Assessment of anthelmintic activity and bio-guided chemical analysis of Persea americana seed extracts

Assessment of anthelmintic activity and bio-guided chemical analysis of Persea americana seed extracts

Veterinary Parasitology 251 (2018) 34–43 Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate...

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Veterinary Parasitology 251 (2018) 34–43

Contents lists available at ScienceDirect

Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar

Research paper

Assessment of anthelmintic activity and bio-guided chemical analysis of Persea americana seed extracts

T

Andressa Soldera-Silvaa, Melina Seyfrieda, Luciano Henrique Campestrinia,1, Selma Faria Zawadzki-Baggioa, Alessandro Pelegrine Minhob, Marcelo Beltrão Molentoc, ⁎ Juliana Bello Baron Maurera, a

Department of Biochemistry and Molecular Biology, Federal University of Paraná, 81531-980, Curitiba, PR, Brazil Embrapa Southeast Livestock, 13560-970, São Carlos, SP, Brazil c Department of Veterinary Medicine, Federal University of Paraná, 80035-050, Curitiba, PR, Brazil b

A R T I C L E I N F O

A B S T R A C T

Keywords: Avocado Haemonchus contortus Larval migration test Epicatechin Rutin 3-O-glucoside quercetin

The aim of this study was to characterize the extracts and fractions of Persea americana Mill. (Avocado) seeds and to determine the composition and the in vitro anthelmintic activity against third-stage larvae (L3) of Haemonchus contortus. The fresh (F) and dried (H) avocado seeds (PA) were subjected to extraction with hot water (W-PAF, W-PAH), ethanol (E-PAF, E-PAH) or methanol 70% (v/v), and partition with solvents of increasing polarity [nhexane (H-PAF, H-PAH), chloroform (C-PAF, C-PAH), ethyl acetate (Ea-PAF, Ea-PAH), and n-butanol (B-PAF, BPAH)], yielding a total of 14 extracts/fractions. After considering the yield, water solubility, and the preliminary results of the larval migration test (LMT), the E-PAF, E-PAH, H-PAF, and H-PAH were selected for further experiments. E-PAH presented an efficiency concentration of 50% (EC50) of 36 μg/mL on the LMT. E-PAH showed the greatest efficiency when its EC50 was compared to the other fractions (E-PAF = 147 μg/mL; HPAF = 801 μg/mL; H-PAH = 77 μg/mL). After that, the E-PAH was chemically characterized, considering its quantitative polyphenolic and flavonoid contents by colorimetric and chromatographic techniques. E-PAH presented 50, 38, and 24 mg/g of dry matter of total phenol, condensed tannins (CT), and flavonoid contents, respectively. Using high performance liquid chromatography (HPLC) analysis, E-PAH had shown to have epicatechin (4.7 μg/mL), rutin (2.8 μg/mL), and chlorogenic acid (1.4 μg/mL) as its main constituents besides quercetin. These isolated compounds were evaluated using the LMT in order to relate the composition to the anthelmintic activity observed for E-PAH. Quercetin (EC50 = 7.8 μg/mL) and epicatechin (EC50 = 10 μg/mL) presented a higher efficiency than rutin (EC50 = 30 μg/mL). Chlorogenic acid was also tested with the LMT but did not present a significant efficiency. According to the results, the phenolic composition of E-PAH and the EC50 values obtained for the isolated phenols, it can be suggested that, besides the CT content, the presence of epicatechin and rutin contributed to the larvicidal activity of E-PAH. In conclusion, avocado seeds may be used as a source of polyphenols with promising anthelmintic applications.

1. Introduction Gastrointestinal nematode infections lead to major economic losses in ruminant production across the world (Calvete et al., 2014). Among the parasites that affect small ruminants, Haemonchus contortus, a hematophagous parasite, is ranked as one of the most important cause for reducing animal performance and productivity, and increasing animal morbidity and mortality (Kumarasingha et al., 2016). The use of medicinal plants as a source of bioactive compounds for the treatment of endoparasite and ectoparasite infections in livestock,



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has become the target of many studies and holds promise for the development of future therapeutic drugs (Githiori et al., 2006; Athanasiadou et al., 2007). One of the main reasons underlying the search for alternative drug therapy is the mitigation of problems associated with the use of semi-synthetic anthelmintic drugs. The alarming parasite resistance situation, environmental pollution, and animal byproduct drug residues (Molento, 2009; Hoste et al., 2015) are some of production impeding factors. The antiparasitic properties of plant extracts are commonly associated with the presence of secondary metabolites, such as essential oils

Corresponding author. E-mail address: [email protected] (J.B.B. Maurer). Department of Biochemistry, Federal University of Santa Catarina, 88040-900, Florianópolis, SC, Brazil.

https://doi.org/10.1016/j.vetpar.2017.12.019 Received 23 November 2017; Received in revised form 21 December 2017; Accepted 23 December 2017 0304-4017/ © 2017 Elsevier B.V. All rights reserved.

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Every 2 days, 100 mL of the respective solvent was removed and 100 mL of new solvent was added, in order to optimize the extraction. The ethanolic extracts were vacuum filtered, concentrated in a rotary evaporator under reduced pressure at 40 °C, freeze-dried, stored at – 4 °CC in the dark, and named E-PAF and E-PAH. The methanolic extracts (M-PAF and M-PAH) were also filtered, concentrated and freezedried under the same conditions as described before. The freeze-dried methanolic extracts were dissolved in distilled water and mixed with nhexane in a 1:1 ratio (v/v). The solution was placed in a separation funnel, and after vigorous agitation, the hexane fractions (H-PAF and HPAH) were separated. This procedure was repeated twice. Subsequently, the residual extract was fractioned using organic solvents of increasing polarity [chloroform (1:1), ethyl acetate (1:1), and n-butanol (1:1)] in a similar manner as described for n-hexane. The chloroform fractions (C-PAF and C-PAH), ethyl acetate fractions (EaPAF and Ea-PAH), and butanol fractions (B-PAF and B-PAH) were thus obtained. The extraction and fractionation procedures produced 14 extracts and fractions in total, and their yields and water solubility characteristics are described in Fig. 1.

or tannins (Molan et al., 2002; Hoste et al., 2006; Pavela, 2015; Pavela and Benelli, 2016). Tannins, which are polyphenol compounds, have been described as oligomeric compounds with high molecular weight (≥ 2000 Da) and are subdivided into two groups: condensed tannins (CT) and hydrolyzable tannins (Craft et al., 2012). CT, also known as procyanidins and prodelphinidins, are oligomers and polymers of flavan-3-ol, while hydrolyzable tannins are compounds that contain a central core of glucose or another polyol esterified with gallic acid, also called gallotannins, or with hexahydroxydiphenic acid, is also called ellagitannins (Craft et al., 2012). However, it has been noted that the anthelmintic activity of plant extracts may also be attributable to other polyphenolic compounds, including phenolic acids (hydroxycinnamic and hydroxybenzoic acids) and flavonoids (flavanols, flavonols) (Barrau et al., 2005; Klongsiriwet et al., 2015, Díaz et al., 2017). The seeds of Persea americana Mill. (avocado tree) are a source of bioactive compounds and the species is cultivated in various parts of the world, including Brazil. The fruit has a great commercial value because of its nutritional quality (Dabas et al., 2013). The seed represents a considerable percentage of the total fruit (12–16%, w/w) and is an under-utilized resource and a waste issue for the avocado industry (Kosińska et al., 2012). From the chemical point of view, avocado seeds contain high levels of phenolic compounds (more than 50%), such as hydroxybenzoic acids, hydroxycinnamic acids, flavanols (such as catechin, epicatechin), flavonols (such as rutin), and procyanidins (Pahua-Ramos et al., 2012). A common approach for studying crude plant extracts and their fractions is by using bio-guided chemical analysis. The current study aimed to characterize the extracts and fractions of the avocado (Persea americana Mill.) seed and to relate its composition to the anthelmintic activity tested. The effect of avocado extracts and fractions, as well as, that of the isolated compounds such as chlorogenic acid, quercetin, rutin, and epicatechin, on third-stage larvae (L3) of Haemonchus contortus were evaluated using an in vitro larval migration test (LMT).

2.3. Colorimetric determination of total phenolic, flavonoid, and condensed tannin contents

2. Material and methods

The total phenolic content was determined using a microassay and the Folin-Ciocalteu reagent, which was adapted from Singleton and Rossi (1965). The calibration curve was prepared using gallic acid (GA) as a standard. The total phenolic content was expressed as milligrams of gallic acid equivalents per gram of dry extract. The flavonoid content was determined by the aluminum chloride method (Vennat et al., 1992), using rutin (RUT) as a standard and was expressed as milligrams of RUT equivalents per gram of dry extract. The condensed tannin (CT) content was determined by the sulfuric vanillin method (Queiroz et al., 2002). Results were expressed as milligrams of epicatechin (EPI) equivalents per gram of dry extract.

2.1. Chemicals and reagents

2.4. High-performance liquid chromatography (HPLC) analysis

Water was treated in an ultra-purifier MS 2000 system from Gehaka (São Paulo, Brazil). HPLC grade acetonitrile was obtained from JT Baker (Xalostoc, Mexico), and HPLC grade formic acid was obtained from TEDIA (Darmstadt, Germany). trans-cinnamic acid, chlorogenic acid, gallic acid, epicatechin, quercetin, rutin, and levamisole were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ivermectin (1% w/v solution, Ivomec, Merial Limited, Duluth, GA, USA), and FolinCiocalteu reagent was obtained from Chromate (São Paulo, Brazil). All other used reagents were of analytical grade.

The phenolic content of the E-PAH extract was determined using an Agilent 1200 Series high-performance liquid chromatography (HPLC) system (Agilent Co., St. Clara, CA, USA) equipped with a vacuum degasser (G1322A), quaternary pump (G1311A), manual injector (Rheodyne, 7725i), and a multi UV–vis wavelength detector (G1365D) operating at a wavelength of 235, 280, 320, 365, 375, and 525 nm. The chromatographic separation was performed using an Agilent Eclipse XDB-C-18 column (150 mm × 4.6 mm, 5 μm particle size) with 20 μL of injected sample. The mobile phase used was formic acid (1%, v/v) (A) and acetonitrile (B) set with the following gradient: 0–5 min, 5% B; 5–10 min, 10% B; 10–20 min, 30% B; 20–30 min, 50% B; and 30–35 min, 5% B, with a flow rate of 1 mL/ min, at 25 °C (adaptated from Jiménez et al., 2017). The extract was diluted with methanol: water (1:4 v/v) to a concentration of 5 mg/mL and filtered through a 0.22-μm membrane filter (JetBiofil, Guangzhou, China) before injection. The standards (trans-cinnamic acid, chlorogenic acid, epicatechin, quercetin, and rutin) were prepared individually as stock solutions in methanol at a concentration of 1 mg/mL, and a diluted solution (100 μg/mL, in aqueous methanol 10%, v/v) was prepared freshly before the analysis. The phenolics were identified by comparing their retention times with those of standards. The quantification of the main compounds was determined by comparing the analytical curves of the standards (rutin, chlorogenic acid and epicatechin). To ascertain the linearity, stock solutions of the standards were prepared individually in methanol at a concentration of 1 mg/mL, and six different concentrations (2.5 to 200 μg/mL) were injected (20 μL) in triplicate to the HPLC system and the analytical curve obtained by plotting peak area versus concentration for each standard presented the square of the correlation coefficient R2 ≥ 0.99 as indicative of the measure of linearity [(rutin, y = 43154 × + 162195, R2 0.9979); (chlorogenic acid,

2.2. Extraction and fractionation of Persea americana seeds Mature avocado fruits (cv Fortuna) were obtained from a commercial organic market. The seeds were manually removed and cut into small pieces (2 × 2 cm). Part of the seeds (315 g) was subjected to an air-drying procedure at 40 °C, until a constant weight was attained. Avocado seeds showed average moisture content of 75 ± 1.5%. The other part (193 g) was used in natura (fresh). The dried seeds (33 g) were submitted to hot aqueous extraction with 400 mL of water for 60 min at 70 °C (Bovo et al., 2013). The aqueous extract was vacuum filtered, concentrated in a rotary evaporator under reduced pressure at 40 °C, freeze-dried, stored at −4 °C in the dark, and designated as WPAH. The same procedure was carried out for the fresh seeds (85 g of seeds and 500 mL of water) and the sample was designated as W-PAF, as shown in Fig. 1. The seeds (20 g dried and 54 g fresh) were submitted to hydroalcoholic extraction with aqueous ethanol 70% (v/v) (400 mL) or with aqueous methanol 70% (v/v) (400 mL), at 4 °C, in the dark, for 10 days. 35

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Fig. 1. Schematic of extraction and fractionation procedures, identification of their respective extracts and fractions, yields, and solubility characteristics.

y = 39116 × + 46220, R2 0.9979); (epicathechin, y = 25331 × + 67358, R2 0.9993)]. System control and data analysis were performed using the EZChrom Elite program on Windows 7.

Prichard, 2001) modified by Demeler et al. (2010). For this experiment, L3 were used fresh (until 30-day old). To remove the cuticle, the larvae were incubated in a solution of 0.3% (v/v) sodium hypochlorite for 1 h. The larvae were washed three times with distilled water, counted by optical microscopy (SM-LUX model, Ernst Leitz GmbH, Wetzlar, Germany) and distributed into 24-multiwell plates (200 larvae per well). Extracts, fractions or isolated compounds were prepared at a concentration of 1 mg/mL (w/v), in distilled water, and dilutions were performed in order to get different concentrations (as indicated in each experiment), in a final volume of 1 mL. Extracts, fractions or isolated compounds were added in the plates, and these were incubated in a BOD-type incubator at 27 °C, for 6 h. Test-groups of a negative control (consisting of distilled water), and a positive control [consisting of levamisole (40 μg/mL) or ivermectin (100 μg/mL)], were performed in parallel. Levamisole was used for the preliminary LMT and ivermectin was used in the experiments performed for EC determinations. After the first incubation, the material was transferred to plates with apparatus with a 22 μm-mesh nylon membrane sieve, and also incubated under the same conditions but with a source of light (150 W) positioned below the plates to stimulate larval migration. After 18 h, the apparatus was removed and the larvae that migrated, as well as the larvae that did not migrate, were counted. Three experiments with three replicates for each concentration and control were performed. The average of the number of larvae that migrated, at each tested concentration, was transformed into the percentage of migration, according to the formula: % Efficiency = B − A B x100 , where B corresponds to the negative control (distilled water) and A corresponds to the average number of larvae that migrated after incubation.

2.5. Light stability of E-PAH This test was carried out to evaluate the light stability of E-PAH when exposed to intense light, since light is required for the LMT. EPAH (5 mg/mL, 200 μL) was placed in a 96-well plate, in triplicate. The plate was exposed to light (150 W), and after 2-, 4.5-, and 6-h incubation periods, aliquots of the extract were analyzed by HPLC, under the same conditions as described before. The phenolic content was compared to that in the previous analysis of E-PAH. 2.6. In vitro anthelmintic activity Prior authorization for the use of laboratory animals was obtained from the Ethical Committee for the Use of Animals of the Federal University of Parana (n° 065/2015). Feces of goats naturally infected with gastrointestinal nematodes were collected directly from the rectal ampoule into a plastic bowl and processed to elaborate fecal cultures. The feces were maintained in a biological oxygen demand (BOD)-type incubator (Eletrolab, São Paulo, Brazil) at 27 °C, for 10 days. The result of fecal culture analysis showed the presence of H. contortus as the abundant (95%) nematode species.

(

2.6.1. Larval migration test (LMT) The objective of the LMT was to determine the effect of the extracts on the mobility of exsheathed third-stage larvae (L3) (Molento and 36

)

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chemical characterization by colorimetric analysis of CT content. Table 1 shows the results for the quantitative analysis of CT content of E-PAH, E-PAF, H-PAH and, H-PAF. The CT content was 23 ± 0.4, 38 ± 1.1, 27 ± 0.1, and 17 ± 1.0 mg EPI/g, respectively for E-PAF, E-PAH, H-PAF, and H-PAH. Higher CT content (47.7 mg/g) has been described for avocado fresh seeds (Dabas et al., 2013). However, the levels of compounds in the seeds vary according to the variety of avocados, conditions of growth and stage of maturation; in addition, the measured levels may also be influenced by the type of sample, the method of extraction, and the techniques employed in the experiments (Dabas et al., 2013). Besides that, many varying factors affect bioavailability and bioefficacy of the plant extracts, such as bioaccessibility, transporters, molecular structures and metabolizing enzymes (Rein et al., 2013).

2.7. Statistical analysis The results of the LMT were analyzed by One-Way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test using the GraphPad Prism 5 program. The differences were considered statistically significant at p ≤ 0.05. The concentrations of the extracts that inhibited 20, 50, and 90% of the larvae migration, expressed as EC20, EC50, and EC90, respectively, were calculated using linear equations (R2 ≥ 0.95) obtained by linearizing the activity curves using Microsoft Excel 2016. 3. Results and discussion 3.1. Preliminary selection and characterization of the extracts and fractions by a bioassay-guided approach

3.2. LMT and EC50 determination of E-PAH, E-PAF, H-PAH and, H-PAF The extraction and partition procedures yielded 14 extracts/fractions (Fig. 1). The C-PAF and C-PAH fractions were excluded by considering their yield in relation to the dried extract (less than 0.14%) (Fig. 2). The B-PAF, B-PAH, Ea-PAF, and Ea-PAH fractions were also excluded since these were not soluble in water (at 25 ° C) (Fig. 2). The choice of a solvent to dissolve plant extracts to be evaluated in vitro is very important, as in the case of the present experiment, it can present toxicity to larvae of parasites, generating a false-positive result. In the case of extracts which are not soluble in water, such as essential oils, it is recommended the use of aqueous solutions of ethanol, Tween 80 or dimethyl sulfoxide, however, it is necessary to previously standardize the non-toxic concentration of the selected solvent for the different in vitro tests. In the present work, we chose to use water solubility as a parameter for the extract selection before the LMT. In this way, the step for standardization of the solvent was not necessary. In addition, water-soluble extracts could be of interest for developing future therapeutic products with higher safety and more ecologically acceptance. The remaining extracts and fractions (E-PAH, E-PAF, H-PAH, and HPAF) were subjected to a preliminary LMT at 100, 250, and 500 μg/mL in order to select the fractions with best larvicidal activity (Fig. 3). The W-PAF and W-PAH fractions presented activities similar to the negative control (distilled water) and were thus excluded. The positive control group, consisting of levamisole (40 μg/mL), a cholinergic anthelmintic (Martin et al., 2012), presented an efficacy of 76.9 ± 7.2%. At a concentration of 500 μg/mL, the E-PAH, E-PAF, H-PAH, and H-PAF fractions presented percentage efficacies that were statistically different from that of the negative control, and were submitted to preliminary

Fig. 4 shows the results of the LMT of E-PAH, E-PAF, H-PAH and, HPAF. As expected, the distilled water in the negative control did not affect the migration of larvae. The positive control group, consisting ivermectin (100 μg/mL), presented an efficiency of 81.6 ± 3.5%. Ivermectin is an antiparasitic semi-synthetic drug with a mechanism of action related to a change in the motility of larvae. It acts as an agonist of the gamma-aminobutyric acid (GABA) neurotransmitter in the nervous system cells and of ligands of the chlorine channels activated by glutamate in neuron and muscle cells (Laing et al., 2017). However, an increase in parasite resistance to this drug has been verified and several authors have reported the current global drug resistance situation, in relation to the main families of anthelmintics (Molento 2009; Gaudin et al., 2016; Campos et al., 2017). E-PAH, at a concentration of 10 μg/mL, presented an efficiency of 76%, and reached an efficiency of 86% at the highest concentration tested. For quantification of larvicidal activity, the EC50 values were calculated and are described in Fig. 4. E-PAH presented an EC50 of 36 μg/mL, showing the best efficiency compared to the other fractions tested (E-PAF = 147 μg/mL; H-PAF = 801 μg/mL; H-PAH = 77 μg/ mL). For our records, any literature data about the effect of avocado seed extracts on larval migration were obtained. E-PAH, E-PAF, H-PAH and, H-PAF presented good efficiency compared to other plant extracts from other species tested by the LMT (Hounzangbe-Adote et al., 2005; Alonso-Díaz et al., 2011; Fernex et al., 2012; Acharya et al., 2014; Féboli et al., 2016). For example, Hounzangbe-Adote et al. (2005) verified that the ethanolic extract of Carica papaya seeds presented a Fig. 2. Selection and characterization of the extracts and fractions using a bioassay-guided approach.

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Fig. 3. Efficiency (%) of W-PAF (A), W-PAH (B), E-PAF (C), E-PAH (D), H-PAF (E), and, H-PAH (F) of Persea americana seeds using the larval migration test (LMT).

to their tannin content (Alonso-Díaz et al., 2011; Fernex et al., 2012). In addition to this, other compounds present in these extracts, such as phenolic acids and flavonoids, have also been demonstrated to have anti-parasitic properties (Barrau et al., 2005; Hernández-Villegas et al., 2011; Klongsiriwet et al., 2015, Féboli et al., 2016, Díaz et al., 2017). In the present study, all fractions (E-PAF, E-PAH, H-PAF and HPAH) presented CT, which may be related to their larvicidal activity. The CT content was determined by colorimetric analysis, but this technique did not permit the verification of the biochemical nature of the polyphenolic profile for these extracts/fractions. Chromatographic analysis is a technique that reveals which type of phenolic compounds are present, enabling further speculation about the relation of the polyphenolic profile with the anthelmintic activity. Since E-PAH presented the best larvicidal activity, this extract was selected for chemical characterization of its phenolic and flavonoid quantitative content, by colorimetric and chromatography techniques, for the purpose of correlating its phenolic composition with its activity.

Table 1 Condensed tannin contents of the extracts (E-PAF, E-PAH) and fractions (H-PAF, H-PAH) obtained from the avocado seeds. Extracts

Condensed tannins (mg EPI per g dry extract)a

E-PAF E-PAH H-PAF H-PAH

23 38 27 17

± ± ± ±

0.4 1.1 0.1 1.0

a Determined according to Queiroz et al. (2002); expressed as epicathecin equivalents (EPI).

70% efficiency, at 600 mg/mL. However, no information about the phytochemical profile of this extract was mentioned, and thus, the authors did not conclude which components could be responsible for the activity observed. The anthelmintic activity of plant extracts has usually been related 38

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Fig. 4. Efficiency (%) and EC determination of E-PAF (A, a), E-PAH (B, b), H-PAF (C, c), and, H-PAH (D, d) of Persea americana seeds using the larval migration test (LMT).

g) or higher (292 mg GAE/g, 351 mg GAE/g) than the value observed for E-PAH. The solvent seems to be an important factor that influences the level of phenolic content. Acetone extracts present higher phenolic content than methanolic or ethanolic extracts (Dabas et al., 2013). Fig. 5 shows the HPLC profile of the phenolics present in E-PAH. Comparison between the retention times of the standard peaks with the

3.3. Chemical characterization of E-PAH The phenolic and flavonoid contents of E-PAH were 50 mg GAE/g and 24 mg RUT/g, respectively. Phenolic contents of avocado seed extracts described in the cited literature (Wang et al., 2010; Pahua-Ramos et al., 2012; Rodríguez-Carpena et al., 2011) were similar (51 mg GAE/ 39

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Fig. 5. HPLC profile at 280 nm of phenolic standards (A), E-PAH (B), and E-PAH submitted at 0 h, 2 h, 4.5 h and 6 h of light exposure (C).

catechins, procyanidins (oligomers and polymers), and phenolic acids (Wang et al., 2010; Rodríguez-Carpena et al., 2011; Pahua-Ramos et al., 2012; Kosińska et al., 2012). The presence of catechin and epicatechin (3-O-flavanols) as main constituents of avocado seeds was described by Rodríguez-Carpena et al. (2011). Contrarily, another study showed that protocatechuic acid (hydroxybenzoic acid-type phenolic acid) is the main phenolic compound found in avocado seeds, followed by kaempferide (O-methylated flavonol), vanillic acid (hydroxybenzoic acid-type), and rutin (glycosylated flavonol) (Pahua-Ramos et al., 2012). Kosińska et al. (2012) described hydroxycinnamic acid-type phenolic acids (chlorogenic and coumaric acids) and procyanidin (oligomers and polymers) as the main constituents of avocado seeds. As mentioned before, the levels of compounds in the seeds can be

retention times of the peaks of E-PAH, led to the identification of hydroxycinnamic acid-type (chlorogenic acid), flavanol-type (epicatechin), aglycon (quercetin) and glycosylated flavonol-type (rutin) phenolic acids. The identification of other unnamed peaks was not possible because their retention times did not match any of the standards used. The concentration of the phenolic and flavonoid compounds in E-PAH was calculated using the equation obtained from the standard curves for chlorogenic acid, epicatechin, and rutin. The phenolic constituents of E-PAH were thus determined as: epicatechin (4.7 μg/mL), rutin (2.8 μg/mL), and chlorogenic acid (1.4 μg/mL). Quercetin was not quantified with acceptable accuracy and precision. Some studies describing the HPLC analysis of the phenolic composition of avocado seed extracts showed that these seeds are rich in 40

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Fig. 6. Efficiency (%) and EC determination of epicatechin (A, a), quercetin (B, b), rutin (C, c), and chlorogenic acid (D, d) using the larval migration test (LMT).

acid, epicatechin, and rutin were monitored. The HPLC chromatograms are shown in Fig. 4. For all phenolic compounds mentioned, the content did not present statistical difference among the different periods of light exposure (data not shown).

influenced by the variety of avocados and the method used for extraction or the techniques employed in the analysis (Dabas et al., 2013). 3.4. Influence of light exposure on the phenolic profile of E-PAH

3.5. LMT and EC50 determination of quercetin, rutin, epicatechin and chlorogenic acid

The LMT was carried out in the presence of light because light is a stimulus for larval migration. Given that phenolic compounds can be light-sensitive, E-PAH was analyzed by HPLC after different periods (0 h, 2 h, 4.5 h, and 6 h) of light exposure. The contents of chlorogenic

Fig. 6 presents the results obtained in the LMT of the isolated 41

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(Embrapa Pecuária Sul – Bagé, RS, Brazil) for technical assistance. This work did not receive any specific grant from funding agencies in the public or private sectors.

phenolic compounds (obtained commercially from Sigma-Aldrich Co), which were also identified in the E-PAH. Comparison of the larvicidal activity of the tested compounds indicates that, except for chlorogenic acid, the others present 90% efficiency at 100 μg/mL. The EC50 values for quercetin, rutin, and epicatechin are presented in Fig. 6. Quercetin (EC50 = 7.8 μg/mL) and epicatechin (EC50 = 10 μg/mL) showed greater efficiency than rutin (EC50 = 30 μg/mL). Chlorogenic acid was also tested, but did not show significant efficiency in the LMT, under the conditions tested. In relation to literature data about plant extracts with anthelmintic activity, it was revealed that tannins, specifically CTs, are directly or indirectly involved in antiparasitic activity. However, other compounds with structural features like CTs, namely flavonoids such as 3-O-flavanol monomers and flavonols, have also received attention due to their anthelmintic properties (Molan et al., 2000; Molan et al., 2002, Molan et al., 2003; Barrau et al., 2005; Klongsiriwet et al., 2015). The larvicidal effects of flavan-3-ols, the basic units of CT polymers such as epicatechin, were described on L3 of Trichostrongylus colubriformis (Molan et al., 2003). In addition, literature data suggests that flavonols (glycosylated or aglycone) might also participate in the modulation of their bioactivity (Barrau et al., 2005; Klongsiriwet et al., 2015; Díaz et al., 2017). The mechanisms underlying the effects of polyphenolic compounds on larval migration are still not described (Molan et al., 2000). Since the LMT depends on an active migration process through a sieve, the reduction in migration rate could be due to either larval mortality or to larval paralysis. Results described by Molan et al. (2003) suggested that polyphenols (CT and flavan-3-ols) have inhibitory effects on L3 motility, as evidenced by their ability to immobilize larvae and prevent their passage through the sieve membrane. In addition, 90% of trapped larvae exposed to CT, remained alive despite their low mobility (Molan et al., 2003). According to the results observed in relation to the phenolic composition of E-PAH and the EC50 values obtained for isolated phenols, it can be suggested that, besides the CT content, the presence of epicatechin and rutin, contributed to the anthelmintic activity of E-PAH, which was verified using LMT. The anthelmintic effect of extracts with a mixture of polyphenolic compounds can be an overall action of a sum of the activities of their constituents, which can present synergistic actions (Klongsiriwet et al., 2015).

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4. Conclusion The use of a bio-guided chemical analysis approach was an efficient option to screen extracts or fractions with the best efficiency for the purpose of characterizing the biochemical nature of active compounds present in those extracts or fractions in relation to the anthelmintic activity tested. The selected extract, E-PAH, obtained by aqueous ethanolic 70% (v/v) extraction of dried avocado seeds, presented the best yield and efficiency compared to the other fractions tested. Considering its phenolic chemical analysis and the EC50 values obtained for isolated phenolic compounds, it can be suggested that, besides the CT content, the presence of epicatechin and rutin contribute to the anthelmintic activity of E-PAH, which was verified using the LMT. In conclusion, seeds of Persea americana Mill. could be a promising source of bioactive compounds with anthelmintic properties. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgement The authors thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES of Brazil for the fellowships granted to Andressa Soldera-Silva and Melina Seyfried. We also thank to Rossana L. Granada 42

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