20 potentially new compounds and 11 new bioactive constituents found in Smilacis Glabrae Rhizoma utilizing HPLC-DAD-ESI-IT-TOF-MSn.

INTRODUCTION: Smilacis Glabrae Rhizoma (SGR) is rich in chemical constituents with a variety of pharmacological activities. However, in-depth research has yet to be conducted on the chemical and pharmacodynamic constituents of SGR. MATERIALS AND METHODS: In this study, the chemical constituents of SGR were analyzed using liquid chromatography-mass spectrometry, and the pharmacodynamic compounds responsible for the medicinal effects of SGR were elucidated through a literature review. RESULTS: In total, 20 potentially new compounds, including 16 flavonoids (C19, C20, and C27-C40) and four phenylpropanoids (C107, C112, C113, and C118), together with 161 known ones were identified in the ethanol extract of SGR using liquid chromatography-mass spectrometry, and 25 of them were unequivocally identified by comparison with reference compounds. Moreover, 17 known constituents of them were identified in the plants of genus Smilax for the first time, and 16 were identified in the plant Smilax glabra Roxb. for the first time. Of 161 known compounds, 84 constituents (including isomers) have been reported to have 17 types of pharmacological activities, covering all known pharmacological activities of SGR; among these 84 bioactive constituents, six were found in the plants of genus Smilax for the first time and five were found in S. glabra for the first time, which are new bioactive constituents found in the plants of genus Smilax and the plant S. glabra, respectively. CONCLUSION: The results provide further information on the chemical composition of SGR, laying the foundation for the elucidation of the pharmacodynamic substances of SGR.

[Existence forms of Tiantian Capsules and its raw material Aloe in rats].

This study explored the existence forms(original constituents and metabolites) of Tiantian Capsules, Aloe, and Tiantian Capsules without Aloe in rats for the first time, aiming to clarify the contribution of Aloe to the existence form of Tiantian Capsules. Rats were administrated with corresponding drugs by gavage once a day for seven consecutive days. All urine and feces samples were collected during the seven days of administration, and blood samples were collected 0.5, 1, and 1.5 h after the last administration. UHPLC-Q-TOF-MS was employed to detect and identify the original constituents and metabolites in the samples. A total of 34, 28, and 2 original constituents and 64, 94, and 0 metabolites were identified in the samples of rats administrated with Aloe, Tiantian Capsules, and Tiantian Capsules without Aloe, respectively. The main metabolic reactions were methylation, hydrogenation, hydroxylation, dehydroxylation, glucuronidation, and sulfation. This study clarified for the first time the existence forms and partial metabolic pathways of Aloe, Tiantian Capsules, and Tiantian Capsules without Aloe in rats, laying a foundation for revealing their effective forms. The findings are of great significance to the research on the functioning mechanism and quality control of Aloe and Tiantian Capsules.

Analysis of In Vivo Existence Forms of Nardosinone in Mice by UHPLC-Q-TOF-MS Technique.

Nardosinone, a sesquiterpene peroxide, is one of the main active constituents of the ethnomedicine Nardostachyos Radix et Rhizoma, and it has many bioactivities, such as antiarrhythmia and cardioprotection. To elucidate its in vivo existence forms, its metabolism is first studied using mice. All urine and feces are collected during the six days of oral dosing of nardosinone, and blood is collected at one hour after the last dose. Besides, to validate some metabolites, a fast experiment is performed, in which nardosinone was orally administered and the subsequent one-hour urine is collected and immediately analyzed by UHPLC-Q-TOF-MS. In total, 76 new metabolites are identified in this study, including 39, 51, and 12 metabolites in urine, plasma, and feces, respectively. Nardosinone can be converted into nardosinone acid or its isomers. The metabolic reactions of nardosinone included hydroxylation, hydrogenation, dehydration, glucuronidation, sulfation, demethylation, and carboxylation. There are 56 and 20 metabolites with the structural skeleton of nardosinone and nardosinone acid, respectively. In total, 77 in vivo existence forms of nardosinone are found in mice. Nardosinone is mainly excreted in urine and is not detected in the feces. These findings will lay the foundation for further research of the in vivo effective forms of nardosinone and Nardostachyos Radix et Rhizoma.

[Identification of chemical constituents in ethyl acetate soluble extract of Sinopodophylli Fructus based on HPLC-MS~n].

A high performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry(HPLC-DAD-ESI-IT-TOF-MS~n, HPLC-MS~n) method was established for qualitative analysis of the chemical components of ethyl acetate extract from Sinopodophylli Fructus. The analysis was performed on a Kromasil 100-5 C_(18)(4.6 mm×250 mm, 5 µm) column, with a mobile phase consisted of 0.1% formic acid(A) and acetonitrile(B) for gradient at a flow rate of 1.0 mL·min~(-1). Electrospray ionization ion trap time-of-flight multistage mass spectrometry was applied for qualitative analysis under positive and negative ion modes. With use of reference substance, characteristic fragmentation and their HR-MS data, 102 components were identified, including 67 flavonoids and 35 lignans. Among them, 45 compounds were reported in Sinopodophylli Fructus for the first time and 19 compounds were identified as new compounds. PharmMapper was used to predict the bioactivity of compounds that were first reported in Sinopodophylli Fructus, and 20 compounds of them were identified to have potential anticancer activity. The results showed that there were many isomers in the ethyl acetate extract of Folium Nelumbinis, and a total of 19 groups of isomers were found. Among them, C_(21)H_(20)O_8 had the highest number of isomers(18 compounds), all of which were α-peltatin or its isomers; C_(21)H_(20)O_7 ranked second, with 10 compounds, all of which were 8-prenylquercetin-3-methyl ether or its isomers. In conclusion, an HPLC-MS~n method was established for qualitative analysis of the ethyl acetate extract(with anti-breast cancer activity) from Sinopodophylli Fructus in this study, which will provide the evidence for clarifying pharmacological active ingredients of the ethyl acetate extract from Sinopodophylli Fructus against breast cancer.

The Relative Content and Distribution of Absorbed Volatile Organic Compounds in Rats Administered Asari Radix et Rhizoma Are Different between Powder- and Decoction-Treated Groups.

Asari Radix et Rhizoma (ARR) is an important traditional Chinese medicine. Volatile organic compounds (VOCs) are the main active constituents of ARR. Research on the metabolite profile of VOCs and the difference of absorbed constituents in vivo after an administration of ARR decoction and powder will be helpful to understand the pharmacological activity and safety of ARR. In this study, headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC-MS) was applied to profile the VOCs from ARR in rats in vivo. A total of 153 VOCs were tentatively identified; 101 were original constituents of ARR (98 in the powder-treated group and 43 in the decoction-treated group) and 15 were metabolites, and their metabolic reactions were mainly oxidation and reduction, with only two cases of methylation and esterification, and 37 unclassified compounds were identified only in the ARR-treated group. Of the 153 VOCs identified, 131 were reported in rats after oral administration of ARR for the first time, containing 79 original constituents, 15 metabolites, and 37 unclassified compounds. In the powder-treated group, methyleugenol, safrole, 3,5-dimethoxytoluene (3,5-DMT), 2,3,5-trimethoxytoluene (2,3,5-TMT), and 3,4,5-trimethoxytoluene (3,4,5-TMT) were the main absorbed constituents, the relative contents of which were significantly higher compared to the decoction-treated group, especially methyleugenol, safrole, and 3,5-DMT. In the decoction-treated group, 3,4,5-TMT, 2,3,5-TMT, kakuol, and eugenol were the main constituents with a higher content and wider distribution. The results of this study provide a reference for evaluating the efficacy and safety of ARR.

Exploring the In Vivo Existence Forms (23 Original Constituents and 147 Metabolites) of Astragali Radix Total Flavonoids and Their Distributions in Rats Using HPLC-DAD-ESI-IT-TOF-MSn.

Astragali Radix total flavonoids (ARTF) is one of the main bioactive components of Astragali Radix (AR), and has many pharmacological effects. However, its metabolism and effective forms remains unclear. The HPLC-DAD-ESI-IT-TOF-MSn technique was used to screen and tentatively identify the in vivo original constituents and metabolites of ARTF and to clarify their distribution in rats after oral administration. In addition, modern chromatographic methods were used to isolate the main metabolites from rat urine and NMR spectroscopy was used to elucidate their structures. As a result, 170 compounds (23 original constituents and 147 metabolites) were tentatively identified as forms existing in vivo, 13 of which have the same pharmacological effect with ARTF. Among 170 compounds, three were newly detected original constituents in vivo and 89 were new metabolites of ARTF, from which 12 metabolites were regarded as new compounds. Nineteen original constituents and 65 metabolites were detected in 10 organs. Four metabolites were isolated and identified from rat urine, including a new compound (calycoisn-3'-O-glucuronide methyl ester), a firstly-isolated metabolite (astraisoflavan-7-O-glucoside-2'-O-glucuronide), and two known metabolites (daidzein-7-O-sulfate and calycosin-3'-O-glucuronide). The original constituents and metabolites existing in vivo may be material basis for ARTF efficacy, and these findings are helpful for further clarifying the effective forms of ARTF.

[Identification based on HPLC and anti-inflammatory targets as well as related constituents analysis of Asarum heterotropoides var. mandshuricum and A. sieboldii].

The present work is to establish an HPLC characteristic chromatograms of Asarum heterotropoides var. mandshuricum(AH) and A. sieboldii(AS), combined with cluster analysis for the identification of the two species, and predict their potential anti-inflammatory related targets by network pharmacological method. Eighty-nine samples(12 batches of AS and 77 batches of AH) were analyzed, and 11 characteristic peaks were identified by reference substances, UV spectrum and LC-MS. Cluster analysis showed that AS and AH were divided into two groups, and the ratio of characteristic peak areas can be used to distinguish them. When the ratio of characteristic peak sarisan to kakuol was greater than 5, it was AS, and when the ratio was less than 2, it was AH. The network pharmacological analysis of 119 constituents of Asari Radix et Rhizoma suggested that the anti-inflammatory effect of Asari Radix et Rhizoma might be related to COX-2, COX-1, iNOS, MAPK14, NR3 C1, PPARG and TNF. Among them, COX-2 is a relatively key target, which interacted with the characteristic constituents, asarinin, sesamin, safrole, methyleugenol and sarisan. The characteristic constituents asarinin and sesamin also interacted with the iNOS and MAPK14. Safrole and sarisan can also interact with iNOS, COX-1 and LAT4 H. Methyleugenol also showed interaction with COX-1 and LAT4 H. Since asarinin and sesamin interacted with three targets, COX-2, iNOS and MAPK14, it implied that they were the main active constituents for the anti-inflammatory activity of Asari Radix et Rhizoma. The COX-2 inhibitory activities of asarinin and sesamin were further studied by molecular docking and bioassay. The HPLC method established was simple, feasible and reliable, with predicted anti-inflammatory targets and anti-inflammatory constituents, which could provide a reference for improving the quality evaluation system of Asari Radix et Rhizoma.

S-DNA and RecA/RAD51-Mediated Strand Exchange in Vitro.

S-DNA (stretched DNA) is an elongated base-paired DNA conformation under high tension. Because the RecA/Rad51 family DNA recombinases form helical filaments on DNA and mediate the formation of the DNA triplex (D-loop), in which the DNA is stretched, and because the extension of these nucleoprotein filaments is similar to the extension of S-DNA, S-DNA has long been hypothesized as a possible state of DNA that participants in RecA/Rad51-mediated DNA strand exchange in homologous recombination. Such a hypothesis, however, is still lacking direct experimental studies. In this work, we have studied the polymerization and strand exchange on S-DNA mediated by Escherichia coli RecA, human Rad51, and Saccharomyces cerevisiae Rad51 by single-molecule magnetic tweezers. We report that RecA/Rad51 polymerizes faster on S-DNA than on B-DNA with the same buffer conditions. Furthermore, the RecA/Rad51-mediated DNA triplex forms faster from S-DNA than from B-DNA together with the homologous single-stranded DNA. These results provide evidence that S-DNA can interact with RecA and Rad51 and shed light on the possible functions of S-DNA.

Metabolites of Medicarpin and Their Distributions in Rats.

Medicarpin is a bioactive pterocarpan that has been attracting increasing attention in recent years. However, its metabolic fate in vivo is still unknown. To clarify its metabolism and the distribution of its metabolites in rats after oral administration, the HPLC-ESI-IT-TOF-MSn technique was used. A total of 165 new metabolites (13 phase I and 152 phase II metabolites) were tentatively identified, and 104, 29, 38, 41, 74, 28, 24, 15, 42, 8, 10, 3, and 17 metabolites were identified in urine, feces, plasma, the colon, intestine, stomach, liver, spleen, kidney, lung, heart, brain, and thymus, respectively. Metabolic reactions included demethylation, hydrogenation, hydroxylation, glucuronidation, sulfation, methylation, glycosylation, and vitamin C conjugation. M1 (medicarpin glucuronide), M5 (vestitol-1'-O-glucuronide) were distributed to 10 organs, and M1 was the most abundant metabolite in seven organs. Moreover, we found that isomerization of medicarpin must occur in vivo. At least 93 metabolites were regarded as potential new compounds by retrieving information from the Scifinder database. This is the first detailed report on the metabolism of ptercarpans in animals, which will help to deepen the understanding of the metabolism characteristics of medicarpin in vivo and provide a solid basis for further studies on the metabolism of other pterocarpans in animals.

[Analysis of polarity components of Angelicae Sinensis Radix and its metabolites in rats by HPLC-MS~n].

This experiment aims to explore the metabolites of n-butanol and water soluble fraction of an ethanol extracts from Angelicae Sinensis Radix in rats. The chemical constituents of n-butanol and water extracts from Angelicae Sinensis Radix were identified by HPLC-DAD-ESI-IT-TOF-MS~n,and the in vivo metabolites of n-butanol and water extracts were analyzed. By analyzing n-butanol and water extracts from Angelicae Sinensis Radix,25 compounds were detected and identified,in which 11 phthalide glycosides were firstly reported. And 19 compounds were detected and identified in rat urine,including 2 prototype constituents and 17 metabolites,and the17 metabolites were new compounds. The method can identify the main constituents and metabolites of extracts from traditional Chinese medicine accurately and rapidly,and provide evidence for interpreting effective forms and pharmacodynamics substance( prototype,metabolites,or both) of Angelicae Sinensis Radix.

Investigation of the in vivo metabolism of harpagoside and distribution of its metabolites in rats by HPLC-IT-TOF-MSn.

Harpagoside, an iridoid glycoside, is the major bioactive constituent of the traditional Chinese medicine Scrophulariae Radix. High-performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (HPLC-ESI-IT-TOF-MSn ) was used to profile and identify the metabolites of harpagoside in rats in vivo and to study the distribution of these metabolites in rats for the first time. A total of 45 metabolites were identified, 37 of which were postulated to be new compounds. The number of detected metabolites in the heart, liver, spleen, lung, kidney, stomach and small intestine was 2, 9, 6, 16, 4, 16 and 6, respectively, which indicated that the target organs of harpagoside should be spleen, lung and stomach. The main types of metabolic reactions of harpagoside in rats are hydrolysis, reduction, sulfuric acid addition, hydroxylation, methoxylation, sulfate substitution, methylation, glucose conjugation and amino acid conjugation. Furthermore, 23 metabolites were determined to have bioactivities based on the literature and 'PharmMapper' analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological effects of harpagoside. Moreover, these findings provide a reference for studying the metabolism and distribution of iridoid compounds.

Investigation of the In Vivo Metabolism of Sibirioside A and Angoroside C in Rats by HPLC-ESI-IT-TOF-MSn.

Sibirioside A and angoroside C are two important phenylpropanoid glycosides of the traditional Chinese medicine Scrophulariae Radix. High performance liquid chromatography, coupled with an ion trap time-of-flight multistage mass spectrometry equipped with electrospray ionization source (HPLC-ESI-IT-TOF-MSn), was applied to the profile and we identified the metabolites of sibirioside A and angoroside C in vivo in rats. A total of four metabolites of sibirioside A were identified: SM1, SM2 and SM3 which were known as new compounds. A total of 25 metabolites were detected for angoroside C: AM4, AM5, AM6, AM7, AM16, AM17, AM20, AM21, AM22, AM23 and AM25 which were identified to be new compounds. The main metabolic reactions were hydrolysis, reduction, hydroxylation, methylation, sulfation, and gluconylation. The prototype of sibirioside A was widely distributed in tissues found in the heart, liver, spleen, lung, kidney, stomach and small intestine of rats, and mainly distributed in the stomach, small intestine, kidney and liver. But for angoroside C, nothing was found in the viscera except the stomach and small intestine. The metabolites of sibirioside A were mainly eliminated from feces, while it was urine for the metabolites of angoroside C. Furthermore, 19 metabolites were likely to have bioactivities based on the 'PharmMapper' analysis, which roughly matched the known pharmacological activities of Scrophulariae Radix (SR) and the prototypes. One of the main pharmacological activities of SR in traditional Chinese medicine is anti-diabetes, and the predicted results showed that SM1, SM2, SM3, AM2, AM4, AM5, AM6, AM9, AM10, AM11, AM12, AM13, AM15, AM18, AM19, AM24, and AM25 might be used to cure diabetes. These findings provide a reference for studying the metabolism, distribution and pharmacological actions of phenylpropanoid glycosides in vivo.

[Identification of chemical constituents in Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn].

This experiment was performed to analyze and identify the chemical constituents of Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn. The analysis was performed on an Agilent Zorbax SB-C18 (4.6 mm×250 mm, 5 µm) column.The mobile phase consisted of 0.1% formic acid was used for gradient at a flow rate of 1.0 mL·min⁻¹. Electrospray ionization ion trap time-of-flight multistage mass spectrometry was applied for qualitative analysis under positive and negative ion modes. The results indicated that 54 compounds consisted of 18 lignans and 36 flavonoids from Xiaoyelian had been detected by their HRMS data, the information of literature and reference substance. Among them, 27 compounds were reported in Sinopodophylli Fructus for the first time. In conclusion, an HPLC-DAD-ESI-IT-TOF-MSn method was established to qualitative analysis of Xiaoyelian in this study, which will provide the evidence for evaluating the quality of Xiaoyelian herbs, clarifying the mechanism, and guiding the development of pharmacological active ingredients.

Chemical Constituents from the Roots and Rhizomes of Asarum heterotropoides var. mandshuricum and the In Vitro Anti-Inflammatory Activity.

Anti-inflammatory compounds were investigated from the ethanol extract of the roots and rhizomes of Asarum heterotropoides var. mandshuricum, a traditional Chinese medicine called Xixin and used for pain and inflammatory. Nine new compounds were isolated, including six new lignans, neoasarinin A-C (1-3), neoasarininoside A and B (4 and 5), and asarinin B (7), and one new monoterpene, asarincin A (8), two new amides, asaramid II and III (10 and 11), and one new natural monoterpene, asaricin B (9), along with 37 known compounds (6, 12-47). Their structures and absolute configurations were elucidated on the basis of spectroscopic methods and chemical analyses. This is the first report of the absolute configuration of asarinin A (6). The 8-O-4' neolignans (1-5) were reported in the genus Asarum for the first time. The 15 compounds 17, 19, 22-25, 28, 31, 36, 40, 42, 43, 45-47 were isolated from the genus Asarum, and compounds 16, 32, 33, 37 and 39 were isolated from A. heterotropoides var. mandshuricum for the first time. Thirty-seven of the isolates were evaluated for anti-inflammatory activity against the release of ß-glucuronidase in polymorphonuclear leukocytes (PMNs) induced by the platelet-activating factor (PAF), and compounds 1, 4, 7, 8, 14, 17-19, 22, 24, 25, 29, 30, 32, 33, 40-43, 45, and 46 showed potent anti-inflammatory activities in vitro, with 27.9%-72.6% inhibitions at 10-5 mol/L. The results of anti-inflammatory assay suggested that lignans obtained from the CHCl3 extract might be the main active components of Xixin.

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.

The profiling and identification of the metabolites of 8-prenylkaempferol and a study on their distribution in rats by high-performance liquid chromatography with diode array detection combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry.

8-Prenylkaempferol is a prenylflavonoid that has various bioactivities and benefits for human health. A high-performance liquid chromatography with a diode array detector combined with electrospray ionization ion trap time-of-flight multistage mass spectrometry (HPLC-DAD-ESI-IT-TOF-MS(n) ) method was established to profile and identify the metabolites of 8-prenylkaempferol in rat in vivo and in vitro, and to study the distribution of these metabolites in rats for the first time. A total of 38 metabolites were detected and tentatively identified, 30 of which were identified as new compounds. The new in vivo metabolic reactions in rats of prenylflavonoids of isomerization, polymerization, sulfation, amino acid conjugation, vitamin C conjugation and other known metabolic reactions were found in the metabolism of 8-prenylkaempferol. The numbers of detected metabolites in feces, urine, plasma, small intestine, stomach, kidneys, liver, heart, lungs, spleen and hepatic S9 fraction were 31, 19, 1, 20, 13, 8, 7, 3, 3, 1 and 11, respectively. This indicated that small intestine and stomach were the major organs in which the 8-prenylkaempferol metabolites were distributed. Furthermore, 16 metabolites were determined to have bioactivities based on the literature and 'PharmMapper' analysis. These findings are useful for better comprehension of the effective forms, target organs and pharmacological actions of 8-prenylkaempferol. Moreover, they provide a reference for the study of the metabolism and distribution of prenylflavonoid aglycone compounds.

Metabolites of Siamenoside I and Their Distributions in Rats.

Siamenoside I is the sweetest mogroside that has several kinds of bioactivities, and it is also a constituent of Siraitiae Fructus, a fruit and herb in China. Hitherto the metabolism of siamenoside I in human or animals remains unclear. To reveal its metabolic pathways, a high-performance liquid chromatography-electrospray ionization-ion trap-time of flight-multistage mass spectrometry (HPLC-ESI-IT-TOF-MS(n)) method was used to profile and identify its metabolites in rats. Altogether, 86 new metabolites were identified or tentatively identified, and 23 of them were also new metabolites of mogrosides. In rats, siamenoside I was found to undergo deglycosylation, hydroxylation, dehydrogenation, deoxygenation, isomerization, and glycosylation reactions. Among them, deoxygenation, pentahydroxylation, and didehydrogenation were novel metabolic reactions of mogrosides. The distributions of siamenoside I and its 86 metabolites in rat organs were firstly reported, and they were mainly distributed to intestine, stomach, kidney, and brain. The most widely distributed metabolite was mogroside IIIE. In addition, eight metabolites were bioactive according to literature. These findings would help to understand the metabolism and effective forms of siamenoside I and other mogrosides in vivo.

Detection of 191 Taxifolin Metabolites and Their Distribution in Rats Using HPLC-ESI-IT-TOF-MS(n).

Taxifolin is a ubiquitous bioactive constituent of foods and herbs. To thoroughly explore its metabolism in vivo, an HPLC-ESI-IT-TOF-MS(n) method combined with specific metabolite detection strategy was used to detect and identify the metabolites of taxifolin in rats. Of the 191 metabolites tentatively identified, 154 were new metabolites, 69 were new compounds and 32 were dimers. This is the first report of the in vivo biotransformation of a single compound into more than 100 metabolites. Furthermore, acetylamination and pyroglutamic acid conjugation were identified as new metabolic reactions. Seventeen metabolites were found to have various taxifolin-related bioactivities. The potential targets of taxifolin and 63 metabolites were predicted using PharmMapper, with results showing that more than 60 metabolites have the same five targets. Metabolites with the same fragment pattern may have the same pharmacophore. Thus these metabolites may exert the same pharmacological effects as taxifolin through an additive effect on the same drug targets. This observation indicates that taxifolin is bioactive not only in the parent form, but also through its metabolites. These findings enhance understanding of the metabolism and effective forms of taxifolin and may provide further insight of the beneficial effects of taxifolin and its derivatives.

[Identification of Taenia solium Insulin Receptor TsIR-1316 and Expression of Its Ligand Binding Domain].

Objective: To characterize the structure of insulin receptor of Taenia solium（TsIR-1316） and express its ligand binding domain (LBD). Methods: Primers for TsIR-1316 were designed according to the genomic data of T. solium, and the TsIR-1316 gene was amplified by PCR. The nucleotide and amino acid sequences of TsIR-1316 were aligned using BLASTN and BLASTP, and the putative signal peptide and structure domains were predicted. The LBD fragment of TsIR-1316 was cloned into the pET-30a（+） vector and expressed. The expressed proteins were purified, separated by SDS-PAGE and analyzed with Western blotting using cysticercus cellulosae-positive serum and TsIR-LBD-immunized rabbit serum. Results: The open reading frame of TsIR-1316 was 5 196 bp, encoded a protein of 1 732 amino acids which had a typical conserved domain of tyrosine kinase family, was 84% homologous with Echinococcus multilocularis, and had a "V"-shaped tertiary structure. As expected, SDS-PAGE showed that the expressed protein had a band at Mr 59 000. Western blotting showed that the recombinant protein had specific reactions with cysticercus cellulosae positive serum and TsIR-LBD immunized rabbit serum, resulting in a specific band at M(r) 59 000. Conclusion: The TsIR-1316 gene was successfully cloned and identified. The expressed protein of TsIR-1316 LBD can be recognized by cysticercus cellulosae positive serum, which suggests a good antigenicity of this protein.

[Identification, Expression and Antigenicity Analysis of Serpin B6 of Taenia solium].

Objective: To identify and express serpin B6 of Taenia solium (Tsserpin B6) and explore its possible use as a diagnostic antigen. Methods: Primers for Tsserpin B6 were designed according to T. solium genome and transcriptome data. The Tsserpin B6 gene was amplified from the total RNA of T. solium cysticercus and subsequently analyzed by bioinformatics. Multiple amino acid sequence alignments of Tsserpin B6 and other parasites serpins were created using the Clustal X1.83. Phylogenetic analyses were performed using the MEGA 6.0. The recombinant expression vector pET-30a-Tsserpin B6 was constructed and expressed in E. coli strain BL21 （DE3）. The expressed proteins were purified, isolated by SDS-PAGE, and analyzed by Western blotting using pig serum infected with T. solium cysticerci. Results: The complete reading frame of Tsserpin B6 was 1 131 bp and encoded a protein of 376 amino acids. The encoded protein had a conservative reactive center loop and distinctive domains of NEEGAE and FTVDHPFLF, and harbored 9 potential linear B cell epitopes. The expressed products of Tsserpin B6 mainly existed as an inclusion body, and reacted with pig serum infected with T. solium, resulting in a specific band at the Mr 53 000. Conclusion: The Tsserpin B6 gene was successfully cloned, and its expressed products can be recognized by pig serum infected with T. solium.