In Vitro Digestion Assessment (Standard vs. Older Adult Model) on Antioxidant Properties and Mineral Bioaccessibility of Fermented Dried Lentils and Quinoa.

The growing number of older adults necessitates tailored food options that accommodate the specific diseases and nutritional deficiencies linked with ageing. This study aims to investigate the influence of age-related digestive conditions in vitro on the phenolic profile, antioxidant activity, and bioaccessibility of minerals (Ca, Fe, and Mg) in two types of unfermented, fermented, and fermented dried quinoa and lentils. Solid-state fermentation, combined with drying at 70 °C, significantly boosted the total phenolic content in Castellana and Pardina lentils from 5.05 and 6.6 to 10.5 and 7.5 mg gallic acid/g dry weight, respectively, in the bioaccessible fraction following the standard digestion model, compared to the unfermented samples. The phenolic profile post-digestion revealed elevated levels of vanillic and caffeic acids in Castellana lentils, and vanillic acid in Pardina lentils, while caffeic acids in Castellana lentils were not detected in the bioaccessible fraction. The highest antioxidant potency composite index was observed in digested fermented dried Castellana lentils, with white quinoa samples exhibiting potency above 80%. Mineral bioaccessibility was greater in fermented and fermented dried samples compared to unfermented ones. Finally, the digestive changes that occur with ageing did not significantly affect mineral bioaccessibility, but compromised the phenolic profile and antioxidant activity.

Effect of the seed coating with biomass of Dunaliella salina on early plant growth and in the secondary metabolites content of Coriandrum sativum.

The environmental and health risks associated with the application of synthetic chemical inputs in agriculture increased the demand for technologies that allow higher performance and quality of vegetable crops by implementing synergistic materials with the principles of sustainability. In this work, the seed coating with the biomass of Dunaliella salina incorporated in a bioplastic film of Manihot esculenta (cassava) was evaluated as an initial growth and secondary compounds stimulator of Coriandrum sativum (coriander) plants. The obtained results demonstrated that the coating stimulated an increase in the germination percentage (28.75%) and also in concentration of bioactive compounds, such as the six-fold increment of caffeic acid (13.33 mg 100 g-1). The carbohydrates, lipids, and proteins present in the microalgae biomass seem to be responsible for these increments once they are known for providing energy to the seedling development and coordinating the secondary metabolites synthesis. As conclusion, we consider the coating with biomass of D. salina an alternative for crop improvement that contributes to the development of sustainable agricultural practices.

A reverse catalytic triad Asp containing loop shaping a wide substrate binding pocket of a feruloyl esterase from Lactobacillus plantarum.

Feruloyl esterase is an indispensable biocatalyst in food processing, pesticide and pharmaceutical industries, catalyzing the cleavage of the ester bond cross-linked between the polysaccharide side chain of hemicellulose and ferulic acid in plant cell walls. LP_0796 from Lactobacillus plantarum was identified as a feruloyl esterase that may have potential applications in the food industry, but the lack of the substrate recognition and catalytic mechanisms limits its application. Here, LP_0796 showed the highest activity towards methyl caffeate at pH 6.6 and 40 °C. The crystal structure of LP_0796 was determined at 2.5 Å resolution and featured a catalytic triad Asp195-containing loop facing the opposite direction, thus forming a wider substrate binding pocket. Molecular docking simulation and site-directed mutagenesis studies further demonstrated that in addition to the catalytic triad (Ser94, Asp195, His225), Arg125 and Val128 played essential roles in the function of the active site. Our data also showed that Asp mutation of Ala23 and Ile198 increased the catalytic efficiency to 4- and 5-fold, respectively. Collectively, this work provided a better understanding of the substrate recognition and catalytic mechanisms of LP_0796 and may facilitate the future protein design of this important feruloyl esterase.

Preclinical safety assessment of the crude extract from Sida rhombifolia L. aerial parts in experimental models of acute and repeated-dose 28 days toxicity in rats.

Sida rhombifolia (Malvaceae) is popularly used as a treatment for several pathological conditions; however, there is a lack of studies that identify its compounds and that evaluate comprehensively the safety of its consumption. Therefore, the aim of this study was to determinate the phytochemical constitution of the crude extract of Sida rhombifolia (CESR), and its safety in models of acute and repeated doses (28 days) toxicity. The tested dose for the model of acute toxicity was 2000 mg/kg doses for the repeated dose model were 150, 300 e 600 mg/kg. Hematological, biochemical, histopathological and oxidative markers were investigated. HPLC-DAD-MS analysis evidenced the presence of caffeic acid, coumarin, and rutin. In the acute toxicity model the only altered parameters were tissue ROS, and AST and BUN in serum. As for the repeated dose experiment both hematological and biochemical markers remained within the values of reference for the species. Obtained results demonstrate that the CESR did not present significant toxic effects when administrated orally to male and female rats in acute and repeated doses.

Spent Grain from Malt Whisky: Assessment of the Phenolic Compounds.

In order to extract antioxidant phenolic compounds from spent grain (SG) two extraction methods were studied: the ultrasound-assisted method (US) and the Ultra-Turrax method (high stirring rate) (UT). Liquid to solid ratios, solvent concentration, time, and temperature/stirring rate were optimized. Spent grain extracts were analyzed for their total phenol content (TPC) (0.62 to 1.76 mg GAE/g SG DW for Ultra-Turrax pretreatment, and 0.57 to 2.11 mg GAE/g SG DW for ultrasound-assisted pretreatment), total flavonoid content (TFC) (0.6 to 1.67 mg QE/g SG DW for UT, and 0.5 to 1.63 mg QE/g SG DW for US), and antioxidant activity was measured using 2,2-diphenyl-2-picrylhydrazyl (DPPH) free radical (25.88% to 79.58% for UT, and 27.49% to 78.30% for UT). TPC was greater at a high stirring rate and high exposure time up to a certain extent for the Ultra-Turrax method, and at a high temperature for the ultrasound-assisted method. P-coumaric acid (20.4 ± 1.72 mg/100 SG DW for UT, and 14.0 ± 1.14 mg/100 SG DW for US) accounted for the majority of the phenolic found compounds, followed by rosmarinic (6.5 ± 0.96 mg/100 SG DW for UT, and 4.0 ± 0.76 mg/100 SG DW for US), chlorogenic (5.4 ± 1.1 mg/100 SG DW for UT, and non-detectable for US), and vanillic acids (3.1 ± 0.8 mg/100 SG DW for UT, and 10.0 ± 1.03 mg/100 SG DW for US) were found in lower quantities. Protocatechuic (0.7 ± 0.05 mg/100 SG DW for UT, and non-detectable for US), 4-hydroxy benzoic (1.1 ± 0.06 mg/100 SG DW for UT, and non-detectable for US), and caffeic acids (0.7 ± 0.03 mg/100 SG DW for UT, and non-detectable for US) were present in very small amounts. Ultrasound-assisted and Ultra-Turrax pretreatments were demonstrated to be efficient methods to recover these value-added compounds.

Adsorption and reduction of Cr(VI) from aqueous solution using cost-effective caffeic acid functionalized corn starch.

Herein, a potential bio-adsorbent (DACS-CA) was formed via immobilizing caffeic acid (CA) on dialdehyde corn starch (DACS) for Cr(VI) removal. The characterization techniques such as IR, Raman, XPS and 13C NMR were performed to analyze surface elements and functional groups on the as-prepared sorbents. Batch experiments revealed that the maximum Cr(VI) removal amount (96.45 mg/g) took place at a pH value of 3.0, adsorption temperatures of 333 K and solid-liquid ratio of 0.2. The isotherms studies found that the Cr(VI) removal of DACS-CA was monolayer adsorption, while the kinetics analysis revealed that chemisorption was the main power for removal process. Characterization analysis found that about Cr(VI) (53.02%) and Cr(III) (46.98%) species co-existed onto the surface of DACS-CA, which implied that a redox reaction may be occurred between Cr(VI) and the bio-adsorbent. Namely, Cr(VI) was first loaded on DACS-CA via electrostatic interaction, subsequently Cr(VI) was partially transformed into Cr(III) by reductive functional groups, meanwhile the resulting Cr(III) was immobilized by the carboxyl groups of DACS-CA. Thus, this bio-adsorbent could serve as an efficient sorbent for the removal of Cr(VI) from wastewater in environmental pollution cleanup.

The ameliorative effects of caffeic acid phenethyl ester in cisplatin-induced nephrotoxicity: assessment of the oxidative stress an inflammation / Los efectos de mejora del éster fenetílico del ácido cafeico en la nefrotoxicidad inducida por cisplatino: evaluación del estrés oxidativo y la inflamación

SUMMARY: The aim of this study is to determine the potential therapeutic effects of CAPE in CP-induced nephrotoxicity in rats. Cisplatin (CP) is an antineoplastic chemotherapeutic used for treatment of many cancer types but its applications may induce nephrotoxicity. Caffeic acid phenethyl ester (CAPE) is an active component of propolis and it has several important physiological activities. Rats were divided into four groups: Control, CAPE (10 µmol/kg/i.p), CP (7 mg/kg/i.p), and CP+CAPE (7 mg/kg/i.p, CP and 10 µmol/kg/i.p, CAPE). After administrations, animals were sacrificed, and kidney tissues were extracted. Histopathological changes were evaluated and TNF-α and IL-6 immunostaining were performed. Moreover, tissue SOD, CAT and MDA levels were measured by ELISA assay to assessment of oxidative stress and lipid peroxidation. CP group showed histopathological deterioration compared to the Control group and CAPE treatment attenuated this damage. When compared with Control and CAPE group, an increase in TNF-α and IL-6 immunoreactivities and tissue MDA levels were observed in the CP group while a decrease in tissue SOD and CAT levels were detected. Furthermore, an improvement was observed in the CP+CAPE compared to the CP group. We suggest that CAPE can be used as a therapeutic agent to attenuate the toxic effects of cisplatin, thanks to its antioxidant and anti-inflammatory properties.

RESUMEN: El objetivo de este estudio fue determinar los posibles efectos terapéuticos de éster fenetílico del ácido cafeico (EFAC) en la nefrotoxicidad inducida por cisplatino (CP) en ratas. El CP es un quimioterapéutico antineoplásico utilizado para el tratamiento de muchos tipos de cáncer, sin embargo sus aplicaciones pueden inducir nefrotoxicidad. El EFAC es un componente activo del propóleo y tiene varias actividades fisiológicas importantes. Para el estudio las ratas se dividieron en cuatro grupos: Control, EFAC (10 µmol / kg / ip), CP (7 mg / kg / ip) y CP + EFAC (7 mg / kg / ip, CP y 10 µmol / kg / ip, EFAC). Después de las administraciones, se sacrificaron los animales y se extrajeron los tejidos renales. Se evaluaron los cambios histopatológicos y se realizó inmunotinción de TNF-α e IL-6. Además, los niveles tisulares de SOD, CAT y MDA se midieron mediante un ensayo ELISA para evaluar el estrés oxidativo y la peroxidación lipídica. El grupo CP mostró deterioro histopatológico en comparación con el grupo Control y el tratamiento con EFAC atenuó este daño. En comparación con el grupo de control y EFAC, se observó un aumento en las inmunorreactividades de TNF-α e IL-6 y los niveles de MDA en el tejido en el grupo de CP, mientras que se detectó una disminución en los niveles de SOD y CAT en los tejidos. Además, se observó una mejora en el CP + EFAC en comparación con el grupo CP. Sugerimos que EFAC puede utilizarse como agente terapéutico para atenuar los efectos tóxicos del cisplatino, gracias a sus propiedades antioxidantes y antiinflamatorias.

Lipase-catalyzed Synthesis of Feruloylated Lysophospholipid in Toluene-Ionic Liquids and Its Antioxidant Activity.

In this study, Novozym 435-catalyzed interesterification of ethyl ferulate (EF) with phosphatidylcholine (PC) in a two-phase system consisting of an ionic liquid (IL) and toluene was optimized to prepare feruloylated lysophospholipids (FLPs). Optimum conditions for the interesterification process were found to be [Bmim][Tf2N]/toluene ratio of 1:1 (v/v), solvent volume of 4 mL, molecular sieves (4 Å) concentration of 80 mg/mL, reaction temperature of 55°C, substrate molar ratio of 5:1 (PC/EF), Novozym 435 concentration of 50 mg/mL. Under these conditions, two FLPs products (1-FLP and 2-FLP) with total conversion rate of 50.79% were obtained. Because the formation of 1-FLP was significantly higher than 2-FLP, 1-FLP was purified and characterized by LC-MS and NMR. In addition, 1-FLP showed DPPH scavenging activity comparable with those of EF and BHT. Therefore, this study provides a good method for transformation of ferulic acid to improve its solubility and promote its application as functional ingredient in the food and pharmaceutical industries.

Valorisation of kitul, an overlooked food plant: Phenolic profiling of fruits and inflorescences and assessment of their effects on diabetes-related targets.

Caryota urens L. has long been valued as a traditional food, the edible fruits being eaten raw and the inflorescences commonly used on sweet sap and flour production. In the current work, the phenolic profile of methanol extracts obtained from the inflorescences and fruits was unveiled for the first time, nine caffeic acid derivatives being identified and quantified. Since kitul products have been reported for their antidiabetic properties, extracts radical scavenging activity and α-amylase, α-glucosidase and aldose reductase inhibitory activity were assessed. The inflorescences' extract was particularly active against yeast α-glucosidase (IC50 = 1.53 µg/mL), acting through a non-competitive inhibitory mechanism. This activity was also observed in enzyme-enriched homogenates obtained from human Caco-2 cells (IC50 = 64.75 µg/mL). Additionally, the extract obtained from the inflorescences showed no cytotoxicity on HepG2, AGS and Caco-2 cell lines. Our data suggest that C. urens inflorescences can support the development of new functional foods with α-glucosidase inhibitory activity.

Assessment of Antioxidant and Cytotoxic Activities and Identification of Phenolic Compounds of Centaurea solstitialis and Urospermum picroides from Turkey

HIGHLIGHTS The phenolic composition, antioxidant activity and cytotoxic potential of the extracts of C. solstitialis and U. picroides were investigated. Caffeic acid was found as the most abundant phenolic compound in the extracts. Both species showed promising antioxidant activity towards different assays. The highest cytotoxic potential was observed in the extract of C. solstitialis.

Abstract It is known that some genera of the Asteraceae family are commonly used in Turkish folk medicine. Several studies have investigated the biological effects of different extracts of Centaurea and Urospermum species, but studies involving the phenolic composition of C. solstitialis and U. picroides extracts are very limited. This study aimed to investigate the phenolic composition and antioxidant activity of C. solstitialis and U. picroides and evaluate their possible cytotoxic effect. RP-HPLC analysis was used to elucidate the phenolic profiles of the ethanolic extracts of flowering parts of C. solstitialis and U. picroides.The both ethanolic extracts were assessed for their antioxidant properties using DPPH, FRAP, phosphomolybdenum and metal chelating assays. Furthermore, the effect of the extracts on cell viability was evaluated against MCF-7 and PC-3 cancer cells and HEK293 cell line using the MTT assay. The most abundant phenolic compound in both extracts was determined to be caffeic acid, and the amount of this compound was 24078.03 and 14329.59 µg g-1 in the extracts of C. solstitialis and U. picroides, respectively. The antioxidant activity of the extracts was found similar. Compared with U. picroides extract, C. solstitialis extract had higher potential on the inhibition of cell viability. The IC50 value of C. solstitialis on MCF cells was found as 58.53 µg mL-1. These data suggest that the extracts of C. solstitialis and U. picroides may be considered as novel and alternative natural antioxidant and anticancer sources.

Safety assessment of a newly synthesized copolymer for micellar delivery of hydrophobic caffeic acid phenethyl ester.

Caffeic acid phenethyl ester (CAPE), a major pharmacologically active component of poplar type propolis, is known for its proapoptotic, anti-inflammatory, antioxidant, antiviral, and enzyme inhibiting activities. The aim of this study was to perform an in vitro and in vivo safety assessment of a micellar system based on a newly synthesized copolymer, consisting of polyglycidol and poly(allyl glycidyl ether) (C12-PAGE-PG) as a drug delivery platform for CAPE. The in vitro studies on HepG2 and L929 cells by MTT and LDH assays after treatment with the empty and CAPE-loaded micelles showed no cytotoxic effects of the empty micelles and retained cytotoxic activity of CAPE loaded in the micelles. No hemolysis or stimulation of mouse lymphocytes or macrophages was observed in vitro. In vivo hematological, biochemical, and histological assays on rats, treated with the empty (2580 and 5160 µg/kg) or CAPE-loaded (375 and 750 µg CAPE/kg) micelles did not reveal pathological changes of any of the parameters assayed after 14-days' treatment. In conclusion, initial toxicological data characterize C12-PAGE-PG as a non-toxic and promising copolymer for development of micellar drug delivery systems, particularly for a hydrophobic active substance as CAPE.

Rapid analysis of the Tanreqing injection by near-infrared spectroscopy combined with least squares support vector machine and Gaussian process modeling techniques.

Near-infrared spectroscopy (NIRS) combined with chemometrics was used to analyze the main active ingredients including chlorogenic acid, caffeic acid, luteoloside, baicalin, ursodesoxycholic acid, and chenodeoxycholic acid in the Tanreqing injection. In this paper, first, two hundred samples collected in the product line were divided into the calibration set and prediction set, and the reference values were determined by the High Performance Liquid Chromatography- Diode Array Detector/Evaporative Light Scattering Detector (HPLC-DAD/ELSD) method. Partial least squares (PLS) analysis was implemented as a linear method for models calibrated with different preprocessing means. Wavelet transformation (WT) was introduced as a variable selection technique by means of multiscale decomposition, and wavelet coefficients were employed as the input for modeling. Furthermore, two nonlinear approaches, least squares support vector machine (LS-SVM) and Gaussian process (GP), were applied to exploit the complicated relationship between the spectra and active ingredients. The optimal models for each ingredient were obtained by LS-SVM and GP methods. The performance of the final models was evaluated by the root mean square error of calibration (RMSEC), root mean square error of cross-validation (RMSECV), root mean square error of prediction (RMSEP) and correlation coefficient (R). All of the models in the paper give a good calibration ability with an R value above 0.92, and the prediction ability is also satisfactory, with an R value higher than 0.85. The overall results demonstrate that nonlinear models are more stable and predictable than linear ones, and they will be more suitable for the CHM system when high accuracy analysis is required. It can be concluded that NIRS with the LS-SVM and GP modeling methods is promising for the implementation of process analytical technology (PAT) in the pharmaceutical industry of Chinese herbal injections (CHIs).

Assessment of the Antigenotoxic Activity of Poly(d,l-lactic- co-glycolic acid) Nanoparticles Loaded with Caffeic Acid Phenethyl Ester Using the Ames Salmonella/Microsome Assay.

In the present study, the antigenotoxic activity of poly(d,l-lactic- co-glycolic acid) (PLGA) nanoparticles (NPs) loaded with caffeic acid phenethyl ester (CAPE) was investigated in comparison to free CAPE using the Ames Salmonella/microsome assay. Additionally, to elucidate the impacts of the type of solvent effect on antigenotoxic activity, the following systems were tested: CAPE in water (poor solvent), ethyl alcohol (good solvent), and PLGA NPs (unknown). The effect of the NP system on solubility was investigated for the first time by assessing the antigenotoxic potential. In this study, the CAPE/PLGA NPs were synthesized using an oil-in-water (o/w) single-emulsion solvent evaporation method with an average size of 206.2 ± 1.2 nm, ζ potential of -19.8 ± 2.5 mV, encapsulation efficiency of 87.2 ± 2.5%, and drug loading of 53.3 ± 1.8%. According to the results of the antigenotoxic activity, the highest antimutagenic activity in both applied strains was found for CAPE in ethanol, and the lowest activity was detected for CAPE in water. Our study has shown that NP systems exhibit high antigenotoxic activity, which is similar to the results of CAPE dissolved in ethanol. These results have shown that NP systems increase biological activity of hydrophobic substances by increasing their solubility and that the use of PLGA instead of organic solvents in drug production may provide an increase in their medical utility.

Colonic fermentation of polyphenolics from Sea buckthorn (Hippophae rhamnoides) berries: Assessment of effects on microbial diversity by Principal Component Analysis.

The present study investigates the stability of polyphenolic in Sea buckthorn berries juice (SBJ) during different phases of digestion and its effect on colonic microbial diversity. At each stage, the Total polyphenolic content (TPC), Total antioxidant activity (TAA) and polyphenolic profile was determined. A 1.64 and 2.20 folds increase in TPC with 4.88 and 9.61 folds increase in TAA were observed during gastric and small intestine digestion (p<0.05) with the release of quercetin from food matrix. The digestion resulted in deformation of intact crystalline structure as indicated by scanning electron micrographs. The colonic fermentation resulted in an increase in quercetin, caffeic acid with decrease in rutin and chlorogenic acid after 36h of fermentation (p<0.05). The Shannon diversity index (H) of beneficial groups including Lactic acid bacteria (LAB), Bacteroides/Prevotella and Bifidobacteria was increased by 35%, 71% and 17%, respectively (p<0.05). The PCA analysis indicated that the presence and digestion of polyphenolics promote the proliferation of Bacteroides/Prevotella group as well as Lactic acid bacteria and Bifidobacteria. The results suggest that SBJ is good source of prebiotic substrate in terms of the proliferation of beneficial gut microbiota.

Minor compounds of the high purity salvianolic acid B freeze-dried powder from Salvia miltiorrhiza and antibacterial activity assessment.

The study explored the isolation and characterisation of three compounds of high purity salvianolic acid B freeze-dried powder extracted from Salvia miltiorrhiza Bunge. A new salvianolic acid, salvianolic acid V (2) together with two known compounds (3-4) was identified. The antibacterial activity tests showed that compound 2 combined with clinical antibiotics such as Levofloxacin or Colistin sulphate together exhibited potent effects against MRSA or Acinetobacter baumanii. This report has considerably extended our knowledge about the diversity and bioactivity of caffeic acid derivatives from S. miltiorrhiza.

NIR Spectroscopy of Actaea racemosa L. rhizome - En Route to Fast and Low-Cost Quality Assessment.

Rhizomes of Actaea racemosa L. (formerly Cimicifuga racemosa) gained increasing interest as a plant-derived drug due to its hormone-like activity and the absence of estrogenic activity. According to the Current Good Manufacturing Practices guidelines and pharmacopeial standards, quality assessment of herbal starting materials includes tests on identity and substitution, as well as quantification of secondary metabolites, usually by HPTLC and LC methods. To reduce the laboratory effort, we investigated near-infrared spectroscopy for rapid species authentication and quantification of metabolites of interest.Near-infrared spectroscopy analysis is carried out directly on the milled raw plant material. Spectra were correlated with reference data of polyphenols and triterpene glycosides determined by LC/diode array detection and LC/evaporative light scattering detection, respectively. Quantification models were built and validated by cross-validation procedures. Clone plants, derived by vegetative propagation, and plants of a collection from different geographical origins cultivated in Berlin were analysed together with mixed batches from wild harvests purchased at wholesalers.Generally, good to excellent correlations were found for the overall content of polyphenols with coefficients of determination of R2 > 0.93. For individual polyphenols such as fukinolic acid, only models containing clone plants succeeded (R2 > 0.92). For the total content of triterpene glycosides, results were generally worse in comparison to polyphenols and were observed only for the mixed batches (R2 = 0.93).Next to quantitative analysis, near-infrared spectroscopy was proven as a rapid alternative to other, more laborious methods for species authentication. Near-infrared spectroscopy was able to distinguish different Actaea spp. such as the North American Actaea cordifolia and the Asian Actaea cimicifuga, Actaea dahurica, Actaea heracleifolia, and Actaea simplex.

Rapid assessment of bioactive phenolics and methylxanthines in spent coffee grounds by FT-NIR spectroscopy.

Spent coffee grounds (SCGs) are a great source of bioactive compounds with interest to pharmaceutical and cosmetic industries. Phenolics and methylxanthines are the main health related compounds present in SCG samples. Content estimation of these compounds in SCGs is of upmost importance in what concerns their profitable use by waste recovery industries. In the present work, near-infrared spectroscopy (NIRS) was proposed as a rapid and non-destructive technique to assess the content of three main phenolics (caffeic acid, (+)-catechin and chlorogenic acid) and three methylxanthines (caffeine, theobromine and theophylline) in SCG samples obtained from different coffee brands and diverse coffee machines. The content of these compounds was determined for 61 SCG samples by HPLC coupled with diode-array detection. Partial least squares (PLS) regression based models were calibrated to correlate diffuse reflectance NIR spectra against the reference data for the six parameters obtained by HPLC. Spectral wavelength selection and number of latent variables were optimized by minimizing the cross-validation error. PLS models showed good linearity with a coefficient of determination for the prediction set (Rp(2)) of 0.95, 0.92, 0.88, 071 and 0.84 for caffeine, caffeic acid, (+)-catechin, chlorogenic acid and theophylline, respectively. The range error ratio (RER) was higher for caffeine (17.8) when compared to other compounds (12.0, 10.1, 7.6 and 9.2, respectively for caffeic acid, (+)-catechin, chlorogenic acid and theophylline). Moreover, the content of caffeine could be used to predict the antioxidant properties of SCG samples (R=0.808, n=61), despite not presenting this property itself. The results obtained confirmed that NIRS is a suitable technique to screen SCG samples unveiling those with high content of bioactive compounds, which are interesting for subsequent extraction procedures.

Combination of immunoprecipitation (IP)-ATP_Glo kinase assay and melanogenesis for the assessment of potent and safe PAK1-blockers in cell culture.

Cucurbitacin I (CBI) is a triterpene from a bitter melon called Goya grown in Okinawa, Japan, and directly inhibits both the Tyr-kinase JAK2 and the G protein RAC, leading to the inactivation of PAK1 (RAC/CDC42-activated kinase 1). Bio 30, a propolis produced in New Zealand, contains CAPE (caffeic acid phenethyl ester) as the major anti-cancer ingredient which directly down-regulates RAC, leading to the inactivation of PAK1. Since PAK1 is essential for the growth of RAS cancer cells such as A549 cell line which carry an oncogenic K-RAS mutant, and the melanogenesis in skin cells, here using these PAK1-blockers as model compounds, we introduce a new approach to the quick assessment of PAK1-blockers in cell culture. First, combining the immuno-precipitation (IP) of PAK1 from cell lysate and the in vitro ATP_Glo kinase assay kit (called "Macaroni-Western" assay), we confirmed that both CBI and Bio 30 inactivate PAK1 in A549 lung cancer cells in 24 h, and inhibit their PAK1-dependent growth in 72 h. Furthermore, we verified that CBI inhibits the PAK1/PAK4-dependent melanogenesis in melanoma cells by far more than 50%, while Bio 30 inhibits the melanogenesis only by 50%, with only a merginal effect on their growth per se. Since the "Macaroni-Western" kinase assay and melanogenesis are both rather simple and quick, the combination of these two cell culture assays would be highly useful for selecting both "potent" (highly cell-permeable) and "safe" (non-toxic) natural or synthetic PAK1-blockers.

Isoprenyl caffeate, a major compound in manuka propolis, is a quorum-sensing inhibitor in Chromobacterium violaceum.

The emergence of antibiotic-resistant bacterial pathogens, especially Gram-negative bacteria, has driven investigations into suppressing bacterial virulence via quorum sensing (QS) inhibition strategies instead of bactericidal and bacteriostatic approaches. Here, we investigated several bee products for potential compound(s) that exhibit significant QS inhibitory (QSI) properties at the phenotypic and molecular levels in Chromobacterium violaceum ATCC 12472 as a model organism. Manuka propolis produced the strongest violacein inhibition on C. violaceum lawn agar, while bee pollen had no detectable QSI activity and honey had bactericidal activity. Fractionated manuka propolis (pooled fraction 5 or PF5) exhibited the largest violacein inhibition zone (24.5 ± 2.5 mm) at 1 mg dry weight per disc. In C. violaceum liquid cultures, at least 450 µg/ml of manuka propolis PF5 completely inhibited violacein production. Gene expression studies of the vioABCDE operon, involved in violacein biosynthesis, showed significant (≥two-fold) down-regulation of vioA, vioD and vioE in response to manuka propolis PF5. A potential QSI compound identified in manuka propolis PF5 is a hydroxycinnamic acid-derivative, isoprenyl caffeate, with a [M-H] of 247. Complete violacein inhibition in C. violaceum liquid cultures was achieved with at least 50 µg/ml of commercial isoprenyl caffeate. In silico docking experiments suggest that isoprenyl caffeate may act as an inhibitor of the violacein biosynthetic pathway by acting as a competitor for the FAD-binding pockets of VioD and VioA. Further studies on these compounds are warranted toward the development of anti-pathogenic drugs as adjuvants to conventional antibiotic treatments, especially in antibiotic-resistant bacterial infections.

Assessment of total (free and bound) phenolic compounds in spent coffee extracts.

Spent coffee is the main byproduct of the brewing process and a potential source of bioactive compounds, mainly phenolic acids easily extracted with water. Free and bound caffeoylquinic (3-CQA, 4-CQA, 5-CQA), dicaffeoylquinic (3,4-diCQA, 3,5-diCQA, 4,5-diCQA), caffeic, ferulic, p-coumaric, sinapic, and 4-hydroxybenzoic acids were measured by HPLC, after the application of three treatments (alkaline, acid, saline) to spent coffee extracts. Around 2-fold higher content of total phenolics has been estimated in comparison to free compounds. Phenolic compounds with one or more caffeic acid molecules were approximately 54% linked to macromolecules such as melanoidins, mainly by noncovalent interactions (up to 81% of bound phenolic compounds). The rest of the quantitated phenolic acids were mainly attached to other structures by covalent bonds (62-97% of total bound compounds). Alkaline hydrolysis and saline treatment were suitable to estimate total bound and ionically bound phenolic acids, respectively, whereas acid hydrolysis is an inadequate method to quantitate coffee phenolic acids.