Adna Prado Massarioli

Winery by-products: extraction optimization, phenolic composition and cytotoxic evaluation to act as a new source of scavenging of reactive oxygen species.

Nearly 20 million tons of winery by-products, with many biological activities, are discarded each year in the world. The extraction of bioactive compounds from Chenin Blanc, Petit Verdot, and Syrah grape by-products, produced in the semi-arid region in Brazil, was optimized by a Central Composite Rotatable Design. The phenolic compounds profile, antioxidant capacity against synthetic free radicals (DPPH and ABTS), reactive oxygen species (ROS; peroxyl radical, superoxide radical, hypochlorous acid), cytotoxicity assay (MTT) and quantification of TNF-α production in RAW 264.7 cells were conducted. Gallic acid, syringic acid, procyanidins B1 and B2, catechin, epicatechin, epicatechin gallate, quercetin 3-β-d-glucoside, delfinidin 3-glucoside, peonidin 3-O-glucoside, and malvidin 3-glucoside were the main phenolic compounds identified. In general, rachis showed higher antioxidant capacity than pomace extract, especially for Chenin Blanc. All extracts showed low cytotoxicity against RAW 264.7 cells and Petit Verdot pomace suppressed TNF-α liberation in vitro. Therefore, these winery by-products can be considered good sources of bioactive compounds, with great potential for application in the food and pharmaceutical industries.

Bioassay - guided isolation of proanthocyanidins with antioxidant activity from peanut (Arachis hypogaea) skin by combination of chromatography techniques.

Purification and bioassay-guided fractionation were employed to isolate proanthocyanidins with antioxidant activity from peanut skin (Arachis hypogaea Runner 886). The crude extract was prepared with acetone (60% v/v) and purified using chromatographic methods, including a semipreparative HPLC technique. As a result, two proanthocyanidins were isolated and identified using NMR, epicatechin-(2 β → O → 7, 4 β → 8)-catechin (proanthocyanidin A1) and epicatechin-(β → 2 O → 7, 4 β → 8)-epicatechin (proanthocyanidin A2). Despite the structural similarity, differences were observed in their antioxidant activity. Proanthocyanidin A1 proved to be more active, with EC50 value for DPPH radical scavenging of 18.25 μg/mL and reduction of Fe(3+)-TPTZ complex of 7.59 mmol/g, higher than that of synthetic antioxidant BHT. This compound evaluated by ABTS(+) was similar to that of natural quercetin. Therefore, peanut skin is an important source of bioactive compounds that may be used as a mild antioxidant for food preservation.

Composição fenólica e atividade antioxidante de resíduos agroindustriais

Nowadays, the agro-industrial processing produces millions of tons of wastes. Many of them are rich in bioactive compounds, being a potential natural source of these substances. This study aimed to evaluate the content of total phenolics, antioxidant activity and phenolic composition of residues generated by three Brazilian agribusiness: Isabel grape pomace (PI) (Vitis labrusca), Verdejo grape pomace (PV) (Vitis vinifera) and guava pomace (PG) (Psidium guajava). The results of total phenolics content (mg GAE g-1) found in the ethanol and aqueous extracts of residues were: PV (20.94±0.46; 8.03±0.43)>PI (16.57±0.19; 4.41±0.01)>PG (3.41±0.09; 1.88±0.06). High antioxidant activity of these extracts, particularly PV and PI, was found by the methods DPPH ●, ABTS ● and beta-carotene bleaching method. A strong positive correlation between antioxidant activity and content of total phenolic compounds was found. The following phenolic compounds were found by gas chromatography with mass spectrometry (GC-MS): gallic acid, epicatechin, quercetin (PV, PI and PG); isovanilic acid (PI, PG), p-coumaric acid (PI), caffeic acid and resveratrol (PV, PI). The results show that these residues, particularly the wineries, are rich in bioactive substances and should be exploited by the food industry and pharmaceuticals.

Anti-Inflammatory, Anti-Osteoclastogenic and Antioxidant Effects of Malva sylvestris Extract and Fractions: In Vitro and In Vivo Studies.

Given their medical importance, natural products represent a tremendous source of drug discovery. The aim of this study was to investigate Malva sylvestris L. extract and fractions and their pharmacological activities followed by chemical identification. The aqueous fraction (AF) was identified as the bioactive fraction in the in vitro and in vivo assays. The AF controlled the neutrophil migration to the peritoneal cavity by 66%, inhibited the antiedematogenic activity by 58.8%, and controlled IL-1β cytokine expression by 54%. The in vitro viability tests showed a concentration-dependent effect, where the MSE and fractions at concentrations under 10 μg/mL were non-toxic to cells. Transcriptional factors of carbonic anhydrase II (CAII), cathepsin K (Ctsk) and tartrate-resistant acid phosphatase (TRAP) were analyzed by qPCR in RAW 264.7 cell lines. The gene expression analysis showed that the AF was the only treatment that could downregulate all the study genes: CAII, Ctsk and TRAP (p<0.05). TRAP staining was used to evaluate osteoclast formation. AF treatments reduced the number of osteoclastogenesis 2.6-fold compared to the vehicle control group. Matrix metalloproteinase 9 (MMP-9) activity decreased 75% with the AF treatment. Moreover, the bioactive fraction had the ability to regulate the oxidation pathway in the ABTS (2,2-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) assay with an activity equivalent to 1.30 μmol Trolox/g and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals 1.01 g/L. Positive ion ESI-mass spectrometry for molecular ions at m/z 611 and 633 confirmed rutin as the major compound in the AF. The AF of M. sylvestris presented anti-inflammatory, controlled osteoclastogenic mechanisms and antioxidant abilities in different in vitro and in vivo methods. In addition, we suggest that given its multi-target activity the bioactive fraction may be a good candidate in the therapy of chronic inflammatory diseases.

Inhibition of DMBA‐induced Oral Squamous Cells Carcinoma Growth by Brazilian Red Propolis in Rodent Model

We investigated the effect of oral administration of hydroalcoholic extract of Brazilian red propolis (HERP) on DMBA‐induced oral squamous cell carcinomas (OSCC) in rodents. The chemical components of the HERP were assessed by high‐performance liquid chromatography (HPLC). Carcinogenesis was topically induced in the lower lip of 25 rats using 9,10‐dimethyl‐1,2‐benzanthracene (DMBA); the tumour was treated with saline (TUM1) and Tween 80 (TUM2) as well as HERP at 10, 50 and 100 mg/kg (HERP10, HERP50 and HERP100, respectively) for 20 weeks. Topical application of saline and oral administration of 100 mg/kg HERP was used in five rats as a control group (CTR). After 26 weeks, the histological malignancy grading and immunohistochemical expression of Ki‐67 and p16INK4A were assessed in the tumours/tissue samples. The compounds identified were propyl gallate, daidzein, catechin, epicatechin, formononetin and biochanin A. Formononetin, daidzein and biochanin A showed concentration of 23.29, 0.38 and 0.67 mg/g of HERP, respectively. HERP at doses of 50 and 100 mg/kg inhibited 40% of OSCC growth and promoted a 3‐week delay in development of clinically detectable tumours. Epithelial dysplasia was observed in all samples with no clinical tumour, except in CTR. No significant difference in the immunoexpression of Ki‐67 and p16INK4A was observed between HERP‐treated and saline/Tween 80‐treated groups (p > 0.05). Our results suggest that HERP exerts chemopreventive activity on the progression of DMBA‐induced epithelial dysplasia to OSCC in an experimental model of labial carcinogenesis; however, this effect is not associated with Ki‐67 and p16INK4A immunoexpression.

Evaluation of the release profile, stability and antioxidant activity of a proanthocyanidin-rich cinnamon (Cinnamomum zeylanicum) extract co-encapsulated with α-tocopherol by spray chilling

Cinnamon has many health improving compounds such as proanthocyanidins, which also have potential for the prevention of damages caused by diabetes. Similarly, α-tocopherol is a natural antioxidant with important role on protection of fatty acids in membranes and lipoproteins. However, the addition of antioxidants in food may result in interaction with food matrix, low stability and unpleasant taste. In the present study, a proanthocyanidin-rich cinnamon extract (PRCE) (Cinnamomum zeylanicum) was co-encapsulated with α-tocopherol into solid lipid microparticles (SLMs) by spray chilling. The microparticles were characterized with regard to the physical and chemical properties, morphology, proanthocyanidin stability and release profile. SLMs were spherical with an average diameter of ca. 80μm. Proanthocyanidins were highly stable in SLMs stored for up to 90days at 5, 25 and 37°C. Moreover, SLMs gradually released proanthocyanidins in simulated gastrointestinal fluids by a diffusional process, following a Korsmeyer-Peppas kinetic. Analyses of the antioxidant compounds indicated that PRCE components exhibited a higher scavenging capacity against reactive oxygen species (ROS) and reactive nitrogen species (RNS). Thus, the SLMs produced in the present study have potential for application in the development of new functional foods and nutraceuticals, also providing an alternative for the controlled release of proanthocyanidins and α-tocopherol into the intestine.

Exploration of avocado by-products as natural sources of bioactive compounds

This study aimed to evaluate the antioxidant, anti-inflammatory, and cytotoxic properties and phenolic composition of peel and seed of avocado varieties Hass and Fuerte using green solvents. Ethanol soluble compounds were identified in peel and seed of both varieties using HPLC-MS/MS and quantified using HPLC-DAD. Agro-industrial by-products of both varieties exhibited high radical scavenging activity against synthetic free radicals (DPPH and ABTS) and reactive oxygen species (peroxyl, superoxide, and hypochlorous acid) and high ability to reduce Fe3+ to Fe2+. The main compounds with significant contribution to the antioxidant activity determined by online HPLC-ABTS●+ analyses were procyanidin B2 and epicatechin in the peel and trans-5-O-caffeoyl-D-quinic acid, procyanidin B1, catechin, and epicatechin in the seed. Peel of Fuerte significantly suppressed TNF-α and nitric oxide (NO) release (459.3 pg/mL and 8.5 μM, respectively), possibly because of the high phenolic content and antioxidant activity detected. Avocado agro-industrial by-products can be used for food and pharmaceutical purposes due to their antioxidant and anti-inflammatory properties.

Volatile and non-volatile/semi-volatile compounds and in vitro bioactive properties of Chilean Ulmo (Eucryphia cordifolia Cav.) honey

Ulmo honey originating from Eucryphia cordifolia tree, known locally in the Araucania region as the Ulmo tree is a natural product with valuable nutritional and medicinal qualities. It has been used in the Mapuche culture to treat infections. This study aimed to identify the volatile and non-volatile/semi-volatile compounds of Ulmo honey and elucidate its in vitro biological properties by evaluating its antioxidant, antibacterial, antiproliferative and hemolytic properties and cytotoxicity in Caco-2 cells. Headspace volatiles of Ulmo honey were isolated by solid-phase microextraction (SPME); non-volatiles/semi-volatiles were obtained by removing all saccharides with acidified water and the compounds were identified by GC/MS analysis. Ulmo honey volatiles consisted of 50 compounds predominated by 20 flavor components. Two of the volatile compounds, lyrame and anethol have never been reported before as honey compounds. The non-volatile/semi-volatile components of Ulmo honey comprised 27 compounds including 13 benzene derivatives accounting 75% of the total peak area. Ulmo honey exhibited weak antioxidant activity but strong antibacterial activity particularly against gram-negative bacteria and methicillin-resistant Staphylococcus aureus (MRSA), the main strain involved in wounds and skin infections. At concentrations >0.5%, Ulmo honey reduced Caco-2 cell viability, released lactate dehydrogenase (LDH) and increased reactive oxygen species (ROS) production in a dose dependent manner in the presence of foetal bovine serum (FBS). The wide array of volatile and non-volatile/semi-volatile constituents of Ulmo honey rich in benzene derivatives may partly account for its strong antibacterial and antiproliferative properties important for its therapeutic use. Our results indicate that Ulmo honey can potentially inhibit cancer growth at least partly by modulating oxidative stress.

Can we conserve trans-resveratrol content and antioxidant activity during industrial production of chocolate?: Trans-resveratrol contents during production of chocolate

BACKGROUND Cocoa exhibits high content of phenolic compounds, among which trans-resveratrol stands out, associated with several bioactive activities such as antioxidant properties. Chocolate contains reduced amounts of these bioactive compounds due to losses during the production process. Therefore, this study aimed to assess changes in total phenolic content, and specifically trans-resveratrol, as well as changes in the antioxidant activity of cocoa and its products during industrial production of chocolate. RESULTS A total of ten different cocoa products were analyzed. The processes of fermentation and roasting caused significant loss of total phenolic compounds and antioxidant activity. The high temperature of roasting had a major influence on this loss (71% for total phenolic compounds and 53-77% for antioxidant activity), except for trans-resveratrol. The trans-resveratrol content formed after fermentation (9.8 μg kg-1 ) showed little variation during the processes, and it was detected in higher concentrations both in natural (11.4 μg kg-1 ) and in alkalized cocoa powder (13.5 μg kg-1 ). Alkalization of cocoa products led to loss of capacity of deactivating superoxide radical. CONCLUSION These findings contribute to the optimization of the production process of chocolate and other food products containing cocoa and its derivatives, aiming to better preserve their bioactive compounds.