Search Results (20)

Plant flowers are recognized as a rich source of bioactive phenolic compounds. In this study, for the first time, the recovery of antioxidant phenolic compounds from Cytisus striatus flowers (CF) was optimized using microwave-assisted extraction (MAE). The variables (% of ethanol, temperature, and time) were studied using a response surface methodology (RSM). Extraction efficiency was assessed by total phenol content, total flavonoid content, and the antioxidant capacity through DPPH, ABTS, FRAP, and CUPRAC assays. Additionally, cytotoxicity and anti-inflammatory properties were evaluated in different cell lines. The optimal extraction conditions (87.6% ethanol, 160.8 °C and 8.76 min) yielded extracts rich in phenolics (85.9 mg GAE/g CF) and flavonoids (120.3 mg RE/g CF), with strong antioxidant capacity. LC-MS/MS analysis identified 27 phenolic compounds, including chrysin, apigenin, and quercetin derivatives. Cytotoxicity tests showed that CF extract maintained high viability (>80%) in human embryonic kidney (HEK293T) and human lung adenocarcinoma (A549) cells up to 2000 µg/mL, indicating low cytotoxicity. The anti-inflammatory potential was evidenced by a decrease in IL-1β levels and an increase in IL-10 cytokine production in LPS-stimulated macrophages. These results highlight the great potential of CF as a promising bioresource to obtain value-added compounds for the development of functional foods, nutraceuticals, and cosmetic products. Full article

Bladder cancer, which has a rising incidence, is the 10th most common cancer. The transitional cell carcinoma histotype is aggressive and often current therapies are ineffective. We investigated the anti-proliferative effect of quercetin, a natural flavonoid, in combination with the alkylating agent mafosfamide (MFA) on two human bladder cancer cell lines, namely RT112 and J82, representing the progression from low-grade to high-grade tumors, respectively. In both cell types, the combined treatment led to a synergic reduction in cell viability confirmed by a combination index of less than one, though different biological responses were noted. In J82 cells, MFA alone and, to a lesser extent, with quercetin caused cell cycle arrest in the G2/M phase, but only the combined treatment triggered apoptotic cell death. In contrast, in RT112 cells, quercetin induced autophagy, evidenced by the autophagosome formation and the increase in LC-3 lipidation. Interestingly, the synergistic effect was observed only when cells were pre-treated with MFA for 24 h before adding quercetin, not in the reverse order. This suggests that quercetin may help overcome MFA resistance to apoptosis. Although further studies are needed, investigating the combined effects of quercetin and MFA could help elucidate the mechanisms of drug resistance in bladder cancer treatment. Full article

Objectives: We have previously shown that inhibition of the mTORC1 nutrient-sensing complex by rapamycin and mTORC1/mTORC2 inhibition by either Torin-2 or RapaLink-1 have differential effects on the global untargeted metabolomics in in vivo and in vitro cell culture models. Methods: In this study, we leveraged the mummichog Python algorithm to analyze the high-dimension untargeted metabolomics data to model the biochemical pathways and metabolic networks and predict their functional activity. We used pancreatic beta-cell culture (Beta TC6) and incubated the cells with either Rapalink-1, Rapamycin or the vehicle control for 24 h. Cells were harvested and flush-frozen in liquid nitrogen. Cells were extracted in ethanol, and the supernatant was collected. The untargeted metabolomics was performed using the high-resolution mass spectrometry LC-MS/MS HILIC peak detection of ESI-positive and -negative polarity modes. The data were collected using Bruker’s maXis-II ESI-Q-q-TOF coupled to Dionex Ultimate-3000 U(H)PLC system using Sequant ZIC-HILIC 150 × 2.1 mm column (Bruker, Hamburg, Germany). We compared the high-resolution untargeted precision metabolomics (LC-MS/MS) between groups using positive and negative polarity modes to capture both hydrophilic and hydrophobic metabolites. We employed the XCMS plus bioinformatics platform to link mTOR-regulated metabolites to the predicted biological pathways. Statistical significance (p < 0.001) was assessed by ANOVA and Ranked order data by Whitney-Cox followed by ad hoc unpaired t-test. Results: The cluster heatmap deconvolution and cloud plot analysis show the differential pattern of metabolites between Rapamycin and Rapalink-treated pancreatic beta cell lines. Mapping the downstream metabolites data onto predictive metabolic pathways and activity networks revealed that the top pathways affected included the pentose phosphate pathway, dopamine and ubiquinol degradation pathways in the ESI-positive polarity mode, and creatine synthesis/glycine degradation and nicotine degradation pathways in the ESI negative polarity mode. Conclusions: The high-resolution untargeted metabolomics can be leveraged as a proxy of the internal exposome yielding high-dimensional data that provide mechanistic insights into metabolic and signaling pathways, and the underlying biology. This approach will have beneficial applications of the internal exposome in determining the optimal precision nutrition pathways for personalized medicine. Full article

Emerging evidence supports the role of rice bran in cancer prevention. Studies were conducted on multiple rice cultivars. However, limited studies were conducted on rice cultivars in the Middle East. In this study, rice bran growing in Iraq (O. sativa ssp. Japonica, cultivars: Amber Barka) was evaluated for its effect on preventing cancer and stimulating the immune system. Rice bran was collected from local mills in Al-Najaf (south of Iraq). Several solvent extracts (ethanol, methanol, n-hexane, and water) were prepared by maceration. MTT assay was used to measure the antiproliferative effects of extracts against a panel of cancer cell lines. The ability of each extract to induce apoptosis and inhibit angiogenesis was measured using standard ELISA kits. The effect of extracts on the immune system was evaluated using a lymphocyte proliferation assay, a pinocytic activity assay, a phagocytic activity assay, and a Th1/Th2 cytokine detection kit. A microbroth dilution method was used to detect the antimicrobial activity of each extract against different microbial strains. LC–MS analysis was used to detect the phytochemical composition of extracts, while DPPH assay was used to determine the antioxidant activity. For the in vivo study, rice bran was added to mouse fodder at 10% and 20%. Mice were treated for two weeks using mouse fodder supplemented with rice bran. In the third week of the experiment, EMT6/P breast cancer cells (1 × 10⁶ cells/mL) were injected subcutaneously into the abdominal area of each mouse. The dimensions of the grown tumors were measured after 14 days of tumor inoculation. A microbroth dilution method was used to evaluate the antimicrobial activity of rice bran extracts against three bacterial strains. The highest antiproliferative activity was observed in ethanol and n-hexane extracts. Ethanol and methanol extract showed the highest activity to induce apoptosis and inhibit angiogenesis. Both extracts were also effective to enhance immunity by activating lymphocytes and phagocytes proliferation with modulations of cytokine levels. The incorporation of rice bran in mice food caused a 20% regression in tumor development and growth compared with the negative control. All extracts exhibited limited antimicrobial activity against tested microorganisms. Methanol extract showed antioxidant activity with an IC₅₀ value of 114 µg/mL. LC–MS analysis revealed the presence of multiple phytochemicals in rice bran including apiin, ferulic acid, and succinic acid. Rice bran is a rich source of active phytochemicals that may inhibit cancer and stimulate the immune system. Rice bran’s biological activities could be due to the presence of multiple synergistically active phytochemicals. Further studies are needed to understand the exact mechanisms of action of rice bran. Full article

Valorizing the wastes of the food industry sector as additives in foods and beverages enhances human health and preserves the environment. In this study, pomegranate pomace (PP) was obtained from the company Schweppes and exposed to the production of polyphenols and fiber-enriched fractions, which were subsequently included in a strawberry-yogurt smoothie (SYS). The PP is rich in carbohydrates and fibers and has high water-absorption capacity (WAC) and oil-absorption capacity (OAC) values. The LC/MS phenolic profile of the PP extract indicated that punicalagin (199 g/L) was the main compound, followed by granatin B (60 g/L) and pedunculagin A (52 g/L). Because of the high phenolic content of PP extract, it (p ≤ 0.05) has high antioxidant activity with SC50 of 200 µg/mL, besides scavenging 95% of DPPH radicals compared to ascorbic acid (92%); consequently, it reduced lung cancer cell lines’ viability to 86%, and increased caspase-3 activity. Additionally, it inhibited the growth of pathogenic bacteria and fungi i.e., L. monocytogenes, P. aeruginosa, K. pneumonia, A. niger, and C. glabrata, in the 45–160 µg/mL concentration range while killing the tested isolates with 80–290 µg/mL concentrations. These isolates were selected based on the microbial count of spoiled smoothie samples and were identified at the gene level by 16S rRNA gene sequence analysis. The interaction between Spike and ACE2 was inhibited by 75.6%. The PP extract at four levels (0.4, 0.8, 1.2, and 1.4 mg/mL) was added to strawberry-yogurt smoothie formulations. During 2 months storage at 4 °C, the pH values, vitamin C, and total sugars of all SYS decreased. However, the decreases were gradually mitigated in PP-SYS because of the high phenolic content in the PP extract compared to the control. The PP-SYS3 and PP-SYS4 scored higher in flavor, color, and texture than in other samples. In contrast, acidity, fat, and total soluble solids (TSS) increased at the end of the storage period. High fat and TSS content are observed in PP-SYS because of the high fiber content in PP. The PP extract (1.2 and 1.6 mg/mL) decreases the color differences and reduces harmful microbes in PP-SYS compared to the control. Using pomegranate pomace as a source of polyphenols and fiber in functional foods enhances SYS’s physiochemical and sensory qualities. Full article

Atopic dermatitis (AD) is a chronic inflammatory disease characterized by hyperactivated immune reactions in the skin. AD is a prevalent health concern in developing countries, with a particularly high incidence among children. Despite ongoing research on AD, prevention and treatment strategies for patients remain inadequate. In this study, the inhibitory effects of Cornus walteri on AD were investigated. C. walteri (Cornaceae), commonly known as “Walter’s dogwood,” is a deciduous shrub used as a traditional medicine to treat dermatologic inflammation caused by lacquer poisoning. However, the role of C. walteri in AD remains largely unknown. To evaluate its anti-AD potential, we investigated the anti-inflammatory activity of the MeOH extract of C. walteri stems (CWS) using the HaCaT human keratinocyte cell line. CWS reduced the secretion of AD-related chemokines, eotaxin-3/CCL26 and thymus and activation-regulated chemokine (TARC/CCL17). In addition, CWS also inhibited the mRNA expression of macrophage-derived chemokine (MDC/CCL22) and upregulated filaggrin, which plays an essential role in skin barrier functions. To identify the bioactive constituents of CWS, phytochemical investigation of CWS led to the isolation of potential bioactive constituents (1–6), including four triterpenoids, one steroid and one diterpene analog, the structures of which were identified as lupeol (1), betulinic acid (2), 5α-stigmast-3,6-dione (3), 3-O-acetylbetulin (4), betulinic acid methyl ester (5) and norphytan (6) through nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography (LC)–mass spectrometry (MS) analysis. The isolated compounds (1–6) were evaluated for their inhibitory activities against eotaxin-3 expression. Compounds 1, 2 and 3 significantly reduced the levels of eotaxin-3. These findings provided experimental evidence that CWS, particularly active compounds 1, 2 and 3, could be further utilized as potential therapeutic agents to treat AD. Full article

Ginsenoside F1, the metabolite of Rg1, is one of the most important constituents of Panax ginseng. Although the effects of ginsenosides on amyloid beta (Aβ) aggregation in the brain are known, the role of ginsenoside F1 remains unclear. Here, we investigated the protective effect of ginsenoside F1 against Aβ aggregation in vivo and in vitro. Treatment with 2.5 μM ginsenoside F1 reduced Aβ-induced cytotoxicity by decreasing Aβ aggregation in mouse neuroblastoma neuro-2a (N2a) and human neuroblastoma SH-SY5Y neuronal cell lines. Western blotting, real-time PCR, and siRNA analysis revealed an increased level of insulin-degrading enzyme (IDE) and neprilysin (NEP). Furthermore, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis confirmed that ginsenoside F1 could pass the blood–brain barrier within 2 h after administration. Immunostaining results indicate that ginsenoside F1 reduces Aβ plaques in the hippocampus of APPswe/PSEN1dE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mice. Consistently, increased levels of IDE and NEP protein and mRNA were observed after the 8-week administration of 10 mg/kg/d ginsenoside F1. These data indicate that ginsenoside F1 is a promising therapeutic candidate for AD. Full article

This study aimed to define a consortium of lactic acid bacteria (LAB) that will bring added value to dried fresh cheese through specific probiotic properties and the synthesis of bioactive peptides (biopeptides). The designed LAB consortium consisted of three Lactobacillus strains: S-layer carrying Levilactobacillus brevis D6, exopolysaccharides producing Limosilactobacillus fermentum D12 and plantaricin expressing Lactiplantibacillus plantarum D13, and one Enterococcus strain, Enterococcus faecium ZGZA7-10. Chosen autochthonous LAB strains exhibited efficient adherence to the Caco-2 cell line and impacted faecal microbiota biodiversity. The cheese produced by the LAB consortium showed better physicochemical, textural and sensory properties than the cheese produced by a commercial starter culture. Liquid chromatography coupled with matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (LC-MALDI-TOF/TOF) showed the presence of 18 specific biopeptides in dried fresh cheeses. Their identification and relative quantification was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM). The results also showed that their synthesis resulted mainly from β-casein and also α-S1 casein degradation by proteolytic activities of the LAB consortium. The designed LAB consortium enhanced the functional value of the final product through impact on biopeptide concentrations and specific probiotic properties. Full article

Rearranged during transfection (RET) is a tyrosine kinase oncogenic receptor, activated in several cancers including non-small-cell lung cancer (NSCLC). Multiple kinase inhibitors vandetanib and cabozantinib are commonly used in the treatment of RET-positive NSCLC. However, specificity, toxicity, and reduced efficacy limit the usage of multiple kinase inhibitors in targeting RET protein. Thus, in the present investigation, we aimed to figure out novel and potent candidates for the inhibition of RET protein using combined in silico and in vitro strategies. In the present study, screening of 11,808 compounds from the DrugBank repository was accomplished by different hypotheses such as pharmacophore, e-pharmacophore, and receptor cavity-based models in the initial stage. The results from the different hypotheses were then integrated to eliminate the false positive prediction. The inhibitory activities of the screened compounds were tested by the glide docking algorithm. Moreover, RF score, Tanimoto coefficient, prime-MM/GBSA, and density functional theory calculations were utilized to re-score the binding free energy of the docked complexes with high precision. This procedure resulted in three lead molecules, namely DB07194, DB03496, and DB11982, against the RET protein. The screened lead molecules together with reference compounds were then subjected to a long molecular dynamics simulation with a 200 ns time duration to validate the inhibitory activity. Further analysis of compounds using MM-PBSA and mutation studies resulted in the identification of potent compound DB07194. In essence, a cell viability assay with RET-specific lung cancer cell line LC-2/ad was also carried out to confirm the in vitro biological activity of the resultant compound, DB07194. Indeed, the results from our study conclude that DB07194 can be effectively translated for this new therapeutic purpose, in contrast to the properties for which it was originally designed and synthesized. Full article

Efforts to direct the specification of human pluripotent stem cells (hPSCs) to therapeutically important somatic cell types have focused on identifying proper combinations of soluble cues. Yet, whether exosomes, which mediate intercellular communication, play a role in the differentiation remains unexplored. We took a first step toward addressing this question by subjecting hPSCs to stage-wise specification toward cardiomyocytes (CMs) in scalable stirred-suspension cultures and collecting exosomes. Samples underwent liquid chromatography (LC)/mass spectrometry (MS) and subsequent proteomic analysis revealed over 300 unique proteins from four differentiation stages including proteins such as PPP2CA, AFM, MYH9, MYH10, TRA2B, CTNNA1, EHD1, ACTC1, LDHB, and GPC4, which are linked to cardiogenic commitment. There was a significant correlation of the protein composition of exosomes with the hPSC line and stage of commitment. Differentiating hPSCs treated with exosomes from hPSC-derived CMs displayed improved efficiency of CM formation compared to cells without exogenously added vesicles. Collectively, these results demonstrate that exosomes from hPSCs induced along the CM lineage contain proteins linked to the specification process with modulating effects and open avenues for enhancing the biomanufacturing of stem cell products for cardiac diseases. Full article

(1) Background: Ellagitannins are natural products occurring in pomegranate and walnuts. They are hydrolyzed in the gut to release ellagic acid, which is further metabolized by the microflora into urolithins, such as urolithin A (UA). Accumulation of damaged mitochondria is a hallmark of aging and age-related neurodegenerative diseases. In this study, we investigated the neuroprotective activity of the metabolite UA against mitochondrial dysfunction in a cellular model of early Alzheimer disease (AD). (2) Methods: In the present study we used SH-SY5Y-APP695 cells and its corresponding controls (SH-SY5Ymock) to assess UA’s effect on mitochondrial function. Using these cells we investigated mitochondrial respiration (OXPHOS), mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) production, autophagy and levels of reactive oxygen species (ROS) in cells treated with UA. Furthermore, we assessed UA’s effect on the expression of genes related to mitochondrial bioenergetics, mitochondrial biogenesis, and autophagy via quantitative real-time PCR (qRT-PCR). (3) Results: Treatment of SH-SY5Y-APP695 cells suggests changes to autophagy corresponding with qRT-PCR results. However, LC3B-I, LC3B-II, and p62 levels were unchanged. UA (10 µM) reduced MMP, and ATP-levels. Treatment of cells with UA (1 µM) for 24 h did not affect ROS production or levels of Aβ, but significantly increased expression of genes for mitochondrial biogenesis and OXPHOS. Mitochondrial Transcription Factor A (TFAM) expression was specifically increased in SH-SY5Y-APP695. Both cell lines showed unaltered levels of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), which is commonly associated with mitochondrial biogenesis. Results imply that biogenesis might be facilitated by estrogen-related receptor (ESRR) genes. (4) Conclusion: Urolithin A shows no effect on autophagy in SH-SY5Y-APP695 cells and its effect on mitochondrial function is limited. Instead, data suggests that UA treatment induces hormetic effects as it induces transcription of several genes related to mitochondrial biogenesis. Full article

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities. Full article

High-density lipoprotein (HDL) plays a vital role in lipid metabolism and anti-inflammatory activities; a dysfunctional HDL impairs cholesterol efflux pathways. To understand HDL’s role in patients with Alzheimer’s disease (AD), we analyzed the chemical properties and function. HDL from AD patients (AD-HDL) was separated into five subfractions, H1–H5, using fast-protein liquid chromatography equipped with an anion-exchange column. Subfraction H5, defined as the most electronegative HDL, was increased 5.5-fold in AD-HDL (23.48 ± 17.83%) in comparison with the control HDL (4.24 ± 3.22%). By liquid chromatography mass spectrometry (LC/MS^E), AD-HDL showed that the level of apolipoprotein (apo)CIII was elevated but sphingosine-1-phosphate (S1P)-associated apoM and anti-oxidative paraoxonase 1 (PON1) were reduced. AD-HDL showed a lower cholesterol efflux capacity that was associated with the post-translational oxidation of apoAI. Exposure of murine macrophage cell line, RAW 264.7, to AD-HDL induced a vibrant expression of ganglioside GM1 in colocalization with apoCIII on lipid rafts alongside a concomitant increase of tumor necrosis factor-α (TNF-α) detectable in the cultured medium. In conclusion, AD-HDL had a higher proportion of H5, an apoCIII-rich electronegative HDL subfraction. The associated increase in pro-inflammatory (apoCIII, TNF-α) components might favor Amyloid β assembly and neural inflammation. A compromised cholesterol efflux capacity of AD-HDL may also contribute to cognitive impairment. Full article

The development of antibiotic resistance by microbials has long been acknowledged. The major challenge worldwide is to develop novel, natural, and potent antibiotics against the multidrug resistant bacteria. In this study, our aim was to develop the method for a highly sensitive instrument, ultra-high performance liquid chromatograph-high resolution mass spectrometer (UHPLC-HRMS), to evaluate the antibacterial property of a natural product. Aechmea magdalenae (Andre) Andre ex Baker, a plant belonging to the family Bromeliaceae, a native of Central America was used in this study. Based on the available literature, it was hypothesized that Aechmea magdalenae has antibacterial activity. In addition, the profiling done on A. magdalenae using gas chromatography-mass spectrometry (GC-MS) also revealed the presence of medicinally important chemical compounds, such as acetic acid. Minimum inhibitory concentration (MIC) of dried Aechmea plant extract was determined for the first time using 96-well plate assay, followed by determination of antibacterial potential using LC-MS. The reason being that other dried methanolic plant extracts, such as Vismia macrophylla, lined up for antibacterial testing have dark extracts, for which determining the antibacterial potential and reading the results with the naked eye would be challenging. To overcome the situation of dark plant extracts, a generalized novel LC-MS method was developed that was used for the plant A. magdalenae, and would be used further for other plants. A blue indicator called resazurin was added to the wells; resazurin, upon incubation with the living cells, got reduced to resorufin (which was pink), while it remained blue with bacterial growth inhibition. The mass difference created due to reduction of resazurin to resorufin was detected by using LTQ Orbitrap Discovery in positive ion mode to determine the antibacterial activity of the plant extract. The sample preparation for LC-MS assay included centrifugation of the samples taken from 96-well plate, followed by filtration of the supernatant, before exposing them to C-18 column. The results obtained from full scan LC-MS spectrum consistently demonstrated the presence of resorufin from wells with bacterial growth, and resazurin from wells with inhibition through peaks of relevant masses. Full article

One of several mechanisms that leads to the development of dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) is called antibody-dependent enhancement (ADE). Monocytes can be infected by the ADE phenomenon, which occurs in dengue secondary infection. This study aimed to investigate the proteins involved in ADE of DENV infection in the human monocytic cell line U937. The phosphoproteins were used to perform and analyze for protein expression using mass spectrometry (GeLC-MS/MS). The differential phosphoproteins revealed 1131 altered proteins compared between isotype- and DENV-specific antibody-treated monocytes. The altered proteins revealed 558 upregulated proteins and 573 downregulated proteins. Protein disulfide isomerase (PDI), which is an enzyme that had a high-ranking fold change and that catalyzes the formation, breakage, and rearrangement of disulfide bonds within a protein molecule, was selected for further study. PDI was found to be important for dengue virus infectivity during the ADE model. The effect of PDI inhibition was also shown to be involved in the early stage of life cycle by time-of-drug-addition assay. These results suggest that PDI is important for protein translation and virion assembly of dengue virus during infection in human monocytes, and it may play a significant role as a chaperone to stabilize dengue protein synthesis. Full article