[Determination of eight cannabinoids in foods with enhanced matrix removal-lipid adsorbent by ultra performance liquid chromatography-tandem mass spectrometry].

A novel method was developed for the simultaneous determination of eight cannabinoids in six types of food matrices, including chocolate, fondant, biscuit, beverage, cookie and baijiu, using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The sample extraction and cleanup steps were optimized, and various purification methods were investigated to remove the oil matrix and glue in chocolate and fudge, respectively. Enhanced matrix removal-lipid adsorbent (EMR-Lipid) provided efficient, selective cleanup of the evaluated matrices. The sample was extracted using acetonitrile, followed by EMR-Lipid cleanup, and then dried using anhydrous sodium sulfate. The acetonitrile layer was concentrated by nitrogen to near-dry after 100 µL 10% glycerol in methanol was added to improve the recovery by reducing loss during concentration under the stream of nitrogen gas. Eight cannabinoids were separated using a Waters ACQUITY UPLC BEH Shield RP18 column (100 mm×3.0 mm, 1.7 µm). The responses of the cannabinoids in the positive and negative ionization modes were investigated and optimized, and the responses were superior in the negative ion mode compared to those in the positive ion mode. MS detection was performed in the multi-reaction monitoring (MRM) mode using an electrospray source in the negative ion mode. The cannabinoids were quantified using an external standard with matrix calibration curves to reduce the influences of the matrix effects on the quantitative results. The developed method was verified, and the conditions of sample pretreatment were also optimized. The calibration curves of tetrahydrocannabinol, cannabidivarin, tetrahydrocannabivarin, and cannabigerol and those of cannabidiol, cannabinol, cannabidiolic acid, and tetrahydrocannabinolic acid exhibited good linearities, with r>0.995, in the ranges of 2-200 and 0.4-40 ng/mL, respectively. The respective limits of detection (LODs, S/N=3) and quantification (LOQs, S/N=10) of tetrahydrocannabinol, cannabidivarin, tetrahydrocannabivarin, and cannabigerol were 4 and 10 µg/kg, and those of cannabidiol, cannabinol, cannabidiolic acid, and tetrahydrocannabinolic acid were 0.8 and 2 µg/kg. The average recoveries of the cannabinoids were 82.0%-114.9% under three spiked levels with corresponding relative standard deviations (RSDs) of <15% (n=6). EMR-Lipid provided efficient, selective cleanups of food matrices with good accuracy. The method is sensitive, rapid, accurate, simple to execute, and it is suitable for the determination of cannabinol compounds in typical food matrices.

Feasibility study on grouting material prepared from red mud and metallurgical wastewater based on synergistic theory.

A new type of red mud/slag/wastewater-based geopolymeric grouts (RSW) was prepared to solve the problem of wastewater and red mud to environment and promoting the safe construction of geotechnical engineering. The applicability of RSW was investigated using different red mud, alkali activator and wastewater dosage. Fourier transform infrared spectrum (FTIR), X-ray diffraction (XRD), semi-calorimetry, Scanning electron microscope-energy dispersive spectrometer (SEM-EDS), and 29Si Magic Angle Spinning (MAS) Nuclear Magnetic Resonance (NMR) were conducted to study the effect of wastewater on RSW. The results showed that wastewater has an accelerating effect on the geopolymerization process of RSW, the mechanical strength increased first and then decreased with the increment of wastewater dosage, the 28 d compressive strength of RSW was 30.2 MPa, which is higher than the cement-based grouts. The leaching of heavy metals were lower than 0.4 mg/L, which demonstrates that the RSW has a good immobilization effect on the heavy metals. The FTIR and SEM-EDS analysis results showed that the ions in wastewater could participated in the geopolymerization process and the hydrated products has a immobilization effect on the heavy metals. Overall, this contribution explores utilizing red mud and wastewater, and preparing high performance grouts for underground engineering.

Author Correction: Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ginsenoside Rg3 regulates CaM gene expression through PI3K/AKT signal system to promote apoptosis of gastric cancer BGC-823 cell / 中国肿瘤生物治疗杂志

@#Objective: To investigate Ginsenoside Rg3 interfering the expression of CaM through PI3K/AKT signaling pathway to affect the biological activity of gastric cancer BGC-823 cells. Methods: After the culture and passage of gastric cancer BGC-823 cells, Western blotting was used to detect the expression of p-AKT and CaM protein in gastric cancer BGC-823 cells treated with IGF-1 and/ or Rg3; The effect of IGF-1 and/or Rg3 on the proliferation of BGC-823 cells was detected by MTT assay; The effect of IGF-1 and/or Rg3 on the invasion of BGC-823 cells was detected by Transwell assay; Effect of IGF-1 and/or Rg3 on apoptosis of BGC-823 cells was detected by Flow Cytometry. Results: Western blotting results showed that the expression of p-AKT and CaM protein increased in BGC-823 cells with the prolongation of IGF-1 treatment (all P<0.05); Compared with the blank control group, Rg3 significantly inhibited the proliferation of BGC-823 cells, while IGF-1 and IGF-1+Rg3 significantly promoted the cell proliferation (all P<0.05); Compared with the blank control group, Rg3 significantly reduced the invasion of BGC-823 cells, while IGF-1 and IGF-1+Rg3 significantly promoted the invasion of BGC-823 cells (all P<0.05);Flow cytometry showed that compared with the blank control group, Rg3 significantly promoted the apoptosis of BGC-823 cells, while IGF-1 and IGF-1+Rg3 significantly inhibited the apoptosis of BGC-823 cells (all P< 0.05). Conclusion: Ginsenoside Rg3 inhibits the expression of CaM by blocking PI3K/AKT signaling pathway, thereby promoting the apoptosis of gastric cancer BGC-823 cells.

Experimental Study on Conductivity Anisotropy of Limestone Considering the Bedding Directional Effect in the Whole Process of Uniaxial Compression.

Experimental studies were conducted on the changes of the potential differences in different directions during the uniaxial compression on limestone samples parallel and normal to the bedding plane. In the test, electric current was supplied at both ends of the samples, and concurrent measurement was conducted in four measuring lines at a 45-degree angle to each other. First, the change laws of the potential differences in different directions and the similarities and differences of rock samples were summarized. In regards to the uniaxial compression properties and crack growth, the above-mentioned similarities and differences were further analyzed. Then, the anisotropy factor was introduced to further explore the response characteristics. It was found that the anisotropic changes of rock samples went through three stages during the uniaxial compression process, providing a reference for describing the properties in different failure stages of rock samples and obtaining precursory information about the fracture. Besides, the relationship between the peak stress and initial potential difference in a direction normal to the current direction was obtained by means of data fitting, providing a new method of predicting the uniaxial compressive strength of rock samples. According to the preceding analysis, this paper studied rock anisotropy by considering the bedding directional effect in terms of conductivity and provided a reference for subsequent study on rock materials' properties and engineering practices.

A servo controlled gradient loading triaxial model test system for deep-buried cavern.

A servo controlled gradient loading model test system is developed to simulate the gradient geostress in deep-buried cavern. This system consists of the gradient loading apparatus, the digital servo control device, and the measurement system. Among them, the gradient loading apparatus is the main component which is used for exerting load onto the model. This loading apparatus is placed inside the counterforce wall/beam and is divided to several different loading zones, with each loading zone independently controlled. This design enables the gradient loading. Hence, the "real" geostress field surrounding the deep-buried cavern can be simulated. The loading or unloading process can be controlled by the human-computer interaction machines, i.e., the digital servo control system. It realizes the automation and visualization of model loading/unloading. In addition, this digital servo could control and regulate hydraulic loading instantaneously, which stabilizes the geostress onto the model over a long term. During the loading procedure, the collision between two adjacent loading platens is also eliminated by developing a guide frame. This collision phenomenon is induced by the volume shrinkage of the model when compressed in true 3D state. In addition, several accurate measurements, including the optical and grating-based method, are adopted to monitor the small deformation of the model. Hence, the distortion of the model could be accurately measured. In order to validate the performance of this innovative model test system, a 3D geomechanical test was conducted on a simulated deep-buried underground reservoir. The result shows that the radial convergence increases rapidly with the release of the stress in the reservoir. Moreover, the deformation increases with the increase of the gas production rate. This observation is consistence with field observation in petroleum engineering. The system is therefore capable of testing deep-buried engineering structures.

Structural Stability Monitoring of a Physical Model Test on an Underground Cavern Group during Deep Excavations Using FBG Sensors.

Fiber Bragg Grating (FBG) sensors are comprehensively recognized as a structural stability monitoring device for all kinds of geo-materials by either embedding into or bonding onto the structural entities. The physical model in geotechnical engineering, which could accurately simulate the construction processes and the effects on the stability of underground caverns on the basis of satisfying the similarity principles, is an actual physical entity. Using a physical model test of underground caverns in Shuangjiangkou Hydropower Station, FBG sensors were used to determine how to model the small displacements of some key monitoring points in the large-scale physical model during excavation. In the process of building the test specimen, it is most successful to embed FBG sensors in the physical model through making an opening and adding some quick-set silicon. The experimental results show that the FBG sensor has higher measuring accuracy than other conventional sensors like electrical resistance strain gages and extensometers. The experimental results are also in good agreement with the numerical simulation results. In conclusion, FBG sensors could effectively measure small displacements of monitoring points in the whole process of the physical model test. The experimental results reveal the deformation and failure characteristics of the surrounding rock mass and make some guidance for the in situ engineering construction.

Anti-psoriatic effects of Honokiol through the inhibition of NF-κB and VEGFR-2 in animal model of K14-VEGF transgenic mouse.

Honokiol (HK), a biphenolic neolignan isolated from Magnolia officinalis, has been reported to possess anti-inflammatory and anti-angiogenic activaties. In this study, our aim was to investigate anti-psoriatic activities of HK and the involved mechanisms. In vitro, the effects of HK on the regulation of Th1/Th2 and TNF-α-induced NF-κB (p65) activation were analyzed by respective FCS and immunofluorescence. Additionally, the K14-VEGF transgenic model was used for the in vivo study. ELISA and Q-PCR were performed to evaluate serum levels of Th1/Th2 cytokines and their corresponding mRNA expressions. Effects on VEGFR-2 and p65 activation, as well as other angiogenic and inflammatory parameters were studied by immunostainings. Importantly, we found that HK significantly decreased the ratio of Th1/Th2-expression CD4(+) T cells and inhibited TNF-α-induced activation of NF-κB. The morphology and histological features of psoriasis were effectively improved by HK treatment. The expression of TNF-α and IFN-γ, and their corresponding mRNA levels were down-regulated and the expression of nuclear p65, VEGFR-2, as well as related phosphorylated proteins (p-VEGFR-2, p-ERK1/2, p-AKT and p-p38) were also suppressed. Overall, these results in our study suggested that HK exhibits anti-psoriatic effects through the inhibition of NF-κB and VEGFR-2.

Isogambogenic acid induces apoptosis-independent autophagic cell death in human non-small-cell lung carcinoma cells.

To overcome drug resistance caused by apoptosis deficiency in patients with non-small cell lung carcinoma (NSCLC), there is a need to identify other means of triggering apoptosis-independent cancer cell death. We are the first to report that isogambogenic acid (iso-GNA) can induce apoptosis-independent autophagic cell death in human NSCLC cells. Several features of the iso-GNA-treated NSCLC cells indicated that iso-GNA induced autophagic cell death. First, there was no evidence of apoptosis or cleaved caspase 3 accumulation and activation. Second, iso-GNA treatment induced the formation of autophagic vacuoles, increased LC3 conversion, caused the appearance of autophagosomes and increased the expression of autophagy-related proteins. These findings provide evidence that iso-GNA induces autophagy in NSCLC cells. Third, iso-GNA-induced cell death was inhibited by autophagic inhibitors or by selective ablation of Atg7 and Beclin 1 genes. Furthermore, the mTOR inhibitor rapamycin increased iso-GNA-induced cell death by enhancing autophagy. Finally, a xenograft model provided additional evidence that iso-GNA exhibited anticancer effect through inducing autophagy-dependent cell death in NSCLC cells. Taken together, our results demonstrated that iso-GNA exhibited an anticancer effect by inducing autophagy-dependent cell death in NSCLC cells, which may be an effective chemotherapeutic agent that can be used against NSCLC in a clinical setting.

Millettia pachycarpa exhibits anti-inflammatory activity through the suppression of LPS-induced NO/iNOS expression.

The present study was designed to investigate the in vitro and in vivo anti-inflammatory activity of flavonoids isolated from Millettia pachycarpa Benth. The seeds of M. pachycarpa Benth were extracted with ethanol and subjected to chromatographic separation for the isolation of bioactive compounds. Their structures were elucidated by spectroscopic methods. The anti-inflammatory activity of the compounds was investigated by evaluating the inhibition ability of NO production, iNOS activity and iNOS protein expression induced by LPS-stimulated RAW264.7 macrophages in vitro and the carrageenan-induced hind paw edema model in vivo. Molecular docking simulation was also employed to obtain the binding parameters in the binding pocket of iNOS. Thirteen compounds (1-13) were isolated from Chinese herbal medicine M. pachycarpa Benth. Among them, 4-hydroxylonchocarpin (6) and deguelin (7) exhibited remarkable inhibitory rates of 66.5% and 57.7%, respectively, compared with that of 52.5% of indomethacin in LPS-induced macrophages cells. 4-hydroxylonchocarpin (6) with low toxicity (IC50 > 100 µm) exhibited better inhibitory effects to positive control of 1400W on iNOS activity at the concentration of 10 µm. Western blot assay revealed that 4-hydroxylonchocarpin (6) inhibited iNOS protein expression in RAW264.7 cells and molecular docking simulation showed that 4-hydroxylonchocarpin (6) fit well into the binding pocket of iNOS. In the carrageenan-induced paw edema model, our data revealed that the anti-inflammatory potential of 4-hydroxylonchocarpin (6) at 10 mg/kg showed comparable inhibitory ability to indomethacin at 5 h while a higher concentration of 4-hydroxylonchocarpin (6) at 50 mg/kg showed higher inhibitory activity than indomethacin, which was further confirmed by plasma levels of nitrite. The overall results suggest that 4-hydroxylonchocarpin (6) might be used as a potential therapeutic agent for inflammation-associated disorders.

Comparison of counter-current chromatography and preparative high performance liquid chromatography applied to separating minor impurities in drug preparations.

Drug impurity profiling and identification are carried out along with the drug discovery process. Due to its inherent low concentration in drug products, the isolation and purification of impurities present a challenge to drug development processes. In our development of honokiol and quercetin as anticancer drug candidates, counter-current chromatography (CCC) and preparative HPLC were used for the impurity profiling and identification of honokiol and quercetin. Several performance parameters such as separation column volume, maximum sample loading, separation time, solvent consumption and sample throughput were investigated in order to compare the separation efficiency. We found that the sample loading capacity and therefore the throughput of preparative HPLC were not satisfactory, while CCC provided larger sample loading (especially for a sample with poor solubility), consumed less solvent and produced higher throughput than preparative HPLC. Six impurities of honokiol including one new compound were isolated in the present work.

Gambogic acid exhibits anti-psoriatic efficacy through inhibition of angiogenesis and inflammation.

BACKGROUND: Psoriasis is a chronic T cell-mediated inflammatory skin disease. Studies have shown that angiogenesis plays an important role in the pathogenesis of psoriasis. Studies have also indicated that Gambogic acid (GA) inhibits angiogenesis and may be a viable drug candidate in anti-angiogenesis therapies. OBJECTIVE: The aim of this study was to investigate the anti-psoriatic effect of GA and the possible mechanisms. METHODS: MTT test on HaCaT cells and immunofluorescence on HUVEC cells were processed. An O/W cream of GA was prepared and topically applied to the ears of K14-VEGF transgenic mice and psoriasis-like guinea-pigs, and the tail skin of Balb/C mice independently. Furthermore, hematoxylin-eosin staining of tissues from three models and immunohistochemistry staining of ear samples from K14-VEGF mice were performed. RESULTS: In vitro, GA inhibited proliferation of HaCaTs and TNF-α-induced activation of NF-κB in HUVECs. In vivo, animals treated with GA showed significant morphological and histological improvements. Immunohistochemical analysis of K14-VEGF transgenic mice revealed that hyperplastic and inflamed vessels were suppressed with GA treatment. The expression of adhesion molecules such as ICAM-1 and E-selectin was significantly decreased. GA inhibited angiogenesis and the expression of VEGFR2 and p-VEGFR2. T lymphocyte infiltration and the expression of IL-17 and IL-22 were also reduced by GA treatment. CONCLUSION: Our results suggest that GA has anti-psoriatic efficacy through inhibition of angiogenesis and inflammation. Therefore, GA is attractive and offers future potential for application in patients with psoriasis.

Bioactivity-guided isolation of anti-inflammation flavonoids from the stems of Millettia dielsiana Harms.

Bioactivity-guided isolation of the EtOAc extract of the stems of Millettia dielsiana Harms yielded two new isoflavones together with nine known ones. Their structures were elucidated by analysis of the spectroscopic data including 2D NMR. All of the isolates were evaluated for their potential to inhibit the LPS-induced production of nitric oxide and TNF-α in murine macrophage RAW 264.7 cells. Among the tested compounds, Millesianin C (1) had the most potent anti-inflammatory effect decreasing NO production similar to that of dexamethasone and decreasing TNF-α secretion better than that of dexamethasone. Their structure-activity relationship was also analyzed.

The onset of double diffusive convection in a viscoelastic fluid-saturated porous layer with non-equilibrium model.

The onset of double diffusive convection in a viscoelastic fluid-saturated porous layer is studied when the fluid and solid phase are not in local thermal equilibrium. The modified Darcy model is used for the momentum equation and a two-field model is used for energy equation each representing the fluid and solid phases separately. The effect of thermal non-equilibrium on the onset of double diffusive convection is discussed. The critical Rayleigh number and the corresponding wave number for the exchange of stability and over-stability are obtained, and the onset criterion for stationary and oscillatory convection is derived analytically and discussed numerically.

In vitro and in vivo antiangiogenic activity of caged polyprenylated xanthones isolated from Garcinia hanburyi Hook. f.

Eleven known caged polyprenylated xanthones 1-11 were isolated from the resin of Garcinia hanburyi Hook. f., and their structures were identified by their MS, NMR and UV spectra. These xanthones showed significant cytotoxicities against four human cancer cell lines (HeLa, A549, HCT-116, and HepG-2) and strong inhibition against the proliferation of the HUVEC cell line in vitro by the MTT method. Furthermore, in an in vivo zebrafish model, xanthones 3 (morellic acid), 7 (gambogenin) and 9 (isogambogenic acid) showed comparable antiangiogenic activities with less toxicities than xanthone 1 (gambogic acid), as evaluated by death and heart rates of treated zebrafish. Xanthone 7 exhibited antiangiogenic activity with no toxicity at concentrations ranging from 8 µM to 16 µM. Meanwhile, xanthones 1, 3, 7 and 9 strongly inhibited the migration of HUVEC at a low concentration of 0.5 µM in HUVEC cell migration assay in vitro. Taken together, these findings strongly suggest that xanthone 7 might be a novel angiogenesis inhibitor.

Identification of honokiol metabolites in rats by the method of stable isotope cluster technique and ultra-high performance liquid chromatography/quadrupole-time-of-flight mass spectrometry.

Honokiol, a natural molecule isolated from Magnolia officinalis Rehd. et Wils., is widely known as an antitumor agent. In present work, an analysis of in vivo biotransformation and metabolites of honokiol has been performed by a combined method based on stable isotope cluster technique with honokiol-[(13)C6]-labeled and ultra-high performance liquid chromatography/quadrupole-time-of-flight-mass spectrometry (UHPLC/Q-TOF-MS). The metabolites could be easily identified by the determination of a chromatographically co-eluted pair of isotopomers (MS doublet peaks) with similar peak intensities and mass difference corresponding to that between isotope-labeled and non-isotope-labeled honokiol. A total of eighteen metabolites were detected and tentatively identified, fourteen of which were reported for the first time. The results indicated that the main metabolic pathways of honokiol in rats were hydroxylation, methylation, sulfation and glucuronidation. This study provided the first essential information on biotransformation and metabolites of honokiol in rats, which was very useful for further pharmacological and clinical studies of honokiol as a potent drug candidate.

Anti-inflammatory activity of ethyl acetate fraction of the seeds of Brucea Javanica.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Brucea javanica (L.) Merr. (Yadanzi in Chinese) have been used for the treatment of inflammation, dysentery, malaria, and cancer in Chinese traditional medicine. However, the anti-inflammatory mechanism of Brucea javanica has not been fully elucidated. This study examined the anti-inflammatory activity of ethyl acetate fraction of the seeds of Brucea javanica (EA-BJ) in vitro and in vivo. MATERIALS AND METHODS: The anti-inflammatory activity of EA-BJ and its ability to modulate the production of NO, PGE2, TNF-α, IL-1ß, IL-6 and IL-10 inflammatory mediators in lipopolysaccharide-activated RAW 264.7 macrophage were evaluated. Moreover, the anti-inflammatory activity of EA-BJ was also in vivo assayed by carrageenan induced paw edema in mice. RESULTS: In vitro assays showed remarkable anti-inflammatory activity of EA-BJ, through the inhibition of production of NO, PGE2, TNF-α, IL-1ß and IL-6 inflammatory mediators and induction of production of IL-10 anti-inflammatory cytokine. In vivo assays showed anti-inflammatory activity for decrement of the paw edema in carrageenan induced paw edema test. CONCLUSION: The results obtained in vitro and in vivo showed that possible anti-inflammatory effects of EA-BJ may be attributed to inhibition pro-inflammatory mediators production, NO, PGE2, TNF-α, IL-1ß and IL-6 and to increase production of IL-10 anti-inflammatory cytokine. The seeds of Brucea javanica may thus prove beneficial in the treatment of inflammatory diseases.

Cytotoxic and apoptotic effects of constituents from Millettia pachycarpa Benth.

The aim of this study is to investigate the cytotoxic and apoptotic effects of constituents from the seeds of Millettia pachycarpa Benth. Fourteen compounds (1-14) including one novel chalcone (10) were isolated as active principles from Chinese herbal medicine M. pachycarpa Benth. Their structures were identified by using spectroscopic methods. All isolates were then evaluated for their cytotoxic effects against several cancer cell lines (HepG2, C26, LL2 and B16) with cisplatin as a positive control. And their apoptosis-inducing effects were tested against HeLa-C3 cells with taxol as a positive control. Both studies showed that compounds 1, 2, 7 and 10 demonstrated significant cytotoxic and apoptotic effects against cancer cells. Moreover, in the apoptosis assay the novel chalcone (10) showed strong apoptosis inducing effects at a concentration of 2µM within 36h. It was found to be the most potent apoptotic inducer of the compounds isolated from M. pachycarpa Benth.