Modulating effect of Eunkyo-san on expression of inflammatory cytokines and angiotensin-converting enzyme 2 in human mast cells.

Eunkyo-san is widely used in the treatment of severe respiratory infections. Mast cells not only serve as host cells for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but also they also exacerbate Coronavirus disease in 2019 (COVID-19) by causing a cytokine storm. Here we investigated whether Eunkyo-san and its active compound naringenin regulate the expression of inflammatory cytokines and factors connected to viral infection in activated human mast cell line, HMC-1 cells. Eunkyo-san and naringenin significantly reduced levels of inflammatory cytokines including interleukin (IL)-1ß, IL-6, IL-8, thymic stromal lymphopoietin, and tumor necrosis factor-α without impacting cytotoxicity. Eunkyo-san and naringenin reduced levels of factors connected to SARS-CoV-2 infection such as angiotensin-converting enzyme 2 (ACE2, SARS-CoV-2 receptor), transmembrane protease/serine subfamily member 2, and tryptase in activated HMC-1 cells. Treatment with Eunkyo-san and naringenin considerably reduced expression levels of ACE2 transcription factor, AP-1 (C-JUN and C-FOS) by blocking phosphatidylinositide-3-kinase and c-Jun NH2-terminal kinases signaling pathways. In addition, Eunkyo-san and naringenin effectively suppressed activation of signal transducer and activator of transcription 3, nuclear translocation of nuclear factor-κB, and activation of caspase-1 in activated HMC-1 cells. Furthermore, Eunkyo-san and naringenin reduced expression of ACE2 mRNA in two activated mast cell lines, RBL-2H3 and IC-2 cells. The overall study findings showed that Eunkyo-san diminished the expression levels of inflammatory cytokines and ACE2, and these findings imply that Eunkyo-san is able to effectively mitigating the cytokine storm brought on by SARS-CoV-2 infection.

Insights into the Vastly Different Effects of Eutectic Solvents on the Stability of Phenolic Compounds.

Eutectic solvents (ESs) have shown stabilizing effects on several molecules. Due to the potential applicability of bioactive compounds, understanding how ESs stabilize them is of great interest in pharmaceutical and related fields. Here, among various ESs, CTU, which comprise thiourea and choline chloride (ChCl), exerted remarkably high stabilizing effects on various phenolic compounds, whereas CU consisting of urea and ChCl exhibited the opposite effects. Using a potent polyphenol, (-)-epigallocatechin gallate (EGCG), as a model compound, we conducted experimental and in silico studies to unravel the underlying mechanisms of the two very similar ESs for the contrasting effects. The results suggest that ESs can affect with great diversity the stability of EGCG by complicated interactions arising from the unique properties of both ESs and their components.

Assembly of a GPCR-G Protein Complex.

The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters. Recent structures of GPCR-G protein complexes obtained by crystallography and cryoelectron microscopy (cryo-EM) reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms. While some G protein subtype-specific differences are observed, there is no clear structural explanation for G protein subtype-selectivity. All of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. In an effort to better understand the structural basis of coupling specificity, we used time-resolved structural mass spectrometry techniques to investigate GPCR-G protein complex formation and G-protein activation. Our results suggest that coupling specificity is determined by one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures.

Deep eutectic solvent-based valorization of spent coffee grounds.

Spent coffee grounds (SCGs) are viewed as a valuable resource for useful bioactive compounds, such as chlorogenic acids and flavonoids, and we suggest an eco-friendly and efficient valorization method. A series of choline chloride-based deep eutectic solvents (DESs) were tested as green extraction solvents for use with ultrasound-assisted extraction. Extraction efficiency was evaluated based on total phenolic content (TPC), total flavonoid content, total chlorogenic acids, and/or anti-oxidant activity. A binary DES named HC-6, which was composed of 1,6-hexanediol:choline chloride (molar ratio 7:1) was designed to produce the highest efficiency. Experimental conditions were screened and optimized for maximized efficiency using a two-level fractional factorial design and a central composite design, respectively. As a result, the proposed method presented significantly enhanced TPC and anti-oxidant activity. In addition, phenolic compounds could be easily recovered from extracts at high recovery yields (>90%) by adsorption chromatography.

One-step sample preparation for convenient examination of volatile monoterpenes and phenolic compounds in peppermint leaves using deep eutectic solvents.

Deep eutectic solvents (DESs) were investigated as an extraction medium for one-step sample preparation for chemical characterization of peppermint (Mentha piperita L.). Rather than applying discontinuous, time-consuming extraction methods to prepare two types of extracts, peppermint leaves were extracted efficiently into a DES, which was composed of choline chloride and d-(+)-glucose at a 5:2 molar ratio. The produced peppermint extracts contained volatile monoterpenes and phenolics at levels sufficient for direct chemical examination of peppermint leaves. The extracted monoterpenes in DES could be quantified via a newly developed method based on headspace solid-phase microextraction coupled to gas chromatography. The same extracts were also directly used to assess phenolics in terms of total phenolic content, total flavonoid content, and antioxidant activity. The proposed method allowed one-step sample preparation for extraction of volatile monoterpenes and phenolics of peppermint leaves and could be applied to various peppermint samples obtained from different origins.

Development and Validation of an Analytical Method Readily Applicable for Quality Control of Tabebuia impetiginosa (Taheebo) Ethanolic Extract.

The dried inner bark of Tabebuia impetiginosa, known as taheebo or red lapacho, has numerous beneficial effects on human health. This study presents the first simple and reliable quantitative method that could serve for the QC of taheebo. The method uses LC-UV spectroscopy to determine the veratric acid (VA; 3,4-dimethoxybenzoic acid) content of taheebo extracts (TEs). Sample preparation entailed the dissolution of TE in methanol (MeOH), facilitated by ultrasonic radiation for 10 min. The optimized conditions included chromatographic separation on an Agilent Eclipse Plus C18 column (4.6 × 150 mm, 5 µm) at 30°C. The mobile phase consisted of 1% acetic acid in water and MeOH, which was eluted under gradient mode at a flow rate of 1.0 mL/min. The detection wavelength was 254 nm. Using these conditions, VA was selectively resolved, and the entire chromatographic analysis time was 27 min. The method was linear in the range of 50-500 µg/mL (r2 = 0.9995), precise (≤3.97% RSD), and accurate (97.10-102.72%). The validated method was applied to three batches of TE samples, yielding an estimated VA content range of 14.92-15.58 mg/g. Thus, the proposed method could serve as an easy and practical method for the QC of TEs or related products containing TEs.

Identification of Major Flavone C-Glycosides and Their Optimized Extraction from Cymbidium kanran Using Deep Eutectic Solvents.

Cymbidium kanran, an orchid exclusively distributed in Northeast Asia, has been highly valued as a decorative plant and traditional herbal medicine. Here, C. kanran extracts were prepared in 70% aqueous methanol using ultrasound-assisted extraction (UAE) and subjected to liquid chromatography-photodiode array detection and ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry analysis, which were used for quantitative and qualitative analysis, respectively. It was found that the extracts were rich in flavone C-glycosides including vicenin-2, vicenin-3, schaftoside, vitexin, and isovitexin. Ten deep eutectic solvents (DESs) were synthesized by combining choline chloride (hydrogen bond acceptor) with various polyols and diols (hydrogen bond donors) and were tested as a medium for the efficient production of extracts enriched with potentially bioactive flavone C-glycosides from C. kanran. A DES named ChCl:DPG, composed of choline chloride and dipropylene glycol at a 1:4 molar ratio, exhibited the best extraction yields. Then, the effects of extraction conditions on the extraction efficiency were investigated by response surface methodology. Lower water content in the extraction solvent and longer extraction time during UAE were desirable for higher extraction yields. Under the statistically optimized conditions, in which 100 mg of C. kanran powder were extracted in 0.53 mL of a mixture of ChCl:DPG and water (74:26, w/w) for 86 min, a total of 3.441 mg g-1 flavone C-glycosides including 1.933 mg g-1 vicenin-2 was obtained. This total yield was 196%, 131%, and 71% more than those obtained using 100% methanol, water, and 70% methanol, respectively.

Simple and rapid determination of zaltoprofen in human plasma by manual-shaking-assisted dispersive liquid-liquid microextraction followed by liquid chromatography with ultraviolet detection.

A readily applicable method was developed to determine the concentration level of zaltoprofen, a non-steroidal antiinflammatory drug from the propionic acid family, in human plasma. This method is based on manual-shaking-assisted dispersive liquid-liquid microextraction coupled with liquid chromatography with ultraviolet detection. Factors affecting the extraction efficiency were screened and optimized by experimental design using fractional factorial and central composite designs, respectively. Optimal conditions were: 220 µL of C2 H4 Cl2 (extraction solvent), 5 mL of 3.75% w/v NaCl aqueous solution at pH 2.0, and manual shaking for 13 s (65 times). The resulting extraction method yielded a reasonable enrichment factor of 18.0 (±0.6, n = 3) and extraction recovery of 86.0% (±3.3%, n = 3). The established method was validated for selectivity, linearity, precision, accuracy, matrix effect, recovery, dilution integrity, and stability, and it met the acceptable criteria for all of the tested parameters. Specifically, the method was linear in the range of 0.16-50.0 mg/L, precise (< 8.8% RSD), accurate (-7.5-5.6% deviation), and showed negligible matrix effects (96.1-106.4%) with high absolute recovery (94.5-97.7%). Compared with previous methods involving labor-intensive liquid-liquid extraction or non-specific protein precipitation, our method allows the simple, rapid, and efficient determination of zaltoprofen using the most affordable analytical instrument, liquid chromatography with ultraviolet detection.

Determination of enantiomeric vigabatrin by derivatization with diacetyl-l-tartaric anhydride followed by ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry.

Vigabatrin, one of the most widely used antiepileptic drugs, is marketed and administered as a racemic mixture, while only S-enantiomer is therapeutically effective. In the present study, diacetyl-l-tartaric acid anhydride was used as an inexpensive and effective chiral derivatization reagent to produce tartaric acid monoester derivatives of vigabatrin enantiomers that could be readily resolved by reversed phase chromatography. Derivatization conditions were statistically optimized by response surface methodology, resulting in an optimal reaction temperature of 44°C and an optimal reaction time of 30min. The derivatized diastereomers of vigabatrin and internal standard (gabapentin) were analyzed using ultra-high performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry. For this analysis, an Agilent ZORBAX Rapid Resolution High Definition Eclipse Plus C18 column (100mm×2.1mm, 1.8µm) was employed for chromatographic separation using 10mM ammonium formate (pH 3.0) and methanol as mobile phase at a flow rate of 0.2mLmin-1. The established method was validated in terms of specificity, linearity, precision, accuracy, dilution integrity, recovery, matrix effect, stability, and incurred sample reanalysis. It was linear over a range of 0.25-100.0mgL-1 for both S- and R-enantiomers (R2≥0.9987 for both). Intra- and inter-day precisions and accuracies were within acceptable ranges. The method was successfully applied to determine the levels of vigabatrin enantiomers in mouse serum after administration of vigabatrin racemate.

A simple and reliable analytical method based on HPLC-UV to determine oleanonic acid content in Chios gum mastic for quality control.

A reliable analytical method based on high-performance liquid chromatography-ultraviolet detection was established for the determination of oleanonic acid (OA) content in Chios gum mastic (CGM). A simple method involving methanol extraction of CGM powder followed by basification and ether extraction was developed to isolate the triterpenic fraction including OA. The triterpenic fraction was chromatographed on a Phenomenex Gemini C18 column (150 × 4.6 mm, 5 µm) under a simple gradient elution of a mobile phase containing acetonitrile and water at a flow rate of 1.0 mL min-1. The detection wavelength was set at 210 nm. Good linearity was achieved in the range of 100.0-1000.0 µg mL-1 with r2 > 0.9993, and the limit of quantification was 32.22 µg mL-1. Accuracy measured at three concentration levels was in the range of 93.72-99.56%, while intra-day and inter-day precisions estimated using both OA standard and CGM samples were no more than 2.83 and 4.57% RSD, respectively. Finally, this method was applied to real CGM samples from various batches, revealing that the OA contents were between 88.13 and 100.83 µg mg-1. These results suggest that the current method can be applied as an efficient analytical method for quality control of CGM.

Indirect enantioseparation of fluoxetine in mouse serum by derivatization with 1R-(-)-menthyl chloroformate followed by ultra high performance liquid chromatography and quadrupole time-of-flight mass spectrometry.

Here we describe a simple and sensitive analytical method for the enantioselective quantification of fluoxetine in mouse serum using ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry. The sample preparation method included a simple deproteinization with acetonitrile in 50 µL of serum, followed by derivatization of the extracts in 50 µL of 2 mM 1R-(-)-menthyl chloroformate at 45ºC for 55 min. These conditions were statistically optimized through response surface methodology using a central composite design. Under the optimized conditions, neither racemization nor kinetic resolution occurred. The derivatized diastereomers were readily resolved on a conventional sub-2 µm C18 column under a simple gradient elution of aqueous methanol containing 0.1% formic acid. The established method was validated and found to be linear, precise, and accurate over the concentration range of 5.0-1000.0 ng/mL for both R and S enantiomers (r(2) > 0.993). Stability tests of the prepared samples at three different concentration levels showed that the R- and S-fluoxetine derivatives were relatively stable for 48 h. No significant matrix effects were observed. Last, the developed method was successfully used for enantiomeric analysis of real serum samples collected at a number of time points from mice administered with racemic fluoxetine.

Tailoring and recycling of deep eutectic solvents as sustainable and efficient extraction media.

The present study demonstrates that deep eutectic solvents (DESs) with the highest extractability can be designed by combining effective DES components from screening diverse DESs. The extraction of polar ginseng saponins from white ginseng was used as a way to demonstrate the tuneability as well as recyclability of DESs. A newly designed ternary DES (GPS-5) composed of glycerol, l-proline, and sucrose at 9:4:1 was used as a sustainable and efficient extraction medium. Based on the anti-tumor activity on HCT-116 cancer cells, it was confirmed that GPS-5 was merely an extraction solvent with no influence of the bioactivity of the ginsenosides extracted. Excellent recovery of the extracted saponins was easily achieved through solid-phase extraction (SPE). Recycling of the DES was accomplished by simple freeze-drying of the washed solutions from the SPE. The extraction efficiencies of the DESs recycled once, twice, and thrice were 92%, 85%, and 83% of that of the freshly synthesized solvent.

Highly efficient extraction of anthocyanins from grape skin using deep eutectic solvents as green and tunable media.

Deep eutectic solvents (DESs) were investigated as tunable, environmentally benign, yet superior extraction media to enhance the extraction of anthocyanins from grape skin, which is usually discarded as waste. Ten DESs containing choline chloride as hydrogen bond acceptor combined with different hydrogen bond donors were screened for high extraction efficiencies based on the anthocyanin extraction yields. As a result, citric acid, D-(+)-maltose, and fructose were selected as the effective DES components, and the newly designed DES, CM-6 that is composed of citric acid and D-(+)-maltose at 4:1 molar ratio, exhibited significantly higher levels of anthocyanin extraction yields than conventional extraction solvents such as 80% aqueous methanol. The final extraction method was established based on the ultrasound-assisted extraction under conditions optimized using response surface methodology. Its extraction yields were double or even higher than those of conventional methods that are time-consuming and use volatile organic solvents. Our method is truly a green method for anthocyanin extraction with great extraction efficiency using a minimal amount of time and solvent. Moreover, this study suggested that grape skin, the by-products of grape juice processing, could serve as a valuable source for safe, natural colorants or antioxidants by use of the eco-friendly extraction solvent, CM-6.

Combination of a sub-3 µm superficially porous particle packed column with charged aerosol detection for the simple and sensitive measurement of nine macrolides in human urine.

Due to the lack of chromophores in many macrolides, analytical methods based on mass spectrometry and electrochemical detection coupled to liquid chromatography have been suggested to be suitable for the quantification of macrolides in complex matrices. In this study, a simple and sensitive analytical method was established for the simultaneous measurement of nine macrolides in human urine by combining a sub-3 µm superficially porous particle packed column with charged aerosol detection. After thorough investigation of various sample preparation methods, including two liquid-liquid extraction methods and four solid-phase extraction methods, HLB solid-phase extraction was selected and further optimized. Absolute recovery of the optimized sample preparation method ranged from 99.5-110.2%, indicating its very high extraction/clean-up efficiency. For chromatography, parameters influencing macrolide separation were systematically optimized, and the resulting conditions allowed baseline separation of nine macrolides within 24 min using a very simple mobile phase. The established method was validated for linearity, limit of detection, limit of quantification, absolute recovery, and precision. Based on its limit of detection (0.025-0.100 µg/mL), the method had similar or greater sensitivity than most methods based on electrochemical detection. It was found that the current method was appropriate for application to real human urine samples after drug administration.

A new analytical method to determine non-steroidal anti-inflammatory drugs in surface water using in situ derivatization combined with ultrasound-assisted emulsification microextraction followed by gas chromatography-mass spectrometry.

Because of the high stability and potential toxic effects of non-steroidal anti-inflammatory drugs (NSAIDs), it is important to closely monitor their concentrations in the environment using a sensitive analytical method. In this study, a simple, rapid, efficient, and sensitive analytical method based on gas chromatography-mass spectrometry (GC-MS) was developed to determine the levels of seven common NSAIDs in various types of surface water. To simplify sample preparation, in situ derivatization using methyl chloroformate was combined with ultrasound-assisted emulsification microextraction. For selection and optimization of significant variables, experiments were statistically designed using Plackett-Burman design and central composite design. The resulting optimal conditions for derivatization and extraction were 100 µL of chloroform (extraction solvent), 10.0 mL of sample, and 240 µL of pyridine (catalyst as a base in derivatization). The optimized sample preparation coupled with optimized GC-MS analysis in selected ion monitoring mode provided good linearity from 0.010 to 5.0 ng mL(-1), and a limit of detection between 0.0050 and 0.010 ng mL(-1), good intra-day and inter-day precision (0.30-6.3% and 5.1-9.5%, respectively), and good accuracy (relative recovery; 91-117% at 0.20 ng mL(-1) and 77-105% at 2.5 ng mL(-1)). Compared with previously reported methods, the current method requires a small volume of sample and simple sample preparation steps for sensitive determination of NSAID levels using a conventional GC-MS system. The method was successfully applied to determine the levels of seven common NSAIDs in various types of surface water.

Monitoring performance of the cameras under the high dose-rate gamma ray environments.

CCD/CMOS cameras, loaded on a robot system, are generally used as the eye of the robot and monitoring unit. A major problem that arises when dealing with images provided by CCD/CMOS cameras under severe accident situations of a nuclear power plant is the presence of speckles owing to the high dose-rate gamma irradiation fields. To use a CCD/CMOS camera as a monitoring unit in a high radiation area, the legibility of the camera image in such intense gamma-radiation fields should therefore be defined. In this paper, the authors describe the monitoring index as a figure of merit of the camera's legibleness under a high dose-rate gamma ray irradiation environment. From a low dose-rate (10 Gy h) to a high dose-rate (200 Gy h) level, the legible performances of the cameras owing to the speckles are evaluated. The numbers of speckles generated by gamma ray irradiation in the camera image are calculated by an image processing technique. The legibility of the sensor indicator (thermo/hygrometer) owing to the number of speckles is also presented.

Effect of sauchinone, a lignan from Saururus chinensis, on bacterial phagocytosis by macrophages.

AMP-activated protein kinase (AMPK) plays an important role in inflammation in various cells and increases the phagocytic ability of macrophages. In this study, we found that sauchinone increased the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC), a downstream target of AMPK, in mouse peritoneal macrophages. Sauchinone increased macrophage phagocytosis of fluorescent Escherichia coli, which was blocked by compound C, an AMPK inhibitor. Sauchinone also increased the phosphorylation of p38 mitogen activated protein kinase (MAPK) in cultured macrophages in a concentration-dependent fashion, which was not blocked by compound C. However, the increase of sauchinone-induced phagocytosis was prevented by SB203580. An inhibitor of the upstream kinase TGF-beta-activated kinase (TAK1), (5z)-7-oxozeaenol, abolished the phosphorylation of ACC and p38 MAPK. Systemic administration of sauchinone to mice led to increased phosphorylation of AMPK and p38 MAPK in the lung, and enhanced phagocytosis of fluorescent E. coli in bronchoalveolar lavage fluid as compared with control mice. These results suggest sauchinone to be a useful adjunctive treatment for bacterial infection.