In situ extraction/encapsulation of olive leaves antioxidants in zein for improved oxidative stability of edible oils.

This study presents a sustainable and cost-effective method for preserving the bioactivity of phenolic compounds in olive leaves (OLE) during their application. The extraction and nanoencapsulation of OLE were performed in a single-step process using a rotor-stator system with zein as the encapsulating agent. The nanoprecipitation step was carried out using an aqueous sodium caseinate solution, resulting in spherical particles with an average diameter of about 640 nm, as confirmed by Transmission Electron Microscopy. Thermal characterization showed that the produced nanoparticles were more thermally stable than free OLE until 250 °C, and FTIR spectra indicated effective interaction between the phenolic compounds and zein. Antioxidant activity was evaluated using TBARS, DPPH, ABTS, and FRAP assays, with results showing that encapsulated OLE had lower antioxidant activity than free OLE. The best antioxidant capacity results were determined by TBARS assay, with IC50 results equal to 43 and 103 µgOLE/mL for free and encapsulated OLE, respectively. No anti-inflammatory potential was detected for both samples using the RAW 264.7 model, and only free OLE showed cytotoxic activity against lung cancer and gastric carcinoma. Encapsulated and free OLE were used as antioxidants in soy, palm, and palm kernel oils and compared to BHT using Rancimat. The Schaal Oven Test was also performed, and the PARAFAC chemometric method analyzed the UV-Vis spectra, which revealed high stability of the oil when 300 mg or the nanoparticles were added per kg oil. Results suggested that zein-encapsulated olive leaf antioxidants can improve the oxidative stability of edible oils.

First report of Colletotrichum tropical causing leaf spot on Cariniana legalis in Brazil.

Cariniana legalis (Mart.) Kuntze (Lecythidaceae), commonly known as "Jequitibá Rosa," an endemic tree from Brazil, is one of the largest trees that grows in the Atlantic Rain Forest. Jequitibá Rosa has been extensively exploited due to the excellent quality of its wood, which has resulted in the species being placed as "vulnerable" on the IUCN red list of threatened species (IUCN, 2021). Thus, conservation of the species is paramount. In October 2018, in the municipality of Ilhéus, Bahia (14º 79' 67" S, 39º 17' 32" W), trees of C. legalis were observed with approximately 80% of leaves presenting light to dark brown necrotic lesions with yellow edges at the tips and margins of the leaves; the lesions gradually expanded through the leaves and coalesced to form wider lesions, which resulted in death of the leaves and weakening of the trees. The symptoms indicate infection caused by a Colletotrichum spp., important pathogens known to cause diseases in several economically important plants. Symptomatic leaves were collected and lesions examined under a stereomicroscope at 20x magnification (Supplementary Figure S1. A). Conidia were transferred directly and aseptically from the leaf spots to potato-dextrose-agar (PDA) and incubated in the dark at 25 ± 1 ºC for 72 h. Subsequently, pure cultures were obtained by monosporic isolation. All fungal structures were measured using a Leica DM500 microscope at 1000x magnification. A pathogenicity test was performed both on hosts in the field and using detached leaves by inoculation with a conidial suspension 106 conidia/mL prepared from a culture grown on PDA incubated in the dark at 25 ± 1 ºC for 10 days. Six healthy and fully developed detached leaves were inoculated with two 10 µL drops of conidial suspension on the adaxial surface. Six leaves were inoculated with two drops of sterile water were used as controls. The inoculated leaves were kept in a humid chamber at 25º C with a 12 h photoperiod. Four trees of C. legalis growing in the field were selected and 120 healthy leaves (30 per tree) sprayed with a conidial suspension and covered with transparent polyethylene plastic bags, with a piece of wet cotton inside, to maintain moisture for 72 h. A further 30 leaves per tree were sprayed with sterile water as controls. DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method following the protocol described by Oliveira et al. (2016). Internal transcribed spacer (ITS), partial histone3 (HIS3), and partial glyceraldehyde-3-phosphatedehydrogenase (GAPDH) gene sequences were amplified by polymerase chain reaction (PCR) using ITS1/ITS4 (White et al. 1990), CYLH3F/CYLH3R (Crous et al. 2004), and GDF1/GDR1 (Guerber et al. 2003) primers. The PCR for the ITS amplification were performed according to Oliveira et al. (2014), and for the HIS3 and GAPDH according Damm et al. (2012). Maximum likelihood analysis (with support estimated by a bootstrap analysis with 1,000 replicates) was performed using PhyML 3.0 (Guindon and Gascuel 2003) and launched from Topali 2.5. The colony produced abundant white to light gray aerial mycelium, and an orange viscous mass of conidia (Supplementary Figure S1. B). Conidia were single-celled, smooth, hyaline and sub-cylindrical with rounded ends, 13 (11-15) × 3.5 (3-4) µm (Supplementary Figure S1. C); appressoria were aseptate, brown, subglobose to clavate, 11 (10-12) × 7 (6-8) µm (Supplementary Figure S1. D). BLASTn analysis revealed that the partial gene sequences of ITS (URM 8381 ‒ MZ158701), GAPDH (URM 8381 ‒ MZ189259) and HIS3 (URM 8381 ‒ MZ189260) were 100% identical to Colletotrichum tropicale (CPO 27.830 ‒MN744296/CBS 129983 ‒ MH865615), (CMM 4071 ‒ KC517181/CPO 27.719 ‒MN737355) and (CBS 124949 ‒ KY856395). A maximum likelihood phylogenetic tree was generated by combining all sequenced loci. The phylogenetic tree revealed that sequences of the isolate URM8381 formed a clade with the sequences from the type species of C. tropicale E.I. Rojas, S.A. Rehner & Samuels (CBS 124949) with a high support value (95), which is distinct from other related species (Supplementary Figure S1. E). The sequences from the isolate were deposited in GenBank under the following accession numbers: ITS: MZ158701; GAPDH: MZ189259 and HIS3: MZ189260. C. tropicale was reisolated from the inoculated leaves, and had the same cultural and morphological characteristics as the original isolate. Both detached leaves and leaves on trees were inoculated in the field and presented leaf spot symptoms (Supplementary Figure S1.F), at 6 and 8 days after inoculation, respectively, which further confirms C.tropicale as the causal agent of the symptoms observed on the leaves of C. legalis. The controls did not develop any symptoms (Supplementary Figure S1. G). C. tropicale belongs to the C. gloeosporioides species complex. In Brazil, C. tropicale causes anthracnose on various hosts, including Annona muricata L. (soursop) and Myrciaria dubia (Kunth) McVaugh ("camu-camu") (Costa et al. 2019; Matos et al. 2020). To our knowledge, this is the first report of C. tropicale causing leaf spot on C. legalis. References: Costa, J. F. O., et al. 2019. Eur. J. Plant Pathol. 153:1119. Crous, P. W., et al. 2004. Stud. Mycol. 50:415. Damm, U., et al. 2012. Stud. Mycol. 73:1. Guerber, J. C., et al. 2003. Mycol. 95:87. Guindon, S.; Gascuel, O. 2003. Syst Biol 52(5): 696-704. https://doi.org/10.1080/10635150390235520. IUCN. 2021. http://www.iucnredlist.org. Matos, K. S., et al. 2020. Plant Dis. 104. https://doi.org/10.1094/PDIS-04-19- 0882-PDN Oliveira R. J. V., et al. 2016. N. Hedw. 103:185. White, T. J., et al. 1990. In: Innis, M.A., et al. (Eds.) Academic Press, San Diego, pp. 315-322. http://dx.doi.org/10.1016/b978-0-12-372180-8.50042-1.

Choline chloride-based deep eutectic solvents as potential solvent for extraction of phenolic compounds from olive leaves: Extraction optimization and solvent characterization.

This study evaluates the use of deep eutectic solvents (DES) prepared with choline chloride ([Ch]Cl) and carboxylic acids for phenolic compound extraction from olive leaves. These extracts were then compared to those obtained using ethanol. The effects of temperature and water addition during DES- and ethanol-based extractions were analyzed using response surface methodology. Due to the lack of solid-liquid equilibrium (SLE) data for [Ch]Cl + acetic acid, SLE, and DES density and viscosity with and without water addition were measured and analyzed. [Ch]Cl:acetic acid (54.1 °C, 50.0% water addition) extracted 15% more phenolic compounds than ethanol (54.1 °C, 0.5% water addition), according to UHPLC-MS based analyses. SLE analyses showed that [Ch]Cl + acetic acid presented a eutectic region at close to a 1:2 molar ratio. DES precursors and water addition influenced solvent physical properties and phenolic compound yield. DES was confirmed to be an innovative, strong solvent for phenolic compound extraction from olive leaves.

Dietary oleuropein and its acyl derivative ameliorate inflammatory response in peritoneal macrophages from pristane-induced SLE mice via canonical and noncanonical NLRP3 inflammasomes pathway.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease without an effective and safe treatment. Besides, macrophages are the major components of the innate immune system and play a critical role in the inflammation process in SLE. Secoiridoids from olive tree are phenolic compounds which have shown important pharmacological effects. Particularly, oleuropein (OL) has shown antioxidant, anti-inflammatory and immunomodulatory properties suggesting a potential application in a large number of inflammatory and reactive oxygen species (ROS)-mediated diseases. In addition, different studies have shown the importance of acyl derivatives of natural phenols due to their better hydrophilic/lipophilic balance.

Partially defatted olive cake in finishing pig diets: implications on performance, faecal microbiota, carcass quality, slurry composition and gas emission.

One of the key factors to improve swine production sustainability is the use of agro-industrial by-products in feeds, such as olive by-products. However, it is necessary to assess its effects on the overall production process, including the animal and the environment. With this aim, an experiment was conducted to determine the effects of including a partially defatted olive cake (PDOC) in pig diets on growth performance, faecal microbiota, carcass quality and gas emission from the slurry. Two finishing diets were formulated, a control (C) diet and a diet with PDOC included at 120 g/kg. Eighty finishing male pigs Duroc-Danbred × (Landrace × Large White) of 60.4 ± 7.00 kg BW were divided between these two treatments. During the finishing period (60 to 110 kg BW, 55 days) average daily gain, average daily feed intake and feed conversion ratio were recorded. Faecal samples from the rectum of 16 animals per treatment were incubated for bacteria enumeration. At the end of finishing period, backfat thickness and loin depth (LD) were measured. Animals were slaughtered to obtain carcass weight and carcass composition parameters, and subcutaneous fat was sampled to analyse the fatty acid (FA) profile. In addition greenhouse gas and ammonia emissions were measured during pig slurry storage using the methodology of dynamic flux chambers. An initial slurry characterisation and biochemical methane potential (B0) were also determined. No significant differences between treatments were found in performance, carcass quality and microbial counts with the exception of LD, which was lower in PDOC compared with C animals (45.5 v. 47.5 mm, SEM: 0.62; P = 0.020). The FA profile of the subcutaneous fat did not differ between treatments, but the monounsaturated FA (MUFA) concentration was higher and the polyunsaturated FA was lower in the animals fed PDOC (50.9 v. 48.3, SEM: 0.48, P < 0.001; 17.6 v. 19.3, SEM: 0.30, P < 0.001 in mg/100 g of Total FA, for PDOC and C animals, respectively). The initial pig slurry characterisation only showed differences in ADF concentration that was higher (P < 0.05) in the slurry from PDOC treatment. Regarding gas emission, slurries from both treatments emitted similar amounts of ammonia (NH3), carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O), as well as B0 values. The results obtained suggest that PDOC may be included in balanced pig diets at rates of up to 120 g/kg without negative effects on performance, carcass quality, gut microflora and slurry gas emission, while improving the MUFA concentration of subcutaneous fat.

Effects of Olive and Pomegranate By-Products on Human Microbiota: A Study Using the SHIME® in Vitro Simulator.

Two by-products containing phenols and polysaccharides, a "pâté" (OP) from the extra virgin olive oil milling process and a decoction of pomegranate mesocarp (PM), were investigated for their effects on human microbiota using the SHIME® system. The ability of these products to modulate the microbial community was studied simulating a daily intake for nine days. Microbial functionality, investigated in terms of short chain fatty acids (SCFA) and NH4+, was stable during the treatment. A significant increase in Lactobacillaceae and Bifidobacteriaceae at nine days was induced by OP mainly in the proximal tract. Polyphenol metabolism indicated the formation of tyrosol from OP mainly in the distal tract, while urolithins C and A were produced from PM, identifying the human donor as a metabotype A. The results confirm the SHIME® system as a suitable in vitro tool to preliminarily investigate interactions between complex botanicals and human microbiota before undertaking more challenging human studies.

Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

Butyrate is a short-chain fatty acid derived from the metabolism of indigestible carbohydrates by the gut microbiota. Butyrate contributes to gut homeostasis, but it may also control inflammatory responses and host physiology in other tissues. Butyrate inhibits histone deacetylases, thereby affecting gene transcription, and also signals through the metabolite-sensing G protein receptor (GPR)109a. We produced an mAb to mouse GPR109a and found high expression on podocytes in the kidney. Wild-type and Gpr109a-/- mice were induced to develop nephropathy by a single injection of Adriamycin and treated with sodium butyrate or high butyrate-releasing high-amylose maize starch diet. Butyrate improved proteinuria by preserving podocyte at glomerular basement membrane and attenuated glomerulosclerosis and tissue inflammation. This protective phenotype was associated with increased podocyte-related proteins and a normalized pattern of acetylation and methylation at promoter sites of genes essential for podocyte function. We found that GPR109a is expressed by podocytes, and the use of Gpr109a-/- mice showed that the protective effects of butyrate depended on GPR109a expression. A prebiotic diet that releases high amounts of butyrate also proved highly effective for protection against kidney disease. Butyrate and GPR109a play a role in the pathogenesis of kidney disease and provide one of the important molecular connections between diet, the gut microbiota, and kidney disease.-Felizardo, R. J. F., de Almeida, D. C., Pereira, R. L., Watanabe, I. K. M., Doimo, N. T. S., Ribeiro, W. R., Cenedeze, M. A., Hiyane, M. I., Amano, M. T., Braga, T. T., Ferreira, C. M., Parmigiani, R. B., Andrade-Oliveira, V., Volpini, R. A., Vinolo, M. A. R., Mariño, E., Robert, R., Mackay, C. R., Camara, N. O. S. Gut microbial metabolite butyrate protects against proteinuric kidney disease through epigenetic- and GPR109a-mediated mechanisms.

Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Colonization of the oropharynx is the initial step in Group A Streptococcus (GAS) pharyngeal infection. We have previously reported that the highly virulent M1T1 GAS clone attaches to oral epithelial cells via M1 protein interaction with blood group antigen carbohydrate structures. Here, we have identified that colonization of human oral epithelial cells by GAS serotypes M3 and M12 is mediated by human blood group antigens [ABO(H)] and Lewis (Le) antigen expression. Removal of linkage-specific fucose, galactose, N-acetylgalactosamine, and sialic acid modulated GAS colonization, dependent on host ABO(H) blood group and Le expression profile. Furthermore, N-linked glycans from human salivary glycoproteins, when released and purified, were potent inhibitors of M1, M3, and M12 GAS colonization ex vivo. These data highlight the important role played by human protein glycosylation patterns in GAS attachment to oral epithelial cell surfaces.-De Oliveira, D. M. P., Everest-Dass, A., Hartley-Tassell, L., Day, C. J., Indraratna, A., Brouwer, S., Cleary, A., Kautto, L., Gorman, J., Packer, N. H., Jennings, M. P., Walker, M. J., Sanderson-Smith, M. L. Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Extraction of oleuropein and luteolin-7-O-glucoside from olive leaves: Optimization of technique and operating conditions.

Olive leaves have become a promising source of phenolic compounds and flavonoids with high added value. Phenolic compounds and flavonoids are important sources of antioxidants and bioactives, and one of the processes used to effectively produce them is extraction via solvents, using aqueous ethanol solutions. To obtain the highest extraction yield per kg of biomass, olive leaves were extracted using a conventional technique (dynamic maceration) and an emerging technology, such as pressurized liquid extraction. Studies of the factors that influence these processes were performed: temperature, leaf moisture content, solvent/solid, and aqueous ethanol concentration were optimized using the central composite and Box-Behnken experiment designs. Pressurized liquid extraction resulted in more efficient oleuropein and luteolin-7-O-glucoside extraction than dynamic maceration. The operational conditions for maximizing the recovery of phenolic compounds and flavonoids and antioxidant capacity were determined to be 190 °C, leaf moisture content of 5%, and aqueous ethanol concentration of 80%.

Acute Effect of a Single Dose of Tomato Sofrito on Plasmatic Inflammatory Biomarkers in Healthy Men.

Sofrito is a Mediterranean tomato-based sauce that typically also contains olive oil, onion, and garlic. The preparation of sofrito modifies the bioactive compounds (carotenoids and polyphenols) in the ingredients to more bioavailable forms, promoting cis-lycopene formation and polyphenol bioaccessibility. To evaluate the health benefits of this cooking technique, the effect of consuming an acute dose of sofrito on the inflammatory status was studied. In a clinical trial, 22 healthy male subjects consumed a single dose of sofrito (240 g/70 kg) after three days without ingesting any tomato products and following a low-antioxidant diet the day before the intervention. Plasma carotenoids and total polyphenol excretion (TPE) were evaluated, as well as the inflammatory biomarkers C-reactive protein (CRP), interleukin-6 (IL-6), interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). After the sofrito intake, a significant decrease in CRP (p = 0.010) and TNF-α (p = 0.011) was observed, but only TNF-α was inversely correlated with an increase in TPE and plasma ß-carotene (not the major carotenoid, lycopene). The positive health effects of this tomato-based product may be attributed not only to lycopene, but to the bioactive compounds of all the ingredients.

Alpha-Actinin-3 R577X Polymorphism Influences Muscle Damage and Hormonal Responses After a Soccer Game.

Coelho, DB, Pimenta, EM, Rosse, IC, Veneroso, C, Pussieldi, GDA, Becker, LK, De Oliveira, EC, Carvalho, MRS, and Silami-Garcia, E. Alpha-actinin-3 R577X polymorphism influences muscle damage and hormonal responses after a soccer game. J Strength Cond Res 33(10): 2655-2664, 2019-The purpose of this study was to evaluate indicators of muscle damage and hormonal responses after soccer matches and its relation to alpha-actinin-3 (ACTN3) gene expression (XX vs. RR/RX), considering that the R allele produces alpha-actinin-3 and provides greater muscle strength and power. Thirty players (10 XX and 20 RR/RX) younger than 16 years were evaluated in this study. Blood samples were collected immediately before, after, 2, and 4 hours after the games to assess muscle damage (creatine kinase [CK] and alpha-actin) and hormonal responses (interleukin-6 [IL-6], cortisol, and testosterone). Postgame CK was higher as compared to the pregame values in both groups and it was also higher in the RR/RX (p < 0.05) than in the XX. The concentrations of alpha-actin and IL-6 were similar for both groups and did not change over time. Testosterone was increased after the game only in the RR/RX group (p < 0.05). Cortisol concentrations in group RR/RX were higher immediately after the game than before the game, and 2 and 4 hours after the game the concentration decreased (p < 0.05). The RR and RX individuals presented higher markers of muscle microtrauma and hormonal stress, probably because they performed more speed and power actions during the game, which is a self-regulated activity. From the different responses presented by RR/RX and XX genotypes, we conclude that the genotypic profile should be taken into account when planning training workloads and recovery of athletes.

New species and records of Cheiromyia Dyte from Brazil and French Guiana (Diptera: Dolichopodidae).

Cheiromyia carolina Limeira-de-Oliveira Brooks sp. nov. and C. nordestina Limeira-de-Oliveira Cumming sp. nov. are described from Brazil, and C. fuscipennis Pollet Brooks sp. nov. is described from the Mitaraka Mountains in southwestern French Guiana. New distribution records are reported for C. brevitarsis Brooks, C. palmaticornis (Parent) and C. pennaticornis (Parent), and a revised key to males of the eight known species of Cheiromyia Dyte, 1980 is provided. The female of C. pennaticornis is also described for the first time.

Comparative study of adjuvants for allergen-specific immunotherapy in a murine model.

AIM: To compare the immunological and clinical changes induced by allergen-specific immunotherapy (AIT) using different adjuvants. MATERIALS & METHODS: Olea europaea pollen-sensitized mice were treated with olea plus aluminum hydroxide, calcium phosphate, monophosphoryl lipid A (MPL) or immunostimulatory sequences (ISS). RESULTS: Aluminum hydroxide seems to drive initially to a Th2-type response. Bacteria-derived adjuvants (MPL and ISS) skew the immune response toward Th1 and Treg pathways. Specific-IgE production was lower after AIT with MPL and ISS. Moreover, IgG2a production significantly increased in ISS-treated mice. Bacteria-derived adjuvants also improved the Th1 cytokine response due to IFN-γ higher secretion. In addition, they improved bronchial hyper-reactivity and lung inflammation. CONCLUSION: Bacteria-derived adjuvants may enhance the efficacy of AIT.

Virgin olive oil and its phenol fraction modulate monocyte/macrophage functionality: a potential therapeutic strategy in the treatment of systemic lupus erythematosus.

Monocytes and macrophages are critical effectors and regulators of inflammation and innate immune response, which appear altered in different autoimmune diseases such as systemic lupus erythematosus (SLE). Recent studies suggested that virgin olive oil (VOO) and particularly its phenol compounds might possess preventive effects on different immune-inflammatory diseases, including SLE. Here, we evaluated the effects of VOO (and sunflower oil) on lipopolysaccharide (LPS)-activated peritoneal macrophages from a model of pristane-induced SLE in BALB/c mice, as well as those of the phenol fraction (PF) from VOO on the immune-inflammatory activity and plasticity in monocytes and monocyte-derived macrophages from healthy volunteers. The release of nitrite and inflammatory cytokines was lower in LPS-treated peritoneal macrophages from pristane-SLE mice fed the VOO diet when compared with the sunflower oil diet. PF from VOO similarly decreased the secretion of nitrite and inflammatory cytokines and expression of inducible nitric oxide, PPARγ and Toll-like receptor 4 in LPS-treated human monocytes. PF from VOO also prevented the deregulation of human monocyte subset distribution by LPS and blocked the genetic signature of M1 macrophages while favouring the phenotype of M2 macrophages upon canonical polarisation of naïve human macrophages. For the first time, our study provides several lines of in vivo and in vitro evidence that VOO and PF from VOO target and counteract inflammatory pathways in the monocyte-macrophage lineage of mice with pristane-induced SLE and of healthy subjects, which is a meaningful foundation for further development and application in preclinical and clinical use of PF from VOO in patients with SLE.

Optimization of operating conditions for the valorization of olive mill wastewater using membrane processes.

The main goal of this work was to assess the performance of an integrated membrane system for the treatment and valorization of wastewater generated from an olive mill (OMW) coming from a traditional extraction press. Two different lots of this effluent were firstly processed in an ultrafiltration (UF) pilot unit after a pretreatment by screening. The effect of transmembrane pressure (TMP) and temperature on the chemical oxygen demand (COD) reduction and removal of total phenolic (TPh) content was investigated. Under optimal conditions (TMP = 1.5 bar and T = 20 °C), 20.6 and 26.8% for COD and TPh removal were achieved, respectively. The permeate from UF was then treated by nanofiltration (NF) in order to obtain a retentate enriched in phenolic compounds. The influence of pressure drop (ΔP), temperature, and pH on the removal of COD, TPh, and permeate flow was examined using a 23 full-factorial experimental design. The effect of the independent variables and their interactions on the process performance was analyzed with Pareto charts. Multivariable regression models allowed to built 3D surface plots. The best conditions that maximize the COD abatement (83.3%) and TPh removal (93.1%) were ΔP = 18 bar, T = 20 °C, and a pH 2.7. It can be concluded that the integration of UF and NF processes investigated in this work is a promising strategy for the treatment and valorization of OMW.

Improving in vivo conversion of oleuropein into hydroxytyrosol by oral granules containing probiotic Lactobacillus plantarum 299v and an Olea europaea standardized extract.

This study reports novel food-grade granules for co-delivery of L. plantarum 299v and a standardized extract of Olea europaea leaves (Phenolea®) as oral carrier of probiotics and hydroxytyrosol. Different granule formulations containing either L. plantarum 299v (Lac), or the olive leave extract (Phe) or their combination (Lac-Phe) have been successfully produced through wet granulation employing excipients generally regarded as safe as granulating/binding agents. L. plantarum cells withstood the manufacturing process and were stable upon storage at 4 °C for more than 6 months. In vitro dissolution studies in simulated gastro-intestinal fluids showed the capability of the granules to rapidly dissolve and deliver both olive leave phenols and living L. plantarum cells. In simulated digestion conditions, Lac and Lac-Phe granules protected L. plantarum against the harsh environment of the gastro-intestinal tract. Co-administration of Lac and Phe oral granules to healthy mice provided for higher amounts of hydroxytyrosol in urines as compared to Phe granules alone, suggesting that L. plantarum 299v boosted in vivo conversion of oleuropein to hydroxytyrosol. On the other hand, PCR-assisted profiling of the Lactobacillus population in faeces obtained from mice treated with Lac or Lac plus Phe confirmed that the probiotic arrived alive to colon and was there able to exert a sort of perturbing effect on the climax colonic microflora. Overall, these results pave the way towards the development of a nutraceutical useful for combined delivery of bioactive hydroxytyrosol and probiotics to colon site.

Effects of aging and resistance training in rat tendon remodeling.

In elderly persons, weak tendons contribute to functional limitations, injuries, and disability, but resistance training can attenuate this age-related decline. We evaluated the effects of resistance training on the extracellular matrix (ECM) of the calcaneal tendon (CT) in young and old rats and its effect on tendon remodeling. Wistar rats aged 3 mo (young, n = 30) and 20 mo (old, n = 30) were divided into 4 groups: young sedentary, young trained, old sedentary (OS), and old trained (OT). The training sessions were conducted over a 12-wk period. Aging in sedentary rats showed down-regulation in key genes that regulated ECM remodeling. Moreover, the OS group showed a calcification focus in the distal region of the CT, with reduced blood vessel volume density. In contrast, resistance training was effective in up-regulating connective tissue growth factor, VEGF, and decorin gene expression in old rats. Resistance training also increased proteoglycan content in young and old rats in special small leucine-rich proteoglycans and blood vessels and prevented calcification in OT rats. These findings confirm that resistance training is a potential mechanism in the prevention of aging-related loss in ECM and that it attenuates the detrimental effects of aging in tendons, such as ruptures and tendinopathies.-Marqueti, R. C., Durigan, J. L. Q., Oliveira, A. J. S., Mekaro, M. S., Guzzoni, V., Aro, A. A., Pimentel, E. R., Selistre-de-Araujo, H. S. Effects of aging and resistance training in rat tendon remodeling.

Phenol-enriched olive oils improve HDL antioxidant content in hypercholesterolemic subjects. A randomized, double-blind, cross-over, controlled trial.

At present, high-density lipoprotein (HDL) function is thought to be more relevant than HDL cholesterol quantity. Consumption of olive oil phenolic compounds (PCs) has beneficial effects on HDL-related markers. Enriched food with complementary antioxidants could be a suitable option to obtain additional protective effects. Our aim was to ascertain whether virgin olive oils (VOOs) enriched with (a) their own PC (FVOO) and (b) their own PC plus complementary ones from thyme (FVOOT) could improve HDL status and function. Thirty-three hypercholesterolemic individuals ingested (25 ml/day, 3 weeks) (a) VOO (80 ppm), (b) FVOO (500 ppm) and (c) FVOOT (500 ppm) in a randomized, double-blind, controlled, crossover trial. A rise in HDL antioxidant compounds was observed after both functional olive oil interventions. Nevertheless, α-tocopherol, the main HDL antioxidant, was only augmented after FVOOT versus its baseline. In conclusion, long-term consumption of phenol-enriched olive oils induced a better HDL antioxidant content, the complementary phenol-enriched olive oil being the one which increased the main HDL antioxidant, α-tocopherol. Complementary phenol-enriched olive oil could be a useful dietary tool for improving HDL richness in antioxidants.

Impacto da incorporação de ômega 3 na funcionalidade da lipoproteína de alta densidade: ensaio clínico aleatorizado, paralelo e controlado / Impact of omega 3 incorporation on the functionality of high-density lipoprotein randomized, parallel and controlled clinical

Tese de DoutoradoDocumentoTese de DoutoradoAutorCartolano, Flavia de Conti (Catálogo USP)Nome completoFlavia de Conti CartolanoE-mailE-mailUnidade da USPFaculdade de Saúde PúblicaÁrea do ConhecimentoNutrição em Saúde PúblicaData de Defesa2018-09-26ImprentaSão Paulo, 2018OrientadorDamasceno, Nágila Raquel Teixeira (Catálogo USP) Rogero, Marcelo Macedo - (Coorientador) (Catálogo USP) Banca examinadora Damasceno, Nágila Raquel Teixeira (Presidente) Lottenberg, Ana Maria Pita Mancini, Márcio Corrêa Izar, Maria Cristina de Oliveira Quintao, Eder Carlos Rocha Título em portuguêsImpacto da incorporação de ômega 3 na funcionalidade da lipoproteína de alta densidade: ensaio clínico aleatorizado, paralelo e controladoPalavras-chave em português DHA EPA Lag Time Lipoproteína de Alta Densidade Tamanho de Partícula Resumo em portuguêsINTRODUÇÃO: Apesar de classicamente a lipoproteína de alta densidade (HDL) ser descrita como um fator de risco independente para as doenças cardiovasculares (DCV), devido ao seu papel no transporte reverso de colesterol (TRC), atualmente sabe-se que esta partícula exerce múltiplos mecanismos biológicos que podem contribuir para esta cardioproteção, com estudos indo além do conteúdo de colesterol associada à HDL (HDL-C). OBJETIVO: Avaliar o impacto da suplementação de ?-3 no perfil de ácidos graxos, tamanho e capacidade antioxidante da HDL. MÉTODOS: Estudo clínico, aleatorizado, controlado, duplo cego e paralelo, onde foram incluídos 147 indivíduos adultos, com fatores de risco cardiovascular, que foram alocados no grupo ?-3 (n=77 - 3,0 g/dia contendo 1,8 g de EPA+DHA) ou no grupo ?-6 (n=70 - 3,0 g/dia de óleo de girassol contendo 1,95 g de ácido linoleico). No início (T0) e após 8 semanas (T8) amostras de sangue foram coletadas e, a partir do plasma ou soro, foram analisados o perfil lipídico (CT, HDL-C, LDL-C, TG), as apolipoproteínas AI, CII e CIII, as subfrações da HDL (Lipoprint®), a atividade da paraoxonase (PON1) e da proteína transportadora de éster de colesterol (CETP), a capacidade antioxidante da HDL (método experimental) e o perfil de ácidos graxos e conteúdo de AGNEs da HDL. Os resultados do efeito do tempo, da intervenção e das interações entre os parâmetros monitorados e os desfechos foram realizados com o auxílio do programa SPSS® versão 20.0. O valor de significância considerado foi de p<0,05. RESULTADOS: Os grupos ?-3 e ?-6 eram semelhantes entre si em relação a idade, sexo, tabagismo e uso de medicamentos, ambos apresentando alta prevalência de hipertensão e dislipidemia. Nos dois grupos, houve redução em todos os marcadores lipídicos, exceto a concentração de HDL-C, que aumentou. Observou-se o efeito do tempo no conteúdo de HDLAGNEs (?=-16,2%), no percentual de HDLGRANDE (?=20,1%) e de HDLPEQUENA (?=-5,0%). O aumento de EPA na HDL se associou à menor chance de ter elevação da atividade da PON1 (OR=0,446; IC=0,200-0,994), da concentração de HDLAGNEs (OR=0,275; IC=0,113-0,660) e do percentual de HDLPEQUENA (OR=0,337; IC=0,146-0,782). Observou-se ainda que o EPA se associou a, aproximadamente, 3,5 mais chances de aumento no percentual da HDLGRANDE (OR=3,522; IC=1,652-7,507). Quanto ao aumento de DHA na HDL, este esteve associado de maneira significativa à diminuição da concentração de Apo AI (OR=0,351; IC=0,150-0,821), além da atividade da PON1 (OR=0,226; IC=0,110-0,639) e da concentração de HDLAGNEs (OR=0,275; IC=0,113-0,668). Resultados similares aos obtidos com o EPA foram observados para o DHA e o tamanho da HDL. Não foi constatado efeito da incorporação de EPA e DHA na resistência à oxidação. CONCLUSÃO: A intervenção com ?-3 promoveu mudanças na composição da partícula de HDL, aumentando o percentual das subfrações maiores, sem, contudo, modificar sua capacidade antioxidante.

BACKGROUND: Although high-density lipoprotein (HDL) is classically described as an independent risk factor for cardiovascular disease (CVD), because of its role in reverse cholesterol transport (RCT), currently, it is known that this particle exerts multiple biological mechanisms that may contribute to this cardioprotection, with studies going beyond HDL cholesterol content (HDL-C). OBJECTIVE: To evaluate the impact of ?-3 supplementation on the fatty acid profile, size and antioxidant capacity of HDL. METHODS: A randomized, controlled, double-blind, parallel clinical study involving 147 adult subjects with cardiovascular risk factors, who were allocated into the ?-3 group (n=77 -3.0 g/day containing 1.8 g EPA + DHA) or the ?-6 group (n=70 -3.0 g/day of sunflower oil containing 1.95 g of linoleic acid). In the beginning (T0) and after 8 weeks (T8) blood samples were collected and, from plasma or serum, were analyzed lipid profile (TC, HDL-C, LDL-C, TG), apolipoproteins AI, CII and CIII, HDL subfractions (Lipoprint®), paraoxonase (PON1) and cholesterol ester carrier protein (CETP) activities, antioxidant capacity of HDL (experimental method), the fatty acid profile and NEFAs content HDL. Results of the effect of time, intervention and interactions between monitored parameters and outcomes were performed with the aid of SPSS® software version 20.0. The significance level considered was p<0.05. RESULTS: The ?-3 and ?-6 groups were similar in relation to age, sex, smoking and medication use, both presenting high prevalence of hypertension and dyslipidemia. In both groups, there was a reduction in all lipid markers except HDL-C concentration, which increased. The effect of time on HDLNEFAs content (?=-16.2%), and on the percentage of HDLLARGE (?=20.1%) and HDLSMALL (?=-5.0%) were observed. Increasing EPA in HDL was associated with a lower chance of enhance PON1 activity (OR=0.446, CI=0.200-0.994), HDLNEFAs concentration (OR=0.275, CI=0.113-0.660), and HDLSMALL (OR=0.337, CI=0.146- 0.782). It was also observed that EPA was associated with approximately 3.5 more chances of raising the percentage of HDLLARGE (OR=3.522, CI=1.652-7.507). Increasing DHA in HDL was significantly associated with a decrease in Apo AI concentration (OR=0.351, CI=0.150- 0.821), and also PON1 activity (OR=0.226, CI=0.110-0.639) and concentration of HDLNEFAs (OR=0.275, CI=0.113-0.668). Similar results obtained with EPA were observed for DHA and HDL size. No effect of the incorporation of EPA and DHA on oxidation resistance was observed. CONCLUSION: The ?-3 intervention promoted changes in the composition of the HDL particle, increasing the percentage of the larger subfractions, without, however, modifying its antioxidant capacity.

Generation of nitric oxide by olive (Olea europaea L.) pollen during in vitro germination and assessment of the S-nitroso- and nitro-proteomes by computational predictive methods.

Nitric oxide is recognized as a signaling molecule involved in a broad range of physiological processes in plants including sexual reproduction. NO has been detected in the pollen grain at high levels and regulates pollen tube growth. Previous studies demonstrated that NO as well as ROS are produced in the olive reproductive tissues in a stage- and tissue-specific manner. The aim of this study was to assess the production of NO throughout the germination of olive (Olea europaea L.) pollen in vitro. The NO fluorescent probe DAF-2DA was used to image NO production in situ, which was correlated to pollen viability. Moreover, by means of a fluorimetric assay we showed that growing pollen tubes release NO. GSNO -a mobile reservoir of NO, formed by the S-nitrosylation of NO with reduced glutathione (GSH) - was for the first time detected and quantified at different stages of pollen tube growth using a LC-ES/MS analysis. Exogenous NO donors inhibited both pollen germination and pollen tube growth and these effects were partially reverted by the specific NO-scavenger c-PTIO. However, little is known about how NO affects the germination process. With the aim of elucidating the putative relevance of protein S-nitrosylation and Tyr-nitration as important post-translational modifications in the development and physiology of the olive pollen, a de novo assembled and annotated reproductive transcriptome from olive was challenged in silico for the putative capability of transcripts to become potentially modified by S-nitrosylation/Tyr-nitration according to well-established criteria. Numerous gene products with these characteristics were identified, and a broad discussion as regards to their potential role in plant reproduction was built after their functional classification. Moreover, the importance of both S-nitrosylation/Tyr-nitrations was experimentally assessed and validated by using Western blotting, immunoprecipitation and proteomic approaches.