Human Intestinal Bacteria Research Articles

Molecular basis of an agarose metabolic pathway acquired by a human intestinal symbiont

In red algae, the most abundant principal cell wall polysaccharides are mixed galactan agars, of which agarose is a common component. While bioconversion of agarose is predominantly catalyzed by bacteria that live in the oceans, agarases have been discovered in microorganisms that inhabit diverse terrestrial ecosystems, including human intestines. Here we comprehensively define the structure–function relationship of the agarolytic pathway from the human intestinal bacterium Bacteroides uniformis (Bu) NP1. Using recombinant agarases from Bu NP1 to completely depolymerize agarose, we demonstrate that a non-agarolytic Bu strain can grow on GAL released from agarose. This relationship underscores that rare nutrient utilization by intestinal bacteria is facilitated by the acquisition of highly specific enzymes that unlock inaccessible carbohydrate resources contained within unusual polysaccharides. Intriguingly, the agarolytic pathway is differentially distributed throughout geographically distinct human microbiomes, reflecting a complex historical context for agarose consumption by human beings.

Intestinal metabolism of Polygonum cuspidatum in vitro and in vivo.

Rhizoma et Radix Polygoni Cuspidati (RRPC) is commonly prescribed for the treatment of amenorrhea, arthralgia, jaundice and abscess in traditional Chinese medicine. Previous pharmacological studies have indicated that polyphenols are the main pharmacological active ingredients in RRPC. Meanwhile, the poor bioavailability of polyphenols in RRPC implies that those components are probably metabolized by intestinal bacteria before absorption. However, there is rather limited information about RRPC''s metabolites produced by intestinal bacteria and the intestinal absorbed constituents. In the present study, the metabolites were characterized after the aqueous extract of RRPC was incubated with the crude enzyme of human intestinal bacteria in vitro. The metabolic characteristics of glycosides in RRPC were figured out by comparing the metabolic profiles of emodin-8-O-β-d-glucopyranoside and polydatin between aqueous extract of RRPC and equivalent amounts of these two glycosides. The transitional constituents absorbed into blood were investigated in rats via intraduodental administration and portal vein intubation. A total of 38 prototype components and 43 metabolites were detected and characterized in vivo. The overall results demonstrated that the intestinal bacteria played an important role in the metabolism of RRPC, and the main metabolic pathways were hydrolysis in vitro, glucuronidation and sulfation in vivo.

Biotransformasi Tartrazin oleh Bakteri Usus Manusia

The aimed of this research was to investigated human intestinal bacteria (Streptococcus faecalis, Eschericia coli, Salmonella Sp., and Vibrio fischeri), to investigated dose of tartrazine, and to investigated biotransformation of tartrazine by human intestinal bacteria (Streptococcus faecalis, Eschericia coli, Salmonella Sp., and Vibrio fischeri). The method of this research was experiment with significant correlation to investigated biotransformation of tartrazine by human intestinal bacteria (Streptococcus faecalis, Eschericia coli, Salmonella Sp., and Vibrio fischeri). This study is primarily focussed on made bacteria medium, made standart inoculums of bacteria, and evaluation for activity of human intestinal bacteria (Streptococcus faecalis, Eschericia coli, Salmonella Sp., and Vibrio fischeri). The results obtained were attributed to the human intestinal bacteria was used in this research was biotransformated tartrazine. From the results that had been increase dose of tartrazine correlation with increase biotransformation by human intestinal bacteria. Highest dose of tartrazine was 15 mg, and highest biotransformation by Vibrio fischeri, which rate of zone decolourisation diameter was 1,22 cm, and rate of zone decolourisation was 1,44 cm2. Statistically showed significantly in correlation dose of tartrazine with rate of zone decolourisation (0,001 < P < 0,05), but statistically showed non significantly in correlation bacteria with rate of zone decolourisation (0,05 < P < 0,268).

"Omic" investigations of protozoa and worms for a deeper understanding of the human gut "parasitome".

The human gut has been continuously exposed to a broad spectrum of intestinal organisms, including viruses, bacteria, fungi, and parasites (protozoa and worms), over millions of years of coevolution, and plays a central role in human health. The modern lifestyles of Western countries, such as the adoption of highly hygienic habits, the extensive use of antimicrobial drugs, and increasing globalisation, have dramatically altered the composition of the gut milieu, especially in terms of its eukaryotic “citizens.” In the past few decades, numerous studies have highlighted the composition and role of human intestinal bacteria in physiological and pathological conditions, while few investigations exist on gut parasites and particularly on their coexistence and interaction with the intestinal microbiota. Studies of the gut “parasitome” through “omic” technologies, such as (meta)genomics, transcriptomics, proteomics, and metabolomics, are herein reviewed to better understand their role in the relationships between intestinal parasites, host, and resident prokaryotes, whether pathogens or commensals. Systems biology–based profiles of the gut “parasitome” under physiological and severe disease conditions can indeed contribute to the control of infectious diseases and offer a new perspective of omics-assisted tropical medicine.

Human Gastrointestinal Metabolism of the Cistanches Herba Water Extract in Vitro: Elucidation of the Metabolic Profile Based on Comprehensive Metabolite Identification in Gastric Juice, Intestinal Juice, Human Intestinal Bacteria, and Intestinal Microsomes.

Cistanches Herba is taken orally as a health food supplement and medicinal plant in Asian countries. It consists of the stems of Cistanche deserticola (CD) and Cistanche tubulosa (CT). The gastrointestinal metabolism of the multiple components contained in Cistanches Herba is crucial for the discovery of bioactive constituents. This study aims to elucidate the comprehensive metabolic profile of the Cistanches Herba water extract by simulating human gastrointestinal metabolism in vitro independently and sequentially using four models: gastric juice, intestinal juice, human intestinal bacteria, and human intestinal microsomes. A total of 35 and 18 metabolites were characterized from CD and CT water extracts, respectively. These metabolites were formed through reduction, methylation, dimethylation, deglycosylation, decaffeoyl, derhamnose, dehydrogenation, and glucuronidation. The difference in metabolites of the Cistanches Herba water extract and single compounds and the difference in metabolites of CD and CT water extracts were caused by the oligosaccharides and polysaccharides in Cistanches Herba.

The activity of Hou-Po-Da-Huang-Tang is improved through intestinal bacterial metabolism and Hou-Po-Da-Huang-Tang selectively stimulate the growth of intestinal bacteria associated with health

Hou-Po-Da-Huang Tang (HPDHT) was used for the treatment of intestinal tract diseases in China. However, the underlying mechanisms via the intestinal bacteria remain largely unclear. Therefore, the aim of this study was to evaluate the metabolism of HPDHT by the human intestinal bacteria and its modulating effect on the intestinal bacteria. As a result, a total of 34 compounds were identified in HPDHT and transformed HPDHT (T-HPDHT). Among them, 12 metabolites were proved to be transformed by human intestinal bacteria. In vitro assays showed that T-HPDHT exhibited more significant elevation of free radical scavenging activity and suppression on the production of nitric oxide (NO) and TNF-α when comparing to HPDHT. Additionally, in vivo experiment confirmed that HPDHT significantly increased activity of superoxide dismutase (SOD), attenuated the malondialdehyde (MDA) and TNF-α levels in the conventional rats compared with that of pseudo germ-free (PGF) rats. In addition, HPDHT could significantly enhance the mean counts of Bifidobacterium and Lactobacillus and inhibit the growth of Clostridium, and Enterobacteriaceae, relative to controls. Due to the transformation of HPDHT being dependent on the bacterial strain, the effect of HPDHT on the selective growth of Bifidobacterium bifidum 29521 and Lactobacillus plantarum 8014 was evaluated. The kinetic parameters of microbial growth and prebiotic activity scores indicated that HPDHT could selectively stimulate the growth of the strains Bifidobacterium bifidum 29521 and Lactobacillus plantarum 8014. Taken together, metabolism of HPDHT by intestinal bacteria is a critical step towards the emergence of their anti-oxidation, anti-inflammation and prebiotic activities. This study provided valuable information for further pharmacological research on HPDHT.

Isolation of Human Intestinal Bacteria Capable of Producing the Bioactive Metabolite Isourolithin A from Ellagic Acid.

Urolithins are intestinal microbial metabolites produced from ellagitannin- and ellagic acid-containing foods such as walnuts, strawberries, and pomegranates. These metabolites, better absorbed than their precursors, can contribute significantly to the beneficial properties attributed to the polyphenols ellagitannins and ellagic acid (EA). However, both the ability of producing the final metabolites in this catabolism (urolithins A, B and isourolithin A) and the health benefits associated with ellagitannin consumption differ considerably among individuals depending on their gut microbiota composition. Three human urolithin metabotypes have been previously described, i.e., metabotype 0 (urolithin non-producers), metabotype A (production of urolithin A as unique final urolithin) and metabotype B (urolithin B and/or isourolithin A are produced besides urolithin A). Although production of some intermediary urolithins has been recently attributed to intestinal species from Eggerthellaceae family named Gordonibacter urolithinfaciens and Gordonibacter pamelaeae, the identification of the microorganisms responsible for the complete transformation of EA into the final urolithins, especially those related to metabotype B, are still unknown. In the present research we illustrate the isolation of urolithin-producing strains from human feces of a healthy adult and their ability to transform EA into different urolithin metabolites, including isourolithin A. The isolates belong to a new genus from Eggerthellaceae family. EA transformation and urolithin production arisen during the stationary phase of the growth of the bacteria under anaerobic conditions. The HPLC-DAD-MS analyses demonstrated the sequential appearance of 3,8,9,10-tetrahydroxy-urolithin (urolithin M6), 3,8,9-trihydroxy-urolithin (urolithin C) and 3,9-dihydroxy-urolithin (isourolithin A) while 3,8-dihydroxy-urolithin (urolithin A) and 3-hydroxy-urolithin (urolithin B) were not detected. For the first time isourolithin A production capacity of pure strains has been described. The biological activity attributed to urolithins A and B and isourolithin A (anti-inflammatory, anti-carcinogenic, cardioprotective, and neuroprotective properties) explains the relevance of identifying these urolithin-producing bacteria as potential novel probiotics with applications in the development of functional foods and nutraceuticals. Their human administration could improve the health benefits upon ellagitannin consumption, especially in metabotype 0 individuals. However, further research is necessary to probe well-established beneficial effects on the host and safety requirements before being considered among the next-generation probiotics.

Simultaneous quantification and semi-quantification of amentoflavone and its metabolites in human intestinal bacteria by liquid chromatography Orbitrap high-resolution mass spectrometry.

A quick, easy, effective method followed by ultra-high-pressure liquid chromatography coupled with linear ion trap-Orbitrap tandem mass spectrometry (UHPLC-LTQ-Orbitrap MS) was developed for the simultaneous identification and quantification of the metabolites produced by amentoflavone (AMF) in human intestinal bacteria from human feces. The method validated for quantification of AMF concerning precision, accuracy, recovery, matrix effect, stability and limits showed acceptable results. Compared with blank human intestinal bacteria chromatography, three metabolites were identified based on high-accuracy protonated precursors and multi-stage mass spectrometry (MSn ) using the proposed strategy. At the same time, a new method was developed for semi-quantification of three metabolites. We describe the trend over 24 h of concentration-time curves for AMF and its metabolites. Moreover, the main metabolic pathway of AMF was clarified in human intestinal bacteria. The method was validated and successfully applied to the detection and quantification of AMF and its metabolites.

Comprehensive evaluation of the bactericidal activities of free bile acids in the large intestine of humans and rodents

In addition to functioning as detergents that aid digestion of dietary lipids in the intestine, some bile acids have been shown to exhibit antimicrobial activity. However, detailed information on the bactericidal activities of the diverse molecular species of bile acid in humans and rodents is largely unknown. Here, we investigated the toxicity of 14 typical human and rodent free bile acids (FBAs) by monitoring intracellular pH, membrane integrity, and viability of a human intestinal bacterium, Bifidobacterium breve Japan Collection of Microorganisms (JCM) 1192T, upon exposure to these FBAs. Of all FBAs evaluated, deoxycholic acid (DCA) and chenodeoxycholic acid displayed the highest toxicities. Nine FBAs common to humans and rodents demonstrated that α-hydroxy-type bile acids are more toxic than their oxo-derivatives and β-hydroxy-type epimers. In five rodent-specific FBAs, β-muricholic acid and hyodeoxycholic acid showed comparable toxicities at a level close to DCA. Similar trends were observed for the membrane-damaging effects and bactericidal activities to Blautia coccoides JCM 1395T and Bacteroides thetaiotaomicron DSM 2079T, commonly represented in the human and rodent gut microbiota. These findings will help us to determine the fundamental properties of FBAs and better understand the role of FBAs in the regulation of gut microbiota composition.

Curcuminoid Demethylation as an Alternative Metabolism by Human Intestinal Microbiota.

Curcumin and other curcuminoids from Curcuma longa are important bioactive compounds exhibiting various pharmacological activities. In addition to the known reductive metabolism of curcuminoids, an alternative biotransformation of curcuminoids by human gut microbiota is reported herein. A curcuminoid mixture, composed of curcumin (1), demethoxycurcumin (2), and bisdemethoxycurcumin (3), was metabolized by the human intestinal bacterium Blautia sp. MRG-PMF1. 1 and 2 were converted to new metabolites by the methyl aryl ether cleavage reaction. Two metabolites, demethylcurcumin (4) and bisdemethylcurcumin (5), were sequentially produced from 1, and demethyldemethoxycurcumin (6) was produced from 2. Until now, sequential reduction of the heptadienone backbone of curcuminoids was the only known metabolism to occur in the human intestine. In this study, a new intestinal metabolism of curcuminoids was discovered. Demethylation of curcuminoids produced three new colonic metabolites that were already known as promising synthetic curcumin analogues. The results could explain the observed beneficial effects of turmeric.

1104 - Interaction between the Herbal Preparation STW 5 and Human Intestinal Bacteria in Vitro

1104 - Interaction between the Herbal Preparation STW 5 and Human Intestinal Bacteria in Vitro

The Effect of Pullulan on the Growth and Acidifying Activity of Selected Stool Microflora of Human.

Pullulan is a microbial polysaccharide of low energy value, which can component of low-calorie foods and in dietary snacks for diabetics. The objective of the study was to determine the effect of pullulan on the growth and fermentation activity of selected human intestinal bacteria. Commercial pullulan purchased from Focubase (China) of a molecular weight of 100,000 Da constituted as experimental material. Food grade pullulan 99% purity. Two control media were prepared: the first standard RCM composed of (g/100 ml): 0.5 glucose, 0.1 soluble starch, 1.0 peptone, 1.0 meat extract, 0.3 yeast extract, 0.3 sodium acetate, 0.05 cysteine hydrochloride, 0.5 NaCl, pH 6.8; and the second modified RCM, wherein the soluble starch was replaced by increased glucose concentration to 2.0% (RCM+G). Experimental medium was the modified RCM medium, wherein the soluble starch and glucose were replaced by pullulan at a concentration of 2.0% (RCM+P). Stool suspensions were prepared from fresh stool samples (1 g) in peptone water (9 g), which were previously homogenized. Then, suspensions at a volume of 300 μl were transferred to the media (RCM, RCM+G, and RCM+P). After mixing, flasks were placed in anaerobic tubes with AnaeroGenTM 2.5 l sachets. Incubation of samples was carried out at 37°C for 48 h. The number of Bifidobacterium, Lactobacillus, and Escherichia coli bacteria, as well as pH and total acidity of the culture during 0, 24, and 48 h were measured. It was found that the numbers of bacteria of the Bifidobacterium and Lactobacillus genus in medium with pullulan were one logarithmic cycle lower in comparison to their numbers in the control media. Higher total acidity (1.48 g/100 ml) of pullulan culture in comparison to the control media was obtained (1.10 and 0.60 g/100 ml), and lower pH values than RCM medium, particularly 4.15 and 4.70, respectively. Pullulan exhibited selective effect on the natural microflora of the colon. Increase in the fermentation activity of bacteria in medium with pullulan favorably influenced modification of the composition of gut microbiota. In summary, pullulan exhibited a selective effect on the natural microflora of the infants' colon. Although no stimulating effect of pullulan on the growth of Bifidobacterium and Lactobacillus was observed, their increased acidifying activity, which probably was the cause of reduction in the number of E. coli bacteria, was confirmed.

Metabolic profile and underlying improved bio-activity of Fructus aurantii immaturus by human intestinal bacteria.

Fructus aurantii immaturus (FAI) is the dried young fruit of Citrus aurantium L. or Citrus sinensis L. Osbeck. The purpose of this paper was to investigate the metabolic fate of FAI upon incubation with human intestinal bacteria, meanwhile to evaluate the antioxidant and anti-inflammatory activities of FAI and the transformed Fructus aurantii immaturus (TFAI). The water extract of FAI was anaerobically incubated with human intestinal bacterial suspensions for 48 h at 37 °C. Liquid chromatography-hybridised with quadrupole-time-of-flight mass spectrometry (LC-Q-TOF/MS) was applied to identify FAI metabolites. A total of 45 compounds were identified in FAI, eleven of which were metabolized by human intestinal bacteria. Nine major metabolites were identified as eriodictyol, naringenin, hesperetin, luteolin, apigenin, chryseriol, isosakuranetin, phloretin and diosmetin. The metabolic profile of FAI was elucidated on the basis of metabolite information. We found that the concentrations of acetic, propionic and butyric acids in FAI culture were all increased during fermentation relative to those of the control. Further bioactive evaluations showed that TFAI exhibited more potent antioxidant and anti-inflammatory abilities than FAI in vitro. Additionally, in vivo experiment confirmed that FAI significantly attenuated the blood endotoxin and TNF-α levels in the conventional rats compared to those of pseudo-germ-free (PGF) rats. This study revealed that metabolites may play a key role in the antioxidant and anti-inflammatory capacities of FAI.

Cow Milk Contamination with Heavy Metals (Mercury and Lead) and the Possibility of Heavy Metals Disintegration by the Human Intestinal Bacteria

Metal pollution in the environment and human diet represents a perennial problem and is detrimental to human health. Developed countries are seeking ways to find a solution for this problem. However, in developing countries such as Iran largely due to the lack of appropriate infrastructures, all people are exposed to the risk of heavy metals and nobody pays due attention to this problem. Bioremediation projects, in which bacteria are used, are currently an active field to solve the problem. The present study aimed to identify bacteria that might be used for disintegration of heavy metals in foods (cow's milk) in the intestines of humans. First, the amount of mercury and lead in cow's milk was determined by atomic absorption spectrophotometry and then 25 bacterial strains from the human intestine were purified and identified. After that, MIC test was conducted on the basis of resistance against heavy metals for 25 samples. To confirm the disintegration of heavy metals, bacterial biomass was isolated, and the atomic absorptions were assessed. Finally, bacterial DNAs were extracted and sequenced. The collected milk samples were contaminated with mercury and lead and 2 out of 25 collected samples were able to disintegrate heavy metals. The DNAs for the two bacteria were isolated, sequenced, and compared with other sequences in the GeneBank. The results revealed that the two isolated bacteria were new strains of E. coli. Finally, the resulting species were labeled and registered with the name of the authors in the NCBI website. Our findings indicated that two strains of E. coli were able to disintegrate heavy metals (mercury and lead) from foodstuffs (cow milk) in the intestinal tract of humans.

Isoflavone metabolism by human intestinal bacteria

Isoflavone metabolism by human intestinal bacteria

The Metabolic Profiling of Isorhamnetin-3-O-Neohesperidoside Produced by Human Intestinal Flora Employing UPLC-Q-TOF/MS.

Isorhamnetin-3-O-neohesperidoside is the major active substance of Puhuang, a traditional herb medicine widely used in clinical practice to tackle many chronic diseases. However, little is known about the interactions between this ingredient and intestinal flora. In this study, ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry together with automated data analysis software (Metabolynx™) was used for analysis of the metabolic profile of isorhamnetin-3-O-neohesperidoside by the isolated human intestinal bacteria. The parent and three metabolites isorhamnetin-3-O-glucoside, isorhamnetin and quercetin were detected and identified based on the characteristics of their deprotonated molecules. These metabolites indicated that isorhamnetin-3-O-neohesperidoside was firstly deglycosylated to isorhamnetin-3-O-glucoside and subsequently to the aglycone isorhamnetin, and the latter was demethylated to quercetin. The majority of bacteria such as Escherichia sp. 23 were capable of converting isorhamnetin-3-O-neohesperidoside to considerable amounts of aglycone isorhamnetin and further to minor amounts of quercetin, while minor amounts of isorhamnetin-3-O-glucoside were detected in minority of bacterial samples such as Enterococcus sp. 30. The metabolic pathway and metabolites of isorhamnetin-3-O-neohesperidoside by the different human intestinal bacteria were firstly investigated. Furthermore, the metabolites of isorhamnetin-3-O-neohesperidoside might influence the effects of traditional herb medicines. Thus, our study is helpful to further unravel how isorhamnetin-3-O-neohesperidoside and Puhuang work in vivo.

Icariin Metabolism by Human Intestinal Microflora.

Icariin is a major bioactive compound of Epimedii Herba, a traditional oriental medicine exhibiting anti-cancer, anti-inflammatory and anti-osteoporosis activities. Recently, the estrogenic activities of icariin drew significant attention, but the published scientific data seemed not to be so consistent. To provide fundamental information for the study of the icaritin metabolism, the biotransformation of icariin by the human intestinal bacteria is reported for the first time. Together with human intestinal microflora, the three bacteria Streptococcus sp. MRG-ICA-B, Enterococcus sp. MRG-ICA-E, and Blautia sp. MRG-PMF-1 isolated from human intestine were reacted with icariin under anaerobic conditions. The metabolites including icariside II, icaritin, and desmethylicaritin, but not icariside I, were produced. The MRG-ICA-B and E strains hydrolyzed only the glucose moiety of icariin, and icariside II was the only metabolite. However, the MRG-PMF-1 strain metabolized icariin further to desmethylicaritin via icariside II and icaritin. From the results, along with the icariin metabolism by human microflora, it was evident that most icariin is quickly transformed to icariside II before absorption in the human intestine. We propose the pharmacokinetics of icariin should focus on metabolites such as icariside II, icaritin and desmethylicaritin to explain the discrepancy between the in vitro bioassay and pharmacological effects.

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols.

The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (+)-taxifolin but not that of (-)-taxifolin. The Km for (+)-taxifolin was 6.4 ± 0.8 μM, and the Vmax was 108 ± 4 μmol min-1 (mg protein)-1 The enzyme also isomerized (+)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439-1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus IMPORTANCE: Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria.

Cloning, expression, and characterization of a four-component O-demethylase from human intestinal bacterium Eubacterium limosum ZL-II.

Eubacterium limosum ZL-II was described to convert secoisolariciresinol (SECO) to its demethylating product 4,4'-dihydroxyenterodiol (DHEND) under anoxic conditions. However, the reaction cascade remains unclear. Here, the O-demethylase being responsible for the conversion was identified and characterized. Nine genes encoding two methyltransferase-Is (MT-I), two corrinoid proteins (CP), two methyltransferase-IIs (MT-II), and three activating enzymes (AE) were screened, cloned, and expressed in Escherichia coli. Four of the nine predicted enzymes, including ELI_2003 (MT-I), ELI_2004 (CP), ELI_2005 (MT-II), and ELI_0370 (AE), were confirmed to constitute the O-demethylase in E. limosum ZL-II. The complete O-demethylase (combining the four components) reaction system was reconstructed in vitro. As expected, the demethylating products 3-demethyl-SECO and DHEND were both produced. During the reaction process, ELI_2003 (MT-I) initially catalyzed the transfer of methyl group from SECO to the corrinoid of ELI_2004 ([CoI]-CP), yielding demethylating products and [CH3-CoIII]-CP; then ELI_2005 (MT-II) mediated the transfer of methyl group from [CH3-CoIII]-CP to tetrahydrofolate, forming methyltetrahydrofolate and [CoI]-CP. Due to the low redox potential of [CoII]/[CoI], [CoI]-CP was oxidized to [CoII]-CP immediately in vitro, and ELI_0370 (AE) was responsible for catalyzing the reduction of [CoII]-CP to its active form [CoI]-CP. The active-site residues in ELI_2003, ELI_2005, and ELI_0370 were subsequently determined using molecular modeling combined with site-directed mutagenesis. To our knowledge, this is the first study on the identification and characterization of a four-component O-demethylase from E. limosum ZL-II, which will facilitate the development of method to artificial synthesis of related bioactive chemicals.

Interaction of effective ingredients from traditional Chinese medicines with intestinal microbiota

A large number and wide varieties of microorganisms colonize in the human gastrointestinal tract. They construct an intestinal microecological system in the intestinal environment. The intestinal symbiotic flora regulates a series of life actions, including digestion and absorption of nutrient, immune response, biological antagonism, and is closely associated with the occurrence and development of many diseases. Therefore, it is greatly essential for the host's health status to maintain the equilibrium of intestinal microecological environment. After effective compositions of traditional Chinese medicines are metabolized or biotransformed by human intestinal bacteria, their metabolites can be absorbed more easily, and can even decrease or increase toxicity and then exhibit significant different biological effects. Meanwhile, traditional Chinese medicines can also regulate the composition of the intestinal flora and protect the function of intestinal mucosal barrier to restore the homeostasis of intestinal microecology. The relevant literatures in recent 15 years about the interactive relationship between traditional Chinese medicines and gut microbiota have been collected in this review, in order to study the classification of gut microflora, the relationship between intestinal dysbacteriosis and diseases, the important roles of gut microflora in intestinal bacterial metabolism in effective ingredients of traditional Chinese medicines and bioactivities, as well as the modulation effects of Chinese medicine on intestinal dysbacteriosis. In addition, it also makes a future prospect for the research strategies to study the mechanism of action of traditional Chinese medicines based on multi-omics techniques.