Research progress on metabolomics in the quality evaluation and clinical study of Panax ginseng.

Panax ginseng, an essential component of traditional medicine and often referred to as the king of herbs, has played a pivotal role in medicine globally for several millennia. Previously, traditional phytochemical methods were mainly used for quality evaluation and pharmacological mechanism studies of ginseng, resulting in the lack of systematicness and innovation and hindering the development and utilization of ginseng resources. Since the beginning of the new century, systems biology technology represented by metabolomics has shown unique advantages in the modernization and internationalization of herbal medicine, establishing a bridge for communication between traditional medicine and modern medicine. P. ginseng, a special herb used in medicine and food, is one of the main research objects for qualitative and quantitative analysis of metabolomics and has gradually become the focus of researchers globally. Here, we conducted a comprehensive summary and analysis of numerous studies published in ginseng metabolomics. This review aims to provide more novel ideas for the quality evaluation, development, and clinical application of ginseng in the future and offer more useful technical references for the modernization and internationalization of herbal medicine based on metabolomics.

[Research progress of Astragali Radix fermentation].

Astragali Radix is one of traditional Chinese medicines with effects in invigorating Qi for consolidating superficies, inducing diuresis to alleviate edema, promoting pus discharge and tissue regeneration. In recent years, the traditional Chinese medicine fermentation technology has received extensive attentions due to its high efficiency and safety. The pharmacological functions of traditional Chinese medicines could be further enhanced after microbial fermentation, which has a broad development prospects. In this paper, we summarized relevant literatures of Astragali Radix fermentation in such aspects as fermentation strains, fermentation forms, process optimization, active ingredients and pharmacological effects, in the expectation of providing a reference for development and utilization of Astragali Radix.

Comparative metabolism study on chlorogenic acid, cryptochlorogenic acid and neochlorogenic acid using UHPLC-Q-TOF MS coupled with network pharmacology.

Chlorogenic acid (5-CQA), neochlorogenic acid (3-CQA), and cryptochlorogenic acid (4-CQA), usually simultaneously exist in many traditional Chinese medicines (TCMs). However, insufficient attentions have been paid to the comparative metabolism study on these three isomeric constituents with similar effects on anti-inflammation until now. In this study, a novel strategy was established to perform comparative analysis of their metabolic fates in rats and elucidate the pharmacological mechanism of anti-inflammation. Firstly, diagnostic product ions (DPIs) deduced from the representative reference standards were adopted to rapidly screen and characterize the metabolites in rat plasma, urine and faeces using UHPLC-Q-TOF MS. Subsequently, Network pharmacology was utilized to elucidate their anti-inflammatory mechanism. Consequently, a total of 73 metabolites were detected and characterized, including 50, 47 and 43 metabolites for 5-CQA, 4-CQA and 3-CQA, orderly. Moreover, the network pharmacology study indicated that these three isomeric constituents and their major metabolites with similar in vivo metabolic pathways exerted anti-inflammatory effects through co-owned 20 biological processes, which involved 10 major signal pathways and 159 potential targets. Our study shed light on the similarities and differences of the metabolic profiling and anti-inflammatory activity among these three isomeric constituents and set an example for the further researches on the active mechanism of isomeric constituents existing in TCMs based on comparative metabolism study.

Comprehensive analysis of the chemical constituents in sulfur-fumigated Lonicerae Japonicae Flos using UHPLC-LTQ-Orbitrap mass spectrometry.

Lonicerae Japonicae Flos (LJF), a kind of traditional Chinese medicines (TCMs), has functions of detoxifying and evacuating heat. In the study, a method based on ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was developed for the chemical constituent analysis of organic acids, flavonoids, iridoids and new-generated compounds in sulfur-fumigated LJF (SF-LJF). Based on the accurate mass measurement (< ± 5 ppm), chromatographic behavior and diagnostic product ions (DPIs), 113 constituents were unambiguously or tentatively characterized from SF-LJF extract, including 46 chlorogenic acids, 19 flavonoids, 29 iridoid glycosides and 19 newly-generated compounds (including 17 sulfur-containing derivatives). In addition, 5-CQA (5-caffeoylquinic acid, chlorogenic acid) was chosen to be sulfur-fumigated for the result validation. It was found that the most significant change of LJF after sulfur fumigation was the occurrence of sulfate or sulfite esterification reactions, which resulted in the emergence of many new sulfur-containing components. Our results demonstrated that the established method was a useful and efficient analytical tool to comprehensively characterize the material basis of SF-LJF, and also an excellent guidance of quality control about LJF.

[UHPLC-HRMS metabonomics to study effect of sulfur-fumigated Ophiopogonis Radix on endogenous metabolites in rats].

The project was launched to analyze the effects of sulfur-fumigated Ophiopogonis Radix on endogenous metabolites in rats by metabonomics. The preparation method of sulfur-fumigated Ophiopogonis Radix in laboratory was established. Then the blood samples of SD rats in blank group,Ophiopogonis Radix extract group and sulfur-fumigated Ophiopogonis Radix extract group were investigated by UHPLC-Q-Exactive. The differential metabolites were screened and identified by PCA(principal component analysis),OPLSDA(orthogonal partial least squares discriminant analysis) and variable importance projection(VIP),and the metabolic pathways were analyzed. Finally,a total of 15 potential biomarkers were identified. Compared with the samples of Ophiopogonis Radix extract group,sulfur-fumigated Ophiopogonis Radix mainly affected the biosynthesis and metabolism of amino acids in normal rats. Its mechanism may be related to the biosynthesis of phenylalanine,tyrosine,tryptophan and aminoacyl-tRNA as well as the metabolism of phenylalanine and tryptophan. Based on UHPLC-HRMS metabonomics,this paper discussed the effects of sulfur-fumigated Ophiopogonis Radix on endogenous metabolites in rats,which provided an idea for the metabolic study of other sulfur-fumigated traditional Chinese medicines.

[Rapid characterization of chemical constituents and rat metabolites of Fufang Gancao Tablets by UHPLC-LTQ-Orbitrap mass spectrometer].

Ultra-high-performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HR-MSn ) method was used to analyze the constituents of Fufang Gancao Tablets and its main metabolites in rat plasma. Rat plasma was collected both before and after oral administration of Fufang Gancao Tablets. After solid phase extraction, ACQUITY UHPLC BEH C18 (2.1 mm×100 mm, 1.7 µm) was used with 0.1% formic acid-acetonitrile solution as the mobile phase for gradient elution. The chemical components in Fufang Gancao Tablets and their prototypes and metabolites in plasma samples were analyzed by LTQ-Orbitrap equipped with an ESI ion source in a positive ion mode. Based on the accurate mass measurements, the retention time and mass fragmentation patterns, a total of 55 compounds were tentatively identified from Fufang Gancao Tablets, including 42 flavonoids, 9 triterpenes and 4 alkaloids. Furthermore, metabolites in rat plasma after oral administration of Fufang Gancao Tablets were also analyzed. A total of 26 compounds were identified, including 20 prototypes and 6 metabolites mainly through metabolic pathways of hydroxylation, glucuronide conjugation, and sulfate conjugation, etc. Our results showed that the ultra-high-performance liquid chromatography coupled with linear ion trap quadrupole Orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap) could comprehensively elucidate the chemical constituents of Fufang Gancao Tablets and their migrating components in rat plasma, providing scientific basis for further studying the metabolism process and pharmacodynamic substance of Fufang Gancao Tablets.

[Identification of metabolites of Danshensu in vivo in rats].

To identify the metabolites of Danshensu in plasma and urine in rats by using UHPLC-LTQ-Orbitrap method. After oral gavage of Danshensu CMC-Na suspension in SD rats, urine and plasma samples were collected and processed by solid phase extraction. ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm) was utilized, with 0.1% formic acid (A)-acetonitrile (B) solution as the mobile phase for gradient elution. Negative electrospray ion mode based data-acquisition method was established to collect the mass spectrometry data of biological samples. As a result, Danshensu and 21 Danshensu Ⅰ phase and Ⅱ phase metabolites were finally identified according to the accurate mass measurements, mass fragmentation behaviors and comparing with the reference standards. The main metabolic pathways included dehydration, methylation, glucuronide conjugation, sulfate conjugation and their composite reactions. Consequently, our study expounded metabolites of Danshensu in rats based on UHPLC-LTQ-Orbitrap method and provided a reference for further researches on therapeutic material basis and mechanism of Danshensu.

ω-3 polyunsaturated fatty acids inhibit the proliferation of the lung adenocarcinoma cell line A549 in vitro.

ω-3 polyunsaturated fatty acids (n-3 PUFA), in particular the marine-derived forms eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been demonstrated to affect cancer cell replication, the cell cycle and cell death. Epidemiological studies have also suggested diets rich in n-3 PUFA were inversely correlated with the development of cancer. In the present study, we explored the effects of DHA and EPA on the proliferation activity and apoptosis of the human lung adenocarcinoma cell line A549. A methyl thiazolyl tetrazolium (MTT) assay was used to detect cell proliferation, apoptosis was detected by flow cytometry and morphological analysis was determined by fluorescence microscopy and transmission electron microscopy. A549 cells were treated with different doses of DHA (40, 45, 50 and 55 µg/ml) or EPA (45, 50, 55 and 60 µg/ml) for 24, 48 and 72 h. The results demonstrated that DHA and EPA significantly suppressed the proliferation of A549 cells and induced apoptosis of A549 cells in a dose- and time-dependent manner. The apoptotic phenomenon was also confirmed by fluorescence microscopy and transmission electron microscopy. Furthermore, compared with the control, the formation of autophagosomes was clearly enhanced in DHA­ or EPA-treated cells. In conclusion, DHA and EPA inhibited the proliferation of A549 cells and induced cell apoptosis and autophagy, which may provide new safe and effective options for the treatment of lung cancer in the future.

Functional endothelial cells derived from embryonic stem cells labeled with HIV transactivator peptide-conjugated superparamagnetic nanoparticles.

BACKGROUND: The development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo, for example, magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Although ES cells have been labeled with SPIO particles, the potential adverse effects of the label have not been fully examined. The objective of this study was to determine whether SPIO labeling affects murine ES cell viability, proliferation, or ability to differentiate into functional endothelial cells (ECs). METHODS: Cross-linked iron oxide (CLIO, an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides, and murine ES cells were labeled with either CLIO-Tat, CLIO, or HIV-Tat. After labeling, ES cells were cultured for 4 days and Flk-1(+) ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS). Flk-1(+) cells were replated on fibronectin-coated dishes, and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF). ES cell viability was determined using trypan blue exclusion, and the proportion of SPIO(+) cells was evaluated using Prussian blue staining and transmission electron microscopy. After differentiation, the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction, flow cytometry, immunocytochemistry, DiI-labeled acetylated low-density lipoprotein (AcLDL) uptake, and Matrigel tube formation assay. RESULTS: CLIO-Tat was a highly effective label for ES cells, with > 96% of cells incorporating the particles, and it did not alter the viability of the labeled cells. ECs derived from CLIO-Tat(+) ES cells were very similar to murine aortic ECs in their morphology, expression of endothelial cell markers, ability to form vascular-like channels, and scavenging of AcLDL from the culture medium. CONCLUSIONS: CLIO-Tat is a highly effective label for ES cells and does not adversely affect cell viability, differentiation, or behavior. CLIO-Tat could be a useful marker for the non-invasive monitoring of transplanted stem cells.

[Study on the chemical constituents from fresh roots of Euphorbia fischeriana].

OBJECTIVE: To study the chemical constituents in ethyl acetate fraction from the Euphorbia fischeriana. METHODS: The compounds were isolated by silica gel column chromatography and HPLC, and their structures were elucidated by means of spectral analyses. RESULTS: Fourteen compounds were identified as 2,4-dihydroxy-6-methoxy-3-methylacetophenone (1), 12-deoxyphorbol-13-hexadecanoate (2), ethylgallate (3), esculetin (4), 2,4-dihydroxy-6-methoxy-acetophenone (5), 12-deoxyphorbol-13-acetate (6), 12-deoxyphorbol-13,20-diacetete (7), jolkinolide B (8), 17-hydroxyjolkinolide B (9), 17-hydroxy-jolkinolide A (10), langduin C (11), 3-methylp-hydroxybenzoic acid (12), methyl gallate (13), 3, 3'-diacetyl-pyridine-4,4'-dimethoxy-2, 2', 6, 6'-tetrahydroxydiphenylmethane (14). CONCLUSION: Compounds 3, 5, 12, 13 are isolated from this plant for the first time.