Cashew apple (Anacardium occidentale L.) extract from a by-product of juice processing: assessment of its toxicity, antiproliferative and antimicrobial activities

Abstract

Cashew apple extract (CAE) is a product with intense yellow color obtained from residual fibers of juice processing. Although CAE is known to be rich in carotenoids and anacardic acids, the biological activities of this potential natural food colorant remain unexplored. The present study is the first to investigate the toxicity, antiproliferative and antimicrobial activities of the lyophilized CAE (L-CAE) and its encapsulated products, using maltodextrin (M-CAE) or cashew gum (CG-CAE) as carriers. In addition to their high carotenoid content, the phenolic contents in all materials was determined using UPLC-QTOF-MS^E. The acute toxicity was performed using adult zebrafish (Danio rerio); antiproliferative activity was assessed using seven different human tumor cell lines [U-251 (glioblastoma), MCF-7 (breast, adenocarcinoma), NCI-ADR/RES (multidrug-resistant ovarian adenocarcinoma), NCI-H-460 (lung, large cell carcinoma), PC-3 (prostate, adenocarcinoma), OVCAR-3 (ovarian adenocarcinoma), and HT-29 (colon, adenocarcinoma)] and an immortalized human keratinocyte (HaCaT) while the antimicrobioal activity was evaluated on Staphylococcus aureus ATCC 25923, Listeria monocytogenes ATCC 19115, Escherichia coli ATCC 25922 and Salmonella Typhimurium ATCC 51812 microorganisms. Both lyophilized and encapsulated CAE samples did not exert acute toxicity against zebrafish neither antiproliferative effect against human tumor and non-tumor cell lines. Further, L-CAE showed potential antimicrobial activity against Listeria monocytogenes, which was confirmed using electron microscopy. The current findings demonstrated that CAE is a potential source of bioactive compounds to use as an additive in the food industry.

Electronic supplementary material

The online version of this article (10.1007/s13197-020-04594-0) contains supplementary material, which is available to authorized users.

Introduction

Cashew apple (Anacardium occidentale L.) is a tropical fruit that was originally found in Brazil, where it is widely cultivated and commercially exploited. The primary product of the cashew tree is the cashew nut (true fruit), which is rich in fats and proteins. After removing the nut from the peduncle, the pseudo-fruits become waste or are processed in industry, mostly, to produce cashew apple juice. Afterwards, approximately 20% to 25% of the pseudo-fruits used in the processing industry remain as residual fibers and are mostly discarded or used as animal food supplements (Abreu et al. 2013).

The number of studies that have explored the efficient utilization of this agro-industrial residue has increased. Abreu et al. (2013) reported a process for obtaining a cashew apple extract (named CAE) using cashew apple fibers as raw material and water as an extractant. The water was able to extract the carotenoids (that presents a lipophilic behavior) because these components, in this specific product/process, are in an emulsified structure. The final product has an intense yellow color, thereby demonstrating its potential use as a food colorant. The optimized process involves enzymatic treatment of the cashew apple fibers, followed by sequential pressing and crossflow microfiltration (Abreu et al. 2013) to generate a product with high carotenoid content, which was concentrated using a microfiltration process. Anacardic acids (AnAc), which are naturally present in all parts of cashew, were, consequently, concentrated. Anacardic acids are phenolic lipids that are recognized for their antibacterial, antitumor, and antioxidant properties (Hamad and Mubofu 2015). However, despite their known biological benefits, higher doses of AnAc demonstrated toxicity in acute and sub-acute assays in mice. Carvalho et al. (2011) observed mild abnormalities (decrease in hematocrit and hemoglobin levels and increase in urea levels) in female BALB/c mice that had received the highest doses of AnAc (600 or 1000 mg/kg). However, no adverse effects were observed at AnAc doses lower than 300 mg/kg, indicating that this dose is safe. Nevertheless, the authors suggested that additional biological studies using AnAc should be performed.

CAE has an intense yellow color because it is rich in carotenoids, where auroxanthin and β-cryptoxanthin represent around 50% of total carotenoids (Abreu et al. 2013). Although CAE is an interesting source of carotenoid pigments, these compounds are susceptible to degradation when isolated (Boon et al 2010). Furthermore, cost-effective processing methods should be employed to preserve the carotenoids and other bioactive compounds (Saini et al. 2015). Carriers, such as maltodextrin or cashew gum, are used to stabilize these carotenoids, which are strongly associated with potential health benefits in humans (Saini et al 2015). Nevertheless, the colorant and biological properties of these bioactive-containing extracts (non-encapsulated and encapsulated) remain to be evaluated and should be explored because of their potential applications, especially in the food industry.

The biological properties of natural products have long attracted the attention of researchers, and studies investigating their specific biological activities, both in vivo and in vitro, have been exploited using cell lines and diverse animal models. To effectively explore the biological mechanisms and effects associated with a disease, the model organism used for the study should provide technical and practical advantages (Kang et al. 2013). The zebrafish (Danio rerio) model system has recently emerged as a highly useful vertebrate model organism for studying genetics, development, environmental toxicology, pharmacology, DNA damage repair, cancer, and other diseases (Dai et al. 2014). To investigate the effects of carotenoids, Saidi et al. (2015) evaluated the effect of zeaxanthin on the visual acuity of adult zebrafish. More recently, zebrafish embryos were used as models of vertebrate development to identify, isolate, and characterize teratogenic metabolites from carotenoid glycosides produced by cyanobacteria (Jaja-Chimedza et al. 2017). However, the authors noted that the toxic effects of anacardic acids or other carotenoids (or materials containing higher levels of these constituents) on adult zebrafish remain to be explored.

The present study aimed to investigate the biological properties of CAE (lyophilized, L-CAE) and its encapsulated materials, M-CAE and CG-CAE, prepared using maltodextrin or cashew gum as a carrier, respectively. In particular, the study focused on their antiproliferative and antimicrobial effects, with the goal of reinforcing or proposing novel applications of these extracts, as a source of bioactive compounds to use as food or further pharmacological purposes. In addition, their acute toxicities to mature adult zebrafish were also evaluated.

Material and methods

Cashew apple extract (CAE) preparation

Cashew apple (Anacardium occidentale L.) peduncles, the CCP-76 variety, was used for juice production by the “NatVita” company in Eusébio (Ceara-Brazil). After juice processing, the residual fibers were frozen at – 18 °C and transported to Embrapa (Fortaleza-Brazil) to obtain the cashew apple extract (CAE). Briefly, the fibers were mixed with water (1:1 w/w), homogenized, and pressed under a continuous helical-type press (Incomap 300, Fortaleza, Brazil), consisting in a helical-shaft with increasing diameter at the end, and surrounded by a 0.5 mm stainless steel sieve, allowing the separation of juice from fibers, with a nominal capacity of 300 kg/h for juice production. After the six cycles of pressing, a suspension with a strong yellow color was obtained. After pre-filtering through a 0.3-mm stainless steel mesh for removing large suspended particles, the resulting suspension was centrifuged (Heraeus Megafuge 40 model, ThermoFisher Scientific, Langenselbold, Germany) at 4415 g for 5 min. The extract was then packed in polyethylene bags, frozen at − 20 °C, and stored for subsequent analyses.

To obtain the CAE, the extract produced by pressing was submitted to a microfiltration system equipped with tubular ceramic membranes. Microfiltration experiments were carried out using laboratory-scale equipment TIA (Techniques Industrielles Appliquées, Bollène, France) using a set comprising four monotubular alumina membranes MEMBRALOX (Pall-Exekia, Bazet, France) with a filtration area of 0.022 m² and average pore diameter of 0.2 μm. The transmembrane pressure was 2.75 bar, and the temperature was controlled at 40 °C (± 2 °C) for all trials. The process was conducted using concentration mode up to a volumetric reduction ratio (VRR of 16). The carotenoid fraction was concentrated in the retention phase and was named cashew apple extract (CAE). Afterwards, CAE was subjected to the different drying processes [lyophilization (L-CAE), spray-dryer encapsulation using maltodextrin as the carrier (M-CAE) and spray-dryer encapsulation using cashew gum as the carrier (CG-CAE)].

Cashew gum and maltodextrin as encapsulating agents

Crude samples of the cashew tree gum exudate were collected from native trees (variety CCP-076) at Pacajús, Ceará, Brazil. The samples were purified according to the methodology described by Torquato et al. (2004) with some modifications. Initially, the crude resin of the cashew was placed in a drying in a stove with air circulation at 45 °C. After drying, the crude resin was ground, sifted (0.3 mm), and dissolved in distilled water (10 g/100 mL). The resulting solution was stirred for 2 h and then filtered. Afterwards, 96% ethanol was added to the filtrate in a ratio of 3:1 (ethanol: filtrate) to precipitate the polysaccharide. The mixture was refrigerated for 12 h until total decantation. The supernatant was drained, and the precipitate was centrifuged at 2825 g for 5 min at 25 °C (Heraeus Megafuge 40 model, ThermoFisher Scientific, Langenselbold, Germain). After centrifugation, the precipitate was placed in a stainless steel tray and placed in an oven with air circulation at 45 °C until complete evaporation of ethanol. The dried material was ground (A-11 Basic Analytical Mill, IKA) and sieved (0.3 mm) to obtain the purified cashew gum (CG). The encapsulating agent maltodextrin (DE 10) was purchased from Cargill Company, Brazil.

Preparation of the encapsulated CAE

CAE samples encapsulated with maltodextrin (M) or cashew gum (CG) were prepared following optimized methods (data not shown). The ratio CAE: encapsulating material was 5:1 (CG:CAE) or 3.5:1 (M:CAE). The CAE was homogenized for 1 min using the carrier (Homogeniser model TE 102 Turratec, Tecnal, Piracicaba, Brazil). After filtration in a stainless steel sieve (0.3 mm mesh) to avoid clogging of the atomizer, the final suspension was spray-dried using in a Spray Dryer (LM MSD 1.0 model; Labmaq do Brasil, Ribeirão Preto, Brazil). The operational conditions were as follows: air inlet temperature, 150 °C; compressed air flow (3.0 L/min), feed flow rate (0.4 L/h), and drying air flow (3.5 m³/min).

Analytical methods

Color

Color analyses were performed by measuring the direct reflectance of the rectangular coordinate system (L*, brightness; a*, intensity of red and green; and b*, intensity of yellow and blue) by applying the CIELAB color scale using a colorimeter (Chroma-Meter CR-410 model, Konica Minolta Sensing Inc., Osaka, Japan). After the instrument was standardized with a white plate (L₀ = 97.51, a₀ = 0.16, and b₀ = 1.75), the colors of samples (50 g, per plate) were evaluated at room temperature (25 °C). The analysis were performed in triplicate.

Total carotenoid contents

Total carotenoid contents were determined according to the method described by Higby (1962). The procedure was a follows: the sample (0.2 g of CAE, 0.5 g M-CAE and 0.5 g of CG-CAE) was added to 30 mL of isopropyl alcohol and 10 mL of hexane. After homogenization for 60 s using a vortex blender, the sample was transferred to a separatory funnel, and the volume was completed with distilled water until 125 mL. After 30 min, the sample was washed and the process was repeated three times. After, the fraction containing the carotenoids was filtered in a 50 mL flask, with 5 mL of acetone, with the volume completed with hexane. All the analysis was performed in triplicate. The reading was performed on a spectrophotometer (SP-220 model, Biospectro, Brazil) at 450 nm. Data were expressed as mg carotenoid/100 g of sample based on the following equation:

$$\text{Total carotenoids}=\left(\text{A}\times 100\right)/\left(250\times \text{L}\times \text{W}\right),$$

where A = absorbance; L = width of the cuvette in cm; and W = the original sample weight in grams divided by the final volume of dilution in mL.

Anacardic acid (AnAc) content

The CAE samples were solubilized at concentration of 1 mg/mL in methanol, directly filtered in 0.45-µm PTFE disc filters into vials, and then injected into the HPLC system in triplicate. The HPLC system comprised a chromatograph (Shimadzu LC-20AB Prominence, Kyoto, Japan) coupled with a diode array detector (Shimadzu SPD-M20A Prominence) and an autosampler (Shimadzu SIL-20AC Prominence). The anacardic acids present in the CAE samples were quantified following a validated method described by Oiram Filho et al. (2018) using a reverse-phase C18 chromatographic column Shim-pack CLC–ODS (M) (150 mm × 4.6 mm × 5 μm, Shimadzu). The mobile phase used was a mixture containing water (Solvent A), acetonitrile (Solvent B), and acetic acid at a ratio of 20:80:1 in isocratic mode. The analysis was performed for 30 min with a flow rate of 1.5 mL/min and temperature of 30 °C using an injection volume of 20 μL. The chromatograms were monitored at a wavelength of 280 nm, and the UV spectra were recorded from 200 to 400 nm.

Structural confirmation of anacardic acids (AnAc) by UPLC–QTOF–MS^E

To confirm the structures of the AnAc, the samples were analyzed on an Acquity UPLC system (Waters, Milford, MA, USA) coupled to a quadrupole/time-of-flight (QToF) mass spectrometer (Waters, Milford, MA, USA). The compounds were separated on an Acquity BEH C18 (150 mm × 2.1 mm, 1.7 μm; Waters, Milford, MA, USA) column operated at 40 °C. The eluent system employed was a mixture of A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) with a flow rate of 0.4 mL/min. The gradient varied linearly from 5 to 95% B (v/v) for 20 min. The sample injection volume was 5 μL. Mass spectra were obtained in the negative-ion mode over a mass range of 50 to 1180 Da. The spectrometer was operated with MS^E centroid programming using a cone voltage of 40 V. The drying gas pressure was set to 35 psi at 370 °C, while the nebulizer gas pressure was set to 40 psi. A capillary voltage of 3500 V and spray shield voltage of a 600 V were used. Identification of AnAc was performed using molecular formulas and m/z values obtained from high-resolution mass spectra using the MassLynx software (Waters Corporation). The data obtained from the present study were compared to those presented in previous studies (Trevisan et al. 2006; Oiram Filho et al. 2018).

Biological evaluation

In vivo evaluation

Zebrafish maintenance

Male and female adult wild zebrafish (Danio rerio) aged 60 to 90 days with similar sizes (3.5 ± 0.5 cm) and weight (0.3 ± 0.2 g) were obtained from Agroquímica: Comércio de Produtos Veterinários LTDA, a supplier located in Fortaleza (Ceará, Brazil). Each group comprised 50 fish, which were acclimated for 24 h in a 9-L glass tank (30 cm × 15 cm × 20 cm) containing dechlorinated tap water (ProtecPlus®) and air pump with submerged filter at 25 °C and pH 7.0 under near-normal circadian rhythm conditions (14:10 h light/dark cycle). Prior to the experiments, fish were provided access to food ad libitum for 24 h. After the experiments, the animals were sacrificed by immersion in ice water (2 to 4 °C) for 10 min until loss of opeanalyses were performedrcular movements was observed (CONCEA 2018). All experimental procedures were approved by the Ethics Committee on Animal Research of the Ceara State University (CEUA-UECE), and the experimental protocol was filed under N. 7210149/2016.

General protocol

The protocols were conducted following the procedures described in Magalhães et al. (2017). On the day of the experiment, zebrafish were randomly selected and transferred to wet sponges for oral treatment (p.o.) with the samples (diluted in distilled water) or controls (vehicle, distilled water) using a 20-μL variable micropipette. After treatment, individual zebrafish were placed in beakers (250 mL) containing 150 mL of water from the fish tank and allowed to recover.

Open-field test

The animals (n = 6/group) were treated with L-CAE or M-CAE or CG-CAE (2, 40, and 200 mg/kg; p.o.) or vehicle (control, distilled water; 20 µL; p.o.) and subjected to the open-field test to determine any alterations in fish motor coordination either through sedation and/or muscle relaxation following the protocol described by Magalhães et al. (2017). A naive group was included. The number of line crossings (LC) for each fish was annotated from 0 to 5 min. For each experimental group, the line crossings in percentage (LC%) were expressed as mean ± standard error relative to the LC value of naive fish (100%) (Magalhães et al. 2017). After confirming the normality of the distribution and data homogeneity, data were analyzed by analysis of variance (ANOVA), followed by Tukey test. P < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism v. 5.01 software.

Acute toxicity

The acute toxicity study was performed using adult zebrafish (Danio rerio) according to the OECD guidelines (OECD 1992). The animals (n = 6/group) were treated with L-CAE, CG-CAE, and M-CAE (2, 40, or 200 mg/kg; p.o) or vehicle (control, distilled water; 20 µL; p.o.). Fish behavior was evaluated visually for 96 h after treatment, after which changes in swimming speed, loss of balance, respiration, and abnormal behavior were evaluated. Fish mortality was recorded daily, and LD₅₀ was determined following the Trimmed Spearman-Karber method with 95% confidence intervals (CONCEA et al. 2018).

In vitro antiproliferative activity assay

Cell culture conditions

Seven different human tumor cell lines [U-251 (glioblastoma), MCF-7 (breast, adenocarcinoma), NCI-ADR/RES (multidrug-resistant ovarian adenocarcinoma), NCI-H-460 (lung, large cell carcinoma), PC-3 (prostate, adenocarcinoma), OVCAR-3 (ovarian adenocarcinoma), and HT-29 (colon, adenocarcinoma)] were kindly provided by the National Cancer Institute at Frederick (NCI-USA). One immortalized human keratinocyte (HaCaT) cell line was kindly provided by Prof. Dr. Ricardo Della Coletta (University of Campinas). For all experiments, the cell lines were maintained in complete medium [RPMI 1640 (Gibco, USA) supplemented with 5% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin:streptomycin (Nutricell, 1000 U/mL:1000 g/mL)] in a humidified atmosphere with 5% CO₂ at 37 °C.

Sample preparation

Aliquots (5 mg each) of doxorubicin (Eurofarma®, 0.5 mg/5 mg, positive control), cashew carotenoid-enriched extract (L-CAE), empty microparticles of cashew gum (CG), and maltodextrin (M) were diluted in DMSO (50 µL) and subsequently added with 950 µL of complete medium (working solution). Final target concentrations were obtained by serial dilution in complete medium. Moreover, microparticles CG-CAE and M-CAE were diluted in DMSO/complete medium as previously described while adjusting the mass samples to 5 mg/L CAE.

Antiproliferative activity assay

The in vitro antiproliferative activity assay was performed as described by Monks et al. (1991). Briefly, after adjusting each cell line density (see Supplementary Material, Table S1), the cell suspensions were plated on 96-well plates (100 μL/well). After 24 h, cells were treated with varying concentrations of L-CAE, empty CG and M microparticles, CG-CAE, and M-CAE [0.25, 2.5; 25; 250 μg/mL (100 μL/well)] in triplicate for 48 h. For doxorubicin, the final concentrations ranged from 0.025 to 25 μg/mL. Before (T₀ plate) and after (T₁ plate) treatment, the cells were fixed with trichloroacetic acid (50%, 50 μL/well, Sigma®), and protein quantification was performed using sulforhodamine B assay (λ = 540 nm). Results were plotted as cell growth profile for each cell line as a function of each sample concentration. The effective concentration, named GI₅₀ (sample concentration required to elicit 50% of cell growth inhibition) was determined by sigmoidal regression analysis using the software ORIGIN 8.0 (Origin Lab Corporation).

Antimicrobial activity

Microorganisms

Antimicrobial activity was evaluated on Gram-positive (Staphylococcus aureus ATCC 25923 and Listeria monocytogenes ATCC 19115) and Gram-negative microorganisms (Escherichia coli ATCC 25922 and Salmonella Typhimurium ATCC 51812).

Inoculum preparation

From the stock cultures maintained at 4 °C on lean nutrient agar, one loop of each microorganism was removed and cultivated in solid trypticase soy agar (TSA) (Difco, Sparks, USA) medium, except for Listeria monocytogenes, which was maintained in TSA-enriched yeast extract (YE) (Becton, Dickinson, Sparks, USA). After incubation at 35 °C for 24 h, the colony morphology was macroscopically observed (aspect and size) to verify the purity of the strains. Using an inoculating loop, one colony of each microorganism was transferred to one 15 mL-tube containing 5 mL of sterile tryptic soy broth (TSB) (Difco, Sparks, USA). The tubes were incubated at 35 °C for 24 h to obtain a microbial suspension with a final concentration of 1.5 × 10⁸ CFU/mL.

Agar diffusion method

The antimicrobial activities of the extracts were determined using the agar diffusion technique according to the protocols of the Clinical and Laboratory Standards Institute (CLSI 2015) with modifications. All bacterial suspensions (10⁸ CFU/mL) were seeded on the surface of Mueller–Hinton agar (MH), except for Listeria monocytogenes, which was seeded in Mueller–Hinton agar enriched with yeast extract (MH-YE). After 5 to 15 min, 5-mm diameter wells were aseptically perforated over the agar surface, after which each well was added with 50 µL of serial dilutions of L-CAE, M-CAE, and CG-CAE (100, 50, 25, and 12.5 mg/mL) in Tween 80 (2%, in water). After 30 min of incubation at 25 °C, all plates were incubated at 35 °C for 24 h. Antimicrobial activity was visually evaluated based on the sizes of the inhibition zones surrounding the wells. Sterile solutions of Tween 80 (2%, in water) and 300,000 U/mL benzylpenicillin benzathine (Eurofarma) were used as the negative and positive controls, respectively.

Scanning transmission electron microscopy (STEM) of bacterium

Bacterial images were obtained following the methodology described by Hooton et al. (2011) with modifications. To prepare the inoculum strain, Listeria monocytogenes ATCC 19,115 was grown in the trypticase soya agar (TSA) medium (Difco, Sparks, USA) enriched with yeast extract (TSA-YE) and subsequently incubated at 35 °C for 24 h. After incubation, sample dilutions (10^–1) were prepared using 9 mL of 0.1% peptone water (Difco, Sparks, USA) as the diluent to obtain a final bacterial concentration of 1.5 × 10⁷ CFU/mL. Equivalent volumes of the sample (100 μL) at the concentration of 100 mg/mL, after which 100 μL of each inoculum was added to a sterile Eppendorf and subsequently homogenized. The samples were incubated at 35 °C for 24 h. Afterward, the samples were treated for 4 h with 100 μL of the Karnovsky fixative solution in cacodylate according to the methods of Karnovsky (1965). A small drop of washed bacterial suspension was spotted onto a carbon-coated copper mesh grid and allowed to sit for 3 min. The excess bacterial suspension was then removed using filter paper. For negative staining, one drop of phosphotungstic acid (pH 7.4) was added to each grid, and the excess stain was removed after 1 min using filter paper. Each grid was covered and allowed to dry for 15 min. The grids were visualized in SEM coupled to STEM detector (model Vega 3 SBU, TESCAN, Czech Republic), at a suitable voltage acceleration of 30 kV.

Results and discussion

The results of the characterization of L-CAE (lyophilized cashew apple extract), M-CAE (cashew apple extract encapsulated with maltodextrin), and CG-CAE (cashew apple extract encapsulated with cashew gum) are summarized in Table 1.

Table 1.

Characterization of L-CAE, M-CAE and CG-CAE

Samples	Anacardic acids (mg/g)				Carotenoids (mg/100 g)	Color
	Total	C15:3	C15:2	C15:1		L*	a*	b*
L-CAE	41.74 ± 0.05a	9.42 ± 0.06a	10.84 ± 0.01a	21.48 ± 0.02a	96.28 ± 3.41a	36.87 ± 0.04c	8.28 ± 0.02a	36.52 ± 0.12c
M-CAE	7.80 ± 0.01b	2.04 ± 0.00b	2.21 ± 0.01b	3.55 ± 0.01b	25.95 ± 0.80b	81.69 ± 0.02a	− 3.34 ± 0.02c	46.02 ± 0.03a
CG-CAE	7.12 ± 0.01c	1.86 ± 0.00c	2.03 ± 0.00c	3.23 ± 0.01c	24.59 ± 0.71b	78.67 ± 0.01b	− 2.01 ± 0.01b	38.31 ± 0.01b

The averages followed by the same letter in columns do not differ statistically from each other. The Tukey's test was applied with a confidence interval of 95%

L-CAE lyophilized cashew apple extract; M-CAE cashew apple extract encapsulated with maltodextrin, and CG-CAE cashew apple extract encapsulated with cashew gum

AnAc was consisted by a mixture of viz. 6-[8′(Z),11′(Z),14′-pentadecatrienyl]salicylic acid (1, known as anacardic acid C15:3), 6-[8′(Z),11′(Z)-pentadecadienyl]salicylic acid (2, anacardic acid C15:2), and 6-[8′(Z)-pentadecenyl]salicylic acid (3, anacardic acid C15:1) (Kubo et al. 2006). The long alkyl chains of anacardic acids come from the condensation of saturated or unsaturated fatty acids with phenolic compounds generated through acetate-malonate-derived pathways (Morais et al. 2017); C15:3, C15:2, and C15:1 were identified in all materials tested (Fig. 1 and Supplementary Material, Table S2 and Figure S5–S7).

Fig. 1.

a Chromatography profile of CAE (1 mg/mL), (I) AnAc triene, (II) AnAc diene, (III) AnAc monoene, analyzed at 280 nm. b. The UV-spectra of (I) AnAc triene, (II) AnAc diene, (III) AnAc monoene and its respectively molecular structure

L-CAE showed higher concentrations of bioactive compounds (carotenoids and anacardic acids, AnAc) relative to the treatments with the encapsulated materials. However, the CAE encapsulation is an interesting strategy to protect the bioactive compounds, as reported in other studies on microencapsulation using bixin (Barbosa et al. 2005) and phenolic compounds (Nunes et al. 2015), for example. The different encapsulation wall materials (maltodextrin or cashew gum) showed similar carotenoid contents (P > 0.05) but significant differences in the color L*, a*, and b* coordinates values. The above results are expected, given that cashew gum has a natural yellow color (L* = 91.87 ± 0.01; a* = 1.49 ± 0.01; and b* = 8.60 ± 0.01), whereas maltodextrin treatment produced the following values: L* = 103.22 ± 0.01; a* = –0.25 ± 0.01, b* = 2.88 ± 0.01.

As previously described, all the materials tested (L-CAE, M-CAE, and CG-CAE) contain AnAc (41.74 ± 0.05, 7.80 ± 0.0, and 7.12 ± 0.01 mg/g for L-CAE, M-CAE, and CG-CAE, respectively). The encapsulation process, although considered a protection to the biological components, presents much lower concentration of AnAc, due to wall material addition. The purified AnAc, extracted of the cashew nut shell liquid (CNSL), shows important biological activities, such as gastroprotective and antitumor agents, and exhibit antimicrobial and antioxidant properties (Sukumari-Ramesh et al. 2011; Carvalho et al. 2011). The CAE presents AnAc in its composition. However, the concentration was not enough to promote an antiproliferative activity since this effect was observed when the AnAc were used in higher concentrations than that in CAE experiments. AnAc are present in cashew apple, nut, and CNSL in varying concentrations; in particular, the highest concentrations were detected in CNSL (353.6 mg/g), followed by cashew fiber (6.1 mg/g) and roasted cashew nut (0.65 mg/g) (Trevisan et al. 2006; Agostini-Costa et al. 2004). Although previous studies reported significant biological effects of CNSL, which contains higher levels of AnAc, presents moderately toxicity when tested on rats acutely (Harlita et al. 2016). Therefore, we evaluated the acute toxicity of CAE and its encapsulated materials (M-CAE and CG-CAE) to individual adult zebrafish, considering that the concentration of AnAc in the membrane process can cause toxicity.

The mortalities of sexually mature zebrafish (Danio rerio), exposed to different concentrations of L-CAE, M-CAE, and CG-CAE are presented in Supplementary Material (Table S3). Treatment with L-CAE, M-CAE, or CG-CAE (2, 40, or 200 mg/kg; p.o.) for 96 h exerted no toxicity to the adult zebrafish (LD₅₀ > 200 mg/kg). In addition, treatment with L-CAE, M-CAE, and CG-CAE did not alter the locomotor activities (P > 0.05 vs. naive or vehicle) of the adult zebrafish based on the open-field test (Fig. 2).

Fig. 2.

Effect of L-CAE or M-CAE or CG-CAE (2 or 40 or 200 mg/kg; p.o.) on locomotor activity of adult zebrafish, analyzed individually during 0–5 min in the open field test. Each column represents the mean ± standard errors of the mean (n = 6/group). Numbers above the bars indicate the percentage of locomotor activity. ANOVA with post-hoc Tukey’s test (#p < 0.05 vs. L-CAE 40 mg/Kg). Control: vehicle (Distilled water). Naive: untreated group (Control)

Locomotor activity is one of the parameters in behavioral analysis that are used to evaluate the effect of drugs on the central nervous systems of adult zebrafish and cause locomotor or non-locomotor impairment (Taylor et al. 2017; Benneh et al. 2017). Recently, we adapted the open-field test in adult zebrafish following the methods of Ahmad and Richardson (2013) to investigate the effects of analgesic drugs (Magalhães et al. 2017). We employed the same methods using L-CAE, M-CAE, or CG-CAE to evaluate their effects on the adult zebrafish locomotor system. Results showed no significant alterations in fish motor coordination, sedation, or muscle relaxation.

Adult zebrafish have been used as animal models to complement rodent studies for investigating genetics, developmental biology, neurobiology, and toxicology (Caballero and Candiracci 2018) because of advantages, including low cost, diverse adaptability, short reproductive cycle, high fecundity, and transparency of their embryos (Dai et al. 2014). In the present study, adult zebrafish were utilized as an alternative model to evaluate the acute toxicities of L-CAE, M-CAE, and CG-CAE. Results indicated that extracts were non-toxic to adult zebrafish for up to 96 h of incubation at all concentrations tested. However, additional in vivo studies should be conducted to evaluate the sub-chronic and chronic effects of CAE using zebrafish as the assay model.

CAE is a source of AnAc, and these compounds have been reported to possess anticancer and antibacterial properties (Sukumari-Ramesh et al. 2011; Zhao et al. 2018). To evaluate the potential biological properties of CAE, tests were conducted using all three materials (L-CAE, CG-CAE, and M-CAE). As demonstrated by the cell growth profile in presence of doxorubicin (Fig. 3a, Table 2), all cells (seven human tumor and one non-tumor cell lines) presented a particular response to one antiproliferative agent. Considering CAE samples, including the empty capsules of CG and M, nor free neither encapsulated CAE were not able to inhibit the proliferation of all tested human cells resulting in concentration required to promote 50% of cell growth inhibition higher than the higher tested concentration (GI₅₀ > 250 µg/mL, Fig. 3 and Table 2). More, both wall materials CG and M did not affect cell proliferation despite been present in higher concentration than CAE in CG-CAE, and M-CAE samples (Fig. 3 and Table 2).

Fig. 3.

Effect of doxorubicin, L-CAEW, M-CAE, CG-CAE and empty CG- and M-microparticles on tumor and non-tumor cell lines after 48 h of treatment. Samples: a Doxorubicin = positive control; b L-CAE = lyophilized cashew apple extract; c CG-CAE = cashew apple extract encapsulated with cashew gum; d M-CAE = cashew apple extract encapsulated with maltodextrin; e CG = empty cashew gum microcapsules; f M = empty maltodextrin microcapsules. Human tumour cell lines: U-251 (glioblastoma), MCF-7 (breast, adenocarcinoma), NCI-ADR/RES (multidrug-resistant ovarian adenocarcinoma), NCI-H-460 (lung, large cell carcinoma), PC-3 (prostate, adenocarcinoma), OVCAR-3 (ovarian adenocarcinoma), and HT-29 (colon, adenocarcinoma). Non-tumour cell line: HaCaT (immortalized human keratinocyte)

Table 2.

Concentration of doxorubicin, L-CAE, M-CAE, CG-CAE and empty CG and M microparticles necessary to cause 50% of cell growth inhibition (GI₅₀ values), in μg/mL, after 48 h of treatment

Cell lines	Samples
	Doxorubicin	L-CAE	CG-CAE	M-CAE	CG	M
U251	0.026	> 250	> 250	> 250	> 250	> 250
MCF7	< 0.025	> 250	> 250	> 250	> 250	> 250
NCI-ADR/RES	0.16	> 250	> 250	> 250	> 250	> 250
NCI-H460	< 0.025	> 250	> 250	> 250	> 250	> 250
PC-3	0.23	> 250	> 250	> 250	> 250	> 250
OVCAR-03	0.14	> 250	> 250	> 250	> 250	> 250
HT29	0.26	> 250	> 250	> 250	> 250	> 250
HaCaT	< 0.025	> 250	> 250	> 250	> 250	> 250

Samples: Doxorubicin = positive control; L-CAE = lyophilized cashew apple extract; CG-CAE = cashew apple extract encapsulated with cashew gum; M-CAE = cashew apple extract encapsulated with maltodextrin; CG = empty cashew gum microcapsules; M = empty maltodextrin microcapsules

Human tumour cell lines: U-251 (glioblastoma), MCF-7 (breast, adenocarcinoma), NCI-ADR/RES (multidrug-resistant ovarian adenocarcinoma), NCI-H-460 (lung, large cell carcinoma), PC-3 (prostate, adenocarcinoma), OVCAR-3 (ovarian adenocarcinoma), and HT-29 (colon, adenocarcinoma). Non-tumour cell line: HaCaT (immortalized human keratinocyte)

GI₅₀: Growth Inhibition 50—sample concentration required to elicit 50% of cell growth inhibition

According to literature (Sukumari-Ramesh et al. 2011; Zhao et al. 2018), the saturated AnAc derivative 2-hydroxy-6-pentadecylbenzoic acid has been reported as antiproliferative agent in different tumor cell lines. Our results may suggest that the presence of unsaturated lateral chain in the AnAcs identified in CAE could affect the antiproliferative potential. Further, as a complex mixture of several compounds, the AnAcs concentration in CAE, ranging from 7.12 to 41.74 mg/g (Table 1), perhaps was insufficient to promote antiproliferative effect in the experimental conditions used.

Furthermore, the absence of antiproliferative effect on non-tumor cells (HaCat, immortalized human keratinocytes) suggested that these samples may not interfere in the proliferation of the cells in normal tissues, corroborating the results on zebrafish model. As already described, free and encapsulated CAE did not significantly affect the central nervous system or neuromotor activities besides lethal doses higher than those tested on zebrafish model. These results indicated for a potential safe use of CAE (lyophilized or encapsulated) as industrial natural dye expanding the naturally derived alternatives of colorants. Driven by the consumer perception, the demand for natural dyes has increased in latter years (Sigurdson et al. 2017). Therefore, the use of CAE (lyophilized or encapsulated) as a natural source of yellow food colorant can be explored.

Results of antimicrobial activity assays showed that only L-CAE exerted antimicrobial activity against Listeria monocytogenes; treatment with 50 and 100 mg/mL L-CAE produced zones of inhibition around the colonies with sizes of 11 and 13 mm, respectively (Supplementary material, Table S4). In contrast, CG-CAE and M-CAE did not exhibit antibacterial activity. Likewise, the lower amount of AnAc from the CG-CAE and M-CAE treatments, presented in Table 1, would be indicative of the lower antimicrobial activity, suggesting a relation between antibacterial activity and the AnAc amount, around 5.5 × higher in L-CAE, when compared to the encapsulated ones (CG-CAE and M-CAE). Our hypothesis is that the AnAc, present in CAE, is able to exert its antimicrobial effect. Regarding to the CG-CAE treatment, it is observed that cashew gum (CG) did not contribute with an amount of AnAc, as expected, due to its purification for use as an encapsulating material. Torquato et al. (2004) showed that CG exerted only weak activity against Saccharomyces cerevisiae but no activity against all other microorganisms tested, and concluded that the negative results can be explained by the AnAc remotion during CG purification. Interestingly, L-CAE was found to be effective against Listeria monocytogenes, a Gram-positive bacterium. As shown in Table 1, L-CAE contained the highest concentration of AnAc, which could explain its antimicrobial activity. Moreover, previous studies have demonstrated the antimicrobial effects of AnAc, which were found to be effective against methicillin-resistant Staphylococcus aureus (Muroi and Kubo 1996; Kubo et al. 2003). However, as the authors known, this is the first reports demonstrating the antimicrobial activity of CAE, a rich-AnAc material, against Listeria monocytogenes.

Electron microscopy is a powerful tool for investigating the effects of stressors on bacterial cells (Xing et al. 2009). Transmission electron microscopy experiments were carried out to directly observe membrane damage induced in Listeria monocytogenes cells following exposure to L-CAE. Changes to the structure of the L. monocytogenes cells after 24 h of treatment with the L-CAE were examined based on the TEM images (Fig. 4). Untreated L. monocytogenes cells showed smooth and well-defined outer walls (Fig. 4a). L-CAE-treated cells presented a slight wrinkling and roughness of the outer wall (Fig. 4b). Therefore, the concave collapses and gaps observed using TEM indicated the possible formation of local pores that might have resulted in the reduced cell viability. Consistent with previous findings, the current results indicated that L-CAE was effective against Listeria monocytogenes. L-CAE contained higher concentrations of bioactive compounds (anacardic acid and carotenoids) relative to the encapsulated materials (CG-CAE or M-CAE), which supported its antimicrobial effects. Therefore, our current findings demonstrated the effects of L-CAE against L. monocytogenes and will encourage future antimicrobial studies using L-CAE.

Fig. 4.

Transmission electron micrographs of Listeria monocytogenes (ATCC 19115): a untreated control cell at 27,400 × magnification; and b cell after exposure to L-CAE at 23,900 × magnification. Control L. monocytogenes cells grown at 35 °C/24 h have an intact cell wall. There is a loss of cell wall in Listeria monocytogenes (ATCC 19115) after exposure to L-CAE

Conclusion

The extract obtained from fibers of cashew apple (Anacardium occidentale) is a source of anacardic acids and carotenoids. Both lyophilized and encapsulated samples of the cashew apple extract (CAE) did not exert acute toxicity on the zebrafish (Danio rerio) model neither showed antiproliferative activity against the tumor and non-tumor cell lines tested. In addition, the current findings demonstrated that lyophilized CAE is a potential source of antimicrobial compounds. However, further studies should investigate the cellular mechanisms underlying the antimicrobial activity of this extract in addition to its potential use as a colorant.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.