Identification and Quantification of Bioactive Molecules Inhibiting Pro-inflammatory Cytokine Production in Spent Coffee Grounds Using Metabolomics Analyses.

In this study, we assessed the anti-inflammatory properties of spent coffee grounds. Methanolic extracts of spent coffee grounds obtained from 3 Arabica cultivars possess compounds that exerted inhibitory effects on the secretion of inflammatory mediators (TNF-α, IL-6, and IL-10) induced by a human pro-monocytic cell line differentiated with PMA and stimulated with lipopolysaccharide (LPS). Our results indicated that the cytokine suppressive activities of the spent coffee ground (SCG) extracts were different among coffee cultivars tested. Hawaiian Kona extracts exhibited inhibitory effects on the expression of 3 examined cytokines, Ethiopian Yirgacheffe extracts reduced the secretion of TNF-α and IL-6, and Costa Rican Tarrazu extracts decreased the secretion of IL-6 only. Untargeted metabolomics analyses of SCG extracts led to the putative identification of 26 metabolites with known anti-inflammatory activities. Multiple metabolites (i.e., chrysin, daidzein, eugenol, naringenin, naringin, oxyresveratrol, pectolinarin, resveratrol, tectochrysin, theaflavin, vanillic acid, and vitexin rhamnoside) identified in the SCGs represent possible novel anti-inflammatory compounds. Of the 26 identified metabolites, the 12 compounds that had high relative intensities in all of the extracts were successfully quantified using liquid chromatography-tandem mass spectrometry analyses. Results from the targeted analyses indicated that caffeine and 5-caffeoylquinic acid (CQA) were the most abundant compounds in the SCG extracts. The contents of caffeine ranged from 0.38 mg/g (Ethiopian Yirgacheffe) - 0.44 mg/g (Costa Rican Tarrazu), whereas 5-CQA concentrations were in the range of 0.24 mg/g (Costa Rican Tarrazu) - 0.34 mg/g (Ethiopian Yirgacheffe). The presence of multiple anti-inflammatory compounds in SCGs provides a promising natural source for cosmetic and pharmaceutical industries.

Black Walnut (Juglans nigra) Extracts Inhibit Proinflammatory Cytokine Production From Lipopolysaccharide-Stimulated Human Promonocytic Cell Line U-937.

Black walnut (Juglans nigra L.) is an excellent source of health-promoting compounds. Consumption of black walnuts has been linked to many health benefits (e.g., anti-inflammatory) stemming from its phytochemical composition and medicinal properties, but these effects have not been systematically studied or characterized. In this study, potential anti-inflammatory compounds found in kernel extracts of 10 black walnut cultivars were putatively identified using a metabolomic profiling analysis, revealing differences in potential anti-inflammatory capacities among examined cultivars. Five cultivars were examined for activities in the human promonocytic cell line U-937 by evaluating the effects of the extracts on the expression of six human inflammatory cytokines/chemokines using a bead-based, flow cytometric multiplex assay. The methanolic extracts of these cultivars were added at four concentrations (0.1, 0.3, 1, and 10 mg/ml) either before and after the addition of lipopolysaccharide (LPS) to human U-937 cells to examine their effect on cytokine production. Results from cytotoxicity and viability assays revealed that the kernel extracts had no toxic effect on the U-937 cells. Of the 13 cytokines [interleukin (IL)-1ß, tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein (MCP)-1, IL-6, IL-8, IL-10, IL-12, IL-17, IL-18, IL-23, IL-33, interferon (IFN)-α, IFN-γ] measured, only six were detected under the culture conditions. The production of the six detected cytokines by phorbol 12-myristate 13-acetate (PMA)-differentiated, LPS-stimulated U-937 was significantly inhibited by the kernel extracts from two cultivars Surprise and Sparrow when the extracts were added before the addition of LPS. Other cultivars (Daniel, Mystry, and Sparks) showed weak or no significant effects on cytokine production. In contrast, no inhibitory effect was observed on the production of cytokines by PMA-differentiated, LPS-stimulated U-937 when the kernel extracts were added after the addition of LPS. The findings suggest that the extracts from certain black walnut cultivars, such as Sparrow and Surprise, are promising biological candidates for potentially decreasing the severity of inflammatory disease.

Puralpha and Purbeta collaborate with Sp3 to negatively regulate beta-myosin heavy chain gene expression during skeletal muscle inactivity.

Adult skeletal muscle retains the capability of transcriptional reprogramming. This attribute is readily observable in the non-weight-bearing (NWB) soleus muscle, which undergoes a slow-to-fast fiber type transition concurrent with decreased beta-myosin heavy chain (betaMyHC) gene expression. Our previous work showed that Sp3 contributes to decreased betaMyHC gene expression under NWB conditions. In this study, we demonstrate that physical and functional interactions between Sp3, Puralpha, and Purbeta proteins mediate repression of betaMyHC expression under NWB conditions. Binding of Puralpha or Purbeta to the single-stranded betaMyHC distal negative regulatory element-sense strand (dbetaNRE-S) element is markedly increased under NWB conditions. Ectopic expression of Puralpha and Purbeta decreased betaMyHC reporter gene expression, while mutation of the dbetaNRE-S element increased expression in C2C12 myotubes. The dbetaNRE-S element conferred Pur-dependent decreased expression on a minimal thymidine kinase promoter. Short interfering RNA sequences specific for Sp3 or for Puralpha and Purbeta decreased endogenous Sp3 and Pur protein levels and increased betaMyHC reporter gene expression in C2C12 myotubes. Immunoprecipitation assays revealed an association between endogenous Puralpha, Purbeta, and Sp3, while chromatin immunoprecipitation assays demonstrated Puralpha, Purbeta, and Sp3 binding to the betaMyHC proximal promoter region harboring the dbetaNRE-S and C-rich elements in vivo. These data demonstrate that Pur proteins collaborate with Sp3 to regulate a transcriptional program that enables muscle cells to remodel their phenotype.