As a treasure resource of novel drug lead compounds, how to rapidly and high-efficiently screen and isolate active components from natural products is critical. Thanks to its high resolution, high automation and flexible integration, online two-dimensional liquid chromatography has great potential for screening active ingredients from complex matrices by integrating a highly specific bio-recognition process into a two-dimensional liquid chromatography system before, on or after the column separation. This review comprehensively summarized recent developments, applications and shortcomings of online two-dimensional liquid chromatography for natural product screening from different integration modes, including pre-column, on-column and post-column screening methods.

The goal of this work was to investigate the antidepressant fraction from Radix Paeoniae Alba and identify its major chemical constituents. Corticosterone injured rat phaeochromocytoma (PC12) cells and behavioral despair depression models of mice were used to evaluate the antidepressant effects of Radix Paeoniae Alba (Bai-Shao) ethanol extract (BS-E) and its three fractions (BS-10E, BS-60E, BS-95E) isolated by macroporous resin column chromatography. Animal experimental procedures were approved by the Animal Ethics Committee of the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College (approval No.: SLXD-20210618051). The results showed that BS-E, BS-10E and BS-60E had protective effects against PC12 cells injury induced by corticosterone, among which BS-60E had the strongest protective effect. BS-60E could significantly shorten the time of forced swimming and tail suspension in despair depression models of mice, and was identified as the antidepressant fraction of Radix Paeoniae Alba. The major chemical constituents in the antidepressant fraction were identified by ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), and their proposed fragmentation pathways in MS spectra were deduced. A total of 79 chemical constituents were identified from BS-60E, including 36 monoterpenes, 34 polyphenols, 6 oligosaccharides, and 3 other constituents, and monoterpenes and polyphenols may be major effective constituents of BS-60E.

As one kind of v-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factors, R1-MYB (MYB-related) family plays an important role in plant growth and development, as well as environmental stress and hormone signal transduction. In this study, R1-MYB family genes in Rheum palmatum L. were systematically screened based on full-length transcriptome sequencing analysis. Firstly, the physicochemical, protein domain and molecular evolution characteristics of the coding proteins were analyzed. Furthermore, the tissue expression levels of R1-MYB genes were analyzed by RNA-seq. We also investigated the expression pattern of RpMYB24 in response to various hormones and abiotic stresses. The results showed that a total of 49 R1-MYB genes were identified, which mainly encoded thermally stable hydrophilic proteins. Most of the deduced proteins were predicted to locate in nucleus. Each protein had a large proportion of random curl and α helix, and also had the W-type conserved amino acids which were the signature of MYB. R1-MYB family members were distributed in five subgroups, including circadian clock associated 1 (CCA1)-like, I-box (GATAAG)-like, CAPRICE (CPC)-like, telomere repeat binding factor (TRF)-like and TATA binding protein (TBP)-like, and the number of CCA1-like was the majority. RNA-seq revealed that 49 R1-MYB genes were differentially expressed in roots, rhizomes and leaves of R. palmatum, and the expression levels of 15 and 23 genes in roots and rhizomes were higher than those in leaves, respectively. RpMYB24 transcript was induced by abscisic acid (ABA), salicylic acid (SA), and methyl jasmonate (MeJA) treatment, and could also significantly respond to injury, low temperature and high temperature stresses except drought stress. This study systematically identified the R1-MYB family genes and their molecular characteristics, better for further gene functional validation, and then provide a scientific basis for the transcriptional regulation mechanism research into rhubarb quality formation.

In order to investigate the effects of asiaticoside (Ass) on H9C2 cardiomyocytes, the present study examined the potential intervention of Ass on the proliferation and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/Bcl-2 homology domain protein (Beclin-1) signaling pathway in H9C2 cardiomyocytes following oxygen and glucose deprivation/reperfusion (OGD/R) injury. H9C2 cardiomyocytes were selected as the research objects, and the activity of H9C2 was detected by cell counting kit-8 (CCK-8). H9C2 cells were divided into control group, OGD/R group, Ass low concentration group (10 μmol·L^-1), Ass high concentration group (80 μmol·L^-1) and Ass high concentration + chloroquine group (80 μmol·L^-1 + 50 μmol·L^-1). The control group was cultured under normal conditions, and the other groups were treated with oxygen and glucose deprivation for 4 h and reperfusion for 2 h. The activity and content of aspartic aminotransferase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK) in the supernatant of H9C2 cardiomyocytes were detected by enzyme-linked immunosorbent assay. Autophagy staining assay kit with monodansylcadaverine (MDC) method to observe cellular autophagy; molecular docking technique to identify the molecular targets of Ass. Immunofluorescence was used to observe the effect of the drug on cell number. The expression levels of PI3K, Akt, selective autophagy adaptor protein (P62) and Beclin-1 were detected by Western blot. Compared with OGD/R group, Ass group had a protective effect from 10-80 μmol·L^-1, and the activities and contents of AST, LDH and CK were decreased. The protein expression levels of PI3K, Akt, P62 and Beclin-1 were decreased. Compared with the administration group, the activities and contents of AST, LDH and CK in Ass high-concentration + chloroquine group were significantly decreased, and the protein expression levels of PI3K, Akt, Beclin-1 and P62 were significantly decreased. Immunofluorescence showed that the inhibitor group and each administration group had different degrees of protective effect compared with the model group. Asiaticoside can reduce the injury of H9C2 cardiomyocyte induced by OGD/R, reduce the content of AST, LDH and CK, reduce the expression level of P62 protein, and reduce autophagy, which may be closely related to the inhibition of PI3K/Akt/Beclin-1 signaling pathway activation.

Our studies were aimed to explore the effect and mechanism of the inhibition of the formation of vasculogenic mimicry (VM) in human glioblastoma cells by Xihuang pill (XHP) medicated serum through regulating the hypoxia inducible factor-1α (HIF-1α)/vascular endothelial growth factor A (VEGFA)/vascular endothelial growth factor receptor 2 (VEGFR2) signaling pathway. The medicated serum of XHP was prepared by gavage for 7 days to male SD rats (approval number of animal experiment ethics: 202105A051). The hypoxia model of U251 cells was established using 200 μmol·L^-1 of CoCl₂. After treatment with XHP-medicated serum, cell viability and proliferation of U251 cells were detected by CCK-8 and cell cloning experiment. Cell apoptosis and cell cycle of U251 cells were determined by flow cytometry. Cell migration and invasion were evaluated by wound healing and Transwell invasion assay. The formation of VM was assessed by three-dimensional cell culture of U251 cells. The protein expression levels of HIF-1α, VEGFA, VEGFR2, phosphorylated-VEGFR2 (p-VEGFR2), vascular endothelial-cadherin (VE-cadherin), Eph receptor tyrosine kinases A2 (EphA2), matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 14 (MMP14) and laminin γ2 in U251 cells were detected by Western blot. The results showed that 10% XHP-medicated serum had little effect on the cell viability, proliferation, apoptosis and cell cycle of U251 cells under hypoxia. Compared with the model group, 10% XHP-medicated serum at 1.0, 1.5 and 2.0 h significantly decreased the migration rate (P < 0.01) and the number of invading U251 cells (P < 0.01). 10% XHP-medicated serum at 2.0 h significantly suppressed the formation of VM tubular structures in U251 cells under the condition of hypoxia (P < 0.01). Western blot experiment showed that 10% XHP-medicated serum significantly down-regulated the expression of HIF-1α, VEGFA, phospho-VEGFR2, VE-cadherin, EphA2 and MMP14 proteins (P < 0.05). In conclusion, XHP could inhibit the formation of VM in human glioblastoma U251 cells to suppress the angiogenesis by down-regulating the HIF-1α/VEGFA/VEGFR2 signaling pathway.

This study started from the effect of baicalin (BC), the main active component of the labiaceae plant Scutellaria baicalensis, on collagen-induced arthritis (CIA) in rats, to explore the mechanism of glucose metabolism reprogramming in fibroblast like synoviocytes (FLSs), a key effector cell of synovial inflammation in rheumatoid arthritis (RA). First of all, CIA rats and tumor necrosis factor-α (TNF-α)-induced RASFs in vitro and in vivo models were established, the arthritis index (AI) score and histopathological changes of CIA rats after BC administration were observed, and the levels of inflammatory factors in serum and cell supernatant were quantified by ELISA, immunocytochemistry and Western blot were used to detect the expression of G-protein-coupled receptor 81 (GPR81) and pyruvate dehydrogenase kinase 1 (PDK1) proteins. In addition, the kit was used to measure the levels of key products and enzyme activities in glucose metabolism reprogramming. The results showed that BC (50, 100 and 200 mg·kg^-1) could alleviate the symptoms of arthritis in CIA rats in a dose-dependent manner, inhibit synovial hyperplasia, alleviate the infiltration of inflammatory cells, down-regulate the levels of pro-inflammatory factors TNF-α and interleukin (IL)-1β, and up-regulate the levels of anti-inflammatory factor IL-10 in CIA rats. At the same time, the secretion levels of lactate, pyruvate, acetyl-CoA, citrate and the activity of lactate dehydrogenase B (LDH-B) were decreased, and the expressions of GRP81 and PDK1 were down-regulated, suggesting that BC mediated the reprogramming process of glucose metabolism. However, when GPR81 inhibitor 3-OBA inhibited lactate uptake, the activity of LDH-B was significantly increased, suggesting that BC inhibited the expression of PDK1, a key enzyme in the reprogramming metabolism from glycolysis to oxidative phosphorylation. All animal experiments in this study were conducted in accordance with the ethical standards of the Laboratory Animal Care Center of Anhui University of Chinese Medicine (approval number: AHUCM-rats-2021049). These studies revealed that baicalin mediated metabolic reprogramming of RASFs from glycolysis to oxidative phosphorylation by inhibiting PDK1 protein expression, and alleviated joint inflammation in CIA rats.

A hydrophilic interaction chromatography tandem mass spectrometry method was developed for simultaneous quantification of 35 components in gualoupi injection. The analytes were separated with an ACQUITY XBridge Amide column using 20 mmol·L^-1 ammonium formate aqueous solution (pH 3.0) as mobile phase A and 20 mmol·L^-1 ammonium formate (pH 3.0)∶acetonitrile (1∶9) as mobile phase B for gradient elution. Mass spectrometry with dynamic multiple reaction monitoring and external standard method were used for quantitative analysis. A total of 35 components were determined in 10 batches of gualoupi injection. The results showed that the 35 compounds had a good linear relationship within their respective concentration ranges with the correlation coefficients (R² > 0.998 0), the recoveries ranged from 76.6% to 118.5%. The results showed that γ-aminobutyric acid, trigonelline, alanine, threonine, homoserine, citrulline, and leucine were abundant in gualoupi injection, while nicotinamide, methylsuccinic acid, cytosine and choline account for a low percentege. The present study provides an important reference for elucidation of the effective material basis and the improvement of quality standard of gualoupi injection.

To establish an ultra high performance liquid chromatography coupled with pre-column derivatization method for simultaneous determination of 17 kinds of free amino acid concentrations in CHO cell which expressed high levels of programmed death protein-1 (PD-1) antibody, and its amino acid metabolism was analyzed by this method. Using 6-aminoquinoline-N-hydroxysuccinimidyl carbamate (AQC) as a derivatization reagent, the free amino acids in different concentrations of amino acid standards or cell lines were transformed into stable UV-absorbent compounds, which were separated by gradient elution through ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm) column. The flow rate was set at 0.7 mL·min^-1, the column temperature at 55 ℃, the loading amount of sample at 1 µL, the UV detection wavelength at 260 nm, and then the free amino acid concentration in cell lines which expressed PD-1 antibody was determined by external standard method. According to the changes of amino acids concentration during the process of cell culture, the amino acid metabolism was analyzed. The results showed that pre-column derivatization high performance liquid chromatography could completely separate 17 kinds of amino acids within 11 minutes, has good linear relationship (R² ≥ 0.999 3) in the range of 0.75-500 μmol·L^-1, the limits of detection was 0.1-0.3 μmol·L^-1, the limits of quantitative was 0.5-1 μmol·L^-1. The established method is quickly and reproducibility. Amino acid metabolism revealed that methionine, isoleucine and leucine may be the restrictive factors of expression for cell line. This method can be used for detection changes of free amino acid concentration in cell line.

The discovery of drug targets plays a crucial role in drug research. Accurate information of small molecule drug-protein interaction can be provided by label-free target discovery technology without any structural modification at the small molecule. So, the label-free drug target discovery technology had become the powerful tool to discover the targets of drugs. Due to the "multi-component and multi-target" characteristics of traditional Chinese medicines (TCMs), the research on its targets and mechanism had been restricted. Based on potential of the label-free target discovery technology in the research of TCMs, this paper summarized the label-free target discovery technology and its application in TCMs research. It will provide a reference for the discovery of targets of TCMs and a new view for promoting the modernization of TCMs.

Five compounds were isolated from the ethyl acetate fraction of Semen Persicae by using various chromatographic methods, including ODS, Sephadex LH-20, HPLC and semipreparative HPLC. Their structures were identified by 1D-NMR, 2D-NMR, HR-ESI-MS, UV, IR, circular dichroism (CD) and ECD calculation techniques: (2R, 3R)-5, 7, 4′-trihydroxy-3′-methoxy-3-formylflavan-3-ol-5-O-β-D-glucopyranoside (1), (7R, 8S)-dihydrodehydrodiconiferyl 6″-benzoyl alcohol-9-O-β-D-glucopyranoside (2), (7R, 8S)-dihydrodehydrodiconiferyl alcohol-9-β-O-D-glucopyranosid (3), 2-methoxy-4-(2-propenyl)-phenyl-O-β-D-glucopyranoside (4), 2-[4-(3-hydroxypropyl)-2-methoxyphenoxy]-propane-1, 3-diol (5). Compound 1 and 2 are new compounds, and compounds 3-5 were obtained from Prunus davidiana (Carr.) Franch. for the first time.