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Cimicifuga racemosa extracts (CREs) have gained well-established use for the treatment of menopausal symptoms such as hot flushes and excessive sweating, and weight gain. While the clinical effects of CREs have been well documented, the mechanisms underlying these effects are largely unknown. More recently, the metabolic effects of the CRE Ze 450 were demonstrated in cultured cells in vitro and in mouse models of obesity in vivo. At the molecular level, metabolic regulation, enhanced insulin sensitivity, and increased glucose uptake were linked to the activation of AMP-activated protein kinase (AMPK). Therefore, we tested the effects of Ze 450 on AMPK phosphorylation and thus activation in cells from different tissues, i.e., murine C2C12 myoblast cells, human HEPG2 liver cells, mouse HT22 neuronal cells, and in murine 3T3L1 adipocytes. Using a FRET-based HTRF-assay, we found that Ze 450 induced AMPK phosphorylation and the activation of this key enzyme of metabolic regulation in cells from various different tissues including C2C12 (muscle), HEPG2 (liver), HT22 (hippocampal), and 3T3-L1 (adipocyte) cells. In C2C12 muscle cells, enhanced AMPK activation was accompanied by reduced mitochondrial respiration and enhanced glucose uptake. Further, Ze 450 enhanced the resilience of the cells against oxidative death induced by ferroptosis inducers erastin or RSL3. Our findings suggest a general effect of Cimicifuga racemosa on AMPK activation in different tissues and across species. This may have a significant impact on expanded therapeutic applications of Ze 450, since AMPK activation and the related metabolic effects have been previously associated with anti-aging effects and the prevention of the metabolic syndrome.
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Harpagophytum procumbens (Hp) is often used in the supportive treatment of inflammatory and degenerative diseases of the skeletal system. Although the clinical efficacy in osteoarthritis has been demonstrated in clinical trials, the molecular target(s) of Hp are unclear. This study quantified the effects of the ethanol Hp extract (60% v/v ethanol, sole active ingredient of Pascoe®-Agil), on the expression and release of the major pro-inflammatory mediators in LPS-stimulated human monocytes and the intracellular signalling pathways involved in inflammation. The Hp extract dose-dependently inhibited the release of TNFα as well as that of interleukin (IL)-6, IL-1ß and prostaglandin E2 (PGE2). The Hp prevented TNFα and IL-6 mRNA expression in human monocytes and cyclooxygenase-2 (COX-2) in RAW 264.7 cells. Furthermore, the Hp extract inhibited LPS-stimulated AP-1-mediated gene transcription activity and binding to the AP-1 response elements. The extract had no effect on the LPS-induced binding of nuclear factor-κB in RAW 264.7 cells, on LPS-induced degradation of IκBα or on LPS-induced activation of mitogen-activated protein kinases (MAPK), p38MAPK and JNK in human monocytes. The data indicate that a standardized ethanol Hp extract inhibits induction of pro-inflammatory gene expression, possibly by blocking the AP-1 pathway. This is novel evidence of a possible mechanism of action of this antiinflammatory drug.
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Anti-Inflamatórios não Esteroides/farmacologia , Ciclo-Oxigenase 2/genética , Harpagophytum/química , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Fator de Necrose Tumoral alfa/genética , Animais , Anti-Inflamatórios não Esteroides/química , Linhagem Celular Tumoral , Ciclo-Oxigenase 2/imunologia , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/farmacologia , Dinoprostona/imunologia , Avaliação Pré-Clínica de Medicamentos , Etanol/química , Genes Reporter , Humanos , Interleucina-1beta/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Lipopolissacarídeos/efeitos adversos , Lipopolissacarídeos/imunologia , Sistema de Sinalização das MAP Quinases , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , NF-kappa B/imunologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Ligação Proteica , RNA Mensageiro , Fator de Transcrição AP-1/genética , Transcrição Gênica , Transfecção , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Introduction: In the current study, we evaluated the effectiveness of two well-defined probiotic strains, Lactobacillus paracasei LPc-G110 (CCTCC M 2013691) and Lactobacillus plantarum GOS42 (DSM 32131), during an experimental gingivitis challenge. The primary objective was to evaluate clinically the effectiveness of lozenges containing one of the two oral probiotic strains, compared with placebo lozenges, on the gingival bleeding (bleeding on marginal probing; BOMP change) after a two-week experimental gingivitis period. The secondary objectives were to assess the effects of the test products on gingival health (Modified Gingival Index; MGI), dental plaque accumulation and fluorescence, and the dynamics of immunological and microbiological aspects after the wash-in phase, followed by a two-week period refraining from oral hygiene and a two-week wash-out phase. Methods: This single-center challenge intervention study was a triple-blind randomized placebo-controlled clinical trial with three parallel groups. The full study population consisted of 117 healthy 18-55 years old human volunteers. Subjects were instructed to use one lozenge, 3 times daily after each meal, containing either L. plantarum, L. paracasei, or lozenges without probiotics (placebo group). After a 2-week wash-in period, the subjects were requested to refrain from any form of oral hygiene for 2 weeks. Results: There were no differences in the primary outcome (BOMP change) among the groups. However, gingival health (MGI) in individuals from the groups exposed to the test products recovered better from experimental gingivitis than the individuals in the placebo group (p = 0.021, one-way ANOVA). The two test products inhibited pro-inflammatory cytokine IL-1ß production, measured in saliva, during the experimental gingivitis period. Both test strains significantly reduced bacterial DNA in tongue samples and L. paracasei strain showed stronger microbiome-modulating potential than the L. plantarum strain. Conclusions: The two tested lozenges with the L. paracasei or L. plantarum strains did show potential for beneficial effects for the oral health of the host during experimental gingivitis to the oral ecosystem.
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Passiflora incarnata L. (Passifloraceae) is important in herbal medicine for treating anxiety or nervousness, Generalized Anxiety Disorder (GAD), symptoms of opiate withdrawal, insomnia, neuralgia, convulsion, spasmodic asthma, ADHD, palpitations, cardiac rhythm abnormalities, hypertension, sexual dysfunction and menopause. However, the mechanism of action is still under discussion. Despite gaps in our understanding of neurophysiological processes, it is increasingly being recognized that dysfunction of the GABA system is implicated in many neuropsychiatric conditions, including anxiety and depressive disorders. Therefore, the in vitro effects of a dry extract of Passiflora incarnata (sole active ingredient in Pascoflair® 425 mg) on the GABA system were investigated. The extract inhibited [(3) H]-GABA uptake into rat cortical synaptosomes but had no effect on GABA release and GABA transaminase activity. Passiflora incarnata inhibited concentration dependently the binding of [(3) H]- SR95531 to GABA(A) -receptors and of [(3) H]-CGP 54626 to GABA(B) -receptors. Using the [(35) S]-GTPγS binding assay Passiflora could be classified as an antagonist of the GABA(B) receptor. In contrast, the ethanol- and the benzodiazepine-site of the GABA(A) -receptor were not affected by this extract. In conclusion, the first evidence was shown that numerous pharmacological effects of Passiflora incarnata are mediated via modulation of the GABA system including affinity to GABA(A) and GABA(B) receptors, and effects on GABA uptake.
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Antagonistas GABAérgicos/farmacologia , Passiflora/química , Extratos Vegetais/farmacologia , Ácido gama-Aminobutírico/metabolismo , 4-Aminobutirato Transaminase/metabolismo , Animais , Ansiedade/tratamento farmacológico , Ansiedade/metabolismo , Ligação Competitiva , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Depressão/tratamento farmacológico , Depressão/metabolismo , Masculino , Plantas Medicinais/química , Ratos , Ratos Sprague-Dawley , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Sinaptossomos/efeitos dos fármacos , Sinaptossomos/metabolismoRESUMO
Tumor necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) are pro-inflammatory cytokines involved in acute and chronic inflammatory diseases. Indeed, immunotherapy blocking these 2 cytokines has been developed. Micro-immunotherapy (MI) also uses ultra-low doses (ULD) of pro-inflammatory cytokines, impregnated on lactose-sucrose pillules, to counteract their overexpression. The study has been conducted with 2 objectives: examine the anti-inflammatory effect in vitro and the capacity of 2 unitary medicines, TNF-α (27 CH) and IL-1ß (27 CH), to reduce the secretion of TNF-α in human primary monocytes and THP-1 cells differentiated with phorbol-12-myristate-13-acetate, after lipopolysaccharide (LPS) exposure; then, investigate the presence of particles possibly containing starting materials using tunable resistive pulse sensing technique. The results show that the unitary medicines, tested at 3 pillules concentrations (5.5, 11 and 22 mM), have reduced the secretion of TNF-α in both models by about 10-20% vs. vehicle control, depending on concentration. In this exploratory study, particles (150-1000 nm) have been detected in MI ULD-impregnated pillules and a hypothesis for MI medicines mode of action has been proposed. Conscious that more evaluations are necessary, authors are cautious in the conclusions because the findings described in the study are still limited, and future investigations may lead to different hypothesis.
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BACKGROUND: Tumor necrosis factor-α (TNF-α) and IL-1ß are 2 pro-inflammatory cytokines known to be involved in rheumatic diseases. The therapeutic strategy used in micro-immunotherapy (MI) to reduce chronic inflammation and attenuate pain consists in mainly targeting these 2 cytokines. 2LARTH® is a sublingually administered medicine consisting of lactose-saccharose globules impregnated with ethanolic preparations of immune mediators and nucleic acids at ultra-low doses. PURPOSE: The aim of the study is to explore the effect of the MI medicine on TNF-α and IL-1ß secretion in human primary enriched monocytes exposed to lipopolysaccharide (LPS). MATERIALS AND METHODS: Placebo and active globules were diluted in culture medium to test 5 lactose-saccharose globules concentrations (from 1.75 to 22 mM). Freshly isolated enriched monocytes from 6 healthy donors were treated with or without LPS (10 ng/mL), LPS+ placebo, or LPS+ 2LARTH® for 24 hours. IL-1ß, TNF-α, and IL-6 release were evaluated by ELISA. RESULTS: The medicine has significantly decreased the level of IL-1ß secretion compared with placebo at these concentrations: 22 mM (P<0.0001), 11 mM (P=0.0086), 5.5 mM (P= 0.0254), and compared with untreated LPS control at these concentrations: 22 mM, 11 mM (P=0.0008), and 5.5 mM (P=0.002). The effect of active globules on the reduction of TNF-α release is significant compared with placebo at these concentrations: 22 mM (P=0.0018), 11 mM (P=0.0005), 5.5 mM (P=0.0136), and compared with untreated LPS control at these concentrations: 22 mM (P=0.0021), 11 mM (P=0.0017), 5.5 mM (P=0.0052) and 2.25 mM (P=0.0196). Besides, IL-6 secretion decreased compared with placebo at 22 mM (P=0.0177) and 11 mM (P=0.0031). CONCLUSION: The results indicate that the tested product exerts significant anti-inflammatory effects on human LPS-stimulated monocytes.
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Background: Probiotic bacteria with anti-inflammatory properties have the potential to be of therapeutic benefit in gingivitis. Objective: To evaluate the effects of potential probiotic strains on inflammatory mediators involved in early gingivitis using an ex vivo inflammation model. Methods: Strains were screened in viable and attenuated forms for effects on bacterial lipopolysaccharide (LPS)-stimulated release of interleukins (IL)-1ß, -6 and -8, tumor necrosis factor-α, prostaglandin E2 and 8-isoprostane from human primary monocytes, and then, if anti-inflammatory effects were shown, on IL-1ß-stimulated release of inflammatory mediators from primary gingival fibroblasts. Lead strains were evaluated for optimal dosing, batch-to-batch variation and functional consistency in toothpaste. Results: Twenty-one of 73 strains showed anti-inflammatory effects in monocytes; of which, seven showed effects in both viable and attenuated forms. Seven of 14 strains showed effects in fibroblasts. Strains Lactobacillus paracasei LPc-G110(SYBIO-15) and Lactobacillus plantarum GOS42(SYBIO-41) induced statistically significant dose-dependent reductions in the release of multiple inflammatory mediators from monocytes, which were consistent across batches. Viable L. paracasei LPc-G110 tooth paste significantly reduced IL-6, IL-8 and prostaglandin E2 release from monocytes versus placebo. Conclusion: Strains L. paracasei LPc-G110 and L. plantarum GOS42 have potential for use as probiotics in oral care products to reduce gingival inflammation.
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Black chokeberry has been known to play a protective role in human health due to its high polyphenolic content including anthocyanins and caffeic acid derivatives. In the present study, we first characterized the polyphenolic content of a commercial chokeberry concentrate and investigated its effect on LPS-induced NF-κB activation and release of pro-inflammatory mediators in macrophages in the presence or the absence of sodium selenite. Examination of the phytochemical profile of the juice concentrate revealed high content of polyphenols (3.3%), including anthocyanins, proanthocyanidins, phenolic acids, and flavonoids. Among them, cyanidin-3-O-galactoside and caffeoylquinic acids were identified as the major compounds. Data indicated that chokeberry concentrate inhibited both the release of TNFα, IL-6 and IL-8 in human peripheral monocytes and the activation of the NF-κB pathway in RAW 264.7 macrophage cells. Furthermore, chokeberry synergizes with sodium selenite to inhibit NF-κB activation, cytokine release and PGE2 synthesis. These findings suggest that selenium added to chokeberry juice enhances significantly its anti-inflammatory activity, thus revealing a sound approach in order to tune the use of traditional herbals by combining them with micronutrients.
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Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , NF-kappa B/antagonistas & inibidores , Photinia/química , Polifenóis/química , Selênio/química , Animais , Células Cultivadas , Dinoprostona/metabolismo , Sinergismo Farmacológico , Sucos de Frutas e Vegetais , Humanos , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Macrófagos/metabolismo , Camundongos , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , NF-kappa B/metabolismo , Compostos Fitoquímicos/química , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Selenito de Sódio/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
A reliable method, which can be used for the determination of lonazolac and its hydroxylated and O-sulfated metabolites in cell culture media with methyllonazolac as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20 x 4 mm I.D.) extraction pre-column and subsequent gradient separation on an Xterra MS C18-HT (100 x 3 mm I.D., 3.5 microm particles) analytical column in the back-flush mode. Signal monitoring was done by measurement of fluorescence responses at 273 nm for excitation and 385 nm for emission. Structural identity of analyte peaks was confirmed by liquid chromatography coupled to mass spectroscopy (LC-MS-MS) using an electrospray ionization (ESI) source in the selected reaction monitoring (SRM) mode. Mean recoveries of lonazolac, hydroxylonazolac and lonazolac sulfate, respectively, from the biological matrix were 104.2 +/- 3.5, 96.7 +/- 2.2, and 100.9 +/- 3.5%. The limit of detection (LOD) for the three compounds was about 5 ng/ml using a total sample volume of only 50 microl. Linearity of signal responses versus concentration for all three analytes was accomplished in the range 10-600 ng/ml. The mean values of the coefficients of variation (CV) for quality control samples measured in duplicate at three different days at the 10, 40, 100, and 400 ng/ml level were 4.46 +/- 1.15, 3.94 +/- 2.13 and 4.79 +/- 2.07% for lonazolac, hydroxylonazolac and lonazolac sulfate. The target analytes were sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.
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Anti-Inflamatórios não Esteroides/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Pirazóis/metabolismo , Espectrometria de Fluorescência/métodos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A rapid and sensitive RP-HPLC assay for determination of 6beta-hydroxytestosterone in human hepatocytes with corticosterone as the internal standard is described. The procedure employs on-line sample enrichment using a BioTrap 500 MS (20x4 mm I.D.) extraction pre-column and subsequent gradient separation on a Prontosil 60-5 C(18)-H (250x2 mm I.D., 5 micrometer particle size) analytical column in the back-flush mode using a ternary eluent system composed of methanol, tetrahydrofuran and water. Signal monitoring was done by measurement of the responses from liquid chromatography coupled to mass spectroscopy (LC-MS/MS) using an atmospheric pressure chemical ionization (APCI) source conducted in the selected reaction monitoring (SRM) mode. Mean recoveries of 6beta-hydroxytestosterone from an estimate of the biological matrix, i.e., Dulbecco's modified Eagle medium "High Glucose", ranged from 101.8-104.4% for samples containing the target analyte at the 250, 500 and 1000 ng/ml level. The limit of quantitation (LOQ) was 20 ng/ml at an injection volume of 100 microliter determined in the same matrix. Linearity of signal responses versus concentration for all three analytes was accomplished in the range of 100-4000 ng/ml. Mean values of the coefficients of variation (C.V.) for the target analyte obtained for the concentrations 250, 500 and 1000 ng/ml at 5 different days in quintuplicate ranged from 1.5-7.7% (within-day) and 4.8-7.3% (between-day). The corresponding values for the accuracy ranged from 87.7-106.1% for the within-day and from 98.8-102.5% for the between-day measurements. The target analyte was sufficiently stable at both storage and sample preparation conditions because no substantial deviations between analyte concentrations measured before and after subsequently performed freeze and thaw cycles were observed.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Hepatócitos/metabolismo , Espectrometria de Massas/métodos , Testosterona/metabolismo , Humanos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A rapid and sensitive assay for the determination of dihydroergocryptine (DHEC) in human plasma and urine samples with dihydroergotamine (DHET) as the internal standard was developed. The procedure employs on-line sample preparation using an extraction pre-column and an octadecylsilylsilica (ODS) analytical column. After centrifugation human plasma or urine were injected onto the pre-column, concentrated and extracted, back-flushed onto the analytical column and eluted with a binary methanol--aqueous formic acid gradient. Either determination of DHEC as well of its mono- and dihydroxy-metabolites was performed by measurement of the signal responses from MS detection in the selected reaction monitoring (SRM) mode using the transition of the respective parent ions to the common daughter ion at m/z=270.2 amu. The limit of quantitation (LOQ) for determinations of DHEC in both plasma and urine were 25 pg/ml for injected sample volumes of 400 microl. Proportionality of signal responses versus concentration was accomplished within the range of 25-1000 pg/ml. Recovery of target analyte from plasma was 99%. Mean values of the coefficients of variation (CV) for the target analyte in plasma ranged from 1.7 to 13.8% (within-day) and 5.0 to 9.1% (between-day) and accuracy from 91.7 to 102.6% for the within-day and from 95.8 to 98.8% for the between-day measurements. The corresponding values for determinations in urine were 1.7-14.5% (within-day) and 5.3-11.8% (between-day) for CV and 95.8-110.7% (within-day) and 100.1-104.6% (between-day) for accuracy.