Mining for Active Molecules in Probiotic Supernatant by Combining Non-Targeted Metabolomics and Immunoregulation Testing

Chronic respiratory diseases such as asthma are highly prevalent in industrialized countries. As cases are expected to rise, there is a growing demand for alternative therapies. Our recent research on the potential benefits of probiotics suggests that they could prevent and reduce the symptoms of many diseases by modulating the host immune system with secreted metabolites. This article presents the first steps of the research that led us to identify the immunoregulatory bioactivity of the amino acid d-Trp reported in our previous study. Here we analyzed the cell culture metabolic footprinting of 25 commercially available probiotic strains to associate metabolic pathway activity information with their respective immune modulatory activity observed in vitro. Crude probiotic supernatant samples were processed in three different ways prior to untargeted analysis in positive and negative ionization mode by direct infusion ESI-FT-ICR-MS: protein precipitation and solid phase extraction (SPE) using HLB and CN-E sorbent cartridges. The data obtained were submitted to multivariate statistical analyses to distinguish supernatant samples into the bioactive and non-bioactive group. Pathway analysis using discriminant molecular features showed an overrepresentation of the tryptophan metabolic pathway for the bioactive supernatant class, suggesting that molecules taking part in that pathway may be involved in the immunomodulatory activity observed in vitro. This work showcases the potential of metabolomics to drive product development and novel bioactive compound discovery out of complex biological samples in a top-down manner.

Metabolomic Profiling and Immunomodulatory Activity of a Polyherbal Combination in Cyclophosphamide-Induced Immunosuppressed Mice

The study was aimed to develop a characterized polyherbal combination as an immunomodulator containing Phyllanthus emblica L., Piper nigrum L., Withania somnifera (L.) Dunal, and Tinospora cordifolia (Willd.) Miers. Through response surface methodology (RSM), the ratio of aqueous extracts of four plant materials was optimized and comprised 49.76% of P. emblica, 1.35% of P. nigrum, 5.41% of W. somnifera, and 43.43% of T. cordifolia for optimum immunomodulatory activity. The optimized combination showed antioxidant potential and contains more than 180 metabolites, out of which gallic acid, quercetin, ellagic acid, caffeic acid, kaempferitrin, and p-coumaric acid are some common and significant metabolites found in plant extracts and in polyherbal combination. Treatment with the polyherbal combination of different doses in cyclophosphamide-induced immunosuppressed mice significantly (p < 0.01) enhanced the subsets of immune cells such as natural killer (NK) cells (60%), B cells (18%), CD4 cells (14%), and CD8 cells (7%). The characterized polyherbal combination exhibited potent immunomodulatory activity, which can be further explored clinically for its therapeutic applicability.

Phytochemical characterization and biological activities of Plectranthus barbatus Andrews

Plectranthus barbatus Andrews (Lamiaceae) is widely distributed in the world and has a range of popular therapeutic indications. This work aimed to evaluate the phytochemical characterization of two leaf extracts of P. barbatus, and their antimicrobial, antineoplastic and immunomodulatory potential. After collection, herborization and obtainment of the P. barbatus aqueous extract (PBA) and acetone:water 7:3 P. barbatus organic extract (PBO), the phytochemical characterization was performed by high-performance liquid chromatography (HPLC). The antimicrobial activity was performed to determine the minimum inhibitory concentration (MIC) against eight bacterial strains using the microdilution test and the fungus Trichophyton rubrum by disc diffusion assay and microdilution test. Cytotoxicity was assessed by MTT and trypan blue methods in normal peripheral blood mononuclear cells (PBMCs) at concentrations ranged between 0.1 to 100 µg.mL-1 and in neoplastic cell lines Toledo, K562, DU-145 and PANC-1 at 1, 10 and 100 µg.mL-1 . Immunomodulatory activity, was evaluated by sandwich ELISA of proinflammatory cytokines at BALB/c mice splenocytes cultures supernatant. Both extracts presented flavonoids, cinnamic derivatives, steroids and ellagic acid. PBO showed bacteriostatic activity against Acinetobacter baumannii (MIC = 250 µg.mL-1) clinical isolate and PBA fungistatic activity against Trichophyton rubrum (MIC = 800 µg.mL-1). The extracts did not exhibit toxicity to PBMCs and neoplastic cells (IC50 > 100 µg.mL-1). Additionally, PBO at 100 µg.mL-1 significantly inhibited IFN-γ and IL-17A cytokines (p = 0.03). Plectranthus barbatus is a potential candidate for therapeutic use due to its low toxicity in healthy human cells and exhibits biological activities of medical interest as bacteriostatic, fungistatic and immunomodulatory.

Sulfated Chinese yam polysaccharide enhances immunomodulatory activity of RAW 264.7 cells via TLR4-MAPK/NF-κB signaling pathway

In this study, Chinese yam polysaccharide (CYP) was isolated from yam by hydroextraction and alcoholic precipitation. Subsequently, the chlorosulfate-pyridine (CSA-Pyr) method was used to obtain the sulfated Chinese yam polysaccharide...

Role of genistein on the yield, structure and immunomodulatory activity of Monascus exopolysaccharide

Manipulating the structures, physicochemical properties, and monosaccharide compositions of exopolysaccharides (EPS) isolated from microorganisms has been reported to enhance their biological activities. Hence, the aim of this work was to...

Comparison of water- and alkali-extracted polysaccharides from Fuzhuan brick tea and their immunomodulatory effects in vitro and in vivo

In the present study, the purpose is to compare the effect of water extraction and alkali-assisted extraction on the structural characteristics and immunomodulatory activity of polysaccharides from Fuzhuan brick tea...

Macrophage immunomodulatory activity of Acanthopanax senticousus polysaccharide nanoemulsion via activation of P65/JNK/ikkαsignaling pathway and regulation of Th1/Th2 Cytokines

Nanoemulsions (NE) are used widely in pharmaceutical drug formulations and vaccine preparation, and Acanthopanax senticousus polysaccharide (ASPS) is a natural bioactive compound with immunostimulatory activity. Therefore, NE-loaded ASPS is expected to provide immunological enhancement for effective treatment. In the present study, Acanthopanax senticousus polysaccharide (ASPS was encapsulated into nanoemulsions, the resultant ASPS–NE were coated with a negative charge, and the immune enhancement mechanism of these ASPS-NE formulations was analyzed. The immunosuppressive animal models (70 ICR mice, male) for the study were established using cyclophosphamide. In addition, the activation of splenocyte proliferation, phagocytosis of the macrophages, the ratio of CD4+ to CD8+, the concentrations of the cytokines in serum, Western blot analysis was used for the analysis of the P65/JNK/ikk α signaling pathway in the peritoneal macrophage s. The results revealed that the ASPS-NE could stimulated the proliferation of splenocytes and enhance immunity. The ASPS-NE induced the expression of different cytokines (TNF-α, IFN-γ, IL-2, and IL-6), could activate the expressions of P65, JNK, and ikkα, and regulated the Th1/Th2 cytokines. These findings demonstrated the potential of ASPS-NE formulations for drug delivery and to induce potent and sustained immune responses.