Multi-level chemical characterization and anti-inflammatory activity evaluation of the polysaccharides from Prunella vulgaris.

Prunella vulgaris (PV) is a medicine and food homologous plant, but its quality evaluation seldom relies on the polysaccharides (PVPs). In this work, we established the multi-level fingerprinting and in vitro anti-inflammatory evaluation approaches to characterize and compare the polysaccharides of P. vulgaris collected from the major production regions in China. PVPs prepared from 22 batches of samples gave the content variation of 5.76-24.524 mg/g, but displayed high similarity in the molecular weight distribution. Hydrolyzed oligosaccharides with degrees of polymerization 2-14 were characterized with different numbers of pentose and hexose by HILIC-MS. The tested 22 batches of oligosaccharides exhibited visible differences in peak abundance, which failed to corelate to their production regions. All the PVPs contained Gal, Xyl, and Ara, as the main monosaccharides. Eleven batches among the tested PVPs showed the significant inhibitory effects on NO production on LPS-induced RAW264.7 cells at 10 µg/mL, but the exerted efficacy did not exhibit correlation with the production regions. Conclusively, we, for the first time, investigated the chemical features of PVPs at three levels, and assessed the chemical and anti-inflammatory variations among the different regions of P. vulgaris samples.

Comprehensive metabolome characterization and comparison between two sources of Dragon's blood by integrating liquid chromatography/mass spectrometry and chemometrics.

Dragon's Blood (DB) serves as a precious Chinese medicine facilitating blood circulation and stasis dispersion. Daemonorops draco (D. draco; Qi-Lin-Jie) and Dracaena cochinchinensis (D. cochinchinenesis; Long-Xue-Jie) are two reputable plant sources for preparing DB. This work was designed to comprehensively characterize and compare the metabolome differences between D. draco and D. cochinchinenesis, by integrating liquid chromatography/mass spectrometry and untargeted metabolomics analysis. Offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS), by utilizing a powerful hybrid scan approach, was elaborated for multicomponent characterization. Configuration of an XBridge Amide column and an HSS T3 column in offline mode exhibited high orthogonality (A0 0.80) in separating the complex components in DB. Particularly, the hybrid high-definition MSE-high definition data-dependent acquisition (HDMSE-HDDDA) in both positive and negative ion modes was applied for data acquisition. Streamlined intelligent data processing facilitated by the UNIFI™ (Waters) bioinformatics platform and searching against an in-house chemical library (recording 223 known compounds) enabled efficient structural elucidation. We could characterize 285 components, including 143 from D. draco and 174 from D. cochinchinensis. Holistic comparison of the metabolomes among 21 batches of DB samples by the untargeted metabolomics workflows unveiled 43 significantly differential components. Separately, four and three components were considered as the marker compounds for identifying D. draco and D. cochinchinenesis, respectively. Conclusively, the chemical composition and metabolomic differences of two DB resources were investigated by a dimension-enhanced analytical approach, with the results being beneficial to quality control and the differentiated clinical application of DB.

An efficient approach addressing the chemical complexity of Jiawei Fangji Huangqi decoction by integrating ultra-high-performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry and intelligent data processing workflows.

A challenge in the quality control of traditional Chinese medicine is the systematic multicomponent characterization of the compound formulae. Jiawei Fangji Huangqi, a modified form of Fangji Huangqi, is a prescription comprising seven herbal medicines. To address the chemical complexity of the Jiawei Fangji Huangqi decoction, we integrated ion mobility-quadrupole time-of-flight high-definition MSE coupled to ultra-high-performance liquid chromatography and intelligent data processing workflows available in the UNIFI software package. Good chromatographic separation was achieved on CORTECS UPLC T3 column within 52 min, and high-accuracy MS2 data were acquired using high-definition MSE in the negative and positive modes. A chemical library of 1250 compounds was created and incorporated into the UNIFI software to enable automatic peak annotation of the high-definition MSE data. We identified or tentatively characterize 430 compounds in the Jiawei Fangji Huangqi decoction. The potential superiority of high-definition MSE over conventional MS data acquisition approaches was revealed in its spectral quality (MS2 ), differentiation of isomers, separation of coeluting compounds, and target mass coverage. The multiple components of the Jiawei Fangji Huangqi decoction were elucidated, offering insight into its improved pharmacological action compared with that of the Fangji Huangqi formula.

Pseudotargeted Metabolomics Approach Enabling the Classification-Induced Ginsenoside Characterization and Differentiation of Ginseng and Its Compound Formulation Products.

The use of diversified ginseng extracts in health-promoting foods is difficult to differentiate, as they share bioactive ginsenosides among different Panax species (e.g., P. ginseng, P. quinquefolius, P. notoginseng, and P. japonicus) and different parts (e.g., root, leaf, and flower). This work was designed to develop a pseudo-targeted metabolomics approach to discover ginsenoside markers facilitating the precise authentication of ginseng and its use in compound formulation products (CFPs). Versatile mass spectrometry experiments on the QTrap mass spectrometer achieved classified characterization of the neutral, malonyl, and oleanolic acid-type ginsenosides, with 567 components characterized. A pseudo-targeted metabolomics approach by multiple reaction monitoring (MRM) of 262 ion pairs could assist to establish key identification points for 12 ginseng species. The simultaneous detection of 14 markers enabled the identification of ginseng from 15 ginseng-containing CFPs. The pseudo-targeted metabolomics strategy enabled better performance in differentiating among multiple ginseng, compared with the full-scan high-resolution mass spectrometry approach.

Multicomponent Characterization of the Flower Bud of Panax notoginseng and Its Metabolites in Rat Plasma by Ultra-High Performance Liquid Chromatography/Ion Mobility Quadrupole Time-of-Flight Mass Spectrometry.

The flower bud of Panax notoginseng (PNF) consumed as a tonic shows potential in the prevention and treatment of cardiovascular diseases. To identify the contained multi-components and, in particular, to clarify which components can be absorbed and what metabolites are transformed, unveiling the effective substances of PNF is of vital significance. A unique ultrahigh-performance liquid chromatography/ion mobility quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) profiling approach and efficient data processing by the UNIFITM bioinformatics platform were employed to comprehensively identify the multi-components of PNF and the related metabolites in the plasma of rats after oral administration (at a dose of 3.6 g/kg). Two MS2 data acquisition modes operating in the negative electrospray ionization mode, involving high-definition MSE (HDMSE) and data-dependent acquisition (DDA), were utilized aimed to extend the coverage and simultaneously ensure the quality of the MS2 spectra. As a result, 219 components from PNF were identified or tentatively characterized, and 40 thereof could be absorbed. Moreover, 11 metabolites were characterized from the rat plasma. The metabolic pathways mainly included the phase I (deglycosylation and oxidation). To the best of our knowledge, this is the first report that systematically studies the in vivo metabolites of PNF, which can assist in better understanding its tonifying effects and benefit its further development.

[Clinical efficacy and safety of moxibustion as adjuvant therapy for COPD in stable phase: a Meta-analysis].

OBJECTIVE: To systematically evaluate the efficacy and safety of conventional therapy combined with moxibustion in the treatment of chronic obstructive pulmonary disease (COPD) in stable phase based on Meta-analysis medicine. METHODS: The randomized controlled trials (RCTs) of moxibustion as adjuvant therapy for COPD were retrieved from the databases of CNKI, Wanfang, SinoMed, PubMed, Web of Science, Cochrane Library and Ebsco. RevMan5.3 software was used for Meta analysis, and the quality of evidence was evaluated according to GRADE standards. RESULTS: A total of 16 RCTs were included, involving 1425 patients. The results of Meta-analysis showed that: compared with the conventional treatment, ①the adjuvant therapy with moxibustion had advantages in reducing the number of acute exacerbations [MD=-0.31, 95%CI:-0.49--0.13, P=0.0006]; ②the adjuvant therapy with moxibustion improved lung function significantly [FEV1% (MD=4.00, 95%CI:2.63-5.37, P<0.000 01) and FEV1/FVC (MD=3.56, 95%CI:1.69-5.43, P=0.000 2)]; ③the adjuvant therapy with moxibustion could extend the 6 min walking distance (6WMD) (MD=35.00, 95%CI:18.02-51.99, P<0.000 1); ④the adjuvant therapy with moxibustion could improve the modified British Medical Research Council breathing questionnaire (mMRC) classification significantly (MD=-0.62, 95%CI:-1.18--0.05, P=0.03); ⑤no adverse reaction was reported in the included literature. CONCLUSION: The efficacy of moxibustion as adjuvant therapy for COPD in stable phase is better than that of simple conventional therapy. Due to insufficient clinical evidence and the limitations of this study, clinical safety is unclear and further evidence is needed to support the results.

The effects of dietary yeast hydrolysate on growth, hematology, antioxidant enzyme activities and non-specific immunity of juvenile Nile tilapia, Oreochromis niloticus.

The present study was aimed to compare and evaluate the impacts of supplemented diets with different yeast hydrolysate (YH) levels on growth performance, body composition, hematological characteristics, antioxidant enzyme activities, and non-specific immunity (intestinal cytokines) of juvenile Nile tilapia (Oreochromis niloticus). Three isonitrogenous (protein, 33%) and isolipidic (lipid, 6%) experimental diets supplemented graded levels of YH (0% for control; 1% and 3% as tested diets) were fed to juvenile Nile tilapia. A total of 240 fish with initial body weight averaging 3.5 ± 0.02 g were randomly divided into three groups with four replicates per group and 20 fish for each replicate. For apparent satiation, the fish were fed twice daily during eight weeks. The results showed no significant difference in survival among all treatments. The fish fed the diet containing 1% yeast hydrolysate had significantly elevated weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER) compared to the control group and lower feed conversion ratio (FCR). The fish fed 1% and 3% YH showed higher glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT) activity and a significantly lower malondialdehyde (MDA) level in the liver than the control group, indicating enhancement of the anti-oxidant status. Serum lysozyme activity was significantly increased in the diet having 1% and 3% yeast hydrolysate supplementation groups, suggesting an improvement influence on the non-specific immune response. The expression of IL-1ß, IL-10, TNF-α, TGF-ß2, ALP and TLR2 was significantly elevated in fish fed the diet containing 1% YH. In conclusion, dietary supplementation with 1% yeast hydrolysate improves growth performance, and feed utilization enhances the antioxidant status and exerts an adequate stimulus on the non-specific immunity (intestinal cytokines) of Nile tilapia.

Gamabufotalin induces a negative feedback loop connecting ATP1A3 expression and the AQP4 pathway to promote temozolomide sensitivity in glioblastoma cells by targeting the amino acid Thr794.

OBJECTIVES: Temozolomide (TMZ) is one of the most commonly used clinical drugs for glioblastoma (GBM) treatment, but its drug sensitivity needs to be improved. Gamabufotalin (CS-6), the primary component of the traditional Chinese medicine "ChanSu," was shown to have strong anti-cancer activity. However, more efforts should be directed towards reducing its toxicity or effective treatment doses. METHODS: Target fishing experiment, Western blotting, PCR, confocal immunofluorescence and molecular cloning techniques were performed to search for possible downstream signalling pathways. In addition, GBM xenografts were used to further determine the potential molecular mechanisms of the synergistic effects of CS-6 and TMZ in vivo. RESULTS: Mechanistic research revealed a negative feedback loop between ATP1A3 and AQP4 through which CS-6 inhibited GBM growth and mediated the synergistic treatment effect of CS-6 and TMZ. In addition, by mutating potential amino acid residues of ATP1A3, which were predicted by modelling and docking to interact with CS-6, we demonstrated that abrogating hydrogen bonding of the amino acid Thr794 interferes with the activation of ATP1A3 by CS-6 and that the Thr794Ala mutation directly affects the synergistic treatment efficacy of CS-6 and TMZ. CONCLUSIONS: As the main potential target of CS-6, ATP1A3 activation critically depends on the hydrogen bonding of Thr794 with CS-6. The combination of CS-6 and TMZ could significantly reduce the therapeutic doses and promote the anti-cancer efficacy of CS-6/TMZ monotherapy.

The sodium pump α1 subunit regulates bufalin sensitivity of human glioblastoma cells through the p53 signaling pathway.

Bufalin is the primary component of the traditional Chinese medicine "Chan Su," which has been widely used for cancer treatment at oncology clinics in certain countries. Evidence suggests that this compound possesses potent antitumor activities, although the exact molecular mechanism(s) require further elucidation. Therefore, this study aimed to further clarify the in vitro and in vivo antiglioma effects of bufalin and the molecular mechanism underlying the regulation of drug sensitivity. The anticancer effects of bufalin were determined by colony formation assays, apoptosis assays, and cellular redox state tests of glioma cells. Confocal microscopy was performed to determine the expression changes of the DNA damage biomarker γ-H2AX and the nuclear translocation of p53 in glioma cells. Western blotting and RT-PCR were used to detect the protein and gene expression levels, respectively. Here, we report that bufalin induced glioblastoma cell apoptosis and oxidative stress and triggered DNA damage. The critical roles of the sodium pump α1 subunit (ATP1A1) in mediating the XPO1-targeted anticancer effect of bufalin in human glioma were further confirmed. Mechanistic studies confirmed the important roles of Src and p53 signaling in mediating bufalin-induced apoptosis. Importantly, bufalin also inhibited the growth of glioma xenografts. In conclusion, our study indicated that therapies targeting the ATP1A1 and p53 signaling-mediated mitochondrial apoptotic pathways regulated by bufalin might be potential treatments for human glioma, and these findings will provide molecular bases for developing bufalin into a drug candidate for the treatment of malignant glioma.

Bufalin inhibits glioblastoma growth by promoting proteasomal degradation of the Na+/K+-ATPase α1 subunit.

Chansu is a traditional Chinese medicine that is generally recognized as a specific inhibitor of Na+/K+-ATPase. Bufalin, an active component of Chansu, is an endogenous steroid hormone with great potential as a cancer treatment. However, the mechanism by which it exerts its antitumor activity requires further research. Currently, the α1 subunit of Na+/K+-ATPase (ATP1A1) is known to exert important roles in tumorigenesis, and the precise mechanisms underlying the effect of Bufalin on the Na+/K+-ATPase α1 subunit was therefore investigated in this study to determine its role in glioblastoma treatments. The effect of ATP1A1 on the sensitivity of glioblastoma cells to Bufalin was investigated using MTT assays, RT-PCR and siRNA. Western blot was also used to explore the important roles of the ubiquitin-proteasome pathway in the Bufalin-mediated inhibition of ATP1A1. Xenografted mice were used to examine the anti-tumor activity of Bufalin in vivo. LC-MS/MS analysis was performed to determine the ability of Bufalin to traverse the blood-brain barrier (BBB). The results indicated that Bufalin inhibited the expression of ATP1A1 in glioblastoma by promoting the activation of proteasomes and the subsequent protein degradation of ATP1A1, while Bufalin had no effect on ATP1A1 protein synthesis. Bufalin also inhibited the expression of ATP1A1 in xenografted mice and significantly suppressed tumor growth. These data should contribute to future basic and clinical investigations of Bufalin. In conclusion, Bufalin significantly inhibited the expression of ATP1A1 in glioblastoma cells by activating the ubiquitin-proteasome signaling pathway. Bufalin may therefore have the potential to be an effective anti-glioma drug for human glioblastoma in the future.

[The study of cytotoxicity of different intracanal medications and cell rehabilitation on human periodontal ligament fibroblasts].

PURPOSE: To determine the cytotoxicity and reversibility of cytotoxicity of 5 intracanal medications in various concentrations with human periodontal ligament fibroblasts (HPLFs), with the aim to provide a comprehensive reliable scientific evidence for making a good choice of these intracanal medications in clinical practice. METHODS: The experimental medications included formocresol (FC), camphorated paramonochlorophenol(CP), metronidazole, calcium hydroxide and traditional Chinese medicine. HPLFs were obtained from healthy premolars extracted for orthodontic reasons. All samples were placed into DMEM, cultured and harvested from the fourth to eighth passages. MTT, alkalinephosphatase (ALP)activity determination and transmission electron microscope (TEM) were used in this study. The data was analyzed by SPSS11.5 software. Student's t test, analysis of variance was used for the statistical analysis. RESULTS: FC, CP had cytotoxicity and inhibited the growth and ALP activity of HPLFs significantly (P<0.01 and P<0.05). The cytotoxicity of FC was at 3- 4 grade, with cell rehabilitation rate less than 30%. The cytotoxicity of CP was at 1 to 3 grade, with cell rehabilitation rate less than 30%. Metronidazole lightly inhibited the proliferation and ALP activity of HPLFs, its cytotoxicity was at 1 grade, with rehabilitation rate from 32% to 85%. Calcium hydroxide and Chinese medicine didn't inhibit the proliferation and ALP activity of HPLFs significantly (P>0.05). Their cytotoxicity was at 0 to 1 grade with cell rehabilitation rate more than 90%. TEM study showed that severe morphologic alteration of HPLFs induced by FC, CP, whereas the cells in Chinese medicine and calcium hydroxide had little alteration on their morphology. Metronidazole induced minor morphologic alteration of HPLFs. CONCLUSIONS: Calcium hydroxide, traditional Chinese medicine have no cytotoxicity while FC and CP lead to severe cytotoxicity, the cytotoxicity is irreversible. Therefore the clinical application of FC and CP is no longer recommended. Traditional Chinese medicine or calcium hydroxide can be taken as ideal intracanal medications.