Test slides were scored only when the internal controls showed clearly positive or negative results (Greggio et al., 2009). One hundred cells (50 cells from each of two replicate slides of each organ) were selected and analyzed for DNA migration. When selecting the cells, cells around the edges or air bubbles were excluded (Azqueta et al., 2009). The cells were scored selleck compound visually into five classes according to tail length: class 0: undamaged, without

a tail; class 1: with a tail shorter than the diameter of the head (nucleus); class 2: with a tail length 1–2 times the diameter of the head; class 3: with a tail longer than 2 times the diameter of the head; and class 4: comets with no heads. International guidelines and recommendations for the comet assay consider visual scoring of the comets to be a well-validated evaluation method (Burlinson et al., 2007). The genotoxic effects were estimated based on two different parameters: damage index (DI) and damage frequency (DF). The damage index ranged from 0 (completely Metformin undamaged: 100 cells × 0) to 400 (with maximum damage: 100 cells × 4). The damage frequency (%) was calculated based on the number of cells with tails compared to the number of cells with no tails. Levels of Endo III and Fpg-sensitive sites were calculated from the DI score obtained with enzyme treatment minus the score without enzyme treatment (buffered). The vehicle was used as a negative control, and treatment

with 4 × 10−5 M MMS for 1 h was

used as a positive control. Statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The results were expressed as the means ± standard error (SE). All biochemical and coagulation parameters were measured in triplicate. The significant differences between the mean values of two experimental groups were determined using the Student’s t test. When more than two groups were compared, an analysis medroxyprogesterone of variance was used, followed by Bonferroni’s post-hoc test to compare pairs of means. P values less than 0.05 were chosen to establish significance. Between 2 and 6 h after LOBE administration (1 mg/kg, s.c.), the rats presented signs of acute toxicity, including progressive malaise, lethargy, dyspnea, tachycardia, prostration and high sensitivity at the venom injection site. Despite general weakness, the animals showed no clear signs of neuromuscular toxicity, such as muscle trembling, paralysis or convulsions. Most of the envenomed animals displayed hematuria (dark-brown urine at 6–12 h), but no signs of macroscopic skin hemorrhage, petechiae, ecchymosis, suffusions or nasal and eye bleeding were observed. After 48 h, all of the rats had gradually recovered from the clinical symptoms and returned to normal. Until the end of the experiments (96 h), no deaths were registered. The animals in the control group (injected s.c. with PBS solution) exhibited no ill effects.

2009, Soomere et al. 2011) and from measurements near Letipea and the SMB model (Suursaar 2010) are discussed above. The long-term average significant wave height estimated using the WAM model (Soomere et al. 2010) is quite small, normally 0.6–0.65 m in the entire Gulf of Finland (Figure 9). The only exception is the entrance area to the gulf and in the central part of this basin, where the average wave height reaches about 0.7 m. The wave height occurring with a probability of 1% is about 2.5 m in the entire open part of the gulf, from the entrance to the Neva Bay. Nutlin-3a research buy The seasonal variation in the wave activity is clearly evident in both observed and numerically

simulated wave data on the south-eastern coast of the Gulf of Finland. The largest observed waves occur within a four-month period from October to January. The same is largely true for the modelled wave heights, which have a more clearly pronounced maximum Selleckchem CYC202 in December–January. The seasonal courses of modelled waves and wind speeds match each other well, but the observed wave heights show more irregular behaviour, with a secondary maximum in June, and

April being the calmest month. This secondary maximum does not appear for wave fields in the Baltic Proper. There is a secondary maximum in wave intensity in October (which is the overall maximum at Narva-Jõesuu). This feature is not evident in the Baltic Proper either (Räämet & Soomere

2010) and can thus be attributed to the wave climate of the southern Gulf of Finland. The wave model and forcing in use do not reproduce this maximum in the wave activity, which is apparently caused by ageostrophic wind properties. A potential reason is that at times the wind field in the Gulf of Finland contains quite Rho strong easterly and westerly winds blowing along the axis of the gulf (Soomere & Keevallik 2003). This wind system is specific to the Gulf of Finland and does not become evident in other parts of the Baltic Sea; it is much weaker in the eastern part of the gulf. In contrast to the wave directions, wave heights in the Gulf of Finland generally reveal much smaller interannual and decadal variations than those in the Baltic Proper (Kelpšaitė et al. 2009, Soomere et al. 2011). In particular, numerical simulations using one-point wind data suggest that the changes to wave conditions in Tallinn Bay area have been much smaller than those reported for the Baltic Proper (Kelpšaitė et al. 2009). This is not unexpected because the fetch length is relatively short here and the resulting changes to the wave height, especially in the relatively sheltered southern part, should follow the changes in the wind speed, which have been negligible since 1980 (Soomere et al. 2010).

CD56dim and CD56bright cells were distinguished by appropriate gating in the CD56+ region. Whole-blood aliquots with appropriate MAbs were incubated in the dark at room temperature for 20 min. Samples with isotypic control antibodies (IgG1[FITC]/IgG1[PE]/IgG1[PCy-5) were run in parallel with each sample. A minimum of 5000 cells was analyzed on a Coulter XL-MCL (Coulter Corp., Miami, FL), and data analyses were performed using XL System II software. Lymphocyte analyses were performed by gating on the lymphocyte region, based on forward and side light scatter. Counts for each subset were obtained by multiplying the total lymphocyte count by the percentage of the respective subset. Peripheral blood

mononuclear cells (PBMC) were isolated from heparinized whole blood by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density gradient centrifugation. They were then selleck compound diluted in RPMI (GIBCO, Carlsbad, CA) with 5% heat-inactivated fetal calf serum (FCS, Sigma–Aldrich), gentamicin (40 μg mL−1), glutamine (200 mM), and 2-mercaptoethanol (5 × 10−5 M) (complete medium). The lymphocyte proliferative response was measured by 3H-thymidine incorporation after stimulation by phytohemaglutinin (PHA) or muromonabCD3 (OKT3, Janssen, Beerse, Belgium). The freshly isolated PBMC

were adjusted to 2 × 106 cells per milliliter, and 100 μL of the suspension was plated in triplicate wells of a 96-well, round-bottomed microplate (Costar, Cambridge, MA). PHA or OKT3 was diluted to a final concentration of 5 μg mL−1. The plates were incubated at 37 °C for 72 h in an ABT-199 clinical trial atmosphere of 5% CO2 and were then pulsed with 1 μCi per well of Resveratrol 3H-thymidine (6.7 Ci·mmol−1, ICN Biomedicals, Irvine, CA), 18 h before harvesting onto glass-fiber filter paper (Skatron Cell Harvester, Norway). Five milliliters of scintillation fluid were added to the

filters, and they were counted in a β-plate scintillation counter (Wallac Oi, Turku, Finland). The control count was subtracted from the mitogenic count and values were expressed as counts per minute. Natural killer cell cytotoxic activity (NKCA) was measured using the standard NK-sensitive K562 cell line and a radioactive chromium release assay. The human erythromyeloid leukemia-derived cell line K562 was maintained in RPMI 1640, supplemented with 10% FBS, gentamicin (40 μg mL−1), and Hepes buffer (Sigma–Aldrich, São Paulo), kept in 5% CO2 at 37 °C. Freshly isolated Ficoll-purified PBMC were adjusted to 1 × 107 cells per milliliter in complete medium and were then diluted serially at 40:1, 20:1, 10:1, and 5:1 effector-to-target (E:T) ratios. The PBMC were placed into 96-well round-bottom microtiter plates and incubated with radiolabeled K562 cells. K562 cells were labeled with 100 μCi·10−6 cells of sodium 51chromate (51Cr; ICN Biomedicals, Irvine, CA) over a 1-h period in a shaking waterbath at 37 °C. After a further 4 h of incubation at 37 °C and 5% CO2, the plates were centrifuged at 100 g for 5 min.

4(1)). On the other hand, B-cell lymphoma protein-3 (Bcl-3), which is involved in clot retraction, is translated upon thrombin activation

and under mammalian target of rapamycin (mTOR) regulation, as shown in Fig. 4(2). Thrombin activation also increases synthesis of continuously translated proteins, such as plasminogen activator inhibitor (PAI-1). Finally, protein synthesis can also occur via a functional spliceosome, which has been found in platelets [4]. Indeed, pre-mRNAs exist in platelets and are spliced upon platelet activation (Fig. 4(3)). Tissue factors and interleukin 1 β are examples of such regulation. These different regulation mechanisms are facilitated by a strong interaction of mRNAs and protein synthesis machinery with the cytoskeleton, and the presence of translation Ferroptosis activation factors such as protein eukaryotic initiation BTK inhibitor libraries factor, which is constitutively expressed. Platelet activation triggers a drastic cytoskeleton remodeling, which changes the localization of the different partners of protein synthesis. Platelet transcriptome was investigated in the context of the variability of platelet reactivity. RNA expression was assessed in 288 healthy individuals using microarray [57]. The expression level of VAMP8/endobrevin was positively associated with high platelet reactivity, as assessed with light transmission aggregometry. In addition, a SNP

(rs1010) and a microRNA (miRNA-96) were shown to be key players in VAMP8 modulation. Since VAMP8 is a

v-SNARE involved in the targeting and fusion of secretory granules to the plasma membrane, this study linked platelet reactivity variability to granule release. Recent data suggest that microRNA (miRNA) play an important role in mRNA regulation in platelets. These small nucleotides (around 22 base pairs) can induce mRNA degradation and either delay or promote translation [58]. Several mRNAs and their modulating miRNAs were recently associated with platelet reactivity in healthy subjects [59]. Among the 284 miRNAs expressed by platelets, Thymidylate synthase 74 were differentially expressed in different platelet reactivity categories. These data were combined with quantitative transcriptomic results on the same cohort, to obtain a list of paired miRNAs-mRNAs with a binding site at the 3′untranslated region (UTR) of mRNA. Among them, 3 pairs were of particular interest and could be validated at the level of protein expression. Although mRNAs and miRNAs play a role in the modulation of platelet function by transcriptomics, their exact role at the proteomic level, as well as their functional impact, remain unclear. Platelets have been extensively analyzed using proteomics [42] and [60]. Indeed, since platelets are anucleated and contain a limited amount of mRNA, their proteome is interesting for the study of their physiology. Recently, the platelet proteome was dramatically extended to reach almost 4000 proteins and 2500 phosphorylation sites [40].

This data was Ruxolitinib concentration finally compared to AML data from the Hemaexplorer database. DEK was found to exhibit a comparable or reduced level of expression

to the common promyelocyte stage of normal myeloid differentiation, which is indicative of immature myeloblasts that accumulate in leukemia (Supplementary Fig. 2). Furthermore, when levels of DEK expression were normalized to that of myeloblasts (equivalent to the closest normal counterpart of myeloid cells), DEK was significantly under-expressed in AML, as indicated by a relative mean value less than 1, which was particularly prominent in the APL sub-type ( Fig. 2C). This section and Fig. 3 should be in the main text of the Results Section after “DEK expression levels are reduced in AML”. This section and

Figure 3 should be in the main text of the Results Section after “DEK expression levels are reduced in AML”. To validate the in silico results, we measured DEK expression by qRT-PCR in Ku-0059436 order a separate and independent cohort of defined primary AML samples. Patient characteristics of this primary AML sample cohort are outlined in Supplementary Table 1. DEK expression was found to be similar in 30 AML samples and the 5 NBM, with no significant change in the ∆Ct between NBM and AML observed ( Fig. 3A). To establish if DEK expression was independent of varying AML subtypes, samples were further divided into the following subgroups: normal karyotype, promyelocytic leukemia (chromosomal translocation t(15;17)), core binding factor leukemia

(chromosomal aberrations t(18;21) and inv(16)), and others, which included 11q23 translocations and complex karyotypes. DEK expression remained similar across all AML subgroups with no significant change in expression between each AML subtype when compared to each other or between individual subtypes and NBM ( Fig. 3B). Although DEK mRNA levels were reduced or remained unchanged it is possible that this does not correlate with protein levels as little is known about the post-transcriptional cues that regulate DEK mRNA. Since we were particularly interested to validate our findings at the protein level a novel custom-built TMA was assembled. The TMA utilized bone marrow biopsies from 122 AML patients and 20 age-matched bone marrow samples from tumor-free normal bone marrow, which were allocated from the Biobank at the University Clinic of the RWTH Aachen University. Exoribonuclease All samples were spotted in triplicate, including appropriate positive and negative controls, to produce five TMA slides in total. The slides were subjected to immunohistochemistry using a monoclonal DEK-specific antibody (Fig. 4). We observed a strong DEK-specific nuclear signal in a colon biopsy, which served as a positive control for the specificity of the antibody (Fig. 4A-1). In contrast, the DEK antibody produced a rather weak, diffusely cytoplasmic staining, which was seen mainly in myeloid progenitor cells, in 90% of normal bone marrow biopsies from tumor-free patients (Fig. 4A-2 and B).

g. Meier 2006). According to Kjellström et al. (2011), precipitation increases during winter in the CHIR99021 north and decreases during summer in the south. However, the borderline migrates back and forth from a northerly position in summer to a southerly one in winter.

The precipitation increase is partly explained by increased zonality and partly by an amplification of the hydrological cycle, as Kjellström & Lind (2009) found. According to Kjellström et al. (2011), the explained variance based upon spatial variances of SLP and the mean absolute error for temperature and precipitation over land suggest that RCA3 driven with the GCMs Arpege, ECHAM5 (experiment ‘-r3’, for the description see Kjellström et al. 2011), HadCM3_ref and HadCM3_low perform best during the control period. However, in winter MK-2206 datasheet all GCM simulations are too zonal, thus affecting the quality of the other variables due to advection. Focusing on the atmospheric surface fields over sea, our analysis confirms the results by Kjellström et al. (2011). However, it is impossible to rank the models. Depending on the variable, the results are quite different. For instance, ECHAM5 and HadCM3_ref driven simulations showed the best SLP

and air temperature results, respectively, but none of the models is perfect for all variables. In addition to biases of PRKD3 the large-scale circulation induced by the lateral boundary data, atmospheric surface variables over sea also suffer from biases of SST and sea ice data from the GCMs. Therefore, the results of RCA3 could be affected such that the gain

of the higher resolution in the RCM is compensated for by these biases. A quality assessment of atmospheric fields from RCA3 over the sea is more a validation of GCM results for the Baltic Sea than an evaluation of RCA3 performance. Hence, in this study the added value of the coupled atmosphere-ice-ocean model RCAO was investigated. Because of the computational burden we performed transient simulations with only two different driving GCMs selected from the group of models with better performance. We showed that the results from both downscaling experiments improved the 2 m air temperature over the sea during summer but not necessarily during winter. The latter finding was explained by the impact from the lateral boundary data. However, further downscaling experiments with other GCMs are necessary to illuminate the impact from various data sets. In addition, it is important to note that further model development to improve RCAO is necessary. We identified too low a wind speed over sea (although the higher resolution improved the situation) and too high an air temperature over ice covered areas, suggesting perhaps the shortcomings of incoming long-wave radiation during winter.

In the present study, we tested the hypothesis that different types of exercise training could lead to different changes in the natriuretic peptides system. We thought that even the swimming training, if chronically realized, could alter the ANP synthesis, secretion and bioavailability in the circulation. To compare the effect of both training modalities, we maintained both exercises at similar intensities by using the intensity of the maximal lactate steady state [11] and [33] to induce adaptations from this website predominantly aerobic activities. The procedures were carried out in compliance with the guidelines for the ethical use of animals in scientific

research as stated by the Federation of the Brazilian Society of Experimental Biology and were approved by the Ethics Committee for Animal Use of

the Federal University of Espírito Santo. The experiments were conducted on 21 spontaneously hypertensive male rats obtained from the Institute of Biomedical Sciences, University of São Paulo (270–300 g; 14 weeks old). The rats were housed with controlled temperature (22 °C), humidity (40%) and light cycles (12-h light/dark), had free access to tap water and were fed standard rat chow (Purina Labina, SP-Brazil) ad libitum. The animals were randomly divided into three groups: sedentary (SD, n = 8), run trained (RT, n = 7) and swim trained (ST, n = 6). learn more The sedentary rats were handled five days/week to become accustomed to the experimental protocols. Swimming training was performed in an apparatus adapted for rats that contained warm water (30–32 °C) and was kept at a depth of 50 cm. The training consisted of swimming sessions five days/week G protein-coupled receptor kinase for 60 min for 8

weeks. The swimming time on the 1st day was 20 min, which was increased daily by 10 min until it reached 60 min on the 5th day. From the second week onwards, the exercise duration was kept constant and the rats were worn caudal dumbbells that weighed 2% of their body weight. The caudal weight was gradually increased until it was 5% on the 6th week and was thereafter kept constant [11], [12], [17] and [21]. All of the rats were weighed weekly to adjust the weight of the dumbbells. The running training was performed on a motorized treadmill (Insight, São Paulo, Brazil) 5 days/week for 8 weeks, with the speed and duration progressively increased. The rats began training at 15 m/min for 20 min/day. The speed was gradually increased such that by the end of the 1st week, the animals ran at 15 m/min for 60 min/day. Thereafter, the duration was maintained but the speed was gradually increased. By the 6th week, the rats ran at 24 m/min for 60 min/day [33], and this exercise program was maintained until the end of the study.

The organization of the digestion here described is the same as found for other hemipterans such as the seed sucker, D. peruvianus ( Silva and Terra, 1994) and a blood feeder, Rhodnius prolixus (Hemiptera: Reduviidae) ( Ferreira et al., 1988 and Terra and Ferreira, 2012). Quantitative comparisons between salivary and midgut enzymes that include

collagenase assays should be carried out in other predatory bugs. This will permit the evaluation as to whether true pre-oral digestion is actually as common as it is supposed to be or if it is usually only a pre-oral dispersion of prey tissues, as described here. This work was supported by the Brazilian research agencies FAPESP, CNPq, CAPES and FAPEMIG. We thank Dr. C. Ferreira for helpful discussions and W. Caldeira, M.V. Cruz and the Nucleus of Microscopy and Microanalysis-UFV for technical assistance. M.C.Q. Fialho is a research find more fellow of CAPES, N.R. Moreira is a graduate fellow of FAPESP, W.R. Terra is a staff member of his department, research fellow of CNPq and a member of the INCT-Entomologia Molecular, J.C. Zanuncio and J.E. Serrão are staff members

of their departments and research fellows of CNPq. “
“Males of many species can respond to the likely threat of post-mating competition (Parker et al., 1996 and Parker et al., 1997) by altering their behaviour prior to mating (Bretman et al., 2011a) and/or the amount of sperm or seminal fluid proteins allocated to

each partner selleck compound (Wedell et al., 2002 and Wigby et al., 2009). For males to accurately and adaptively match the expression of a trait to their competitive environment they must be able to significantly influence the expression Baf-A1 order of that trait. For apparently male-limited traits such as sperm and seminal fluid production, the degree of control of sex-specific expression should be high. However, this may not be the case for ‘shared’ reproductive traits, such as mating duration, that arise as an emergent property of the interaction between males and females (Arnqvist and Rowe, 2005). Intuitively, the value of shared traits should be influenced by both sexes. However, this need not be true if one sex has evolved predominant control or precise mechanisms for matching the value of the trait to the environment. Determining the relative influence of each sex over shared traits that can exhibit plasticity to the social and sexual environment is important to understand the repertoire of plastic responses that are available to each sex. In order to test whether there is sex specific control of a plastic shared trait we require a system in which the shared trait can be expressed, but where one sex is rendered incapable of exerting any influence over it. In this study we were able to achieve this by adapting methodology from classic studies of courtship in Drosophila melanogaster ( Cook and Cook, 1975, Grossfield, 1972 and Spieth, 1966).

5. The expression of chaperones was then induced with 0.2% arabinose (w/v) at 30 °C overnight. At that point, the OD600 was recorded and cultures were normalized to the same OD600. Cells were pelleted and resuspended in 10 ml ice-cold PPB buffer (30 mM Tris–HCl, pH 8.0, 1 mM EDTA, 20% sucrose) (Teknova,

CA) at 1:4 dilution. Following incubation at 4 °C for 1 h, samples were centrifuged for 30 min and supernatants containing the periplasmic extracts were collected. Pellets were resuspended in 10 ml BugBuster® solution (Novagen, NJ) supplemented with one tablet of complete EDTA-free protease inhibitor cocktail (Roche, IN) and 2500 units benzonase check details nuclease (Novagen) in order to reduce the viscosity of the lysates. Following 1 hour incubation in ice, http://www.selleckchem.com/products/Adrucil(Fluorouracil).html lysates were centrifuged at 16,000 g for 20 min at 4 °C and supernatants containing the cytoplasmic extracts were collected. To prepare periplasmic extracts of cells expressing Fabs together with the chaperones, TG1 cells harboring the Fab and chaperone plasmid constructs (or pAR3 alone as negative control) were grown overnight at 37 °C in 2YT growth media supplemented with 34 μg/ml

chloramphenicol, 100 μg/ml carbenicillin and 2% (w/v) glucose and subcultured in 100 ml flasks at 37 °C until the OD600 reached 0.5. Thirty minutes after the addition of 0.2% arabinose (w/v), isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and cultures were incubated overnight at 30 °C. At that point the OD600

was recorded and cultures were normalized to equal OD600. Cells were pelleted and resuspended in 10 ml ice-cold PPB sucrose buffer (Teknova) at 1:4 dilution and one tablet of complete EDTA-free protease inhibitor cocktail (Roche). Following incubation at 4 °C for 1 h, samples were centrifuged for 30 min and the supernatants containing the periplasmic extracts were collected. Similarly, periplasmic Silibinin extracts from TG1 cells expressing the ING1 Fab and cytFkpA from a single tricistronic vector were generated without chloramphenicol selection (only with carbenicillin) and simultaneous induction of ING1 Fab and cytFkpA with 1 mM IPTG. Samples of periplasmic and cytoplasmic extracts were resuspended in SDS loading buffer with 0.7 M beta-mercaptoethanol, boiled and loaded in NuPAGE® 4–12% Bis–Tris precast gels (Invitrogen, CA) using NuPAGE MOPS SDS running buffer (Invitrogen). Proteins from reduced gels were then transferred to PVDF membranes using the Millipore-SNAP-i.d.® electroblotter (Millipore, CA). The membranes were blocked with 0.

The elemental analysis was carried out using an inductively coupled plasma mass spectrometer (ICP-MS), Perkin–Elmer SCIEX, model ELAN 6000 (Thornhill, Canada) coupled to a cross flow nebulizer and a Scott spray chamber. The operational parameters are listed in Table 1. Red grapes from the V. labrusca L. varieties Concord, Isabel and Bordo, were manually harvested in Videira, South Region of Brazil, and kindly

donated by the Agricultural Research Company of Santa Catarina State (EPAGRI). All the varietal grapes were harvested at the stage of technical maturity, with soluble solids readings between 16 and 18 °Brix. This parameter was determined according Cabozantinib cost to OIV (1990). The ripened grapes from the three cultivars were brought to the laboratory and the fresh grapes were washed with tap water to remove adhering

dust and dirt. Grapes were Silmitasertib kept separately at −12 °C and all varietal grape juices were prepared within a period of sixty days, followed by the analysis. Previously to juice preparation, grape samples were gently defrosted in a thermostatically controlled water bath at 20 °C for 5 min. Grape seeds of each variety were manually collected from the berries and washed separately with ultrapure water. The grape seeds were dried at room temperature (24 °C) for 15 min and weighed, followed by maceration with berries. Samples were then manually crushed and macerated with seeds under agitation in a thermostatic water bath at 24 °C for 5 min. Grape berries were separated from the rachis and 20 g of randomly selected berries of each cultivar were individually weighed in triplicate. Grape seeds of each cultivar were added at concentrations of 50, 100 and 200 g/kg of grape berry. A blank sample for each cultivar was weighed and macerated without the addition of seeds. Samples were macerated under agitation at 24 °C for 5 min, followed by the addition of 50 mL of ultrapure water. The mixture was transferred to 100 mL flasks and sonicated on an ultrasonic bath at 24 °C for 15 min, followed by the addition of the pectinolytic enzyme Pectinex®

Ultra Color at concentration of 1 mL/L and incubated in a thermostatically controlled water bath PAK5 at 50 °C for 60 min. After the enzymatic treatment, the grape mash was manually pressed for 1 min using nylon filter bags, and the extracted juices were heat processed at 80 °C for 5 min. Finally, grape juices were filtered through a Whatman n°1 filter paper and packed in clean amber glass bottles. Total phenolic content of the grape juices was determined spectrophotometrically using the Folin–Ciocalteu colorimetric method (Singleton & Rossi, 1965). The reaction with the Folin–Ciocalteu reagent was carried out at room temperature (24 °C) for 90 min, with the samples kept in dark. The absorbance of the juice samples and the blank was measured at 760 nm.