确定不同抗生素在体外条件下对ICR小鼠肠道菌群转化黄豆苷原能力的差异性,为人类合理使用抗生素提供一些指导。
本试验首次在体内外应用抗生素处理ICR小鼠肠道内的大豆异黄酮转化菌株。当使用正常成人剂量时,11种供试抗生素均完全抑制ICR小鼠肠道微生物菌群体外转化黄豆苷原为雌马酚能力；当加入正常成人剂量1/32浓度时,阿莫西林、氧氟沙星、诺氟沙星几乎不产生抑制作用,多西环素、利君沙和头孢拉定使培养基中雌马酚浓度降低了87.7%,80.5%和65.2%,其余5种抗生素阿奇霉素、吉他霉素、土霉素、利福平和甲硝唑,则完全抑制黄豆苷原向雌马酚的转化；当使用剂量为正常成人剂量1/256时,阿奇霉素、利福平甲硝唑完全抑制了ICR小鼠肠道微生物菌群体外转化能力,继续降低抗生素浓度,测得阿奇霉素、利福平和甲硝唑有效抑制黄豆苷原转化为雌马酚的临界点浓度分别为1/512,1/800和1/1024。
Niu-O16(AY263505)是从牛瘤胃胃液分离的能将黄豆苷原还原为二氢黄豆苷原(DHD)的革兰氏阳性细菌菌株。为了研究菌株Niu-O16能否定植在ICR小鼠肠道内,给ICR小鼠灌服体外试验确定的敏感抗生素,抑制其肠道内大豆异黄酮的转化。为了保护灌服菌株免受胃液中低pH值的破坏,将Niu-O16包裹在海藻酸钠微胶囊中。这种微胶囊在人工胃液中并不溶解,但在人工肠液中能够很好的破溶。体外模拟胃液和肠液试验中,在人工胃液中4h没有破裂溶解,在人工肠液中0.5h时就有部分破裂溶解,到3h海藻酸钠微胶囊则完全破裂溶解。经灌服微胶囊后定时解剖发现,2h后ICR小鼠胃中存在大量微胶囊,而肠道中没有发现；6h后胃中没有发现微胶囊,但是在与胃连接的十二指肠中发现大量的微胶囊；12h后胃中和肠道各段都没有发现微胶囊。在解剖过程中收集的小鼠粪便中没有发现微胶囊。
由于使用抗生素仅能抑制大豆异黄酮转化活性2周,虽然灌服组比对照组的大豆异黄酮转化活性有明显提高,但是仍不能确定是否为灌服的Niu-O16的定植,需要进一步采用菌体标记手段加以确定。
Soybean isoflavones (SI) are the secondary metabolites mainly composed by isoflavone daidzein and genistein. SI are degraded into equol and O-desmethylangolensin (O-Dma) by intestinal microflora in gastra intestinal tract after being absorbed. At present, among all SI metabolites, equol shows the highest bioactivity.However, SI biotransforming microflora are being influeced by many factors, such as dietary habits, age, disease, use of antibiotic, and many other factors and so on.
In this paper, ICR mice were used as experimental animals since their gut microflora can transforme daidzein into dihydrodaidzein(DHD) and equol stablely. The effects of different antibiotics on the biotransformation of daidzein into equol by intestinal microflora of ICR mice in vitro were investigated. In order to provide some guidance for rational useage of antibiotics for human beings, the critical inhibitory concentrctions of sonsitire antibiobics were determined as well.
This is the first study of the influence of different antibiotics on transformation activicty of isoflance by intestinet microflora of ICR mice. All eleven kinds of antibiotics showed completely inhibitive effects on the bioconversion of daidzein into equol under normal adult dose. Three types of antibiotics including amoxicillin, ofloxacin and norfloxacin had no effects under the concentration of 1/32 normal adult dose. On the contrary, another five antibiotics including azithromycin, leucomycin, oxytetracycline, rifampicin, and metronidazole totally inhibited the biotransforming capacity of the intestinal microflora of ICR mice under the same concentration. The biosynthesized equol from daidzein was detected to decrease 87.7%,80.5% and 65.2%, respectively when the same concentration of doxycycline, erythromycin and cephradine was used. Under the concentration of 1/256 normal adult dose, three antibiotics including azithromycin, rifampicin and metronidazole showed completely inhibitive effects. The lowest inhibitive concentration of the three sensitive antibiotics mentioned above were 1/512,1/800 and 1/1024, respectively.
Gram positive bacterial strain Niu-O16(AY263505) isolated from bovine rumen gastric juice is capable of biotransforming daidzein into DHD. In order to study whether bacterial strain Niu-016 was able to combine in gastrointestinal tract of ICR mice or not, the tested ICR mice were treated with sensitive antibiotics determined by in vitro experimats till their daidzein biotransformation activity was totally inhibited. In order to protect bceterial strain Niu-016 from being damaged by low-pH gastric juice of ICR mice, strain Niu-016 were wrapped into sodium microcapsules are not broken or melted in simulated gastric juice, however they can break down by simulated intestinal microcapsules witsowt bacterial stain Niu-016 were anatomy. In order to know whether such kind of microcapsules will experience similer procedures as that of in vitro experiments. The feces of tested ICR mice were collected for detection of eliminated microcapsules after being fed with microcapsules. But no microcapsules were found in any feces. At necropsy, there were a lot of microcapsules in ICR mouse stomach after 2 hours, but in intestine none of the miorocapasules were found. Six hours later, all the microcapsules disappeared in stomach. Microcapsules were not found neither in stomach nor intestine after 12 hours. Since no microcapsules were found in either mice feces by anatoy, which illustratment that all microcapsules can break throw completely 12 hours.
Since the daidzein biotransforming activity was able to be totally inhibited for just about two weeks by using antibiotics. The deteced concentred of DHD by microflora of tested mice fed with bacterial strain Niu-O16 was much higher than that of blank control, it is still not sure whether baeterial strain Niu-016 can plant in intestinal tract of ICR mice or not. A further study in needed to be done.
本试验采用转化黄豆苷原稳定且重现性好的ICR小鼠为试验动物。确定不同抗生素在体外条件下对ICR小鼠肠道菌群转化黄豆苷原能力的差异性,为人类合理使用抗生素提供一些指导。
本试验首次在体内外应用抗生素处理ICR小鼠肠道内的大豆异黄酮转化菌株。当使用正常成人剂量时,11种供试抗生素均完全抑制ICR小鼠肠道微生物菌群体外转化黄豆苷原为雌马酚能力；当加入正常成人剂量1/32浓度时,阿莫西林、氧氟沙星、诺氟沙星几乎不产生抑制作用,多西环素、利君沙和头孢拉定使培养基中雌马酚浓度降低了87.7%,80.5%和65.2%,其余5种抗生素阿奇霉素、吉他霉素、土霉素、利福平和甲硝唑,则完全抑制黄豆苷原向雌马酚的转化；当使用剂量为正常成人剂量1/256时,阿奇霉素、利福平甲硝唑完全抑制了ICR小鼠肠道微生物菌群体外转化能力,继续降低抗生素浓度,测得阿奇霉素、利福平和甲硝唑有效抑制黄豆苷原转化为雌马酚的临界点浓度分别为1/512,1/800和1/1024。
Niu-O16(AY263505)是从牛瘤胃胃液分离的能将黄豆苷原还原为二氢黄豆苷原(DHD)的革兰氏阳性细菌菌株。为了研究菌株Niu-O16能否定植在ICR小鼠肠道内,给ICR小鼠灌服体外试验确定的敏感抗生素,抑制其肠道内大豆异黄酮的转化。为了保护灌服菌株免受胃液中低pH值的破坏,将Niu-O16包裹在海藻酸钠微胶囊中。这种微胶囊在人工胃液中并不溶解,但在人工肠液中能够很好的破溶。体外模拟胃液和肠液试验中,在人工胃液中4h没有破裂溶解,在人工肠液中0.5h时就有部分破裂溶解,到3h海藻酸钠微胶囊则完全破裂溶解。经灌服微胶囊后定时解剖发现,2h后ICR小鼠胃中存在大量微胶囊,而肠道中没有发现；6h后胃中没有发现微胶囊,但是在与胃连接的十二指肠中发现大量的微胶囊；12h后胃中和肠道各段都没有发现微胶囊。在解剖过程中收集的小鼠粪便中没有发现微胶囊。
由于使用抗生素仅能抑制大豆异黄酮转化活性2周,虽然灌服组比对照组的大豆异黄酮转化活性有明显提高,但是仍不能确定是否为灌服的Niu-O16的定植,需要进一步采用菌体标记手段加以确定。
Soybean isoflavones (SI) are the secondary metabolites mainly composed by isoflavone daidzein and genistein. SI are degraded into equol and O-desmethylangolensin (O-Dma) by intestinal microflora in gastra intestinal tract after being absorbed. At present, among all SI metabolites, equol shows the highest bioactivity.However, SI biotransforming microflora are being influeced by many factors, such as dietary habits, age, disease, use of antibiotic, and many other factors and so on.
In this paper, ICR mice were used as experimental animals since their gut microflora can transforme daidzein into dihydrodaidzein(DHD) and equol stablely. The effects of different antibiotics on the biotransformation of daidzein into equol by intestinal microflora of ICR mice in vitro were investigated. In order to provide some guidance for rational useage of antibiotics for human beings, the critical inhibitory concentrctions of sonsitire antibiobics were determined as well.
This is the first study of the influence of different antibiotics on transformation activicty of isoflance by intestinet microflora of ICR mice. All eleven kinds of antibiotics showed completely inhibitive effects on the bioconversion of daidzein into equol under normal adult dose. Three types of antibiotics including amoxicillin, ofloxacin and norfloxacin had no effects under the concentration of 1/32 normal adult dose. On the contrary, another five antibiotics including azithromycin, leucomycin, oxytetracycline, rifampicin, and metronidazole totally inhibited the biotransforming capacity of the intestinal microflora of ICR mice under the same concentration. The biosynthesized equol from daidzein was detected to decrease 87.7%,80.5% and 65.2%, respectively when the same concentration of doxycycline, erythromycin and cephradine was used. Under the concentration of 1/256 normal adult dose, three antibiotics including azithromycin, rifampicin and metronidazole showed completely inhibitive effects. The lowest inhibitive concentration of the three sensitive antibiotics mentioned above were 1/512,1/800 and 1/1024, respectively.
Gram positive bacterial strain Niu-O16(AY263505) isolated from bovine rumen gastric juice is capable of biotransforming daidzein into DHD. In order to study whether bacterial strain Niu-016 was able to combine in gastrointestinal tract of ICR mice or not, the tested ICR mice were treated with sensitive antibiotics determined by in vitro experimats till their daidzein biotransformation activity was totally inhibited. In order to protect bceterial strain Niu-016 from being damaged by low-pH gastric juice of ICR mice, strain Niu-016 were wrapped into sodium microcapsules are not broken or melted in simulated gastric juice, however they can break down by simulated intestinal microcapsules witsowt bacterial stain Niu-016 were anatomy. In order to know whether such kind of microcapsules will experience similer procedures as that of in vitro experiments. The feces of tested ICR mice were collected for detection of eliminated microcapsules after being fed with microcapsules. But no microcapsules were found in any feces. At necropsy, there were a lot of microcapsules in ICR mouse stomach after 2 hours, but in intestine none of the miorocapasules were found. Six hours later, all the microcapsules disappeared in stomach. Microcapsules were not found neither in stomach nor intestine after 12 hours. Since no microcapsules were found in either mice feces by anatoy, which illustratment that all microcapsules can break throw completely 12 hours.
Since the daidzein biotransforming activity was able to be totally inhibited for just about two weeks by using antibiotics. The deteced concentred of DHD by microflora of tested mice fed with bacterial strain Niu-O16 was much higher than that of blank control, it is still not sure whether baeterial strain Niu-016 can plant in intestinal tract of ICR mice or not. A further study in needed to be done.
引文
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