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1.
Molecules ; 26(6)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802139

RESUMO

It is usually a tedious task to profile the chemical composition of a given herbal medicine (HM) using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) due to the time-consuming sample preparation and laborious post-acquisition data processing procedures. Even worse, some labile compounds may face degradation risks when exposed to organic solvents for a relatively long period. As one of the most popular HMs, the promising therapeutic benefits of Epimedii Herba (Chinese name: Yinyanghuo) are well defined; however, the chemical profile, and in particular those flavonoids that have been claimed to be responsible for the efficacy, remains largely unknown. Attempts are devoted here to achieve direct LC-MS measurement and efficient post-acquisition data processing, and chemome comparison among three original sources of Epimedii Herba, such as Epimedium sagittatum (Esa), E. pubescens (Epu), and E. koreanum (Eko) was employed to illustrate the strategy utility. A home-made online liquid extraction (OLE) module was introduced at the front of the analytical column to comprehensively transfer the compounds from raw materials onto the LC-MS instrument. A mass defect filtering approach was programmed to efficiently mine the massive LC-MS dataset after which a miniature database was built involving all chemical information of flavonoids from the genus Epimedium to draw a pentagonal frame to rapidly capture potential quasi-molecular ions (mainly [M-H]-). A total of 99 flavonoids (66 in Esa, 84 in Eko, and 66 in Epu) were captured, and structurally annotated by summarizing the mass fragmentation pathways from the mass spectrometric data of authentic compounds and an in-house data library as well. Noteworthily, neutral loss of 144 Da was firstly assigned to the neutral cleavage of rhamnosyl residues. Significant species-differences didn't occur among their chemical patterns. The current study proposed a robust strategy enabling rapid chemical profiling of, but not limited to, HMs.

3.
Zhongguo Zhong Yao Za Zhi ; 46(3): 505-510, 2021 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-33645013

RESUMO

"Target fishing" strategy was used to investigate the direct targets and mechanism of Shouhui Tongbian Capsules on relaxing bowel. Magnetic beads cross-linked with the chemical constituents from Shouhui Tongbian Capsules were prepared. The potential target proteins were captured from the total protein lysates of rat intestine using the beads. The captured proteins were further identified by LC-MS/MS, and the associated pathways were analyzed by Cytoscape. RESULTS:: showed that 138 potential target proteins were identified, which were involved in eight signaling pathways, including tricarboxylic acid cycle, pyrimidine metabolism, sulfur metabolism, fatty acid degradation, alanine/aspartate/glutamate metabolism, arginine/proline metabolism, valine/leucine/isoleucine degradation, and ß-alanine metabolism. Taken together, Shouhui Tongbian Capsules may exert relaxing bowel effect by acting on multiple signaling pathways to promote intestinal gurgling, inhibit inflammation, as well as improve intestinal barrier function, intestinal water secretion, and intestinal flora.


Assuntos
Intestinos , Espectrometria de Massas em Tandem , Animais , Cápsulas , Cromatografia Líquida , Leucina , Ratos
4.
Zhongguo Zhong Yao Za Zhi ; 46(1): 130-138, 2021 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-33645062

RESUMO

As a precious traditional Chinese medicine(TCM), snake bile has been widely used in numerous Chinese medicine prescriptions. Bile acid(BA) derivatives have been demonstrated as the primary chemical family in snake bile. In-depth chemical characterization of BAs is of great importance towards the establishment of quality standards and clarification of the effective material basis for snake bile. This study firstly employed ~1H-NMR to preliminarily analyze the chemical profiles of snake bile, an automated fraction collector was subsequently implemented to obtain the fractions-of-interest. The fraction was then concentrated and re-analyzed by LC-MS. Based on ~1H-NMR, BAs were found to be the main components of snake bile, and six BAs including CDCA, CA, TCDCA, TCA, TDCA and GCA were tentatively identified from the representative spectrum with the assistance of literature and reference compounds. Whereas the content of TCA in snake bile was too great, resulting in a great obstacle for the detection of trace components, the automated fraction collector was subsequently implemented to obtain the fractions-of-interest for LC-MS analysis. According to matching MS/MS information and retention time with reference compounds as well as database retrieval, a total of 57 BAs were detected and annotated. Because of the combination of ~1H-NMR and LC-MS platforms, the findings are beneficial for the in-depth characterization of BAs in snake bile, which provides references for the establishment of quality control and evaluation methods of snake bile.


Assuntos
Ácidos e Sais Biliares , Espectrometria de Massas em Tandem , Animais , Bile , Cromatografia Líquida , Serpentes
5.
Artigo em Inglês | MEDLINE | ID: mdl-33756451

RESUMO

In comparison of herbal medicines, less attention has been paid onto animal medicines, partially attributing to the protein-enriched property. Particularly, it is still challenging to conduct quality evaluation for the animal medicines because of the lack of a fit-for-purpose analytical tool. Herein, an attempt was made to propose a workflow allowing the quality assessment of animal medicines by LC-MS/MS, and musk that is one of the most precious traditional Chinese medicines was employed as a representative case for utility illustration. After the extraction of protein from musk with a well-defined protocol, tryptic digestion was conducted to hydrolyze proteins into peptides, and the peptide-enriched sample was subjected to nanoLC-Orbitrap MS measurement. The tandem mass spectral dataset was retrieved in Human Swiss-Prot FASTA database, and the sequences together with the sources of 733 tryptic peptides, in total, were annotated. Because of the abundant distributions, eight peptides were chosen as the analytes for quantitative measurements, and their quantitative MS parameters, such as ion transitions and collision energies, were rapid optimized in an authentic compound-free manner using online energy-resolved MS (ER-MS). On the other side, the annotated peptides were structurally consolidated via synthesizing reference peptides. When the synthetic peptides were applied for parameter optimization with the authentic compound-dependent manner, the values were almost identical with those from online ER-MS measurements. After being validated with diverse assays, the developed method was applied for the simultaneous determination of eight peptides in 28 batches of musk samples, including captive (C1-C18) and wild ones (W1-W10). Significant differences took place for the content patterns of concerned tryptic peptides between the captive and wild musk samples. Trace distributions occurred for DVDAAYMNK in most batches. Captive samples were rich of QSLEASLAETEGR, TLLDIDNTR, and EVATNSELVQSGK, whereas wild samples were able to accumulate YLGYLEQLLR. Together, the present study provided a meaningful approach for the quality evaluation of musk, as well as other peptide-enriched animal medicines, even if the absences of authentic peptides.

6.
Phytochemistry ; 185: 112674, 2021 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-33770687

RESUMO

Two undescribed oleanane-type saponins, julibrosides K-L, along with three undescribed oleanane-type prosapogenins, julibrosides M-O, were isolated from the stem bark of Albizia julibrissin Durazz. and the mild alkaline hydrolysate of the total saponin, respectively. Their structures were established by extensive analysis of 1D and 2D NMR experiments (COSY, TOCSY, HSQC, HMBC, and HSQC-TOCSY) and mass spectrometry. Furthermore, the cytotoxic activities of the isolated compounds against BGC-823, A549, HCT-116, and HepG2 cell lines were evaluated, and julibroside L showed significant cytotoxic activities against the four cancer cell lines with IC50 values of 5.77, 4.80, 4.26, and 4.93 µM, respectively.

7.
Zhongguo Zhong Yao Za Zhi ; 46(6): 1301-1326, 2021 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-33787126

RESUMO

Zaoren Anshen prescription preparations(ZRASs), which are prepared from three traditional Chinese herb medicines, namely fried Zizyphi Spinosae Semen, Salvia Miltiorrhizae Radix et Rhizoma and vinegar-processed Schisandrae Chinensis Fructus, are a series of proprietary Chinese medicines for the treatment of insomnia, amnesia and dizzy in clinic. In recent years, pharmacodynamic effect, chemical constituents and quality control of ZRASs had been extensively studied for the purpose of ensuring their safety, efficacy and stability, and a great progress had been made. However, there is no review of the research advance of ZRASs up to date. The present review summarized the research advance of ZRASs in quality control standards, chemical constituents, pharmacodynamic effects, and chemical analysis for the first time, with the aim to provide a reference for further studies on the effective constituents and quality control of ZRASs.


Assuntos
Medicamentos de Ervas Chinesas , Salvia miltiorrhiza , Medicamentos de Ervas Chinesas/farmacologia , Medicina Tradicional Chinesa , Prescrições , Rizoma
8.
Artigo em Inglês | MEDLINE | ID: mdl-33744433

RESUMO

Rapid development of high-throughput technologies has permitted the identification of an increasing number of disease-associated genes (DAGs), which are important for understanding disease initiation and developing precision therapeutics. However, DAGs often contain large amounts of redundant or false positive information, leading to difficulties in quantifying and prioritizing potential relationships between these DAGs and human diseases. In this study, a network-oriented gene entropy approach (NOGEA) is proposed for accurately inferring master genes that contribute to specific diseases by quantitatively calculating their perturbation abilities on directed disease-specific gene networks. In addition, we confirmed that the master genes identified by NOGEA have a high reliability for predicting disease-specific initiation events and progression risk. Master genes may also be used to extract the underlying information of different diseases, thus revealing mechanisms of disease comorbidity. More importantly, approved therapeutic targets are topologically localized in a small neighborhood of master genes on the interactome network, which provides a new way for predicting drug-disease associations. Through this method, 11 old drugs were newly identified and predicted to be effective for treating pancreatic cancer and then validated by in vitro experiments. Collectively, the NOGEA was useful for identifying master genes that control disease initiation and co-occurrence, thus providing a valuable strategy for drug efficacy screening and repositioning. NOGEA codes are publicly available at https://github.com/guozihuaa/NOGEA.

9.
Signal Transduct Target Ther ; 6(1): 71, 2021 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-33602894

RESUMO

Mitochondrial fusion/fission dynamics plays a fundamental role in neuroprotection; however, there is still a severe lack of therapeutic targets for this biological process. Here, we found that the naturally derived small molecule echinacoside (ECH) significantly promotes mitochondrial fusion progression. ECH selectively binds to the previously uncharacterized casein kinase 2 (CK2) α' subunit (CK2α') as a direct cellular target, and genetic knockdown of CK2α' abolishes ECH-mediated mitochondrial fusion. Mechanistically, ECH allosterically regulates CK2α' conformation to recruit basic transcription factor 3 (BTF3) to form a binary protein complex. Then, the CK2α'/BTF3 complex facilitates ß-catenin nuclear translocation to activate TCF/LEF transcription factors and stimulate transcription of the mitochondrial fusion gene Mfn2. Strikingly, in a mouse middle cerebral artery occlusion (MCAO) model, ECH administration was found to significantly improve cerebral injuries and behavioral deficits by enhancing Mfn2 expression in wild-type but not CK2α'+/- mice. Taken together, our findings reveal, for the first time, that CK2 is essential for promoting mitochondrial fusion in a Wnt/ß-catenin-dependent manner and suggest that pharmacologically targeting CK2 is a promising therapeutic strategy for ischemic stroke.

10.
J Ethnopharmacol ; 271: 113899, 2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-33549763

RESUMO

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine Cistanche deserticola Y. C. Ma has effect of "tonifying kidney and strengthening bone". However, the specific active extracts of C. deserticola and mechanisms for treatment of osteoporotic are not clear. AIM OF THE STUDY: We wanted to identify the effective component extracts of C. deserticola for the treatment of osteoporosis and the potential mechanisms. MATERIALS AND METHODS: Our group researched the extracts of C. deserticola with anti-osteoporotic activity, including total glycosides (TGs), polysaccharides (PSs), and oligosaccharides (OSs) in senescence accelerated mouse prone 6 (SAMP6) mice. The Goldner's Trichrome, Van Gieson's (VG), Safranin O-Fast Green staining and Von Kossa staining were performed to investigate the bone structure formation and calcium deposits. Serum was collected for detecting biochemical markers. Bone micro-architecture was detected by micro-CT. Expressions of bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), osteoprotegerin (OPG), receptor activator of nuclear factor-κ B ligand (RANKL), p-glycogen synthetase kinase-3ß (p-GSK-3ß), and p-ß-catenin were analyzed by western blotting and immunohistochemistry. RESULTS: TGs and PSs ameliorated bone histopathological damages, promoted the formation of new bone, collagenous fiber, and chondrocytes, and accelerated the calcium deposits. Moreover, they remarkable altered the biomarkers of bone turnover and effectively ameliorated bone microarchitecture. The further mechanisms study showed that TGs and PSs significantly decreased the expressions of RANKL, p-ß-catenin, as well as up-regulated the expression of BMP-2, OCN, OPG, and p-GSK-3ß (Ser9). CONCLUSION: The findings of this study suggest that TGs and PSs can promote osteoblastogenic bone formation and improve bone microstructure damage in SAMP6 mice, and their therapeutic effect on osteoporosis is via activating Wnt/ß-catenin signaling pathway.

11.
Med Res Rev ; 2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33521978

RESUMO

Cistanches Herba (CH, Chinese name: Roucongrong), is a very precious, tonic Chinese medicine. Cistanche deserticola and Cistanche tubulosa are the two commonly used species and authenticated in Chinese Pharmacopoeia. Due to the parasitic nature of Cistanche plants, the wild source was once endangered and listed in the Appendix II of Convention on International Trade in Endangered Species of Wild Fauna and Flora. However, after continuously struggling in the past decades, CH has grown up to a big brand of Chinese medicine featured with the cultivation area as 1.26 million mu, the annual output as 6000 tons, and the related industrial output value as more than 20 billion China Yuan, attributing to large-scale cultivation and in-depth phytochemical and pharmacological investigations. Noteworthily, great achievements have reached concerning the research and development of relevant products, such as modern drugs, traditional Chinese medicine prescriptions, and dietary supplements. The current review summarizes the research progresses concerning the distribution and cultivation, phytochemistry, pharmacology, metabolism and product development of CH in the past decades, and the emerging challenges and developing prospects are discussed as well.

12.
Chem Biodivers ; 18(3): e2001065, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33576170

RESUMO

Two enantiomeric humulane sesquiterpenes, namely (+)-alashanoid N (1a) and (-)-alashanoid N (1b), along with two known analogs ((2R,3R,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (2) and (2R,3S,5R)-2,3-epoxy-6,9-humuladien-5-ol-8-one (3)), were described from the peeled stems of a folk Mongolian herbal medicine Syringa pinnatifolia. Their structures were characterized based on UV, IR, NMR, and HR-ESI-MS data analyses, and the absolute configurations were determined by data analysis of X-ray diffraction and quantum chemical calculations. (+/-)-Alashanoid N showed inhibition against NO production in RAW 264.7 macrophage cells with IC50 values of 90.1 µM and 71.7 µM, and protective effect against oxygen-glucose deprivation injury to H9c2 cells at a concentration of 20 µM and 5 µM, respectively.

13.
Anal Chem ; 93(4): 2541-2550, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33439008

RESUMO

Direct infusion (DI) has an extraordinary high-throughput advantage. Pseudo-targeted metabolomics (PTM) has been demonstrated integrating the merits of both nontargeted and targeted metabolomics. Herein, we attempted to implant DI into the PTM concept to configure a new strategy allowing shotgun PTM. First, a versatile MS/MSALL program was applied to acquire MS1 and MS2 spectra. Second, online energy-resolved MS (online ER-MS) was conducted to obtain breakdown graph as well as optimal collision energy (OCE) for each ion transition paired by precursor ion and the dominant product ion. Third, selected reaction monitoring (SRM) was responsible to output a quantitative dataset with a constant length. Moreover, breakdown graph also served as orthogonal structural evidence when matching MS2 spectra between DI-MS/MS and an in-house library to strengthen structural annotation confidence. To evaluate and illustrate the utility of the new strategy toward shotgun PTM of medicinal plants, in-depth chemome comparison was conducted within three Cistanche species, all of which are edible medicinal plants and playing essential roles for turning the deserts into the oases. A total of 185 variables participated in the quantitative measurement program. Each diagnostic ion pair was featured with an OCE. Significant species differences occurred, and echinacoside, acteoside, isoacteoside, 2'-acetyl-acteoside, tubuloside B, mannitol, sucrose, betaine, malate, as well as choline were found to be confirmative chemical markers offering primary contributions toward the species discrimination. After cross-validation with LC-MS/MS, DI-MS/MS fortified with the new strategy is an eligible tool for shotgun PTM, beyond Cistanche plants.


Assuntos
Cistanche/química , Metabolômica/métodos , Plantas Medicinais/química , Espectrometria de Massas em Tandem/métodos , Animais , Ensaios de Triagem em Larga Escala/métodos , Plantas Comestíveis/química
14.
Int Immunopharmacol ; 91: 107337, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33401206

RESUMO

CD40 ligand (CD40L) and B-cell activating factor (BAFF) play important roles in the function of B cells. However, the difference of their regulatory effects remains obscure. In this study, we used anti-CD40 to imitate CD40L and investigated the different regulatory effects of CD40L and BAFF on the function of B cells. In the functional analyses, both anti-CD40 and BAFF significantly enhanced the survival and differentiation of B cells, and slightly increased the activation and proliferation. However, in the transcriptome analysis, anti-CD40 and BAFF exerted very different regulation on the gene expression profile of B cells. Anti-CD40 upregulated the expression of genes related to the adaptive immune function of B cells, but BAFF enhanced the genes associated with the innate immune function. Furthermore, the effect analysis of the combination of anti-CD40 or BAFF with anti-IgM also demonstrated that anti-CD40 could cooperate with anti-IgM to promote the proliferation of B cells, but BAFF could not do it. The mechanism study revealed that the different effects of anti-CD40 and BAFF on B cells were resulting from the different modulation on NF-кB, ERK1/2, and PI3K-AKT signaling pathways. Collectively, the results suggest that CD40L mainly promotes adaptive immune function of B cells, but BAFF primarily enhances innate immune function.

15.
Artigo em Inglês | MEDLINE | ID: mdl-33310479

RESUMO

Clarification the existence forms, including prototype and metabolite(s) is the prerequisite for understanding in-depth the therapeutic mechanisms of a given agent, particularly when oral administration. However, it is still a long distance for unambiguous structural identification of metabolites even employing the cutting-edge MS/MS technique, and the determinant obstacle is produced by its inherent isomer-blind disadvantage. To tackle with this drawback, online energy-resolved mass spectrometry (online ER-MS) was introduced to enable isomeric discrimination after that high-resolution MS/MS provided empirical molecular formula as well as substructures. In-depth metabolic characterization of cistanoside F (CF), an effective natural product, was conducted as a proof-of-concept for the new strategy namely three-dimensional MS that was configured by MS1, MS2 and online ER-MS as 1st, 2nd, and 3rd dimensions, respectively. Sensitive metabolite detection was assisted by predictive multiple-reaction monitoring function on Qtrap-MS, and the empirical formulas of all metabolites were calculated from the quasi-molecular ions yielded from IT-TOF-MS. Subsequently, substructures of each metabolite were constructed by combining the calculated element compositions and the well-defined mass fragmentation pathways. Finally, online ER-MS was responsible to generate optimal collision energies for bonds-of-interest, and enabled rational selection among candidate structures. A total of thirteen metabolites were detected and confirmatively identified in rat after oral treatment of CF using LC-3D MS. Acyl-migration, hydrolysis and sulfation played key roles for the metabolic fate of CF. More importantly, LC-3D MS is an eligible tool to achieve confidence-enhanced structural annotation of metabolites in biological matrices because of the unique isomeric differentiation ability from online ER-MS.

16.
ACS Chem Neurosci ; 2020 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-33225685

RESUMO

Cerebral ischemia is accompanied by mitochondrial integrity destruction. Thus, reversion of mitochondrial damage holds great potential for cerebral ischemia therapy. As a crucial Bcl-2 family member, pro-apoptotic Bax protein is a main effector of mitochondrial permeabilization and plays an important role in mitochondrial homeostasis. However, there is still a lack of an effective cerebral protective strategy through selectively targeting Bax. In this study, we reported that natural small-molecule protosappanin A (PTA) showed a significant mitochondrial protective effect on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cells injury through increasing ATP production and maintaining mitochondrial DNA (mtDNA) content. The mechanism study revealed that PTA selectively induced pro-apoptotic protein Bax degradation, without affecting other Bcl-2 family members such as Bcl-2, Bcl-xl, Bad, Puma, Bid, Bim, and Bik. In addition, we found that PTA promoted the association of autophagosomal marker LC3B to Bax for its degradation via an autophagy-dependent manner but not the ubiquitin-proteasome pathway. Collectively, our findings offered a new pharmacological strategy for maintaining mitochondrial function by inducing autophagic degradation of Bax and also provided a novel drug candidate against ischemic neuronal injury.

17.
Fitoterapia ; 147: 104776, 2020 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-33166598

RESUMO

Three pairs of enantiomers mucroniferals A-C (1-3), with a novel skeleton of 1,4-epoxynaphthalene-2,3-dicarboxylic acid first reported from nature source, were isolated from Corydalis mucronifera. Their structures were elucidated based on extensive spectroscopic data analysis of MS, 1D and 2D NMR, and their absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of the experimental and calculated ECD data. Mucroniferals A-C showed broad-spectrum inhibitory activities on seedling growth of all plants tested (Lepidium apetalum, Raphanus sativus, Lactuca sativa, and Arabidopsis thaliana) with a dose-dependent relationship. Additionally, mucroniferals A and B exhibited significant inhibitory effects on germination of most seeds at concentration of 80 µg/mL, and the inhibition was reversible.

18.
Eur J Pharmacol ; : 173723, 2020 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-33159933

RESUMO

Endoplasmic reticulum (ER) stress has been considered as a promising strategy in developing novel therapeutic agents for cardiovascular diseases through inhibiting cardiomyocyte apoptosis. Protocatechualdehyde (PCA) is a natural phenolic compound from medicinal plant Salvia miltiorrhiza with cardiomyocyte protection. However, the potential mechanism of PCA on cardiovascular ischemic injury is largely unexplored. Here, we found that PCA exerted markedly anti-apoptotic effect in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced H9c2 cells (Rat embryonic ventricular H9c2 cardiomyocytes), which was detected by 3-(4, 5-dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT), lactate dehydrogenase (LDH), Hoechst 33258 and acridine orange/ethidium bromide (AO/EB) assays. PCA also obviously protected cardiomyocytes in myocardial fibrosis model of mice, which was determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. Transcriptomics coupled with bioinformatics analysis revealed a complex pharmacological signaling network especially for PCA-mediated ER stress on cardiomyocytes. Further mechanism study suggested that PCA suppressed ER stress via inhibiting protein kinase R-like ER kinase (PERK), inositol-requiring enzyme1α (IRE1α), and transcription factor 6α (ATF6α) signaling pathway through Western blot, DIOC6 and ER-Tracker Red staining, leading to a protective effect against ER stress-mediated cardiomyocyte apoptosis. Taken together, our observations suggest that PCA is a major component from Salvia miltiorrhiza against cardiovascular ischemic injury by suppressing ER stress-associated PERK, IRE1α and ATF6α signaling pathways.

19.
J Agric Food Chem ; 68(44): 12454-12466, 2020 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-33084329

RESUMO

Flavonoids occupy the largest family of natural products and possess a broad spectrum of health benefits. Their metabolites are sometimes the truly effective molecules in vivo. It is still challenging, however, to unambiguously identify flavonoid metabolites using conventional LC-MS/MS. Herein, we aimed to pursue auxiliary structural clues to m/z values in both MS1 and MS2 spectra through LC coupled to three-dimensional MS (LC-3D MS). MS1, as the first dimension, was in charge of suggesting theoretical molecular formulas, MS2, the as second dimension, was responsible for offering substructures, and exactly, online energy-resolved MS (ER-MS), as the third dimension, provided optimal collision energies (OCEs) that reflected the linkage manners among the substructures. Metabolic characterization of a natural sweet taste modulator, namely, (R)-7,3'-dihydroxy-4'-methoxy-8-methylflavane (DHMMF), was conducted as a proof-of-concept. Extensive efforts, such as full MS1 and MS2 scans on IT-TOF-MS and predictive selected-reaction monitoring mode on Qtrap-MS, were made for in-depth metabolite mining. Seventeen metabolites (M1-M17) were captured from DHMMF-treated biological samples, including 17 (M1-M17), 10 (M4-M9, M11, M13, M14, and M16), and 2 (M5 and M10) metabolites from urine, plasma, and feces, respectively. Their structures were configured by integrating MS1, MS2, and OCE information. Except M10, all metabolites were new compounds. LC-MS/MS-guided chromatographic purification yielded three glucuronyl-conjugated metabolites (M5, M8, and M11), and NMR spectroscopic assays consolidated the structures transmitted from LC-3D MS. Demethylation, glucuronidation, and sulfation occurred as the primary metabolic pathways of DHMMF. Above all, LC-3D MS bridged LC-MS/MS from putatively structural annotation toward confidence-enhanced identification, beyond the metabolite characterization of flavonoids.

20.
Org Lett ; 22(21): 8725-8729, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33104367

RESUMO

Pelletierine, a proposed building block of Lycopodium alkaloids (LAs), was demonstrated to be synthesized via the non-enzymatic Mannich-like condensation of Δ1-piperideine and 3-oxoglutaric acid produced by two new type III PKSs (HsPKS4 and PcPKS1) characterized from Huperzia serrata and Phlegmariurus cryptomerianus, respectively. The findings provide new insights for further understanding the biosynthesis of LAs such as huperzine A.

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