PMID: 

Environ Sci Pollut Res Int. 2017 Sep ;24(27):21693-21699. Epub 2017 Jul 29. PMID: 28756602

Abstract Title: 

Anxiety-like behavioural effects of extremely low-frequency electromagnetic field in rats.

Abstract: 

In recent years, extremely low-frequency electromagnetic field (ELF-EMF) has received considerable attention for its potential biological effects. Numerous studies have shown the role of ELF-EMF in behaviour modulation. The aim of this study was to investigate the effect of short-term ELF-EMF (50 Hz) in the development of anxiety-like behaviour in rats through change hypothalamic oxidative stress and NO. Ten adult male rats (Wistar albino) were divided in two groups: control group-without exposure to ELF-EMF and experimental group-exposed to ELF-EMF during 7 days. After the exposure, timeopen field test and elevated plus maze were used to evaluate the anxiety-like behaviour of rats. Upon completion of the behavioural tests, concentrations of superoxide anion (O·), nitrite (NO, as an indicator of NO) and peroxynitrite (ONOO) were determined in the hypothalamus of the animals. Obtained results show that ELF-EMF both induces anxiety-like behaviour and increases concentrations of O·and NO, whereas it did not effect on ONOOconcentration in hypothalamus of rats. In conclusion, the development of anxiety-like behaviour is mediated by oxidative stress and increased NO concentration in hypothalamus of rats exposed to ELF-EMF during 7 days.

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PMID: 

Climacteric. 2018 10 ;21(5):446-453. PMID: 30269660

Abstract Title: 

Effects of red clover on perimenopausal and postmenopausal women's blood lipid profile: A meta-analysis.

Abstract: 

The study aimed to perform a systematic review with meta-analysis of randomized controlled trials (RCTs) to access the effects of red clover isoflavones on the blood lipid profile of both perimenopausal and postmenopausal women. PubMed, Web of Science, Scopus, SciELO, and Cochrane Library were searched for the terms 'red clover', 'Trifolium pratense', and 'randomized controlled trial' during November 2017. Summary measures were reported as weighted mean differences (WMD) with 95% confidence interval (CI). Fixed or random effects models were used for meta-analyses, according to heterogeneity. Risk of bias was measured with the Cochrane tool. Twelve RCTs (totalizing 1284 perimenopausal and postmenopausal women receiving red clover isoflavones for 4 weeks-18 months) resulted in a significant decrease in total cholesterol (WMD = -12.34 mg/dl; 95% CI: -18.21, -6.48), low-density lipoprotein cholesterol (WMD = -10.61 mg/dl; 95% CI: -15.51, -5.72), and triglycerides (WMD = -10.18 mg/dl; 95% CI: -16.23, -4.13) together with asignificant increase in high-density lipoprotein cholesterol (WMD = 1.60 mg/dl; 95% CI: 0.17, 3.03). In conclusion, the results demonstrate that the ingestion of red clover may have a beneficial effect on the lipid profile of perimenopausal and postmenopausal women.

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PMID: 

Cell Physiol Biochem. 2018 ;46(1):389-400. Epub 2018 Mar 23. PMID: 29590658

Abstract Title: 

Effects of Fifty-Hertz Electromagnetic Fields on Granulocytic Differentiation of ATRA-Treated Acute Promyelocytic Leukemia NB4 Cells.

Abstract: 

BACKGROUND/AIMS: Life on Earth is constantly exposed to electromagnetic fields (EMFs) and the effects induced by EMFs on biological systems have been extensively studied producing different and sometimes contradictory results. Extremely low-frequency electromagnetic fields (ELF-EMFs) have shown to play a role in regulating cell proliferation and differentiation, although how EMFs influence these processes remains unclear. Human acute promyelocytic leukemia (APL) cells are characterized by the arrest of differentiation at the promyelocytic stage due to epigenetic perturbations induced by PML/RARα fusion protein (Promyelocytic Leukemia protein – PML/Retinoic Acid Receptor alpha – RARα). Therapeutic administration of all-trans retinoic acid (ATRA) re-establishes the leukemogenic mechanism re-inducing the normal differentiation processes.METHODS: We studied the effects of ELF-EMFs (50 Hz, 2 mT) on the ATRA-mediated granulocytic differentiation process of APL NB4 cells (a cell line established from the bone marrow of a patient affected by the acute promyelocytic leukemia) by monitoring cellular proliferation and morphology, nitrob lue tetrazolium (NBT) reduction and the expression of differentiation surface markers. Finally, we investigated mechanisms focusing on reactive oxygen species (ROS) generation and related molecular pathways.RESULTS: ELF-EMF exposure decreases cellular proliferation potential and helps ATRA-treated NB4 cells to mature. Furthermore, the analysis of ROS production and the consequent extracellular signal regulated kinases (ERK1/2) phosphorylation suggest that a changed intracellular oxidative balance may influence the biological effects of ELF-EMFs.CONCLUSIONS: These results indicate that the exposure to ELF-EMF promotes ATRA-induced granulocytic differentiation of APL cells.

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PMID: 

Sci Rep. 2017 09 13 ;7(1):11470. Epub 2017 Sep 13. PMID: 28904402

Abstract Title: 

Power frequency magnetic field promotes a more malignant phenotype in neuroblastoma cells via redox-related mechanisms.

Abstract: 

In accordance with the classification of the International Agency for Research on Cancer, extremely low frequency magnetic fields (ELF-MF) are suspected to promote malignant progression by providing survival advantage to cancer cells through the activation of critical cytoprotective pathways. Among these, the major antioxidative and detoxification defence systems might be targeted by ELF-MF by conferring cells significant resistance against clinically-relevant cytotoxic agents. We investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field was supported by improved defence towards reactive oxygen species (ROS) and xenobiotics, as well as by reduced vulnerability against both HOand anti-tumor ROS-generating drug doxorubicin. ELF-MF induced a proliferative and survival advantage by activating key redox-responsive antioxidative and detoxification cytoprotective pathways that are associated with a more aggressive behavior of neuroblastoma cells. This was coupled with the upregulation of the major sirtuins, as well as with increased signaling activity of the erythroid 2-related nuclear transcription factor 2 (NRF2). Interestingly, we also showed that the exposure to 50 Hz MF as low as 100 µT may still be able to alter behavior and responses of cancer cells to clinically-relevant drugs.

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PMID: 

Arch Physiol Biochem. 2018 Oct 15:1-8. Epub 2018 Oct 15. PMID: 30320514

Abstract Title: 

Anti-inflammatory potential of Trifolium pratense L. leaf extract in LPS-stimulated RAW264.7 cells and in a rat model of carrageenan-induced inflammation.

Abstract: 

This study evaluated the anti-inflammatory potential of a 40% prethanol extract of Trifolium pratense leaves (40% PeTP) using in vitro (RAW264.7 cells) and in vivo (carrageenan-induced inflammation model) experiments. Pretreatment with 40% PeTP significantly inhibited the LPS-induced expression of nitric oxide (NO), prostaglandin E(PGE), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and inflammatory cytokines, including tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in RAW264.7 cells, without inducing cytotoxicity. The inhibitory effects of 40% PeTP are mediated through suppression of the nuclear translocation of nuclear factor (NF)-κB and the phosphorylation of mitogen-activated protein kinases (MAPKs). Oral administration of 40% PeTP at 50, 100, and 200 mg/kg of body weight suppressed carrageenan-induced oedema in a dose-dependent manner. Collectively, our results suggested that 40% PeTP exerts potential anti-inflammatory effects by suppressing the activation of the NF-κB and MAPK pathways in vitro, and by reducingcarrageenan-induced paw oedema in vivo.

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PMID: 

Nutr Cancer. 2019 ;71(1):128-140. Epub 2018 Dec 29. PMID: 30596276

Abstract Title: 

Antiproliferative Effect of Trifolium Pratens L. Extract in Human Breast Cancer Cells.

Abstract: 

Breast cancer is currently the most common cancer in women worldwide. Tamoxifen is used for the treatment of both early and advanced estrogen receptor positive breast cancer. Trifolium pratense L. has been suggested for cancer treatment in traditional medicine. The effect of T. pratense extract on breast cell lines (MCF-7 and MDA-MB-231) was investigated here. The cells were treated with T. pratense extract and cell viability was evaluated using trypan blue staining, MTT assay and lactate dehydrogenase activity measurement. Apoptosis, autophagy cell death was detected using fluorescent dye staining. Griess assay was performed to nitric oxide (No) measurement. Change in expression level of some apoptotic and autophagic- related genes was detected using real-time PCR. The combination effect of exteract and tamoxifen was evaluated by calculating the combination index and dose reduction index (DRI) values. T. pratense extract reduced the cell viability in dose and time- dependent manner. Apoptosis and autophagy was increased after treatment with T. pratense extract. Nitric oxide production significantly reduced in cells and combination of tamoxifen and T. pratense extract had a synergistic cytotoxic effect on breast cancer cell lines. T. pratense as a dietary supplement can be beneficial for some patients with breast cancer who receive conventional chemotherapy.

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PMID: 

Int J Radiat Biol. 2016 10 ;92(10):596-602. Epub 2016 Jul 21. PMID: 27442448

Abstract Title: 

NADPH oxidase-produced superoxide mediated a 50-Hz magnetic field-induced epidermal growth factor receptor clustering.

Abstract: 

PURPOSE: A 50-Hz magnetic field (MF) was found to induce epidermal growth factor receptor (EGFR) clustering in our previous study. The aim of this work was to investigate the molecular mechanisms that mediated MF-induced EGFR clustering.MATERIALS AND METHODS: Human amniotic epithelial (FL) cells were exposed to a 50-Hz MF. Total reactive oxygen species (ROS), cytoplasmic and mitochondrial superoxide production were detected by DCFH-DA, DHE and MitoSOX, respectively. EGFR clustering was analyzed using confocal microscopy after indirect immunofluorescence staining.RESULTS: Results showed that exposing FL cells to MF at intensity higher than 0.2 mT for 15 min enhanced total ROS production. Additionally, enhanced total ROS and cytoplasmic superoxide production were observed after exposing cells to MF at 0.4 mT for 5, 15, or 30 min, while mitochondrial superoxide production for 15 or 30 min. Pretreatment with Nox inhibitor, DPI, effectively inhibited MF-induced cytoplasmic superoxide production and subsequent EGFR clustering while mitochondrial superoxide production was not affected.CONCLUSIONS: Nox-produced superoxide mediated a 50-Hz magnetic field-induced EGFR clustering.

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PMID: 

Int J Radiat Biol. 2016 ;92(3):148-55. Epub 2016 Feb 5. PMID: 26850078

Abstract Title: 

Exposure to a 50-Hz magnetic field induced mitochondrial permeability transition through the ROS/GSK-3β signaling pathway.

Abstract: 

PURPOSE: To investigate the biological effects of a 50-Hz magnetic field (MF) on mitochondrial permeability.MATERIALS AND METHODS: Human amniotic epithelial cells were exposed to MF (50 Hz, 0.4 mT) for different durations. Mitochondrial permeability, mitochondrial membrane potential (ΔΨm), cytochrome c (Cyt-c) release and the related mechanisms were explored.RESULTS: Exposure to the MF at 0.4 mT for 60 min transiently induced mitochondrial permeability transition (MPT) and Cyt-c release, although there was no significant effect on mitochondrial membrane potential (ΔΨm). Other than decreasing the total Bcl-2 associated X protein (Bax) level, MF exposure did not significantly affect the levels of Bax and B-cell lymphoma-2 (Bcl-2) in mitochondria. In addition, cells exposed to the MF showed increased intracellular reactive oxidative species (ROS) levels and glycogen synthase kinase-3β (GSK-3β) dephosphorylation at 9 serine residue (Ser(9)). Moreover, the MF-induced MPT was attenuated by ROS scavenger (N-acetyl-L-cysteine, NAC) or GSK-3β inhibitor, and NAC pretreatment prevented GSK-3β dephosphorylation (Ser(9)) caused by MF exposure.CONCLUSION: MPT induced by MF exposure was mediated through the ROS/GSK-3β signaling pathway.

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PMID: 

Toxicol Lett. 2010 Feb 15 ;192(3):330-6. Epub 2009 Nov 12. PMID: 19913603

Abstract Title: 

Exposure to ELF magnetic fields modulate redox related protein expression in mouse macrophages.

Abstract: 

The interaction of extremely low frequency (ELF) magnetic fields (MF) with cells can induce alterations in various cell physiological processes. Here, we present evidence that exposure of mouse macrophages to 50 Hz, 1.0 mT MF lead to immune cell activation seen as increased production of reactive oxygen species (ROS), and also to modulation on the expression level of important proteins acting in redox regulatory processes and thus explaining the noted changes in ROS levels seen after exposure. The MF exposure caused slight and transient decreases after short term exposures (2h or less) of clathrin, adaptin, PI3-kinase, protein kinase B (PKB) and PP2A, whereas longer exposures had no effect. The levels of the NAD(P)H oxidase subunit gp91phox oscillated between increased and normal levels compared to controls. The stress proteins Hsp70 and Hsp110 exhibited increased levels at certain time points, but not generally. The effects of MF on protein levels are different from the effects exerted by 12-O-tetradecanolyphobol-13-acetate (TPA) or LPS, although all three factors cause increases in ROS release. This suggests that ELF MF interacts with other cellular constituents than these chemicals, although induced pathways at least partially converge.

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PMID: 

J Food Biochem. 2019 Sep ;43(9):e12981. Epub 2019 Jul 21. PMID: 31489654

Abstract Title: 

Structural characterization and anti-inflammatory activity evaluation of chemical constituents in the extract of Trifolium repens L.

Abstract: 

The chemical constituents in Trifolium repens L. were comprehensively studied by UPLC in this work, and a total number of 308 compounds were detected with 169 ones identified. The possible fragmentation pathways were proposed and fragmentation rules were summarized. On the basis of the concluded strategies, the characterized compounds could be classified into organic acids and their derivatives, alkaloids, amino acids, peptides, flavonoids, oligosaccharides, coumarins, and other types of compounds. This approach provided a rapid way for the identification of constituents in T. repens L., and even in other complex analytes. Among the separation and identification of the constituents, three compounds of great amount were isolated and characterized by NMR. The expression of iNOS and COX-2 in LPS-induced RAW 264.7 cells was suppressed by the pretreatment with three isolated constituents. The results implied they may potentially serve as a remedy for the therapy of inflammation. PRACTICAL APPLICATIONS: This work provided a rapid method for the identification of the complex analyte, which could be used in TCM, natural food and so on. The summarized fragmentation rule could be applied for the analysis of several types of compounds, such as organic acids and their derivatives, alkaloids, amino acids and peptides, flavonoids, oligosaccharides, coumarins, and so on. Most of natural plants contain these kinds of compounds, so these rules could have wide applications. Except the phytochemical investigation, T. repens L. displayed anti-inflammation activity according to the reported literature, and the three isolated constituents may potentially serve as a remedy for the therapy of inflammation referring to the result of this research.

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