Publications by authors named "Yoshihiro Hayakawa"

185 Publications

Crucial contribution of GPR56/ADGRG1, expressed by breast cancer cells, to bone metastasis formation.

Cancer Sci 2021 Oct 10. Epub 2021 Oct 10.

Division of Molecular Bioregulation, Cancer Research Institute, Kanazawa University, Ishikawa, Japan.

From a mouse triple-negative breast cancer cell line, 4T1, we previously established 4T1.3 clone with a high capacity to metastasize to bone after its orthotopic injection into mammary fat pad of immunocompetent mice. Subsequent analysis demonstrated that the interaction between cancer cells and fibroblasts in a bone cavity was crucial for bone metastasis focus formation arising from orthotopic injection of 4T1.3 cells. Here, we demonstrated that a member of adhesion G-protein-coupled receptor (ADGR) family, G-protein-coupled receptor 56 (GPR56)/ adhesion G-protein coupled receptor G1 (ADGRG1), was expressed selectively in 4T1.3 grown in a bone cavity but not under in vitro conditions. Moreover, fibroblasts present in bone metastasis sites expressed type III collagen, a ligand for GPR56/ADGRG1. Consistently, GPR56/ADGRG1 proteins were detected in tumor cells in bone metastasis foci of human breast cancer patients. Deletion of GPR56/ADGRG1 from 4T1.3 cells reduced markedly intraosseous tumor formation upon their intraosseous injection. Conversely, intraosseous injection of GPR56/ADGRG1-transduced 4T1, TS/A (mouse breast cancer cell line), or MDA-MB-231 (human breast cancer cell line) exhibited enhanced intraosseous tumor formation. Furthermore, we proved that the cleavage at the extracellular region was indispensable for GPR56/ADGRG1-induced increase in breast cancer cell growth upon its intraosseous injection. Finally, inducible suppression of Gpr56/Adgrg1 gene expression in 4T1.3 cells attenuated bone metastasis formation with few effects on primary tumor formation in the spontaneous breast cancer bone metastasis model. Altogether, GPR56/ADGRG1 can be a novel target molecule to develop a strategy to prevent and/or treat breast cancer metastasis to bone.
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http://dx.doi.org/10.1111/cas.15150DOI Listing
October 2021

Flavanols and Flavanes from Crinum asiaticum and Their Effects on LPS Signaling Pathway Through the Inhibition of NF-κB Activation.

Planta Med 2021 Sep 2. Epub 2021 Sep 2.

Institute of Natural Medicine, University of Toyama, Toyama, Japan.

Three new flavanols, (2,3)-7-methoxy-flavan-3-ol (1: ), (2,3)-7-hydroxy-flavan-3-ol (2: ), and (2,3)-2'-hydroxy-7-methoxy-flavan-3-ol (3: ), together with two known flavans (4: and 5: ), were isolated from the chloroform extract of . Their structures were elucidated by various spectroscopic methods, including 1D and 2D NMR, HR-ESI-MS, and CD data. The isolated compounds 1: and 3: -5: showed inhibitory activity toward LPS-induced nitric oxide (NO) production. Further investigation of the NF-B pathway mechanisms indicated that 1: and 3: -5: inhibited the LPS-induced IL-6 production and p65 subunit phosphorylation of NF-B in RAW264.7 cells, with an effective dose of 10 µM.
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http://dx.doi.org/10.1055/a-1585-5877DOI Listing
September 2021

STING agonist loaded lipid nanoparticles overcome anti-PD-1 resistance in melanoma lung metastasis via NK cell activation.

J Immunother Cancer 2021 07;9(7)

Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido, Japan

Background: Resistance to an immune checkpoint inhibitor (ICI) is a major obstacle in cancer immunotherapy. The causes of ICI resistance include major histocompatibility complex (MHC)/histocompatibility locus antigen (HLA) class I loss, neoantigen loss, and incomplete antigen presentation. Elimination by natural killer (NK) cells would be expected to be an effective strategy for the treatment of these ICI-resistant tumors. We previously demonstrated that a lipid nanoparticle containing a stimulator of an interferon gene (STING) agonist (STING-LNP) efficiently induced antitumor activity via the activation of NK cells. Thus, we evaluated the potential of reducing ICI resistance by STING-LNPs.

Methods: Lung metastasis of a B16-F10 mouse melanoma was used as an anti-programmed cell death 1 (anti-PD-1)-resistant mouse model. The mice were intravenously injected with the STING-LNP and the mechanism responsible for the improvement of anti-PD-1 resistance by the STING-LNPs was analyzed by RT-qPCR and flow cytometry. The dynamics of STING-LNP were also investigated.

Results: Although anti-PD-1 monotherapy failed to induce an antitumor effect, the combination of the STING-LNP and anti-PD-1 exerted a synergistic antitumor effect. Our results indicate that the STING-LNP treatment significantly increased the expression of CD3, CD4, NK1.1, PD-1 and interferon (IFN)-γ in lung metastases. This change appears to be initiated by the type I IFN produced by liver macrophages that contain the internalized STING-LNPs, leading to the systemic activation of NK cells that express PD-1. The activated NK cells appeared to produce IFN-γ, resulting in an increase in the expression of the PD ligand 1 (PD-L1) in cancer cells, thus leading to a synergistic antitumor effect when anti-PD-1 is administered.

Conclusions: We provide a demonstration to show that a STING-LNP treatment can overcome PD-1 resistance in a B16-F10 lung metastasis model. The mechanism responsible for this indicates that NK cells are activated by stimulating the STING pathway which, in turn, induced the expression of PD-L1 on cancer cells. Based on the findings reported herein, the STING-LNP represents a promising candidate for use in combination therapy with anti-PD-1-resistant tumors.
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http://dx.doi.org/10.1136/jitc-2021-002852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8256839PMC
July 2021

NKG2D defines tumor-reacting effector CD8 T cells within tumor microenvironment.

Cancer Sci 2021 Sep 28;112(9):3484-3490. Epub 2021 Jul 28.

Institute of Natural Medicine, University of Toyama, Toyama, Japan.

For successful immunotherapy for cancer, it is important to understand the immunological status of tumor antigen-specific CD8 T cells in the tumor microenvironment during tumor progression. In this study, we monitored the behavior of B16OVA-Luc cells in mice immunized with a model tumor antigen ovalbumin (OVA). Using bioluminescence imaging, we identified the time series of OVA-specific CD8 T-cell responses during tumor progression: initial progression, immune control, and the escape phase. As a result of analyzing the status of tumor antigen-specific CD8 cells in those 3 different phases, we found that the expression of NKG2D defines tumor-reacting effector CD8 T cells. NKG2D may control the fate and TOX expression of tumor-reacting CD8 T cells, considering that NKG2D blockade in OVA-vaccinated mice delayed the growth of the B16OVA-Luc2 tumor and increased the presence of tumor-infiltrating OVA-specific CD8 T cells.
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http://dx.doi.org/10.1111/cas.15050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409295PMC
September 2021

Design and synthesis of 2-Substituted-4-benzyl-5-methylimidazoles as new potential Anti-breast cancer agents to inhibit oncogenic STAT3 functions.

Bioorg Chem 2021 08 27;113:105033. Epub 2021 May 27.

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, 2152 Alexandria, Egypt; Pharmacy Program, Allied Health Department, College of Health and Sport Sciences, University of Bahrain, Bahrain; Cancer Nanotechnology Research Laboratory (CNRL), Faculty of Pharmacy, Alexandria University, Alexandria 21521, Egypt. Electronic address:

STAT3 signaling is known to be associated with tumorigenesis and further cancer cell-intrinsic activation of STAT3 leads to altered regulation of several oncogenic processes. Given the importance of STAT3 in cancer development and progression particularly breast cancer, it is crucial to discover new chemical entities of STAT3 inhibitor to develop anti-breast cancer drug candidates. Herein, 4-benzyl-2-benzylthio-5-methyl-1H-imidazole (2a) and 4-benzyl-5-methyl-2-[(2,6-difluorobenzyl)thio]-1H-imidazole (2d) from a group of thirty imidazole-bearing compounds showed greater STAT3 inhibition than their lead compounds VS1 and the oxadiazole derivative MD77. Within all tested compounds, ten derivatives effectively inhibited the growth of the two tested breast cancer cells with IC values ranging from 6.66 to 26.02 µM. In addition, the most potent derivatives 2a and 2d inhibited the oncogenic function of STAT3 as seen in the inhibition of colony formation and IL-6 production of breast cancer cell lines. Modeling studies provided evidence for the possible interactions of the synthesized compounds with the key residues of the STAT3-SH2 domain. Collectively, our present study suggests 2-substituted-4-benzyl-5-methylimidazoles are a new class of anti-cancer drug candidates to inhibit oncogenic STAT3 function.
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http://dx.doi.org/10.1016/j.bioorg.2021.105033DOI Listing
August 2021

Synthetic E-guggulsterone derivative GSD-1 inhibits NF-κB signaling and suppresses the metastatic potential of breast cancer cells.

Biomed Pharmacother 2021 Aug 18;140:111737. Epub 2021 May 18.

Section of Host Defences, Institute of Natural Medicine, University of Toyama, Toyama, Japan. Electronic address:

Guggulsterone (GS) [4,17(20)-pregnadiene-3,16-dione], is the main active phytosterol constituent in guggul, the gum resin of Commiphora wightii (Arnott.) Bhand./Commiphora mukul Engl. tree, and is known for its medicinal effects. In this study, we report that GSD-1, a structurally-related synthetic GS derivative, strongly inhibits NF-κB activation induced by TNF-α. GSD-1 prevented the nuclear translocation of p65 through the blockade of IκBα degradation and p65 phosphorylation, and further inhibited the activation of upstream kinases, including transforming growth factor-β activated kinase 1 (TAK1), IκB kinase (IKK) α, and IKKβ. Furthermore, GSD-1 inhibited the cell-intrinsic activation of NF-κB, and exerted its direct anti-cancer and anti-metastatic effects in both murine and human breast cancer cell lines. This study demonstrated GSD-1 to be an attractive compound to target NF-κB activation that has potential for treating breast cancer growth and metastasis.
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http://dx.doi.org/10.1016/j.biopha.2021.111737DOI Listing
August 2021

Inhibition of cell-intrinsic NF-κB activity and metastatic abilities of breast cancer by aloe-emodin and emodic-acid isolated from Asphodelus microcarpus.

J Nat Med 2021 Sep 14;75(4):840-853. Epub 2021 May 14.

Institute of Natural Medicine, University of Toyama, Sugitani 2630, Toyama, 930-0194, Japan.

Anthraquinones are a major class of compounds naturally occurring in Asphodelus microcarpus. The pharmacological actions of anthraquinones in cancer cells are known to induce apoptosis or autophagy, and revert multidrug resistance. In this study, five anthraquinone-type analogs were isolated from the methanol extract of A. microcarpus leaves and identified as, emodin, rhein, physcion, aloe-emodin, and emodic acid. Among them, aloe-emodin and emodic-acid strongly inhibited the proliferation, cells-intrinsic NF-κB activity and metastatic ability of breast cancer. Although aloe-emodin inhibited p38 and ERK phosphorylation, emodic-acid more markedly inhibited JNK, in addition to p38 and ERK phosphorylation. Both aloe-emodin and emodic-acid inhibited the secretion of the pro-tumorigenic cytokines IL-1β and IL-6, and VEGF and MMP expression, and subsequently inhibited the invasive and migratory potential of 4T1 cells. Thus, our study demonstrated the effects of aloe-emodin and emodin-acid in controlling the migratory and invasive ability of 4T1 breast cancer cells, in addition to inhibiting NF-κB activity and the expression of its downstream target molecules.
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http://dx.doi.org/10.1007/s11418-021-01526-wDOI Listing
September 2021

Identification of Ophiocordyceps gracilioides by Its Anti-tumor Effects through Targeting the NFκB-STAT3-IL-6 Inflammatory Pathway.

Biol Pharm Bull 2021 ;44(5):686-690

Institute of Natural Medicine, University of Toyama.

Although more than 400 species of Cordyceps s.l. have been identified, most have not been well explored regarding their potential for medicinal use. In this study, the profiles of constituents of ten different species of Ophiocordyceps, which is an unexplored species of Cordyceps, were analyzed and their anti-tumor effects were further examined. Although all Ophiocordyceps samples exhibited similar peak patterns, Ophiocordyceps gracilioides (O. grac) had a distinct constituent profile from the other samples. Furthermore, O. grac was the most active in suppressing the transcriptional activities of both nuclear factor-kappa B (NF-κB) and signal transducer and activator of transcription (STAT)3, and the production of interleukin (IL)-6 from breast cancer cells. This study demonstrated that O. grac is a relatively unexplored Cordyceps s.l. that may have medicinal potential to inhibit the NFκB-STAT3-IL-6 inflammatory pathway in cancer.
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http://dx.doi.org/10.1248/bpb.b20-01032DOI Listing
January 2021

Anti-metastatic effects of ergosterol peroxide from the entomopathogenic fungus Ophiocordyceps gracilioides on 4T1 breast cancer cells.

J Nat Med 2021 Sep 27;75(4):824-832. Epub 2021 Apr 27.

Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.

Ophiocordyceps gracilioides is an entomoparasitic ascomycetes whose bioactivity has not been examined in detail. In this study, we identified the bioactive compounds ergosterol peroxide (EPO) and ergosterol (ERG) from the MeOH extract of O. gracilioides mycelia related to its anti-cancer effects by targeting the Nuclear Factor kappa B (NF-ĸB)/Signal Transducer and Activator of Transcription 3 (STAT3) inflammatory pathways. Using gene-reporter assays, we demonstrated that EPO markedly inhibits both NF-ĸB and STAT3 activity in 4T1 cells, whereas ERG had limited effect. Consistent with their effects on NF-ĸB and STAT3 activity, EPO, but not ERG, exerted anti-proliferative effects on 4T1 cells. Furthermore, EPO significant inhibited both the migration and invasion of 4T1 cells in vitro, and pre-treatment of 4T1 cells with EPO significantly inhibited the formation of experimental lung metastases in vivo. Collectively, we demonstrated that ERG and EPO can be isolated from O. gracilioides mycelia, and further identified EPO as an active constituent of its anti-metastatic effects through the inhibition of NF-ĸB and STAT3 inflammatory pathways in 4T1 breast cancer cells.
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http://dx.doi.org/10.1007/s11418-021-01520-2DOI Listing
September 2021

Anti-inflammatory effects of Morus alba Linne bark on the activation of toll-like receptors and imiquimod-induced ear edema in mice.

BMC Complement Med Ther 2021 Apr 9;21(1):115. Epub 2021 Apr 9.

Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.

Background: Morus alba L. bark has been widely used in traditional medicine for treating several inflammatory diseases, such as hypertension, diabetes mellitus and coughing; however, the molecular mechanisms underlying its anti-inflammatory effects are not well understood.

Methods: We examined the effects of an extract of Morus alba L. bark (MabE) on Toll-like receptor (TLR) ligand-induced activation of RAW264.7 macrophages using a luciferase reporter assay and immunoassays. For the in vivo experiment, we used an imiquimod-induced ear edema model to examine the anti-inflammatory effects of MabE.

Results: MabE inhibited the TLR ligand-induced activation of NF-κB in RAW264.7 cells without affecting their viability. Consistent with the inhibition of NF-κB activation, MabE also inhibited the production of IL-6 and IL-1β from TLR ligand-treated RAW264.7 cells. In vivo MabE treatment inhibited the ear swelling of IMQ-treated mice, in addition to the mRNA expression of IL-17A, IL-1β and COX-2. The increases in splenic γδT cells in IMQ-treated mice and the production of IL-17A from splenocytes were significantly inhibited by MabE treatment.

Conclusion: Our study suggests that the anti-inflammatory effects of MabE on the activation of the macrophage cell line RAW246.7 by TLRs and IMQ-induced ear edema are through the inhibition of NF-κB activation and IL-17A-producing γδT cells, respectively.
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http://dx.doi.org/10.1186/s12906-021-03291-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8033707PMC
April 2021

Erianthridin suppresses non-small-cell lung cancer cell metastasis through inhibition of Akt/mTOR/p70 signaling pathway.

Sci Rep 2021 03 23;11(1):6618. Epub 2021 Mar 23.

Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai, Wangmai, Pathumwan, Bangkok, Thailand.

Cancer metastasis is a major cause of the high mortality rate in lung cancer patients. The cytoskeletal rearrangement and degradation of extracellular matrix are required to facilitate cell migration and invasion and the suppression of these behaviors is an intriguing approach to minimize cancer metastasis. Even though Erianthridin (ETD), a phenolic compound isolated from the Thai orchid Dendrobium formosum exhibits various biological activities, the molecular mechanism of ETD for anti-cancer activity is unclear. In this study, we found that noncytotoxic concentrations of ETD (≤ 50 μM) were able to significantly inhibit cell migration and invasion via disruption of actin stress fibers and lamellipodia formation. The expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 was markedly downregulated in a dose-dependent manner after ETD treatment. Mechanistic studies revealed that protein kinase B (Akt) and its downstream effectors mammalian target of rapamycin (mTOR) and p70 S6 kinase (p70) were strongly attenuated. An in silico study further demonstrated that ETD binds to the protein kinase domain of Akt with both hydrogen bonding and van der Waals interactions. In addition, an in vivo tail vein injection metastasis study demonstrated a significant effect of ETD on the suppression of lung cancer cell metastasis. This study provides preclinical information regarding ETD, which exhibits promising antimetastatic activity against non-small-cell lung cancer through Akt/mTOR/p70-induced actin reorganization and MMPs expression.
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http://dx.doi.org/10.1038/s41598-021-85675-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7987990PMC
March 2021

Establishment of bioluminescent imaging model using murine T cell lymphoma susceptive to NK cell-dependent immune-surveillance.

J Immunol Methods 2021 04 17;491:112993. Epub 2021 Feb 17.

Institute of Natural Medicine, University of Toyama, Toyama, Japan. Electronic address:

Although the importance of NK cells as immune effector cells in controlling growth and metastatic dissemination of tumor cells has been widely recognized, it is unclear whether NK cells in different organs similarly control tumor cell growth and metastasis. In the present study, we established a bioluminescent imaging model of mouse T cell lymphoma cells, which are highly susceptive to NK cell-dependent immune-surveillance, to monitor the dissemination of lymphoma cells using an in vivo imaging system. The use of this model is expected to be a highly sensitive method to examine the role of NK cells in controlling lymphoma dissemination in a variety of tissues.
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http://dx.doi.org/10.1016/j.jim.2021.112993DOI Listing
April 2021

Development of a simultaneous analytical method for clozapine and its metabolites in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry with linear range adjusted by in-source collision-induced dissociation.

Biomed Chromatogr 2021 Jul 4;35(7):e5094. Epub 2021 Mar 4.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Japan.

Clozapine (CLZ) is a key drug in treatment-resistant schizophrenia. Therapeutic drug monitoring (TDM) of CLZ and its metabolites, N-desmethylclozapine and clozapine N-oxide, is required to monitor and manage the risks of side effects. Although quantification methods for TDM have been developed for CLZ and its metabolites, they were not sufficiently accurate for the quantification of CLZ owing to the upper limits of the calibration curves. An analytical method using high-performance liquid chromatography/electrospray ionization tandem mass spectrometry was developed and validated for the simultaneous measurement of CLZ and its metabolites in human plasma. To expand the concentration range of the calibration curves, we used a linear range shift technique using in-source collision-induced dissociation (CID). Using our approach, the linearity and quantitative range were improved compared to those reported by previous studies, and were sufficient for TDM in clinical practice. The intra- and inter-assay accuracy was 84.6%-114.8%, and the intra- and inter-assay precisions were ≤9.1% and ≤9.9%, respectively. Moreover, all samples from patients with treatment-resistant schizophrenia were successfully quantified. Therefore, our novel analytical method using in-source CID had the appropriate performance to measure the plasma concentrations of CLZ and its metabolites for TDM in clinical practice.
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http://dx.doi.org/10.1002/bmc.5094DOI Listing
July 2021

ASK1 suppresses NK cell-mediated intravascular tumor cell clearance in lung metastasis.

Cancer Sci 2021 Apr 6;112(4):1633-1643. Epub 2021 Mar 6.

Laboratory of Cell Signaling, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan.

Tumor metastasis is the leading cause of death worldwide and involves an extremely complex process composed of multiple steps. Our previous study demonstrated that apoptosis signal-regulating kinase 1 (ASK1) deficiency in mice attenuates tumor metastasis in an experimental lung metastasis model. However, the steps of tumor metastasis regulated by ASK1 remain unclear. Here, we showed that ASK1 deficiency in mice promotes natural killer (NK) cell-mediated intravascular tumor cell clearance in the initial hours of metastasis. In response to tumor inoculation, ASK1 deficiency upregulated immune response-related genes, including interferon-gamma (IFNγ). We also revealed that NK cells are required for these anti-metastatic phenotypes. ASK1 deficiency augmented cytokine production chemoattractive to NK cells possibly through induction of the ligand for NKG2D, a key activating receptor of NK cells, leading to further recruitment of NK cells into the lung. These results indicate that ASK1 negatively regulates NK cell-dependent anti-tumor immunity and that ASK1-targeted therapy can provide a new tool for cancer immunotherapy to overcome tumor metastasis.
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http://dx.doi.org/10.1111/cas.14842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8019214PMC
April 2021

Simultaneous analysis of drugs administered to lung-transplanted patients using liquid chromatography-tandem mass spectrometry for therapeutic drug monitoring.

Biomed Chromatogr 2021 Jun 22;35(6):e5067. Epub 2021 Jan 22.

Department of Pharmaceutical Sciences, Tohoku University Hospital, Sendai, Miyagi, Japan.

Several drugs are administered to lung-transplanted patients, which are monitored using therapeutic drug monitoring (TDM). Therefore, we developed and validated a liquid chromatography-tandem mass spectrometry method to simultaneously analyze immunosuppressive drugs such as mycophenolic acid, antifungal drugs such as voriconazole and itraconazole, and its metabolite hydroxyitraconazole. Chromatographic separation was achieved using a C18 column and gradient flow of mobile phase comprising 20 mM aqueous ammonium formate and 20 mM ammonium formate-methanol solution. A simple protein precipitation treatment was performed using acetonitrile/methanol and mycophenolic acid- H , voriconazole- H , itraconazole- H , and hydroxyitraconazole- H as internal standards. The linearity ranges of mycophenolic acid, voriconazole, itraconazole, and hydroxyitraconazole were 100-20,000, 50-10,000, 5-1000, and 5-1000 ng/mL, respectively. The retention time of each target was less than 2 min. The relative errors in intra- and inter-day were within ±7.6%, the coefficient of variation was 8.9% or less for quality control low, medium, and high, and it was 15.8% or less for lower limit of quantitation. Moreover, the patient samples were successfully quantified, and they were within the linear range of measurements. Therefore, our new method may be useful for TDM in lung-transplanted patients.
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http://dx.doi.org/10.1002/bmc.5067DOI Listing
June 2021

Anti-metastatic Effects of Baicalein by Targeting STAT3 Activity in Breast Cancer Cells.

Biol Pharm Bull 2020 ;43(12):1899-1905

Institute of Natural Medicine, University of Toyama.

Signal transducer and activator of transcription 3 (STAT3) is considered a potential target for cancer treatment because of its relationship with cellular transformation and tumor initiation and progression. In this study, we aimed to identify a new anti-cancer drug candidate from natural products by targeting STAT3 activity. Using STAT3-luciferase reporter cell line, we screened the chemical library of natural products and found that baicalein, a flavone isolated from the roots of Scutelleria baicalensis, strongly suppressed STAT3 activity in breast cancer cells. Baicalein inhibited STAT3 transcriptional activity and its phosphorylation, and further exhibited anti-proliferative effects in breast cancer cells. Moreover, baicalein suppressed the production of interleukin (IL)-6 and the metastatic potential of breast cancer cells both in vitro and in vivo. Collectively, our study suggests baicalein as an attractive phytochemical compound for reducing metastatic potential of breast cancer cells by regulating STAT3 activity.
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http://dx.doi.org/10.1248/bpb.b20-00571DOI Listing
August 2021

Loss of cell wall integrity genes cpxA and mrcB causes flocculation in Escherichia coli.

Biochem J 2021 01;478(1):41-59

Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan.

Flocculation has been recognized for hundreds of years as an important phenomenon in brewing and wastewater treatment. However, the underlying molecular mechanisms remain elusive. The lack of a distinct phenotype to differentiate between slow-growing mutants and floc-forming mutants prevents the isolation of floc-related gene by conventional mutant screening. To overcome this, we performed a two-step Escherichia coli mutant screen. The initial screen of E. coli for mutants conferring floc production during high salt treatment yielded a mutant containing point mutations in 61 genes. The following screen of the corresponding single-gene mutants identified two genes, mrcB, encoding a peptidoglycan-synthesizing enzyme and cpxA, encoding a histidine kinase of a two-component signal transduction system that contributed to salt tolerance and flocculation prevention. Both single mutants formed flocs during high salt shock, these flocs contained cytosolic proteins. ΔcpxA exhibited decreased growth with increasing floc production and addition of magnesium to ΔcpxA suppressed floc production effectively. In contrast, the growth of ΔmrcB was inconsistent under high salt conditions. In both strains, flocculation was accompanied by the release of membrane vesicles containing inner and outer membrane proteins. Of 25 histidine kinase mutants tested, ΔcpxA produced the highest amount of proteins in floc. Expression of cpxP was up-regulated by high salt in ΔcpxA, suggesting that high salinity and activation of CpxR might promote floc formation. The finding that ΔmrcB or ΔcpxA conferred floc production indicates that cell envelope stress triggered by unfavorable environmental conditions cause the initiation of flocculation in E. coli.
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http://dx.doi.org/10.1042/BCJ20200723DOI Listing
January 2021

Anti‑inflammatory compounds moracin O and P from Morus alba Linn. (Sohakuhi) target the NF‑κB pathway.

Mol Med Rep 2020 Dec 20;22(6):5385-5391. Epub 2020 Oct 20.

Department of Research and Development, Institute of Natural Medicine, University of Toyama, Toyama 930‑0194, Japan.

Accumulating evidence suggests that inflammation is linked to multiple pathological processes and induces cellular and molecular damage through the activation of inflammatory signaling pathways, including the NF‑κB pathway. The aim of the present study was to identify natural anti‑inflammatory products that can target NF‑κB activity, in order to establish a novel therapeutic approach for inflammatory diseases. Using a 4T1 breast cancer cell line that expresses the firefly luciferase gene under the control of an NF‑κB response element, 112 natural products were tested for their anti‑inflammatory properties. Sohakuhi (Morus alba Linn. bark) extract was observed to strongly suppress NF‑κB activity without affecting cell viability. To further examine the anti‑inflammatory effect of Sohakuhi, tumor necrosis factor‑related apoptosis‑inducing ligand (TRAIL)‑induced cellular damage of human HaCaT keratinocytes was evaluated. While TRAIL triggered the phosphorylation of the p65 subunit of NF‑κB, leading to cellular damage in HaCaT cells, treatment with Sohakuhi extract protected HaCaT cells against TRAIL‑induced cellular damage. Moreover, Sohakuhi treatment also upregulated the anti‑apoptotic proteins Bcl‑xL and Bcl‑2. Importantly, through chemical fractionation of Sohakuhi extract, moracin O and P were confirmed to mediate its anti‑inflammatory effects. Collectively, the present results indicated that Sohakuhi and moracin may represent potential candidates for the development of novel anti‑inflammatory drugs.
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http://dx.doi.org/10.3892/mmr.2020.11615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7647032PMC
December 2020

An ion-pair free LC-MS/MS method for quantitative metabolite profiling of microbial bioproduction systems.

Talanta 2021 Jan 8;222:121625. Epub 2020 Sep 8.

Graduate School of Science, Technology and Innovation, Kobe University, Japan; Engineering Biology Research Center, Kobe University, Japan.

Data-driven engineering of microbes has been demonstrated for the sustainable production of high-performance chemicals. Metabolic profiling analysis is essential to increase the productivity of target compounds. However, improvement of comprehensive analysis methodologies is required for the high demands of metabolic engineering. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) based methodology was designed and applied to cover a wide target range with high precision. Ion-pair free separation of metabolites on a pentafluorophenyl propyl column enabled high-precision quantification of 113 metabolites. The method was further evaluated for high reproducibility and robustness. Target analytes consisted of primary metabolites and intermediate metabolites for microbial production of high-performance chemicals. 95 metabolites could be detected with high reproducibility of peak area (intraday data: CV<15%), and 53 metabolites could be sensitively determined within a wide dynamic linear range (3-4 orders of magnitude). The developed system was further applied to the metabolomic analysis of various prokaryotic and eukaryotic microorganisms. Differences due to culture media and metabolic phenotypes could be observed when comparing the metabolomes of conventional and non-conventional yeast. Furthermore, almost all Kluyveromyces marxianus metabolites could be detected with moderate reproducibility (CV<40%, among independent extractions), where 41 metabolites were detected with very high reproducibility (CV<15%). In addition, the accuracy was validated via a spike-and-recovery test,and 78 metabolites were detected with analyte recovery in the 80-120% range. Together these results establish ion-pair free metabolic profiling as a comprehensive and precise tool for data-driven bioengineering applications.
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http://dx.doi.org/10.1016/j.talanta.2020.121625DOI Listing
January 2021

Targeting PSMD14 inhibits melanoma growth through SMAD3 stabilization.

Sci Rep 2020 11 5;10(1):19214. Epub 2020 Nov 5.

Department of Cancer Cell Biology, Faculty of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.

Although melanoma therapy is improved by novel molecular targeted reagents, including vemurafenib, aberrant proliferation and early metastasis remain obstacles for melanoma; therefore, novel target molecules for melanoma need to be identified. In this study, we focused on deubiquitinating enzymes, which regulate protein stability through ubiquitin-proteasome systems, and identified 26S proteasome non-ATPase regulatory subunit 14 (PSMD14) as a molecule related to melanoma growth using siRNA library screening. Similar to a previous report, PSMD14 knockdown strongly induced p21 expression and inhibited RB phosphorylation in melanoma. After in silico analysis, TGF-β signaling was identified as a negatively correlated gene set with PSMD14 expression. Although TGF-β signaling is also related to the invasive phenotype of melanoma, PSMD14 knockdown suppressed melanoma migration and reduced SLUG expression, suggesting that targeting PSMD14 suppresses both growth and migration. Furthermore, SMAD3 expression increased in nucleus and SMAD3 degradation was delayed after PSMD14 knockdown. Thus, our present study suggests that targeting PSMD14 can inhibit melanoma growth and migration through either SMAD3 accumulation or SLUG reduction, respectively.
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http://dx.doi.org/10.1038/s41598-020-76373-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7644625PMC
November 2020

Development of a novel comprehensive analytical method for volatile compounds using supercritical fluid chromatography/mass spectrometry with a highly cross-linked styrene divinylbenzene polymer-based column.

J Chromatogr A 2020 Aug 22;1626:461363. Epub 2020 Jun 22.

Division of Metabolomics, Research Center for Transomics Medicine, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka, 812-8582, Japan. Electronic address:

Analytical techniques to determine volatile compounds such as flavor, aroma, and fragrances are in high demand due to their wide range of applications in industry, the chemical properties of them are very diverse. Supercritical fluid chromatography (SFC) is capable of high speed, high peak capacity separation and has a high separation coverage. It is also an advantageous for preparative purifications due to its unique mobile phase conditions. However, there is no column commercially available for SFC that is suitable to comprehensively separate volatile compounds. SFC is limited to the use of silica-based columns due to weak retentions and polymer-based column issues such as pressure, swelling and shrinkage tolerances. This study demonstrated comprehensive analytical method for volatile in SFC using a highly cross-linked styrene divinylbenzene (SDVB) polymer-based column, newly developed for SFC. In this study, 23 typical volatile compounds with a wide variety of chemical properties were selected as model compounds. The newly developed SDVB column showed, compared to conventional silica-based columns (k > 0.3), an excellent overall and substantial improved retentions (k > 1.6) under SFC mobile phase conditions. It was also able to retain esters (hydroxy acetate, pentyl butylate, methyl salicylate) and non-polar terpenes (limonene, pinene) that did not show sufficient retention in any other commercially available silica-based columns. Aldehydes reacting on NH column due to Schiff base formation were also successfully eluted. It was confirmed that SDVB column provided comprehensive separation and wide coverage for volatile compounds.
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http://dx.doi.org/10.1016/j.chroma.2020.461363DOI Listing
August 2020

Antimetastatic effects of thalidomide by inducing the functional maturation of peripheral natural killer cells.

Cancer Sci 2020 Aug 14;111(8):2770-2778. Epub 2020 Jul 14.

Institute of Natural Medicine, University of Toyama, Toyama, Japan.

Thalidomide and its analogues are known as immunomodulatory drugs (IMiDs) that possess direct antimyeloma effects, in addition to other secondary effects, including antiangiogenic, antiinflammatory, and immunomodulatory effects. Although the involvement of natural killer (NK) cells in the antitumor effects of IMiDs has been reported, it is unclear whether IMiDs inhibit cancer cell metastasis by regulating the antitumor function of NK cells. In this study, we examined the protective effects of thalidomide against cancer metastasis by focusing on its immunomodulatory effects through NK cells. Using experimental lung metastasis models, we found that pharmacological effects of thalidomide on host cells, but not its direct anticancer tumor effects, are responsible for the inhibition of lung metastases. To exert the antimetastatic effects of thalidomide, both γ-interferon (IFN-γ) production and direct cytotoxicity of NK cells were essential, without notable contribution from T cells. In thalidomide-treated mice, there was a significant increase in the terminally differentiated mature CD27 NK cells in the peripheral tissues and NK cells in thalidomide-treated mice showed significantly higher cytotoxicity and IFN-γ production. The NK cell expression of T-bet was upregulated by thalidomide treatment and the downregulation of glycogen synthase kinase-3β expression was observed in thalidomide-treated NK cells. Collectively, our study suggests that thalidomide induces the functional maturation of peripheral NK cells through alteration of T-bet expression to inhibit lung metastasis of cancer cells.
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http://dx.doi.org/10.1111/cas.14538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419051PMC
August 2020

Functional characterization of multiple PAS domain-containing diguanylate cyclases in sp. PCC 6803.

Microbiology (Reading) 2020 07;166(7):659-668

Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is a second messenger known to control a variety of bacterial processes. The model cyanobacterium, sp. PCC 6803, has a score of genes encoding putative enzymes for c-di-GMP synthesis and degradation. However, most of them have not been functionally characterized. Here, we chose four genes in (), which encode proteins with a GGDEF, diguanylate cyclase (DGC) catalytic domain and multiple Per-ARNT-Sim (PAS) conserved regulatory motifs, for detailed analysis. Purified DgcA, DgcB and DgcC were able to catalyze synthesis of c-di-GMP from two GTPs . DgcA had the highest activity, compared with DgcB and DgcC. DgcD did not show detectable activity. DgcA activity was specific for GTP and stimulated by the divalent cations, magnesium or manganese. Full activity of DgcA required the presence of the multiple PAS domains, probably because of their role in protein dimerization or stability. mutants carrying single deletions of were not affected in their growth rate or biofilm production during salt stress, suggesting that there was functional redundancy . In contrast, overexpression of resulted in increased biofilm formation in the absence of salt stress. In this study, we characterize the enzymatic and physiological function of DgcA-DgcD, and propose that the PAS domains in DgcA function in maintaining the enzyme in its active form.
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http://dx.doi.org/10.1099/mic.0.000929DOI Listing
July 2020

Pharmacological targeting of natural killer cells for cancer immunotherapy.

Cancer Sci 2020 Jun 5;111(6):1869-1875. Epub 2020 May 5.

Institute of Natural Medicine, University of Toyama, Toyama, Japan.

Natural killer (NK) cells are innate lymphocytes that rapidly respond to cancer cells without prior sensitization or restriction to the cognate antigen in comparison with tumor antigen-specific T cells. Recent advances in understanding NK-cell biology have elucidated the molecular mechanisms underlying the differentiation and maturation of NK cells, in addition to the control of their effector functions by investigating the receptors and ligands involved in the recognition of cancer cells by NK cells. Such clarification of NK-cell recognition of cancer cells also revealed the mechanism by which cancer cells potentially evade NK-cell-dependent immune surveillance. Furthermore, the recent clinical results of T-cell-targeted cancer immunotherapy have increased the expectations for new immunotherapies by targeting NK cells. However, the potential use of NK cells in cancer immunotherapy is not fully understood. In this review, we discuss the current evidence and future potential of pharmacological targeting of NK cells in cancer immunotherapy.
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http://dx.doi.org/10.1111/cas.14418DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293096PMC
June 2020

Anti-inflammatory activities of isopimara-8(9),15-diene diterpenoids and mode of action of kaempulchraols B-D from Kaempferia pulchra rhizomes.

J Nat Med 2020 Mar 31;74(2):487-494. Epub 2020 Jan 31.

Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama, 930-0194, Japan.

Kaempulchraols B-D (2-4), isopimara-8(9),15-diene diterpenoids isolated from Kaempferia pulchra rhizomes collected in Myanmar, were identified as potent NF-κB inhibitors. These compounds were also effective as NO inhibitory agents, with IC values of 47.69, 44.97, and 38.17 μM, respectively, without showing any cytotoxicity against LPS-induced RAW264.7 cells. Investigations of the mechanisms of action of 2-4 revealed that they inhibit the NF-κB-mediated transactivation of a luciferase reporter gene, IL-6 production, and COX-2 expression, with an effective dose of 25 μM. Thus, isopimarane diterpenoids are suggested to be potent inhibitors of NF-κB pathways and could be further explored as potential anti-inflammatory lead compounds.
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http://dx.doi.org/10.1007/s11418-020-01389-7DOI Listing
March 2020

Anti-inflammatory activities of isopimara-8(14),-15-diene diterpenoids and mode of action of kaempulchraols P and Q from Kaempferia pulchra rhizomes.

Bioorg Med Chem Lett 2020 01 3;30(2):126841. Epub 2019 Dec 3.

Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan. Electronic address:

Inflammation is an extensively recognized link to many pathological diseases. It is a host response for protection from infections and tissue damage. Infections trigger acute inflammation; however, persistent infection will contribute to chronic inflammation and higher disease susceptibility. Deregulated inflammatory responses can cause excessive or long-lasting tissue damage, manifested as cancer, immune disorders, diabetes, etc. NF-κB is a central mediator of pro-inflammatory gene induction and functions in both innate and adaptive immune cells; therefore, the anti-inflammatory regulation of NF-κB is needed. Natural products reportedly play an important role in controlling the inflammatory response pathways. However, the anti-inflammatory activities of isopimara-8-(14),15-diene diterpenoids have not yet been fully elucidated. To elucidate the anti-inflammatory activities of the isopimara-8(14),15-diene diterpenoids, we investigated 21 isopimara-8(14),15-diene diterpenoids previously isolated from Kaempferia pulchra rhizomes. Eleven compounds exhibited NO inhibitory activity against lipopolysaccharide (LPS)-induced RAW264.7 cells, with IC values ranging from 30 to 100 μM. Furthermore, the most potent kaempulchraols P and Q, with IC values of 39.88 and 36.05 μM, respectively, inhibited the NF-κB-mediated transactivation of a luciferase reporter gene, IL-6 production, and COX-2 expression, with an effective dose of 25 μM. These findings provide new insights into the anti-inflammatory activities of the isopimara-8(14),15-diene diterpenoids.
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http://dx.doi.org/10.1016/j.bmcl.2019.126841DOI Listing
January 2020

Rational Combination Therapy for Melanoma with Dinaciclib by Targeting BAK-Dependent Cell Death.

Mol Cancer Ther 2020 02 19;19(2):627-636. Epub 2019 Nov 19.

Division of Pathogenic Biochemistry, Institute of Natural Medicine, University of Toyama, Toyama, Japan.

Mutation of the oncogene BRAF is among the most common genetic alterations in melanoma. BRAF inhibitors alone or in combination with MEK inhibitors fail to eradicate the tumor in most patients due to combinations of intrinsic or acquired resistance. Therefore, novel strategies are needed to improve the therapeutic efficacy of BRAF inhibition. We demonstrated that dinaciclib has potent antimelanoma effects by inducing BAK-dependent apoptosis through MCL1 reduction. Contrary to dinaciclib, the inhibitors of BRAF/MEK/CDK4/6 induced apoptosis dominantly through a BAX-dependent mechanism. Although the combination of BRAF and MEK inhibitors did not exhibit additive antimelanoma effects, their combination with dinaciclib synergistically inhibited melanoma growth both and Collectively, our present findings suggest dinaciclib to be an effective complementary drug of BAX-dependent antimelanoma drugs by targeting BAK-mediated apoptosis, and other such rational drug combinations can be determined by identifying complementary drugs activating either BAK or BAX.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7007861PMC
February 2020

Molecular mechanisms of natural compounds in cell death induction and sensitization to chemotherapeutic drugs in lung cancer.

Phytother Res 2019 Oct 10;33(10):2531-2547. Epub 2019 Jul 10.

Preclinical Toxicity and Efficacy Assessment of Medicines and Chemicals Research Cluster, Chulalongkorn University, Bangkok, Thailand.

Cancer remains one of the leading causes of death worldwide, especially lung cancer. Chemotherapeutic drugs are commonly used for lung cancer treatment; nonetheless, undesirable side effects and drug resistance remain major challenges for therapeutic success. Therefore, harmless and effective treatments against lung cancer are urgently required. The use of natural phytochemical products, in single or combinatorial therapy, is an emerging strategy for prevention and cure of cancer because of the various remarkable anticancer properties of these compounds. Cell death, which primarily occurs via apoptosis and nonapoptotic mechanisms (necrosis, autophagy, and cellular senescence), is one of the antineoplastic effects of natural compounds. In this review, we highlight representative plant-derived compounds with cancer chemopreventive and sensitizing effects in combination with chemotherapeutic drugs with various cell death-inducing mechanisms. Relevant molecular mechanisms implicated in the pharmacological effects of these natural compounds are discussed. Overall, this review provides a reference and new perspective for application of phytochemical agents as potential anti-lung cancer drugs for further cancer drug research and development.
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http://dx.doi.org/10.1002/ptr.6422DOI Listing
October 2019

Macrophage-specific hypoxia-inducible factor-1α deletion suppresses the development of liver tumors in high-fat diet-fed obese and diabetic mice.

J Diabetes Investig 2019 Nov 11;10(6):1411-1418. Epub 2019 Apr 11.

First Department of Internal Medicine, University of Toyama, Toyama, Japan.

Aims/introduction: Chronic inflammation of the liver is often observed with obesity or type 2 diabetes. In these pathological conditions, the immunological cells, such as macrophages, play important roles in the development or growth of liver cancer. Recently, it was reported that hypoxia-inducible factor-1α (HIF-1α) is a key molecule for the acquisition of inflammatory M1 polarity of macrophages. In the present study, we examined the effects of altered macrophage polarity on obesity- and diabetes-associated liver cancer using macrophage-specific HIF-1α knockout (KO) mice.

Materials And Methods: To induce liver cancer in the mice, diethylnitrosamine, a chemical carcinogen, was used. Both KO mice and wild-type littermates were fed either a high-fat diet (HFD) or normal chow. They were mainly analyzed 6 months after HFD feeding.

Results: Development of liver cancer after HFD feeding was 45% less in KO mice than in wild-type littermates mice. Phosphorylation of extracellular signal-regulated kinase 2 was also lower in the liver of KO mice. Those effects of HIF-1α deletion in macrophages were not observed in normal chow-fed mice. Furthermore, the size of liver tumors did not differ between KO and wild-type littermates mice, even those on a HFD. These results suggest that the activation of macrophage HIF-1α by HFD is involved not in the growth, but in the development of liver cancer with the enhanced oncogenic extracellular signal-regulated kinase 2 signaling in hepatocytes.

Conclusions: The activation of macrophage HIF-1α might play important roles in the development of liver cancer associated with diet-induced obesity and diabetes.
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http://dx.doi.org/10.1111/jdi.13047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825928PMC
November 2019

Development of a practical online supercritical fluid extraction-supercritical fluid chromatography/mass spectrometry system with an integrated split-flow method.

J Chromatogr A 2019 May 19;1592:161-172. Epub 2019 Jan 19.

Division of Metabolomics, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. Electronic address:

Herein, we describe a practical online supercritical fluid extraction-supercritical fluid chromatography/mass spectrometry (SFE-SFC/MS) system with an integrated split-flow method and a pre-column trap method that is well suited for the continuous extraction and separation of a wide range of compounds, including hydrophilic ones. Although an SFE-SFC system with a splitting method is already commercially available, in this study, we added some new features to this system: 1) a splitting method that further reduces the amount of extractant introduced into SFC, 2) a trap column, connected before the analytical column, with a different separation mechanism than the analytical column in the system with the splitting method, and 3) a system for calculating the recovery rate of SFE during online SFE-SFC/MS. In the above setup, part of the analyzed extract is introduced into the separation section at a higher split ratio owing to the make-up pump flow rate, thus reducing the distortion of the target analyte peak shape caused by the use of a strong extractant. Furthermore, the separation efficiency is improved by the use of an additional pre-column capable of interacting with compounds weakly retained on the analytical column. Finally, we show that equalization of the SFE and autosampler injection conditions allows evaluation of the recovery rate of SFE during online SFE-SFC/MS.
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http://dx.doi.org/10.1016/j.chroma.2019.01.044DOI Listing
May 2019
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