Identification of the Microbial Transformation Products of Secoisolariciresinol Using an Untargeted Metabolomics Approach and Evaluation of the Osteogenic Activities of the Metabolites.

Secoisolariciresinol (SECO) is one of the major lignans occurring in various grains, seeds, fruits, and vegetables. The gut microbiota plays an important role in the biotransformation of dietary lignans into enterolignans, which might exhibit more potent bioactivities than the precursor lignans. This study aimed to identify, synthesize, and evaluate the microbial metabolites of SECO and to develop efficient lead compounds from the metabolites for the treatment of osteoporosis. SECO was fermented with human gut microbiota in anaerobic or micro-aerobic environments at different time points. Samples derived from microbial transformation were analyzed using an untargeted metabolomics approach for metabolite identification. Nine metabolites were identified and synthesized. Their effects on cell viability, osteoblastic differentiation, and gene expression were examined. The results showed that five of the microbial metabolites exerted potential osteogenic effects similar to those of SECO or better. The results suggested that the enterolignans might account for the osteoporotic effects of SECO in vivo. Thus, the presence of the gut microbiota could offer a good way to form diverse enterolignans with bone-protective effects. The current study improves our understanding of the microbial transformation products of SECO and provides new approaches for new candidate identification in the treatment of osteoporosis.

Separation and Enrichment of Alkaloids from Coptidis Rhizoma and Euodiae Fructus by Macroporous Resin and Evaluation of the Effect on Bile Reflux Gastritis Rats.

The Zuojin Pill consists of Coptidis Rhizoma (CR) and Euodiae Fructus (EF). It has been a classic prescription for the treatment of gastrointestinal diseases in China since ancient times. Alkaloids are considered to be its main pharmacologically active substances. The authors of the present study investigated the feasibility of preparing high purity total alkaloids (TAs) from CR and EF extracts separately and evaluated the effect for the treatment of bile reflux gastritis (BRG). Coptis chinensis Franch. and Evodia rutaecarpa (Juss.) Benth. were used in the study. An optimized method for the enrichment and purification of TAs with macroporous resin was established. Furthermore, qualitative analysis by using ultra-high performance liquid chromatography coupled with electrospray ionization and quadrupole-time of flight mass spectrometry (UHPLC-ESI-QTOF-MS) was explored to identify the components of purified TAs. Thirty-one compounds, thirty alkaloids and one phenolic compound, were identified or tentatively assigned by comparison with reference standards or literature data. A method of ultra-high performance liquid chromatography coupled with diode array detector (UHPLC-DAD) for quantitative analysis was also developed. The contents of nine alkaloids were determined. Moreover, a rat model of BRG was used to investigate the therapeutic effect of the combination of purified TAs from CR and EF. Gastric pathologic examination suggested that the alkaloids' combination could markedly attenuate the pathological changes of gastric mucosa.

Characterization of the Anticoagulative Constituents of Angelicae Sinensis Radix and Their Metabolites in Rats by HPLC-DAD-ESI-IT-TOF-MSn.

Angelicae Sinensis Radix is commonly used in traditional Chinese medicine. Pharmacological studies show that Angelicae Sinensis Radix has clear anticoagulant activity. Therefore, in this study, the anticoagulant activity of crude Angelicae Sinensis Radix extracts was investigated by measuring the thrombin times of the extracts. The results revealed that the petroleum ether-soluble fraction of Angelicae Sinensis Radix exhibited significant anticoagulant activity in vitro, and 26 compounds were characterized by high-performance liquid chromatography with diode array detection combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry. In addition, 5 prototype constituents, 24 in vivo metabolites in rat urine and 7 prototype constituents, and 9 in vitro metabolites in the rat hepatic S9 incubation system of the petroleum ether-soluble fraction were tentatively identified. All metabolites were found from Angelicae Sinensis Radix for the first time. Among them, 13 (three ferulic acid-related constituents, six senkyunolide D-related constituents, and four senkyunolide F-related constituents) were identified as new metabolites (new compounds). This study is the first to qualitatively characterize the chemical constituents of the potent anticoagulative extract of Angelicae Sinensis Radix and to explore its metabolism. The result is a notable improvement in the discovery of Angelicae Sinensis Radix metabolites, and it provides the chemical basis for the effective forms and pharmacodynamic substances (prototypes, metabolites, or both) of the anticoagulant activity of Angelicae Sinensis Radix.

[Determination of markers from characteristic HPLC chromatogram of phenols in three official origins of Ephedrae Herba and quantitative analysis of four phenols].

This study is to establish the characteristic HPLC chromatogram of phenols in Ephedrae Herba, from which to pick out the marker peaks, followed by the analysis of the regularity of their distribution and content in the herbaceous stems of Ephedra sinica, E. intermedia and E. equisetina. The HPLC-DAD method for the characteristic chromatogram as well as quantitative analysis was established. The separation was carried out on a YMC-Pack ODS-A column (4.6 mm x 250 mm, 5 µm), eluted with the mobile phases as 0.01% formic acid aqueous solution (A) and acetonitrile (B) in a linear gradient (0-10 min, 17% B; 10-25 min, 17%-19% B; 25- 33 min, 19%-48% B; 33-35 min, 48%-51% B; 35-44 min, 51% B). The flow rate was kept at 1.0 mL · min⁻¹. The column tem- perature was 40 °C, and the detection wavelength was set at 350 nm (0-16 min) and 330 nm (16-44 min). Forty-six batches of collected samples from three official origins of Ephedrae Herba were detected, whose liquid chromatograms proven to be helpful to the differentiation of different origins. With principal component analysis and the analysis of distribution of peak area, twelve key peaks from the chromatogram were discussed in details on their contributions to the characteristics and differences of three official origins of the herb: peak area of peak 10, 11, 12 were found out to be significantly higher in E. equisetina than in other two origins, whose sum (higher than 146 mAU in E. equisetina) was useful for the discrimination between E. equisetina and the other two origins; peak area of 1 and 4 were respectively higher in E. sinica and E. intermedia than in other official origins, indicating their important effect on the differen- tiation of corresponding origins; peak 8 and 9 were picked out as two characteristic common peaks in three official origins of the herb, whose peak area showed little difference among different origins; further, peak area of other key peaks in the chromatogram also showed some difference among three origins, which make contributions to the differentiation of origins as well. Then, four phenols as 2"-O-α- L-rhamnosyl-isovitexin (1), vitexin (2), pollenitin B (5) and herbacetin-7-O-ß-D-glucoside (6) were quantitative analyzed with the above-mentioned method, with good linear relationship and accuracy (recoveries in a range of 97.8%-102.5%). The content of the four phenols were firstly reported in Ephedrae Herba from official origins, which were respectively trace-1.55 (1), trace-0.160 (2), trace-0.284 (5) and trace-0.620 (6) mg · g⁻¹ in all of the tested samples. In addition, the content of these phenols showed differences in three official origins, especially 1, whose content in E. sinica [(0.670 ± 0.88) mg ± g⁻¹] were significantly higher than in other two origins (lower than 0.16 mg ± g⁻¹ besides sample Ei-060630-2-2), and 6, whose average content in E. equisetina [(0.260 ± 0.039 2) mg · g⁻¹] were twice as high as in E. sinica [(0.120 ± 0.270) mg · g⁻¹] and E. intermedia [(0.136 ± 0.485) mg g⁻¹], indicating the important effects of the two constituents on the differentiation among three official origins of the herb. The method established for the characteristic HPLC chromatogram and quantitative analysis of phenols was simple and accurate, and the marker constituents selected may provide new guides for the discrimination of official origins as well as the improvement of quality criteria of EphedraeHerba.

The profiling and identification of the metabolites of (+)-catechin and study on their distribution in rats by HPLC-DAD-ESI-IT-TOF-MS(n) technique.

(+)-Catechin, a potential beneficial compound to human health, is widely distributed in plants and foods. A high-performance liquid chromatography with diode array detector and combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry method was applied to profile and identify the metabolites of (+)-catechin in rats and to study the distribution of these metabolites in rat organs for the first time. In total, 51 phase II metabolites (44 new) and three phase I metabolites were tentatively identified, comprising 16 (+)-catechin conjugates, 14 diarylpropan-2-ol metabolites, 6 phenyl valerolactone metabolites and 18 aromatic acid metabolites. Further, 19 phase II metabolites were new compounds. The in vivo metabolic reactions of (+)-catechin in rats were found to be ring-cleavage, sulfation, glucuronidation, methylation, dehydroxylation and dehydrogenation. The numbers of detected metabolites in urine, plasma, small intestine, kidney, liver, lung, heart, brain and spleen were 53, 23, 27, 9, 7, 5, 3, 2 and 1, respectively. This indicated that small intestine, kidney and liver were the major organs for the distribution of (+)-catechin metabolites. In addition, eight metabolites were found to possess bioactivities according to literature. These results are very helpful for better comprehension of the in vivo metabolism of (+)-catechin and its pharmacological actions, and also can give strong indications on the effective forms of (+)-catechin in vivo.

Non-ephedrine constituents from the herbaceous stems of Ephedra sinica.

Three new flavonoids, ephedroside A (1), ephedroside B (2), ephedroside C (3), together with fifty-four known compounds 4-57 were isolated from the EtOH extract of the herbaceous stems of Ephedra sinica. The structures of these compounds were elucidated by spectroscopic techniques, as well as by comparison with literature data. Thirty-eight of these compounds were isolated from the genus Ephedra for the first time. The antimicrobial activities of eight compounds were tested by measuring the minimum inhibitory concentrations (MIC) against bacteria (both Gram positive and Gram negative) and fungi, and were found to be in the range of 0.105-0.926 mM. Among them, compound 2 showed the best antimicrobial activity against Pseudomonas aeruginosa with MIC value of 0.105 mM.

Two new dihydrobenzofuran lignans from Rabdosia lophanthoides (Buch.-Ham.ex D.Don) Hara.

Two new dihydrobenzofuran lignanosides, (7R,8S)-4,3',9'-trihydroxyl-3-methoxyl-7,8-dihydrobenzofuran-1'-propylneolignan-9-O-(6-O-syringoyl)-beta-D-glucopyranoside, named lophanthoside B (1) and (7R,8S)-4,9,9'-trihydroxyl-3-methoxyl-7,8-dihydrobenzofuran-1'-propylneolignan-3'-O-beta-D-glucopyranoside (2), an enantiomer of umbroside, along with four known dihydrobenzofuran lignans (3-6), were isolated from 50% acetone extract of Rabdosia lophanthoides (Buch.-Ham.ex D.Don) Hara. Their structures were elucidated by NMR and MS experiments.

Chemical constituents from the leaves of Broussonetia papyrifera.

To separate and identify the chemical constituents from the leaves of Broussonetia papyrifera (Linn.) Vent, various columns including Diaion HP-20, Toyopearl HW-40C, Sephadex LH-20, silica gel were employed for the isolation and purification of compounds from the leaves of B. papyrifera. The structures of the compounds were elucidated by their physiochemical characteristics and spectral data. Nineteen compounds were isolated from the leaves of B. papyrifera and their structures were identified as apigenin (1), apigenin-7-O-beta-D-glucopyranoside (2), chrysoerid-7-O-beta-D-glucopyranoside (3), apigenin-7-O-beta-D-glucopyranuronide (4), vitexin-7-O-beta-D-glucopyranoside (5), luteolin (6), 5,7,4'-trihydroxyl-6-C-[a-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranosyl flavone (7), 5,7,4'-trihydroxyl-8-C-[a-L-rhamnopyranosyl (1-->2)]-beta-D-glucopyranosyl flavone (8), saponaretin (9), vitexin (10), benzyl benzoate-2, 6-di-O-beta-D-glucopyranoside (11), (2R, 3R, 5R, 6S, 9R)-3-hydroxy-5,6-epoxy-beta-ionol-2-O-beta-D-glucopyranoside (12), (2R, 3R, 5R, 6S, 9R)-3-hydroxyl-5,6-epoxy-acetyl-beta-ionol-2-O-beta-D-glucopyranoside (13), ficustriol (14), (6S, 9S)-roseoside (15), 3beta-hydroxy-5alpha,6alpha-epoxy-beta-ionone-2alpha-O-beta-D-glucopyranoside (16), icariside B1 (17), sammangaoside A (18), 3-hydroxy-5alpha,6alpha-epoxy-beta-ionone (19). Compounds 11, 12 and 13 are new compounds, the others are isolated from this genus Broussonetia for the first time.

Aristololactams and aporphines from the stems of Fissistigma oldhamii (Annonaceae).

Two aristololactams, aristololactam GI (1) and aristololactam GII (2), and three aporphines, fissistigamide A (3), fissistigamide B (4) and fissistigmine (5), together with nineteen known alkaloids, one flavone and one anthraquinone were isolated from the ethanol extracts of the stems of Fissistigma oldhamii (Annonaceae). Their structures were elucidated primarily by analysis of NMR, IR, UV, MS and CD data. Alkaloid 1 is a chiral aristololactam formed from a phenylpropanoid derivative attached to a 3,4-dihydroxy aristololactam scaffold. The absolute configuration of 1 was determined by comparing experimental and calculated ECD spectra. The anti-inflammatory activity of the crude extracts and the five alkaloids were tested by measuring the amount of TNF-α and IL-6 released from LPS stimulated RAW264 cell via ELISA. The results demonstrated that the CHCl(3)-soluble part and alkaloid 2 exhibited significant anti-inflammatory activity in vitro in both assays.

The profiling and identification of the absorbed constituents and metabolites of Paeoniae Radix Rubra decoction in rat plasma and urine by the HPLC-DAD-ESI-IT-TOF-MS(n) technique: a novel strategy for the systematic screening and identification of absorbed constituents and metabolites from traditional Chinese medicines.

Paeoniae Radix Rubra (PRR, the dried roots of Paeonia lactiflora) is a commonly used traditional Chinese medicine (TCM). A clear understanding of the absorption and metabolism of TCMs is very important in their rational clinical use and pharmacological research. To find more of the absorbed constituents and metabolites of TCMs, a novel strategy was proposed. This strategy was characterized by the following: the establishment and utilization of the databases of parent compounds, known metabolites and characteristic neutral losses; the comparison of base peak chromatograms and ClogPs; and the use of the HPLC-DAD-ESI-IT-TOF-MS(n) technique. This strategy was first applied to screen and identify the absorbed constituents and metabolites of PRR decoction and paeoniflorin in rats. In total, 13 new absorbed constituents and 90 new metabolites of PRR decoction were detected. Among these metabolites, the structures of 70 metabolites were identified, and the conjugation types and structure skeletons of the other 20 metabolites were preliminarily determined. Moreover, 35 new metabolites of some constituents of PRR, i.e., 22 new metabolites of paeoniflorin, 10 new metabolites of gallic acid-related compounds, 1 new metabolite of (epi)catechin-related compounds, and 2 new metabolites of other compounds, were reported for the first time. The results also indicated that (epi)catechin-related compounds, gallic acid-related compounds and paeoniflorin were the main precursors of these metabolites. Phase I reactions (dehydroxylation, decarboxylation, dehydrogenation) and phase II reactions (sulfation, glucuronidation and methylation) were observed as the main metabolic pathways of PRR. According to the literature, the 11 absorbed constituents and 11 metabolites have various bioactivities. This study is the first to explore the absorption and metabolism of PRR decoction, and the result also is a notable improvement in the discovery of paeoniflorin metabolites in vivo. These findings enhance our understanding of the metabolism and Effective forms (the truly active structures) of PRR decoction and paeoniflorin.