A comparative study of three chemometrics methods combined with excitation-emission matrix fluorescence for quantification of the bioactive compounds aesculin and aesculetin in Cortex Fraxini.

Cortex Fraxini is an important traditional Chinese herbal medicine with various medical functions. Aesculin and aesculetin are the main effective components of Cortex Fraxini. The fluorescence signals of the two compounds have a high degree of overlap with each other, making quantitative analysis difficult with conventional analytical methods. In the present study, different chemometrics methods, including lasso regression (LAR), interval partial least squares (iPLS), and multidimensional partial least squares (N-PLS) methods, were employed and combined with excitation-emission matrix (EEM) fluorescence for the purpose of accurate quantification of aesculin and aesculetin in Cortex Fraxini samples. The most satisfactory results were obtained by using the N-PLS method based on the EEM spectra without scatterings, with correlation coefficient of calibration and prediction values higher than 0.9972 and 0.9962, respectively, root mean squared errors for calibration and prediction values lower than 0.0304 and 0.1165, respectively, and recovery values in the range of 83.32%-104.62%. The obtained credible models indicated that the N-PLS method combined with EEM spectra has the advantages of being green, low cost, and accurate and it is a good strategy for the determination of active compounds in complex samples. To further confirm the accuracy of the obtained results, the same samples were analyzed by the recognized ultra-performance liquid chromatography method.

Pien Tze Huang Inhibits Migration and Invasion of Hepatocellular Carcinoma Cells by Repressing PDGFRB/YAP/CCN2 Axis Activity.

OBJECTIVE: To investigate the effects of Pien Tze Huang (PZH) on the migration and invasion of HCC cells and underlying molecular mechanism. METHODS: Cell counting kit-8 (CCK-8) was applied to evaluate the cell viabilities of SMMC-7721, SK-Hep-1, C3A and HL-7702 (6 × 103 cells/well) co-incubated with different concentrations of PZH (0, 0.2, 0.4, 0.6, 0.8 mg/mL) for 24 h. Transwell, wound healing assay, CCK-8 and Annexin V-FITC/PI staining were conducted to investigate the effects of PZH on the migration, invasion, proliferation and apoptosis of SK-Hep-1 and SMMC-7721 cells (650 µ g/mL for SK-Hep-1 cells and 330 µ g/mL for SMMC-7721 cells), respectively. In vivo, lung metastasis mouse model constructed by tail vein injection of HCC cells was used for evaluating the anti-metastasis function of PZH. SK-Hep-1 cells (106 cells/200 µ L per mice) were injected into B-NDG mice via tail vein. Totally 8 mice were randomly divided into PZH and control groups, 4 mice in each group. After 2-d inoculation, mice in the PZH group were administered with PZH (250 mg/kg, daily) and mice in the control group received only vehicle (PBS) from the 2nd day after xenograft to day 17. Transcriptome analysis based on RNA-seq was subsequently used for deciphering anti-tumor mechanism of PZH. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were applied to verify RNA-seq results. Luciferase reporter assay was performed to examine the transcriptional activity of yes-associated protein (YAP). RESULTS: PZH treatment significantly inhibited the migration, invasion, proliferation and promoted the apoptosis of HCC cells in vitro and in vivo (P<0.01). Transcriptome analysis indicated that Hippo signaling pathway was associated with anti-metastasis function of PZH. Mechanical study showed PZH significantly inhibited the expressions of platelet derived growth factor receptor beta (PDGFRB), YAP, connective tissue growth factor (CCN2), N-cadherin, vimentin and matrix metallopeptidase 2 (MMP2, P<0.01). Meanwhile, the phosphorylation of YAP was also enhanced by PZH treatment in vitro and in vivo. Furthermore, PZH played roles in inhibiting the transcriptional activity of YAP. CONCLUSION: PZH restrained migration, invasion and epithelial-mesenchymal transition of HCC cells through repressing PDGFRB/YAP/CCN2 axis.

Synthesis of Melatonin Derivatives and the Neuroprotective Effects on Parkinson's Disease Models of Caenorhabditis elegans.

Melatonin (MT) is a hormone with antioxidant activity secreted by the pineal gland in the human brain, which is highly efficient in scavenging free radicals and plays an important role in the neuro-immuno-endocrine system. Emerging evidence showed that MT supplementation was a potential therapeutic strategy for Parkinson's disease (PD), which inhibits pathways associated with oxidative stress in PD. In this study, we reported a C7-selective olefination of melatonin under rhodium catalysis with the aid of PIII-directing groups and synthesized 10 new melatonin-C7-cinnamic acid derivatives (6a-6j). The antioxidant potential of the compounds was evaluated both by ABTS and ORAC methods. Among these newly synthesized melatonin derivatives, 6a showed significantly higher activity than MT at 10-5 M. In the transgenic Caenorhabditis elegans model of PD, 6a significantly reduces alpha-synuclein aggregation and dopaminergic neuronal damage in nematodes while reducing intracellular ROS levels and recovers behavioral dysfunction induced by dopaminergic neurodegeneration. Further study of the mechanism of action of this compound can provide new therapeutic ideas and treatment strategies for PD.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of six compounds in Guizhi Fuling capsule and tablet samples.

A fast and simple ultra-high performance supercritical fluid chromatography method has been developed for the determination of six analytes, namely (paeonol, coumarin, cinnamic alcohol, cinnamic acid, paeoniflorin, and amygdalin) in Guizhi Fuling capsule and tablet samples. The influence of the key chromatographic parameters for the separation purposes was evaluated. The optimal column was Trefoil CEL1 column. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol at flow rate of 1.0 mL/min. The back pressure of the system was set to 1.38 × 107  Pa and the temperature to 45°C. The six compounds were separated within 11 min by the proposed ultra-high performance supercritical fluid chromatography method with satisfactory resolution. Method validation confirmed that the procedure is accurate with the recovery rates from 87.04 to 104.30%, intraday precision values less than 4.81% and interday precision less than 5.22%, and linear with R2 higher than 0.9967. Therefore, this work provides a simple and novel method for the simultaneous analysis of six compounds in Guizhi Fuling capsule and tablet samples.

Identification of bioactive compounds that contribute to the α-glucosidase inhibitory activity of rosemary.

To investigate the bioactive compounds that contribute to the α-glucosidase inhibitory activity of rosemary, phenolics and triterpene acids were characterized and quantified using quadrupole-Orbitrap mass spectrometry and enzyme assay. Two phenolic diterpenes (carnosol and hydroxy p-quinone carnosic acid) and two triterpene acids (betulinic acid and ursolic acid) were identified as potent α-glucosidase inhibitors. Carnosol, a major diterpene in rosemary, showed significant α-glucosidase inhibitory activity with IC50 value of 12 µg mL-1, and its inhibition mode was competitive. The inhibition mechanism of carnosol on α-glucosidase was further investigated by a combination of surface plasmon resonance (SPR) spectroscopy, fluorescence quenching studies and molecular-modeling techniques. The SPR assay suggested that carnosol had a high affinity to α-glucosidase with equilibrium dissociation constant (KD) value of 72.6 M. Fluorescence quenching studies indicated that the binding between carnosol and α-glucosidase was spontaneous and mainly driven by hydrophobic forces. Molecular docking studies revealed that carnosol bound to the active site of α-glucosidase. Furthermore, the oral administration of carnosol at 30 mg kg-1 significantly reduced the postprandial blood glucose levels of normal mice. This is the first report on the α-glucosidase inhibition and hypoglycemic activity of phenolic diterpenes, and these results could facilitate the utilization of rosemary as a dietary supplement for the treatment of diabetes.