A Comprehensive Study on the Chemical Characterization and Neuroprotective Evaluation of Pracaxi Nuts Extracts Obtained by a Sustainable Approach.

The Amazonian Pentaclethra macroloba (Willd.) Kuntze nuts contain a lipidic fraction with health-promoting effects, but little is known about the bioactivity of other constituents. In this study, the lipidic fraction obtained using supercritical fluid extraction (SFE) with CO2 was chemically characterized by using lipidomics techniques. The SFE-CO2 residue, named as pracaxi cake, was re-extracted by pressurized liquid extraction following a biorefinery approach. Using a response surface methodology and based on the extraction yield and different in vitro assays, two optimum conditions were obtained: 80% and 12.5% of ethanol at 180 °C. Under these conditions, extraction yield and different in vitro measurements related to neuroprotection were assessed. Chemical characterization of these extracts suggested the presence of triterpenoid saponins and spermidine phenolamides, which were not previously reported in pracaxi nuts. These results suggest that pracaxi oil extraction by-products are a valuable source of bioactive compounds with neuroprotective potential.

Nutritional Composition and Bioactive Compounds of Native Brazilian Fruits of the Arecaceae Family and Its Potential Applications for Health Promotion.

The fruits from the Arecaceae family, although being rich in bioactive compounds with potential benefits to health, have been underexplored. Studies on their composition, bioactive compounds, and effects of their consumption on health are also scarce. This review presents the composition of macro- and micronutrients, and bioactive compounds of fruits of the Arecaceae family such as bacaba, patawa, juçara, açaí, buriti, buritirana, and butiá. The potential use and reported effects of its consumption on health are also presented. The knowledge of these underutilized fruits is important to encourage production, commercialization, processing, and consumption. It can also stimulate their full use and improve the economy and social condition of the population where these fruits are found. Furthermore, it may help in future research on the composition, health effects, and new product development. Arecaceae fruits presented in this review are currently used as raw materials for producing beverages, candies, jams, popsicles, ice creams, energy drinks, and edible oils. The reported studies show that they are rich in phenolic compounds, carotenoids, anthocyanins, tocopherols, minerals, vitamins, amino acids, and fatty acids. Moreover, the consumption of these compounds has been associated with anti-inflammatory, antiproliferative, antiobesity, and cardioprotective effects. These fruits have potential to be used in food, pharmaceutical, and cosmetic industries. Despite their potential, some of them, such as buritirana and butiá, have been little explored and limited research has been conducted on their composition, biological effects, and applications. Therefore, more detailed investigations on the composition and mechanism of action based on in vitro and/or in vivo studies are needed for fruits from the Arecaceae family.

Emerging Lipids from Arecaceae Palm Fruits in Brazil.

Arecaceae palm tree fruits (APTFs) with pulp or kernel rich in oil are widely distributed in six Brazilian biomes. APTFs represent a great potential for the sustainable exploitation of products with high added value, but few literature studies have reported their properties and industrial applications. The lack of information leads to underutilization, low consumption, commercialization, and processing of these fruit species. This review presents and discusses the occurrence of 13 APTFs and the composition, physicochemical properties, bioactive compounds, and potential applications of their 25 oils and fats. The reported studies showed that the species present different lipid profiles. Multivariate analysis based on principal component analysis (PCA) and hierarchical cluster analysis (HCA) indicated a correlation between the composition of pulp and kernel oils. Myristic, caprylic, capric, and lauric acids are the main saturated fatty acids, while oleic acid is the main unsaturated. Carotenoids and phenolic compounds are the main bioactive compounds in APTFs, contributing to their high oxidative stability. The APTFs oils have a potential for use as foods and ingredients in the cosmetic, pharmaceutical, and biofuel industries. However, more studies are still necessary to better understand and exploit these species.

High-added value co-products obtained from pecan nut (Carya illinoinensis) using a green extraction technology.

Abstract: The aqueous (AF) and solid (SF) fractions obtained as co-products in the aqueous extraction of pecan nut oil assisted by Alcalase® were evaluated. In the AF, the degree of protein hydrolysis (DH) and the electrophoretic profile of protein hydrolysates, phenolic compounds, and antioxidant capacity (reducing potential of the hydrophilic compounds, RPHC, 2,2-diphenyl-1-picrylhydrazyl, DPPH; and inhibition of lipid peroxidation) were determined. The proximate composition and microstructure were evaluated in SF. The results indicated a DH of 3.9%. The sample treated with the enzyme (ET) showed a molecular weight of proteins lower than 15 kDa. The ET showed higher content of phenolics (726.3 mg GAE/100 g) and antioxidant capacity higher than the sample without enzymatic treatment. The SF showed a residual lipid content rich in oleic and linoleic acids. Furthermore, changes in the proximate composition and the microstructure were observed. The results indicate the potentiality of hydrolyzed fractions for application in food.

Dataset on the phytochemicals, antioxidants, and minerals contents of pecan nut cake extracts obtained by ultrasound-assisted extraction coupled to a simplex-centroid design.

This article contains a dataset related to the research published in "The potential of the pecan nut cake as an ingredient for the food industry" [1]. A three-component simplex-centroid mixture design coupled with response surface methodology (RSM) was applied to generate statistical models and to analyze the dataset. The method was also applied to evaluate the effect of different solvents (ethanol, water, and acetic acid) on the extraction of bioactive compounds of pecan nut cake (PNC) and its antioxidant activity. Furthermore, simultaneous optimization of the solvent mixture was carried out to predict the optimum point with the best combination of solvents to obtain an extract with enhanced phytochemical composition, as well as high in vitro antioxidant activity. The maximization of total phenolic compounds, condensed tannins, and antioxidant activity of the PNC was predicted by the desirability function. A total of 80 interactions were run to provide the best condition for optimization. The combined use of the different solvents enables a higher recovery of the compounds than their isolated use. This dataset may help other researchers on the application of a mixture design to recover phytochemicals from a broad range of co-products such as defatted meals and other nut cakes, which are sometimes discarded as waste by many industries.

Ultrasound-Assisted Extraction for the Recovery of Carotenoids from Guava's Pulp and Waste Powders.

In this work, lycopene- and ß-carotene-rich extracts were obtained from guava's pulp and waste powders using maceration (ME), ultrasonic bath (BUAE, 25 °C for 30 min), and ultrasonic probe (PUAE, 25 °C for 5 min). Extracts were evaluated for total carotenoid content, antioxidant capacity, color, and lycopene and ß-carotene content by HPLC-DAD. Bath-type ultrasound-assisted extraction (BUAE) was the best technique to obtain lycopene from guava pulp (135.0 mg 100 g-1) and waste (76.64 mg 100 g-1), followed by ME (pulp = 107.6, waste = 43.57 mg 100 g-1), and PUAE (pulp = 44.19, waste = 33.83 mg 100 g-1). The total carotenoid content positively affected the extracts' antioxidant capacity. Dehydration of guava fractions showed to be an efficient method to increase carotenoid availability. This is the first work to report the recovery of carotenoids from guava by ultrasonic extraction. Furthermore, this method has shown to be a suitable approach to reduce extraction time and solvent use. These extracts could be further applied to lipid-rich foods as a natural antioxidant and/or as an ingredient in the development of functional foods.

May the superfruit red guava and its processing waste be a potential ingredient in functional foods?

Guava (Psidium guajava L.), a popular fruit in tropical countries, can be consumed in natura or transformed into many products. Guava's processing waste (seed, peel, and pulp leftovers) can represent up to 30% of the fruit's volume. Evidence indicates that this fraction still holds a significant amount of antioxidant substances, such as phenolic compounds. Therefore, the objective of this study was to investigate the phytochemical composition (total phenolics, flavonoids, flavonols, and condensed tannins), phenolic profile, and antioxidant activity (DPPH, modified DPPH, ABTS, and FRAP) of guava's pulp and waste extracts (GPE and GWE) obtained from guava's pulp and waste powders (GPP and GWP). The extraction was performed using a probe-type ultrasound for 2 min at 25 °C, using ethanol:water (30:70, v/v) as the solvent (15 mL for 1 g of sample). The phenolic profile of guava's powders and extracts was performed by LC-ESI-MS/MS analysis. GPE showed the highest concentration of total phenolics (2348.3 mg GAE 100 g-1 by Folin-Ciocalteu and 518.00 mg GAE 100 g-1 by Prussian Blue), and the greater in vitro antioxidant activity among all the assays tested. On the other hand, GWE was found to hold a higher amount of flavonoids (1006.08 mg QE 100 g-1), flavonols (352.59 mg QE 100 g-1), and condensed tannins (1466.9 mg g-1). Overall, 37 phenolic compounds were identified among all the samples, with 14 compounds being reported for the first time for red guava. Ellagic acid was the major phenolic present in all samples. The results showed that guava's pulp and waste are a rich source of phenolics compounds and may be used as an ingredient for the development of functional foods.

Sapucaia nut (Lecythis pisonis Cambess) and its by-products: A promising and underutilized source of bioactive compounds. Part II: Phenolic compounds profile.

In this study, the profile of the bioactive compounds of sapucaia nut (Lecythis pisonis Cambess) and its by-products have been investigated. The phenolic profile by LC-ESI-MS/MS, the total phenolic content, the condensed tannins and the antioxidant activity of the sapucaia nut and shell were determined. 14 phenolic compounds were identified in the sapucaia nut extract, primarily phenolic acids and flavonoids. Catechin, epicatechin, myricetin, ellagic acid and ferulic acid presented significant correlation to the antioxidant activity. The sapucaia shell contained 22 phenolic compounds, 13 of which were quantified. The sapucaia shell extract showed a high content of total phenolic compounds, a high condensed tannins content, and high antioxidant activity. The higher antioxidant activity of the shell can be associated with a higher content of phenolics. Overall, it can be concluded that the sapucaia nut is a raw material rich in phenolic compounds that present high antioxidant activity. The nuts and the cake may be used as a promising raw material for the food industry, while the shells could be an alternative source of natural antioxidants. Further use in the cosmetics and pharmaceutical industry may also be envisaged.

Sapucaia nut (Lecythis pisonis Cambess) and its by-products: A promising and underutilized source of bioactive compounds. Part I: Nutritional composition and lipid profile.

The nutritional composition of the sapucaia nut, cake and shell, the nut and cake minerals content and the lipid profile of the nut oil (fatty acids, tocopherols, phytosterols and triacylglycerols) were determined. The nuts and cake exhibited a high content of lipid (47.9 to 60.8 mg 100 g-1), protein (15.8 to 19.5 mg 100 g-1), dietary fiber (16.5 to 22.6 mg 100 g-1) and provided an excellent source of selenium (26.4 to 46.94 µg g-1). The oil contained a high amount of unsaturated fatty acids (39.7 to 45.4% of oleic and 32.2 to 46.6% of linoleic acids) and presented a high Oxidative Stability Index (8.57-12.95 h) indicating the presence of antioxidant compounds in the oil. The major triacylglycerols in the sapucaia oil were LLO, PLO, LOO, POO, OOO, PLL and LLL. The main bioactive lipids identified in the oil were γ-tocopherol (19.2 to 28.5 mg 100 g-1) and ß-sitosterol (92.8 to 194 mg 100 g-1). The results showed that the sapucaia nut and its by-products are a promising natural source of bioactive and nutritional compounds and when present in the diet can contribute to the maintenance of human health. In addition, the nut and by-product represents a promising raw material for the food industry.

Chemical Characterization and Release of Polyphenols from Pecan Nut Shell [Carya illinoinensis (Wangenh) C. Koch] in Zein Microparticles for Bioactive Applications.

The pecan nut [Carya illinoinensis (Wangenh) C. Koch] is a natural source of polyphenols with antioxidant properties. In this study, the encapsulation of aqueous and hydroalcoholic extracts of pecan nut shell were evaluated for the release of bioactive compounds and antioxidant potential in order to explore food applications using zein as encapsulating agent. The extracts showed high contents of total phenolics, condensed tannins and high antioxidant activity. Concentrations of proanthocyanidins were 9-fold higher in hydroalcoholic extracts. The LC-DAD analysis showed that catechins were the major phenolic compounds in samples, with epigallocatechin levels up to 138.62 mg mL-1. Zein microparticles loaded with aqueous extract released 2.3 times more phenolic compounds than the hydroalcoholic extracts and the DSC thermograms showed that extracts of pecan nut shell remained thermally stable up to 240 °C. The zein microcapsules obtained in this study were efficiently encapsulated and represent an interesting additive due its high antioxidant capacity, physicochemical characteristics and morphology. The use of zein microparticles combined with natural extracts constitute a step forward in the improvement of current technology for delivering phenolic compounds with applications in functional foods and nutraceuticals.

Ultrasonic-assisted extraction combined with sample preparation and analysis using LC-ESI-MS/MS allowed the identification of 24 new phenolic compounds in pecan nut shell [Carya illinoinensis (Wangenh) C. Koch] extracts.

Ultrasonic-assisted extraction combined with statistical tools (factorial design, response surface methodology and kinetics) were used to evaluate the effects of the experimental conditions of temperature, solid-to-solvent ratio, ethanol concentration and time for the extraction of the total phenolic content from pecan nut shells. The optimal conditions for the aqueous and hydroalcoholic extract (with 20% v/v of ethanol) were 60 and 80 °C; solid to solvent ratio of 30 mL·g-1 (for both) and extraction time of 35 and 25 min, respectively. Using these optimize extraction conditions, 426 and 582 mg GAE·g-1 of phenolic compounds, from the aqueous and hydroalcoholic phases respectively, were obtained. In addition, the analysis of the phenolic compounds using the LC-ESI-MS/MS system allowed the identification of 29 phenolic compounds, 24 of which had not been reported in literature for this raw material yet.

Nutritional potential, chemical profile and antioxidant activity of Chichá (Sterculia striata) nuts and its by-products.

The Sterculia striata nut commonly known as chichá is consumed raw or toasted inBrazil but information on its chemical composition and antioxidant activity are scarce in the literature. In this work, the nutritional composition, minerals profile, lipid composition and phenolic compounds profile of chichá nuts and its by-products (pellicle and shell) were determined. The results showed that the nuts and the cake contain considerable amount of fibers and proteins and are rich in minerals (Fe, Mn and Cu). Oleic acid was the main fatty acid (37.8%), and PPO (36.0%) and POP (15.6%) were the main triacylglycerides present in the chichá oil. The phytosterols ß-sitosterol, stigmasteroland campesterol (1848.5µgg-1, 542.2µgg-1 and 186.3µgg-1, respectively), and the γ-, δ-, α- and ß-tocopherol (8.85; 2.10; 1.64, and 0.11mg100g-1, respectively) were identified in the oil. The antioxidant activity (ABTS and FRAP assays) followed the order pellicle>shell>nuts (13.25 and 84.65; 8.71 and 64.3; 5.85 and 36.79µmolTEACg-1, respectively). The main phenolic compounds identified in the extracts were ellagic and ferulic acids; ellagic and protocatechuic acids; and protocatechuic, ellagic and methoxyphenylacetic acids for the nuts, shells and pellicle, respectively. A strong positive correlation was observed between total phenolic content and antioxidant activity determined by ABTS and FRAP assays (r=0.9067, p<0.01; and r=0.9584, p<0.01; respectively). Collectively, the results showed that the chichá is a nut of high nutritional value, rich in bioactive compounds.

Impact of the addition of cocoa butter equivalent on the volatile compounds profile of dark chocolate.

The effect of the partial replacement of cocoa butter (CB) by cocoa butter equivalent (CBE) in the release of volatile compounds in dark chocolate was studied. The fatty acid profile, triacylglyceride composition, solid fat content (SFC) and melting point were determined in CB and CBE. Chocolate with CB (F1) and with different content of CBE (5 and 10%-F2 and F3, respectively) were prepared. Plastic viscosity and Casson flow limit, particle size distribution and release of volatile compounds using a solid phase microextraction with gas chromatography (SMPE-GC) were determined in the chocolate samples. The melting point was similar for the studied samples but SFC indicated different melting behavior. CBE showed a higher saturated fatty acid content when compared to CB. The samples showed similar SOS triglyceride content (21 and 23.7% for CB and CBE, respectively). Higher levels of POS and lower POP were observed for CB when compared to CBE (44.8 and 19.7 and 19 and 41.1%, respectively). The flow limit and plastic viscosity were similar for the studied chocolates samples, as well as the particle size distribution. Among the 27 volatile compounds identified in the samples studied, 12 were detected in significantly higher concentrations in sample F1 (phenylacetaldehyde, methylpyrazine, 2,6-dimethylpyrazine, 2-ethyl-5-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, tetramethylpyrazine, trimethylpyrazine, 3-ethyl-2,5-dimethylpyrazine, phenethyl alcohol, 2-acetylpyrrole, acetophenone and isovaleric acid). The highest changes were observed in the pyrazines group, which presented a decrease of more than half in the formulations where part of the CB was replaced by the CBE.

Aqueous extract from pecan nut [Carya illinoinensis (Wangenh) C. Koch] shell show activity against breast cancer cell line MCF-7 and Ehrlich ascites tumor in Balb-C mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In Brazil many health disorders are treated with the consumption of different varieties of tea. Shell extracts of pecan nut (Carya illinoinensis), which have significant amounts of phenolic compounds in their composition, are popularly taken as tea to prevent diverse pathologies. AIM OF THE STUDY: Phenolic compounds from pecan nut shell extract have been associated with diverse biological effects but the effect on tumor cells has not been reported yet. The aim of the current work was to evaluate the relationship between DNA fragmentation, cell cycle arrest and apoptosis induced by pecan nut shell extract and its antitumor activity. MATERIALS AND METHODS: Cytotoxicity, proliferation, cell death and cell cycle were evaluated in MCF-7 cells by MTT, colony assay, differential coloring and flow cytometry assays, respectively. DNA damage effects were evaluated through intercalation into CT-DNA and plasmid DNA cleavage. Tumor growth inhibition, survival time increase, apoptosis and cell cycle arrest were assessed in Ehrlich ascites tumor in Balb/C mice. RESULTS: The cytotoxic effect of pecan nut shell extracts, the induction of cell death by apoptosis and also the cell cycle arrest in MCF-7 cells have been demonstrated. The survival time in mice with Ehrlich ascites tumor increased by 67%. DNA damage was observed in the CT-DNA, plasmid DNA and comet assays. The mechanism involved in the antitumor effect of pecan nut shell extracts may be related to the activation of key proteins involved in apoptosis cell death (Bcl-XL, Bax and p53) and on the cell cycle regulation (cyclin A, cyclin B and CDK2). CONCLUSIONS: These results were attributed to the phenolic profile of the extract, which presented compounds such as gallic, 4-hydroxybenzoic, chlorogenic, vanillic, caffeic and ellagic acid, and catechin, epicatechin, epigallocatechin and epicatechin gallate. The results indicated that pecan nut shell extracts are effective against tumor cells growth and may be considered as an alternative to the treatment of cancer.

Impact of pecan nut shell aqueous extract on the oxidative properties of margarines during storage.

BACKGROUND: The oxidative properties of margarines supplemented with pecan nut shell extract, rosemary extract and butylated hydroxytoluene (BHT) were investigated. RESULTS: The extracts of pecan nut shell and rosemary showed a high content of total phenolics and condensed tannins (93 and 102.9 mg GAE g-1 and 46 and 38.9 mg CE g-1 respectively) as well as a high antioxidant activity (1257 and 2306 µmol TEAC g-1 and 293 and 856 mg TEAC g-1 by ABTS and DPPH methods respectively). Gallic acid, chlorogenic acid, p-hydroxybenzoic acid, epicatechin and epicatechin gallate were identified in the pecan nut shell extract. Peroxide and p-anisidine values of 3.75-4.43 meq kg-1 and 1.22-2.73 respectively, Totox values of 9.88-10.8 and specific extinction values of 4.38-4.59 and 0.92-0.94 at 232 and 268 nm respectively were observed after 8 months of storage in the studied samples. CONCLUSION: Margarines supplemented with pecan nut shell extract, rosemary extract or BHT during prolonged storage were found to be of equal quality within the degree of confidence limits. The extract of pecan nut shell may be considered as a natural product replacement for the synthetic antioxidant BHT. © 2016 Society of Chemical Industry.

Hepatoprotective effects of pecan nut shells on ethanol-induced liver damage.

The hepatoprotective activity of the aqueous extract of the shells of pecan nut was investigated against ethanol-induced liver damage. This by-product of the food industry is popularly used to treat toxicological diseases. We evaluated the phytochemical properties of pecan shell aqueous extract (AE) and its in vitro and ex vivo antioxidant activity. The AE was found to have a high content of total polyphenols (192.4±1.9 mg GAE/g), condensed tannins (58.4±2.2 mg CE/g), and antioxidant capacity, and it inhibited Fe(2+)-induced lipid peroxidation (LP) in vitro. Rats chronically treated with ethanol (Et) had increased plasmatic transaminases (ALT, AST) and gamma glutamyl transpeptidase (GGT) levels (96%, 59.13% and 465.9%, respectively), which were effectively prevented (87; 41 and 383%) by the extract (1:40, w/v). In liver, ethanol consumption increased the LP (121%) and decreased such antioxidant defenses as glutathione (GSH) (33%) and superoxide dismutase (SOD) (47%) levels, causing genotoxicity in erythrocytes. Treatment with pecan shell AE prevented the development of LP (43%), GSH and SOD depletion (33% and 109%, respectively) and ethanol-induced erythrocyte genotoxicity. Catalase activity in the liver was unchanged by ethanol but was increased by the extract (47% and 73% in AE and AE+Et, respectively). Therefore, pecan shells may be an economic agent to treat liver diseases related to ethanol consumption.

Fatty acid composition in preterm and term breast milk.

The aim of this study was to determine and compare the fatty acid (FA) composition of colostrum and mature milk produced by nursing mothers of preterm and at-term newborns, in Florianópolis, SC, Brazil. Low contents of Eicosapentaenoic acid (EPA) (0.02%/colostrum and 0.01%/mature milk for preterm and term milk) and Docosahexaenoic acid (DHA) (colostrum group: 0.10%/preterm and 0.09%/term; mature milk: 0.05%/preterm and 0.03%/term) were determined. The comparison among the groups showed that the elaidic acid content was significantly higher (1.67%) in mature term milk. The content of rumenic acid (conjugated linoleic acid) was significantly higher in at-term colostrum compared with preterm colostrum. When considering the maturity of the milk, there was a significant increase in the percentage of this FA in the preterm group. The results show that, overall, the greatest differences observed were between the colostrums and mature milks for both groups and not between preterm and at-term mothers.

[Conjugated linoleic acid (CLA) and its relationship with cardiovascular disease and associated risk factors]. / Acido linoléico conjugado (CLA) e sua relação com a doença cardiovascular e os fatores de risco associados.

The term CLA (conjugated linoleic acid) corresponds to a mixture of positional and geometric isomers of linoleic acid, of which two (9/c/, 11/t/ and 10/t/, 12/c/) have biological activity. This review covers aspects related to CLA (sources, synthesis, distribution in human tissues, physiological activity), as well as its relationship with cardiovascular diseases. Most studies attribute the beneficial effects associated to the consumption of CLA to the reduction of risk factors for the development of cardiovascular diseases, such as the reduction of plasmatic triacylglycerols and cholesterol. Other research demonstrates the reduction of atherosclerotic processes. However, many studies indicate that CLA does not present beneficial effects or may even present negative effects. Thus, although there are a great number of studies related to CLA, we consider it premature to make any recommendation for the ingestion of these compounds, apart from those naturally present in a healthy diet.

Composition of w-3 and w-6 fatty acids in freeze-dried chicken embryo eggs with different days of development

No presente trabalho, foi determinada a composição em ácidos graxos w-3, w-6 e, em especial, o DHA em ovos de galinha embrionados e liofilizados com períodos de incubação pré-estabelecidos. De acordo com os resultados obtidos, os ovos férteis e embrionados apresentaram a seguinte composição média de ácidos graxos saturados: palmítico (23,18 + 0,54%) e esteárico (7,70 + 0,28%); insaturados: palmitoléico (3,00 + 0,19%), oléico (36,28 + 0,58%), linoléico (22,18 + 0,34%), linolênico (1,08 + 0,04%), araquidônico (2,04 + 0,03%) e DHA (0,91 + 0,03%); total de ácidos w-3 (2,26 + 0,10%) e total ácidos w-6 (24,62 + 0,33%). Verifica-se que não há diferenças significativas nos teores médios totais de ácido graxos w-3 (p=0,1226), entre os ovos embrionados liofilizados com diferentes dias de incubação (3,5,7,9, e 11 dias) e ovos férteis liofilizados (dia 0). Porém, há diferenças significativas nos teores médios totais de ácidos graxos w-6 (p=0,0001). Também, há uma forte evidência estatística que o modelo quadrático está associado com os valores esperados do teor de DHA (p=0,0013).