Highly selective colorimetric determination of glutathione based on sandwich-structured nanoenzymes composed of gold nanoparticle-coated molecular imprinted metal-organic frameworks.

A sandwich-structured composite nanoenzyme (NH2-MIL-101(Fe)@Au@MIP) was prepared using molecularly imprinted polymers, metal-organic frameworks, and gold nanoparticles and a highly selective glutathione (GSH) colorimetric sensor was constructed. The inner part of the composite nanoenzymes is a metal-organic framework loaded with gold nanoparticles (AuNPs), NH2-MIL-101(Fe)@Au, which has superior peroxidase-like activity compared with  NH2-MIL-101(Fe). This is due to the surface plasmon resonance effect of AuNPs. GSH can form strong Au-S bonds with AuNPs, which can significantly reduce the enzymatic activity of NH2-MIL-101(Fe)@Au, thereby changing the absorbance at 450 nm of the sensing system. The degree of change in absorbance is correlated with the concentration of GSH. In the outer part, the molecularly imprinted polymer with oxidized glutathione (GSSG) as a dummy template provided specific pores, which significantly improved the selectivity of the sensing system. The sensor showed good GSH sensing performance in the range 1 ~ 50 µM with a lower limit of detection (LOD) of 0.231 µM and good sensing performance in fetal bovine serum, indicating its high potential for clinical diagnostic applications.

Ginsenoside 24-OH-PD from red ginseng inhibits acute T-lymphocytic leukaemia by activating the mitochondrial pathway.

Ginsenoside 24-hydroxy-ginsengdiol (24-OH-PD), extracted from red ginseng, is a novel diol-type ginsenoside, strongly inhibits the growth of human T-cell acute lymphoblastic leukaemia (T-ALL) CCRF-CEM cells. Our research aimed at investigating the mechanism underlying this inhibition. Cell viability was determined using the cell counting kit-8 (CCK-8) assay, and NOD/SCID mice bearing CCRF-CEM cells were used to verify the therapeutic effect of 24-OH-PD on T-ALL in vivo. We equally analysed pathways related to 24-OH-PD in CCRF-CEM cells using RNA-Seq analysis. Cell apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and mitochondrial permeability transition pore (mPTP) levels were detected by flow cytometry. The activity of caspase3 and caspase9 was detected by enzyme activity detection kits. The expression levels of apoptosis-related proteins and mRNA were determined through western blotting and quantitative reverse-transcription PCR assays (qRT-PCR). CCK-8 assay and animal xenograft experiments confirmed that 24-OH-PD significantly inhibited T-ALL in a dose-dependent manner, both in vivo and in vitro. RNA-Seq results suggest that mitochondria-mediated apoptosis pathway plays an important role in this process. Furthermore, intracellular ROS levels increased, mPTP opened, and ΔΨm decreased following 24-OH-PD treatment. Pretreatment with the antioxidant, NAC, reversed the effects of 24-OH-PD on apoptosis and ROS generation. Moreover, 24-OH-PD treatment increased the expression of Bax and caspase family members, thereby releasing cytochrome c (Cytc) and inducing apoptosis. Our findings showed that, 24-OH-PD induces apoptosis in CCRF-CEM cells by activating the mitochondrial-dependent apoptosis pathway through ROS accumulation. This inhibitory effect implies that 24-OH-PD could be further developed as treatment of T-ALL.

Fluorescent probes based on the core-shell structure of molecular imprinted materials and gold nanoparticles for highly selective glutathione detection.

Glutathione (GSH) is a polypeptide with important physiological functions. Real-time and accurate detection of GSH is of great significance for clinical diagnosis, disease treatment and pathogen detection. A fluorescent nanosensor based on composite core-shell nanoparticles for the highly selective detection of GSH is reported. In the cores, the fluorescence of rhodamine b was quenched by using gold nanoparticles (AuNPs), and GSH could competitively combine with AuNPs to cause rhodamine b to fall off, thereby recovering the fluorescence. In the shell part, molecularly imprinted materials using oxidized glutathione (GSSG) as a pseudotemplate provide GSH/GSSG specific pores and improve the specificity and anti-interference ability of the sensor. The GSH sensor has a detection range of 0-100 µM and limit of detection (LOD) of 0.18 µM, and robust sensing performance in fetal bovine serum, indicating its great potential for clinical diagnosis.

Rapid Screening of Lipase Inhibitors in Scutellaria baicalensis by Using Porcine Pancreatic Lipase Immobilized on Magnetic Core-Shell Metal-Organic Frameworks.

As for ligand fishing, the current immobilization approaches have some potential drawbacks such as the small protein loading capacity and difficult recycle process. The core-shell metal-organic frameworks composite (Fe3O4-COOH@UiO-66-NH2), which exhibited both magnetic characteristics and large specific surface area, was herein fabricated and used as magnetic support for the covalent immobilization of porcine pancreatic lipase (PPL). The resultant composite Fe3O4-COOH@UiO-66-NH2@PPL manifested a high loading capacity (247.8 mg/g) and relative activity recovery (101.5%). In addition, PPL exhibited enhanced tolerance to temperature and pH after immobilization. Then, the composite Fe3O4-COOH@UiO-66-NH2@PPL was incubated with the extract of Scutellaria baicalensis to fish out the ligands. Eight lipase inhibitors were obtained and identified by UPLC-Q-TOF-MS/MS. The feasibility of the method was further confirmed through an in vitro inhibitory assay and molecular docking. The proposed ligand fishing technique based on Fe3O4-COOH@UiO-66-NH2@PPL provided a feasible, selective, and effective platform for discovering enzyme inhibitors from natural products.

Systems biology analysis of the effect and mechanism of total flavonoids of Astragali Radix against cyclophosphamide-induced leucopenia in mice.

This study aimed to demonstrate the pharmacological mechanism of total flavonoids extracted from Astragali Radix (AR) on cyclophosphamide (Cy)-induced leucopenia in mice. First, flow cytometry, network pharmacology and plasma metabolomics were integrated to investigate the pharmacological mechanism of total flavonoids, the targets from network pharmacology and metabolites from metabolomics were analyzed by DAVID. Then, the key cytokines were validated to confirm the predicted metabolic pathway results. The results showed that total flavonoids significantly increased body weight, routine blood indices, bone marrow DNA cells, and also markedly caused lymphocyte proliferation by increasing the percentages of CD4+ and CD8+. Using network pharmacology and metabolomics methods, the study identified 13 signal-related pathways regulated by total flavonoids including PI3K-Akt signaling pathway, Jak-STAT signaling pathway, Sphingolipid signaling pathway, and so on. Total flavonoids also reversed changes in serum cytokines IL-2, IL-6, and GM-CSF. Total flavonoids exhibits protective effects against leucopenia probably by modulating immunologic functions, promoting cell proliferation, and regulating related metabolic pathways at the system level.

Synthesis of 5-Alkynyltetrandrine Derivatives and Evaluation of their Anticancer Activity on A549 Cell Lines.

BACKGROUND: Lung cancer is one of the most prevalent malignancies and thus the development of novel therapeutic agents for managing lung cancer is imperative. Tetrandrine, a bis-benzyltetrahydroisoquinoline alkaloid isolated from Stephania tetrandra S. Moore, has been found to exert cytotoxic effects on cancerous cells. METHODS: A series of 5-alkynyltetrandrine derivatives was synthesized via the Sonogashira cross-coupling reactions and evaluated as potential anti-tumor agents. The anti-tumor activities of 12 compounds on lung cancer cells (A549) were evaluated using the MTT method. The population of apoptotic cells was measured using a TUNEL assay. Real-time PCR quantified the gene expression levels of Bcl-2, Bax, survivin and caspase-3. The content of Cyt-C was detected using a Human Cyt-C ELISA kit. RESULTS: Most of these compounds exhibited better activities than tetrandrine itself on A549 cells. Among them, compound 7 showed the highest cytotoxicity among the tested compounds against human lung adenocarcinoma A549 cells with an IC50 of 2.94 µM. Preliminary mechanistic studies indicated that compound 7 induced apoptosis of human lung cancer A549 cells and increased the level of the proapoptotic gene Bax, release of Cyt-C from mitochondria and activation of caspase-3 genes. CONCLUSION: The results suggest that compound 7 exerts its antitumor activity against A549 cells through the induction of the intrinsic (mitochondrial) apoptotic pathway. These findings will contribute to the future design of more effective anti-tumor agents in lung cancer therapy.

Synthesis of novel tetrandrine derivatives and their inhibition against NSCLC A549 cells.

A series of novel tetrandrine (Tet) derivatives were synthesized through Suzuki -Miyaura reaction and evaluated for their cytotoxicity against human non-small cell lung carcinoma (NSCLC) A549 cells. Interestingly, most of derivatives showed similar cytotoxicity to Tet against NSCLC A549 cells, and particularly, compounds Y5, Y6, Y9 and Y11 showed the most significant cytotoxic effects with IC50 values ranging from 3.87 to 4.66 mM. The present study is expected to contribute to the future design of more effective anticancer agents in lung cancer chemotherapy.

Nuclear magnetic resonance based metabolomic differentiation of different Astragali Radix.

Astragali Radix (AR) is one of the most popular herbal medicines in traditional Chinese medicine (TCM). Wild AR is believed to be of high quality, and substitution with cultivated AR is frequently encountered in the market. In the present study, two types of ARs (wild and cultivated) from Astragalus membranaceus (Fisch.) Bge. and A. membranaceus var. mongholicus (Bge.) Hsiao, growing in different regions of China, were analyzed by NMR profiling coupled with multivariate analysis. Results showed that both could be differentiated successfully and cultivation patterns or growing years might have greater impact on the metabolite compositions than the variety; the metabolites responsible for the separation were identified. In addition, three extraction methods were compared and the method (M1) was used for further analysis. In M1, the extraction solvent composed of water, methanol, and chloroform in the ratio of 1 : 1 : 2 was used to obtain the aqueous methanol (upper layer) and chloroform (lower layer) fractions, respectively, showing the best separation. The differential metabolites among different methods were also revealed. Moreover, the sucrose/glucose ratio could be used as a simple index to differentiate wild and cultivated AR. Meanwhile, the changes of correlation pattern among the differential metabolites of the two varieties were found. The work demonstrated that NMR-based non-targeted profiling approach, combined with multivariate statistical analysis, can be used as a powerful tool for differentiating AR of different cultivation types or growing years.

5-Aminosalicylic acid inhibits inflammatory responses by suppressing JNK and p38 activity in murine macrophages.

CONTEXT: 5-Aminosalicylic acid (5-ASA), as an anti-inflammatory drug, has been extensively used for the treatment of mild to moderate active ulcerative colitis (UC), but the possible mechanisms of action remain unclear. OBJECTIVE: To investigate the effects of 5-ASA on the production of inflammatory mediators by murine macrophages stimulated with lipopolysaccharide (LPS), and determine the underlying pharmacological mechanism of action. MATERIALS AND METHODS: The levels of nitric oxide (NO) and interleukin-6 (IL-6) were measured by Varioskan Flash and IL-6 Enzyme-Linked Immunosorbent Assay sets. Real time quantitative polymerase chain reaction was used to determine the level of induced nitric oxide synthase (iNOS). The effects of 5-ASA on iNOS, the c-Jun N-terminal kinases (JNKs), p38 and nuclear factor (NF)-κB signaling pathways were examined using western blotting. RESULTS: 5-ASA suppressed the production of NO and IL-6, and also decreased the expression of iNOS in LPS-induced RAW264.7 cells. 5-ASA inhibited the phosphorylation of JNKs and p38, but did not block NF-κB activation at all doses tested. DISCUSSION AND CONCLUSION: The results indicated that the anti-inflammatory effect of 5-ASA was mainly regulated by the inhibition of the JNKs, p38 pathways rather than NF-κB pathway. Further research is required to clarify the detailed mechanism of the action.

NMR-based metabonomics and correlation analysis reveal potential biomarkers associated with chronic atrophic gastritis.

Chronic atrophic gastritis (CAG) is one of the most important pre-cancerous states with a high prevalence. Exploring of the underlying mechanism and potential biomarkers is of significant importance for CAG. In the present work, 1H NMR-based metabonomics with correlative analysis was performed to analyze the metabolic features of CAG. 19 plasma metabolites and 18 urine metabolites were enrolled to construct the circulatory and excretory metabolome of CAG, which was in response to alterations of energy metabolism, inflammation, immune dysfunction, as well as oxidative stress. 7 plasma biomarkers and 7 urine biomarkers were screened to elucidate the pathogenesis of CAG based on the further correlation analysis with biochemical indexes. Finally, 3 plasma biomarkers (arginine, succinate and 3-hydroxybutyrate) and 2 urine biomarkers (α-ketoglutarate and valine) highlighted the potential to indicate risks of CAG in virtue of correlation with pepsin activity and ROC analysis. Here, our results paved a way for elucidating the underlying mechanisms in the development of CAG, and provided new avenues for the diagnosis of CAG and presented potential drug targets for treatment of CAG.

Design and synthesis of new tetrandrine derivatives and their antitumor activities.

A series of tetrandrine derivatives were designed and synthesized using Suzuki coupling reaction. Eleven targeted compounds with over 50% inhibition against HL60 and A549 human cancer cell lines at 10 µM were further evaluated for the in vitro antitumor activities by MTT or SRB assay. The biological results revealed that some compounds exhibited potent antitumor activities. Thiophene derivative 6 and acetylphenyl derivative 5 were the most active ones against HL60 and A549 cell lines, with IC50 values less than 5 µM, which thus could be considered as useful candidate for further development of new antitumor agents.

A metabonomic analysis reveals novel regulatory mechanism of Huangqi injection on leucopenia mice.

CONTEXT: Huangqi injection (HQI), extracted from Astragali Radix, which has capability on treating the leucopenia. However, the potential metabolic mechanism is poorly understood. OBJECTIVE: To investigate the effect of HQI on cyclophosphamide (Cy)-induced leucopenia in mice, the nuclear magnetic resonance (NMR)-based metabolomic profiling technique coupled with multivariate statistical analysis was applied. MATERIALS AND METHODS: NMR analysis was used to identify the various compounds of HQI, and high-performance liquid chromatography was applied to determine the contents of major compounds. A experimental mice model of leucopenia induced by Cy and NMR-based metabolomic approach was used to evaluate the pharmacological effect of HQI and to investigate its probable acting mechanism on leucopenia. RESULTS: HQI increased body weight and elevated the white blood cell (WBC), monocytes (MO), neutrophils (NE), and lymphocyte (LY) levels of Cy-treated mice. In addition, the levels of most perturbed endogenous metabolites could be reversed after HQI treatment. Correlations between WBC, MO, NE, LY, and altered metabolite profiles in spleen were greater than that in serum, and the correlation in MO was more evident than those for WBC, NE, and LY. DISCUSSION AND CONCLUSION: HQI showed obvious efficacy on the mice model of leucopenia. And the drug action of HQI on leucopenia was probably related with regulating metabolic pathways of energy metabolism, amino acids metabolism, oxidative stress, and choline metabolism. However, various compounds were present in the HQI, and the bioactive compounds responsible for the drug actions should be further investigated.

[~1H NMR fingerprints of Huangqi injection solution].

The study established the 1H NMR-based fingerprinting and analyzed 8 batches of Huangqi injection solution.1H NMR-based fingerprinting of both primary and secondary metabolites of Huangqi injection were established, and the 1H-1H chemical shift correlation spectroscopy (COSY) and 1H-13C chemical shift correlation spectroscopy (HSQC) were used to identify the chemical components in Huangqi injection solution. Coupled with similarity analysis and relative content determination,8 batches of Huangqi injection solution were analyzed. Twenty-five metabolites (both primary and secondary) were identified, and the significant differences were found in the chemical composition among these Huangqi samples. The content and content variation of the primary metabolites were much higher than those of the secondary metabolites, which was the major cause of the uniformity of the Huangqi injections. The results on the quality variations of Huangqi injections in this study will serve as a basis for improving the quality control.