[Biotransformation of psoralenoside and isopsoralenoside in Psoraleae Fructus incubated with human intestinal bacteria flora in vitro].

Absorption is crucial to the resultant efficacy of oral drugs where the intestinal bacteria flora functions as one of the first-pass effects.The present study investigated the biotransformation of psoralenoside and isopsoralenoside in Chinese medicine Psoraleae Fructus(the dried fruit of Psoralea corylifolia) with the internationally recognized human intestinal bacteria flora model in vitro.Pso-ralenoside and isopsoralenoside were anaerobically incubated with human intestinal bacteria flora at 37 ℃, respectively, and biotransformation products were analyzed and identified using high-performance liquid chromatography-tandem mass spectrometry(HPLC-MS) and comparison with reference standards.The main biotransformation products of psoralenoside were psoralen and a small amount of 6,7-furano-hydrocoumaric acid, and the main biotransformation products of isopsoralenoside were isopsoralen and a small amount of 5,6-furano-hydrocoumaric acid.

Biotransformation of Ginsenosides (Rb1 , Rb2 , Rb3 , Rc) in Human Intestinal Bacteria and Its Effect on Intestinal Flora.

Ginsenosides, including Rb1 , Rb2 , Rb3 and Rc, belong to protopanaxadiol-type saponins in Panax ginseng C. A. Mey. Their contents are high in P. ginseng. They could inhibit oxidant stress, enhance immunity, lower blood sugar, resist tumor cells and facilitate other physiological activities. This study aimed to explore the interaction between ginsenosides Rb1 , Rb2 , Rb3 and Rc and the intestinal flora of healthy people. It also sought to analyse the biotransformation products and pathways of these ginsenosides in in-vitro human intestinal bacteria and their effects on the diversity of human intestinal flora. Human intestinal bacteria were incubated with ginsenosides Rb1 , Rb2 , Rb3 and Rc at 37 °C under anaerobic conditions. Samples were taken at different timepoints. The transformed products were identified by rapid high-resolution liquid chromatography-quadrupole time-of-flight mass spectrometry. After 48 h of transformation, the transformed product of ginsenosides Rb1 , Rb2 , Rb3 and Rc was ginsenoside compound K. The transformation rates were 83.5 %, 88.7 %, 85.6 %, and 84.2 %. 16S rRNA sequencing technology was applied to the bioinformatic analysis of faecal samples incubated for 48 h. Relative to the blank control, the relative abundance of Firmicutes and Proteobacteria significantly increased at the phylum level. Moreover, the relative abundance of Bacteroidetes significantly decreased in ginsenosides Rb1 , Rb2 , Rb3 and Rc. At the genus level, the relative abundance of Escherichia significantly increased, whereas that of Dorea, Prevotella and Megasphaera significantly decreased in all groups. These results showed that Rb1 , Rb2 , Rb3 and Rc could improve the structure and diversity of human intestinal flora and balance the metabolic process.

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.

Biotransformation and metabolic profile of caudatin-2,6-dideoxy-3-O-methy-ß-d-cymaropyranoside with human intestinal microflora by liquid chromatography quadrupole time-of-flight mass spectrometry.

In our previous studies, caudatin-2,6-dideoxy-3-O-methy-ß-d- cymaropyranoside (CDMC) was for the first time isolated from Cynanchum auriculatum Royle ex Wightand and was reported to possess a wide range of biological activities. However, the routes and metabolites of CDMC produced by intestinal bacteria are not well understood. In this study, ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) technique combined with Metabolynx(TM) software was applied to analyze metabolites of CDMC by human intestinal bacteria. The incubated samples collected for 48 h in an anaerobic incubator and extracted with ethyl acetate were analyzed by UPLC-Q-TOF-MS within 12 min. Eight metabolites were identified based on MS and MS/MS data. The results indicated that hydrolysis, hydrogenation, demethylation and hydroxylation were the major metabolic pathways of CDMC in vitro. Seven strains of bacteria including Bacillus sp. 46, Enterococcus sp. 30 and sp. 45, Escherichia sp. 49A, sp. 64, sp. 68 and sp. 75 were further identified using 16S rRNA gene sequencing owing to their relatively strong metabolic capacity toward CDMC. The present study provides important information about metabolic routes of CDMC and the roles of different intestinal bacteria in the metabolism of CDMC. Moreover, those metabolites might influence the biological effect of CDMC in vivo, which affects the clinical effects of this medicinal plant.

Ardipusilloside-I Metabolites from Human Intestinal Bacteria and Their Antitumor Activity.

Ardipusilloside-I (ADS-I) is a triterpenoid saponin extracted from Ardisia pusilla DC, and has been demonstrated to have potent antitumor activity. However, ADS-I metabolism in humans has not been investigated. In this study, we studied the biotransformation of ADS-I in human intestinal bacteria, and examined the in vitro antitumor activity of the major metabolites. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was used to detect ADS-I biotransformation products, and their chemical structures were identified by high performance liquid chromatography-nuclear magnetic resonance (HPLC-NMR). The antitumor activity of the major metabolites was determined by the MTT assay. Here, we show that main reaction seen in the metabolism of ADS-I in human intestinal bacteria was deglycosylation, which produced a total of four metabolites. The structures of the two major metabolites M1 and M2 were confirmed by using NMR. MTT assay showed that ADS-I metabolites M1 and M2 have the same levels of inhibitory activities as ADS-I in cultured SMMC-7721 cells and MCF-7 cells. In conclusion, this study demonstrates deglycosylation as a primary pathway of ADS-I metabolism in human intestinal bacteria, and suggests that the pharmacological activity of ADS-I may be mediated, at least in part, by its metabolites.

Deglycosylation of isoflavone C-glycosides by newly isolated human intestinal bacteria.

BACKGROUND: Plant isoflavones are mostly present in the glycoside form. Isoflavone aglycones produced by intestinal microflora are reported to be more bioactive than the glycoside form. However, the deglycosylation of isoflavone C-glycosides is known to be rare, and is less studied. RESULTS: Three new bacteria were isolated from human faecal samples, two of which hydrolysed the C-glycosidic bond of puerarin, daidzein-8-C-glucoside. They were identified as two Lactococcus species, herein designated as MRG-IFC-1 and MRG-IFC-3, and an Enterococcus species, herein designated MRG-IFC-2, based on their 16S rDNA sequences. From a reactivity study, it was found that Lactococcus sp. MRG-IFC-1 and Enterococcus sp. MRG-IFC-2 hydrolysed isoflavone C- and O-glycosides, as well as the flavone O-glycoside apigetrin, but could not hydrolyse the flavone C-glycosidic bond of vitexin. The other Lactococcus sp., MRG-IF-3, could not hydrolyse the C-glycosidic linkage of puerarin, while it showed a broad substrate spectrum of O-glycosidase activity similar to the other two bacteria. Puerarin was completely converted to daidzein within 100 min by Lactococcus sp. MRG-IFC-1 and Enterococcus sp. MRG-IFC-2, which is the fastest conversion among the reported human intestinal bacteria. CONCLUSION: Two new puerarin-metabolising human intestinal bacteria were isolated and identified, and the deglycosylation activity for various flavonoid glycosides was investigated. The results could facilitate the study of C-glycosidase reaction mechanisms, as well as the pharmacokinetics of bioactive C-glycoside natural products.

The metabolic profile elucidation of Lonicera japonica flos water extract and the metabolic characteristics evaluation of bioactive compounds in human gastrointestinal tract in vitro.

Lonicera japonica Flos (LJF) is taken orally as a health food and medicinal plant in China for a long time. The gastrointestinal metabolism of LJF was investigated in vitro by three independent models (gastric juice, intestinal juice, and human intestinal bacteria), qualitative analyzed by UPLC-LTQ-Orbitrap-MSn and quantified by HPLC-DAD. 72 prototype compounds were detected in LJF water extraction (LJF-WE), including 14 organic acids, 43 iridoids, 14 flavonoids and one other compound. The prototype and metabolic components of LJF-WE bio-transformed by simulated gastric fluid (70 and 12), intestinal fluid (69 and 12) and human fecal bacteria (29 and 70) were characterized, respectively. The metabolites were formed through desaccharization, isomerization, hydrogenation, methylation, dehydration, and then cyclization, glucuronization and dimethylation followed. 8 bioactive compounds including neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid, sweroside, secoxyloganin, isochlorogenic acid B, isochlorogenic acid A and isochlorogenic acid C were much stable in simulated gastric fluid and intestinal fluid, compared with human fecal bacteria. Especially, sweroside and secoxyloganin with glucoside bonds degradated extraordinarily fast, because of the abundant ß-glucosidases in human fecal bacteria.

Metabolic profile and underlying antioxidant improvement of Ziziphi Spinosae Folium by human intestinal bacteria.

Ziziphi Spinosae Folium, the leaf of Ziziphus jujuba Mill. Var. spinosa (Bunge) Hu ex H. F. Chou (LZJS), is currently used as a healthy tea in China. This study evaluated the chemical components and antioxidant activities of LZJS flavonoid (LZJSF) and fermented LZJSF (FLZJSF) using human intestinal bacteria (HIB) through dynamic fermentation. Eighteen flavonoids were simultaneously identified in LZJSF using UHPLC-Q-Orbitrap-MS method, nine of which were targeted for a HIB metabolism study. Seven small phenolic acids were identified in FLZJSF. Not only at chemical level but also at PC12 cell level, FLZJSF samples fermented for 4 and 6 h showed significant positive correlation between their activities and flavonoid aglycones, which were transformed from LZJSF. However, FLZJSF samples (8 h and longer time) mainly contained phenolic acids and indicated weak activities. Thus, LZJSF was found to result in increased antioxidant activity and could be commercially utilized as a novel functional food.

Bifidobacterium adolescentis P2P3, a Human Gut Bacterium Having Strong Non-Gelatinized Resistant Starch-Degrading Activity.

Resistant starch (RS) is metabolized by gut microbiota and involved in the production of short-chain fatty acids, which are related to a variety of physiological and health effects. Therefore, the availability of RS as a prebiotic is a topic of interest, and research on gut bacteria that can decompose RS is also important. The objectives in this study were 1) to isolate a human gut bacterium having strong degradation activity on non-gelatinized RS, 2) to characterize its RS-degrading characteristics, and 3) to investigate its probiotic effects, including a growth stimulation effect on other gut bacteria and an immunomodulatory effect. Bifidobacterium adolescentis P2P3 showing very strong RS granule utilization activity was isolated. It can attach to RS granules and form them into clusters. It also utilizes high-amylose corn starch granules up to 63.3%, and efficiently decomposes other various types of commercial RS without gelatinization. In a coculture experiment, Bacteroides thetaiotaomicron ATCC 29148, isolated from human feces, was able to grow using carbon sources generated from RS granules by B. adolescentis P2P3. In addition, B. adolescentis P2P3 demonstrated the ability to stimulate secretion of Th1 type cytokines from mouse macrophages in vitro that was not shown in other B. adolescentis. These results suggested that B. adolescentis P2P3 is a useful probiotic candidate, having immunomodulatory activity as well as the ability to feed other gut bacteria using RS as a prebiotic.

Human gastrointestinal metabolism of the anti-rheumatic fraction of Dianbaizhu (Gaultheria leucocarpa var. yunnanensis) in vitro: Elucidation of the metabolic analysis in gastric juice, intestinal juice and human intestinal bacteria by UPLC-LTQ-Orbitrap-MSn and HPLC-DAD.

The anti-rheumatic fraction (ARF), is responsible for the anti-inflammatory and analgesic effects of Dianbaizhu derived from the aerial part of Gaultheria leucocarpa var. yunnanensis (Ericaceae). The gastrointestinal metabolism of ARF was investigated in vitro through simulating a series of models-gastric juice, intestinal juice, and human intestinal bacteria, analyzed by HPLC-DAD and UPLC-LTQ-Orbitrap-MSn. ARF includes three categories: methyl salicylate glycosides, organic acids and the others. The primordial and metabolic components of ARF bio-transformed by simulated gastric fluid (36 and 13), intestinal fluid (29 and 7) and two human fecal bacteria (34 and 34, 40 and 25) were characterized, respectively. The methyl salicylate glycosides, MSTG-B, MSTG-A and gaultherin, with terminal-xylosyl-moiety in sugar chain were always being found in the whole gastrointestinal incubation processing. The metabolites were formed through hydrolysis of ester and glucosidic bond, as well as methylation, hydroxylation, acetylation, sulfation, reduction, decarboxylation, deglycosylation and glucuronidation. The metabolic conversion effect of the four index compounds, MSTG-B, MSTG-A, gaultherin, and chlorogenic acid by human intestinal bacteria exhibited much stronger. Those markers' variation in content-time curve in volunteer A gut flora were faster than that in volunteer B's. These results indicate that ARF is relatively stable in the gastrointestinal tract.

Identification of active compound combination contributing to anti-inflammatory activity of Xiao-Cheng-Qi Decoction via human intestinal bacterial metabolism.

Human intestinal bacteria play an important role in the metabolism of herbal medicines, leading to the variations in their pharmacological profile. The present study aimed to investigate the metabolism of Xiao-Cheng-Qi decoction (XCQD) by human intestinal bacteria and to discover active component combination (ACC) contributing to the anti-inflammatory activity of XCQD. The water extract of XCQD was anaerobically incubated with human intestinal bacteria suspensions for 48 h at 37 °C. A liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-Q-TOF/MS) method was performed for identification of the metabolites. In addition, the anti-inflammatory effects of XCQD and biotransformed XCQD (XCQD-BT) were evaluated in vitro with cytokines in RAW264.7 cells induced by lipopolysaccharide (LPS). A total of 51 compounds were identified in XCQD and XCQD-BT. Among them, 20 metabolites were proven to be transformed by human intestinal bacteria. Significantly, a combination of 14 compounds was identified as ACC from XCQD-BT, which was as effective as XCQD in cell models of inflammation. In conclusion, this study provided an applicable method, based on intestinal bacterial metabolism, for identifying combinatory compounds responsible for a certain pharmacological activity of herbal medicines.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Demethylation of Polymethoxyflavones by Human Gut Bacterium, Blautia sp. MRG-PMF1.

Polymethoxyflavones (PMFs) were biotransformed to various demethylated metabolites in the human intestine by the PMF-metabolizing bacterium, Blautia sp. MRG-PMF1. Because the newly formed metabolites can have different biological activities, the pathways and regioselectivity of PMF bioconversion were investigated. Using an anaerobic in vitro study, 12 PMFs, 5,7-dimethoxyflavone (5,7-DMF), 5-hydroxy-7-methoxyflavone (5-OH-7-MF), 3,5,7-trimethoxyflavone (3,5,7-TMF), 5-hydroxy-3,7-dimethoxyflavone (5-OH-3,7-DMF), 5,7,4'-trimethoxyflavone (5,7,4'-TMF), 5-hydroxy-7,4'-dimethoxyflavone (5-OH-7,4'-DMF), 3,5,7,4'-tetramethoxyflavone (3,5,7,4'-TMF), 5-hydroxy-3,7,4'-trimethoxyflavone (5-OH-3,7,4'-TMF), 5,7,3',4'-tetramethoxyflavone (5,7,3',4'-TMF), 3,5,7,3',4'-pentamethoxyflavone (3,5,7,3',4'-PMF), 5-hydroxy-3,7,3',4'-tetramethoxyflavone (5-OH-3,7,3',4'-TMF), and 5,3'-dihydroxy-3,7,4'-trimethoxyflavone (5,3'-diOH-3,7,4'-TMF), were converted to chrysin, apigenin, galangin, kaempferol, luteolin, and quercetin after complete demethylation. The time-course monitoring of PMF biotransformations elucidated bioconversion pathways, including the identification of metabolic intermediates. As a robust flavonoid demethylase, regioselectivity of PMF demethylation generally followed the order C-7 > C-4' ≈ C-3' > C-5 > C-3. PMF demethylase in the MRG-PMF1 strain was suggested as a Co-corrinoid methyltransferase system, and this was supported by the experiments utilizing other methyl aryl ether substrates and inhibitors.

Isolation and Characterization of a Human Intestinal Bacterium Eggerthella sp. AUH-JLD49s for the Conversion of (-)-3'-Desmethylarctigenin.

Arctiin is the most abundant bioactive compound contained in the Arctium lappa plant. In our previous study, we isolated one single bacterium capable of bioconverting arctigenin, an aglycone of arctiin, to 3'-desmethylarctigenin (3'-DMAG) solely. However, to date, a specific bacterium capable of producing other arctiin metabolites has not been reported. In this study, we isolated one single bacterium, which we named Eggerthella sp. AUH-JLD49s, capable of bioconverting 3'-DMAG under anaerobic conditions. The metabolite of 3'-DMAG by strain AUH-JLD49s was identified as 3'-desmethyl-4'-dehydroxyarctigenin (DMDH-AG) based on electrospray ionization mass spectrometry (ESI-MS) and 1H and 13C nuclear magnetic resonance spectroscopy. The bioconversion kinetics and bioconversion capacity of strain AUH-JLD49s were investigated. In addition, the metabolite DMDH-AG showed an inhibitory effect on cell growth of human colon cancer cell line HCT116 and human breast cancer cell line MDA-MB-231.

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.

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.

Biotransformation and metabolic profile of catalpol with human intestinal microflora by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Traditional Chinese medicine (TCM) has been used in clinical practice for thousands of years. Catalpol, an iridoid glucoside, abundantly found in the root of the common used herb medicine Rehmannia glutinosa Libosch, has been reported to show various biological effects and pharmacological activities. After oral administration, the active ingredient might have interactions with the intestinal bacteria, which could help unravel how the medicine was processed in vivo. In this work, different pure bacteria from healthy human feces were isolated and used to bioconvert catalpol. Ultra performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF/MS) technique combined with Metabolynx(™) software was applied to analyze catalpol metabolites. Compared with blank samples, parent compound (M0) and four metabolites (M1-M4) were detected and tentatively identified based on the characteristics of their protonated ions. The metabolites were likely to be: catalpol aglycone (M1), acetylated catalpol (M2), dimethylated and hydroxylated catalpol aglycone (M3), nitrogen-containing catalpol aglycone (M4). M1 and M4 were generated in the majority of the samples like Bacteroides sp. 45. M3 was obtained in several bacterial samples like Enterococcus sp. 8-2 and M2 was detected only in the sample of Enterococcus sp. 43-1. To our knowledge, the metabolic routes and metabolites of catalpol produced by human intestinal bacteria were all firstly reported.

Screening and identification of three typical phenylethanoid glycosides metabolites from Cistanches Herba by human intestinal bacteria using UPLC/Q-TOF-MS.

Acteoside, isoacteoside, and 2'-acetylacteoside are three representative phenylethanoid glycosides (PhGs), which are widely distributed in many plants and also known as the active components of Cistanches Herba. However, the extremely low oral bioavailability of acteoside in rats implies that these structural similar components may go through multiple sequential routes of hydrolysis in gastrointestinal tract before they are absorbed into blood. Therefore, the metabolites of these three components and other PhGs from gastrointestinal tract such as echinacoside, are supposed to be the bioactive elements. In this study, we established an approach combining ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) with MS(E) technology and MetaboLynx™ software for the rapid metabolic profiling of acteoside, isoacteoside, and 2'-acetylacteoside by human intestinal bacteria. As a result, 11 metabolites of acteoside, 7 metabolites of isoacteoside, and 11 metabolites of 2'-acetylacteoside were identified respectively. 8 metabolic pathways including deglycosylation, de-rhamnose, de-hydroxytyrosol, de-caffeoyl, deacetylation, reduction, acetylation, and sulfate conjugation were proposed to involve in the generation of these metabolites. Furthermore, we found that the degraded metabolites hydroxytyrosol (HT) and 3-hydroxyphenylpropionic (3-HPP) were transformed from acteoside, isoacteoside, and 2'-acetylacteoside by human intestinal bacteria and demonstrated similar bioactivities to their precursors. These findings are significant for our understanding of the metabolism of PhGs and the proposed metabolic pathways of bioactive components might be crucial for further pharmacokinetic evaluations of Cistanches Herba.

Identification of Echinacoside Metabolites Produced by Human Intestinal Bacteria Using Ultraperformance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

Echinacoside (ECH) is one of the representative phenylethanoid glycosides. It is widely present in plants and exhibits various bioactivities. However, the extremely low oral bioavailability of ECH in rats implies that ECH may go through multiple hydrolysis steps in the gastrointestinal tract prior to its absorption into the blood. Therefore, the gastrointestinal metabolites of ECH are more likely to be the bioactive components. This study established an approach combining ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) with MS(E) technology and MetaboLynx software for rapid analysis of the ECH metabolic profile produced by human intestinal bacteria. As a result, 13 ECH metabolites and 5 possible metabolic pathways (including deglycosylation, dehydroxylation, reduction, hydroxylation, and acetylation) were identified. Furthermore, hydroxytyrosol (HT) and 3-hydroxyphenylpropionic acid (3-HPP) were found to be the two bioactive metabolites of ECH produced by human intestinal bacteria.

Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry for rapid analysis of the metabolites of morroniside produced by human intestinal bacteria.

Morroniside, the most abundant iridoid glycoside in the valuable traditional Chinese medicine Fructus Corni, exhibits various pharmacological activities and biological effects. Intestinal flora plays an important role in the metabolism of drug compounds, which might lead to the variation of ethnopharmacological profile of the medicine. However, little is known of the interactions of the morroniside with human intestinal bacteria. In this study, different pure bacteria were isolated from human feces and their capability to convert morroniside were investigated. The metabolites of morroniside were analyzed by ultra high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) technique using Metabolynx™ software. Parent compound and three metabolites were detected and tentatively identified based on the characteristics of their protonated ions. The parent is proposed to be metabolized by three main metabolic pathways including deglycosylation, dehydroxylation and methylation. Morroniside was firstly metabolized to its aglycone (M1), and then was further converted to dehydroxylated aglycone (M2) and methylated aglycone (M3). This is the first report of the metabolism of morroniside by human intestinal bacteria. These metabolites might influence the biological activities of morroniside in vivo, which could affect the clinical effects of medicines. Thus, the study on the metabolism of morroniside by human intestinal bacteria is very helpful to unravel how traditional medicines work.