Leaves of Mauritia flexuosa L. f. (Arecaceae) is effective in vitro and in vivo in the control of Haemonchus contortus

In this study we evaluated the potential of Mauritia flexuosa leaves in the egg hatching and larval development inhibition and for reduction of egg count of this nematode in sheep feces. The leaves of this palm were collected and dehydrated for the production of aqueous and ethanolic extracts with and without tannins. Gas chromatography analysis indicated the presence of fifteen and ten major compounds in the aqueous and ethanolic extracts, respectively, and both showed catechin peaks. The total of condensed tannins for leaves of Mauritia flexuosa was 33.23% ± 2. Aqueous and ethanolic extracts showed 100% anthelmintic activity to inhibit hatchability at 75 mg/ml. The inhibitory concentrations LC90 for aqueous and ethanolic extracts were, respectively, 21.8 and 8.5 mg/ml. The dehydrated leaves powder of M . flexuosa at ≥ 152.08 mg / g of coproculture presented efficiencies greater than 80% for inhibition of larval development. The in vivo administration of aqueous extract at 62.1 mg/kg PC promoted anti-helminthic efficacy of 54.57% and, after 14 days, no clinical signs of toxicity and clinical changes were observed in the treated lambs, indicating potential of this extract for the control alternative of haemonchosis.


Introduction
One of the main limiting factors in sheep farming is the occurrence of gastrointestinal parasitosis caused by Haemonchus contortus. Sheep with haemonchosis present severe anemia and submandibular edema, and in more severe cases, mortality of parturient females and pups (Jackson et al., 2012). The frequent use of synthetic anthelmintics favors the selection of nematode strains that are resistant to the different groups of these drugs (Learmount et al., 2016). The use of plants extracts, such as Cocos nucifera from the Arecaceae family (Oliveira et al., 2009), have demonstrated anthelmintic in vivo efficacy, thus representing sustainable alternatives to the use of conventional anthelmintics.
Mauritia flexuosa L. f. -Arecaceae (buriti) is a non-deciduous palm tree found in Veredas (Ferreira, 2005). Oils extracted from this species fruits are mixed with other herbs and used in the skin diseases treatment (Paniagua-zambrana et al., 2015). However, this species anthelmintic potential in the alternative control of Haemonchus contortus is not known. In this perspective, the present study aimed to evaluate the in vitro and in vivo anthelmintic activity of M. flexuosa leaves on a Haemonchus contortus albendazole-resistant strain.

Collection area and extracts preparation
Mauritia flexuosa leaves samples from young specimens (from six to ten meters height) were collected in October, 2016 (beginning of the rain season), in the most preserved area of the Água Doce vereda (15°13'30" S 44°55'04" W) localized in the Pandeiros Environmental Protection Area (EPA), Pandeiros river, Januária, Minas Gerais, Brazil.
Damaged or deteriorated leaves were discarded, and the selected leaves were washed in running water, dehydrated in forced circulation ovens at 40ºC ± 5 for 72 hours, and ground. The obtained powder was stored in a fresh and dark environment (Nery et al., 2010). Subsamples were used for dry matter (DM) determination, at 105ºC, to calculate the tested concentrations.
The aqueous (AE) and ethanolic (EE) extracts were obtained according to Nery et al. (2010). Both extracts were filtered in a gauze and cotton funnel and individually placed on a forced circulation oven at 40° C for three days. Extracts subsamples were used for DM determination, at 105º C, and to obtain tannin-free extracts according to the method proposed by Nyman (1998).

Extracts characterization and proanthocyanidins quantification
For the derivatization procedure, extracts aliquots (1.0mg) measured in internally conical glass (suitable for this process), were dissolved in 60 µL pyridine and 100 µL BSTFA (N, O-Bis (trimethylsilyl) trifluoroacetamide) containing 1% chlorotrimethylsilane. The reaction mixture was heated at 60 ºC for 30 min. 1 µL of the obtained solution was injected into the Gas chromatography-mass spectrometry (GC-MS) in triplicates.
The chromatographic analysis was performed on a gas chromatograph (Agilent Technologies (GC 7890ª)) equipped with an electron-ionization detector (CG-EM) and DB-5MS capillary column (Agilent Technologies, 30 m long x 0.25 mm internal diameter x 0.25 μm film thickness).
Helium (99.9999% purity) was used as a carrier gas at 1 mL min -1 . An auto-injector (CTC combiPaL) was used to inject 1 µL of the sample into the chromatograph using 1:10 split ratio. The split/splitless injector was maintained at 290ºC. The chromatographic column, initially at 80 ºC, isothermal for five min., heated at 4 ºC min -1 up to 260 º C for 10 min. After the compounds separation, the temperature was elevated up to 300 ºC and maintained for 2 min. (post run). The interface temperature was maintained at 280 ºC, ionization performed by 70 eV impact, and m/z scanning range from 30 to 600 Da.

In vitro parasitological tests
The experimental procedures were conducted according to the Ethics Committee on Animal Use of the Universidade Federal de Minas Gerais (CEUA-UFMG) and approved under the following protocol number: 275/2013.

Hatchability test
Three Santa Inês sheep, six to ten months old, were orally inoculated with 2,000 infective larvae (L3) of H. contortus albendazole-resistant strain (Duarte et al. 2012). After 28 days, 80g of feces were collected directly from the rectal ampulla of each animal and transported to the laboratory. The Gordon & Whitlock (1939) technique, modified by Ueno & Gonçalves (1998), was used to quantify the eggs per gram of feces (EPG). Following an approximate 5 min rest period, the samples were evaluated under an optical microscope with a 10x objective lenses in duplicate.
For the in vitro evaluation of H. contortus larvae eclosion, the modified methodology proposed by the World Association for the Advancement of Veterinary Parasitology (WAAVP) according to Coles et al. (1992) to evaluate inhibition of hatchability was applied. The eggs were retrieved by the Bizimenyera et al. (2006) adapted methodology.
The quantification of blastomerate eggs, larval eggs and first-instar larvae (L1) were performed in an optical microscope with the 10x objective lens. The experiment was performed with five replicates per treatment, in a completely randomized design.
The mean hatchability inhibition efficiency was calculated by the Coles et al. (1992) adapted formulae: In the hatchability test, the number of L1 and eggs not hatched was converted into values relative to the initial amount in each replicate. The data obtained were submitted to the variance and means analysis, compared by the Tukey post-hoc test with 5% probability and probit regression analysis was applied to determine the concentration to inhibit 90% (LC90) of hatchability, using the SAEG® 9.1 (2007) statistical package.

Larval development inhibition (LDI)
The larval development inhibition test was performed according to Nery et al., (2010), adapted by Borges (2003) For the statistical analysis, the LPG number was transformed via the Y= log (x + 10) equation, submitted to the variance analysis and compared by the Duncan test, considering the significance level at 5%. The probit regression analysis was performed to determine the concentration to inhibit 90% of the larval development, following a 7-day incubation period. All analyses were conducted in the SAEG® 9.1 (2007) statistical package.

In vivo analysis
The extracts effect of reducing the number of eggs in the feces was evaluated in twelve Santa Inês lambs (6 male and 6 female), five to six months old, weighing in average 25.5 Kg. During the 14-days adaptation period, the animals were individually confined and fed a diet containing sorghum silage, concentrate, mineral premix, and water ad libitum. The lambs presenting zero EPG in two counting procedures were infected with 800 H. contortus L3 (infective larvae as reported above) per 10 kg of body weight.
Twenty-eight days after infection, the lambs were divided into two homogeneous groups based on EPG, weight, and gender (3 male and 3 female per group). One group with non-treated lambs represented the negative control, and the other group The EPG was evaluated three times with weekly intervals. Each period covered in average three days, where daily means were obtained, totalizing six EPG measurements per animal (Morais-Costa et al., 2016). The EPG means were registered Research, Society and Development, v. 10, n. 12, e276101220493, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i12.20493 two days before the treatment and at the administration day. Posteriorly, the mean EPG was calculated in days 7, 8 and 9 (second period); Days 14, 15 and 16 (third period). The MacMaster technique was performed with saturated NaCl and a minimum sensitivity of 25 eggs/g of feces (Gordon e Whitlock, 1939). The treatment efficiency was calculated by the following formula adapted from Coles et al. (1992): % EPG reduction =100 × [1-(mean EPG per treated group / mean EPG per non-treated group)].
The obtained EPG data was transformed into log10 (x + 10) and submitted to variance analysis in the two evaluated periods. The means were compared by the Scott-Knott test (P <0.05).

In vitro anthelmintic activity
Mauritia flexuosa AE and EE at 75 mg/mL presented 100% of anthelmintic activity in the hatchability test, and over 90% at 37.5 mg/mL, with statistically significant differences as compared to the negative control (sterile water) (P < 0.05) ( Table   2). The LC90 for M. flexuosa leaves AE and EE were, respectively, 21.8 ± 3 and 8.5 ± 3 mg/ml.
The tannin-free EE presented 61.5% efficiency at 75 mg/ml, which is comparatively lower than the EE with tannins efficiency at the same concentration (Table 2). Different letters in the columns indicate a significant difference (P <0.05) by Tukey test at 5%. *% efficacy = 100 x (1 -L1 / initial egg numbers). Source: Authors (2020).
Mauritia flexuosa tannin-free AE LC90 was 25.48 mg/ml. It was not possible to calculate the tannin-free EE 90% lethal concentrations due to its low efficiency.
Mauritia flexuosa powder from dehydrated leaves at 304 mg/g of coproculture, presented 86.54% of anthelmintic efficiency for LDI (Table 3) and differed statistically from the control with sterile water (P <0.05). The AE at the highest concentration (12.83 mg/g) was 53% effective to inhibit larval development (Table 3). Different letters indicate significant difference by the Duncan test (P < 0.05). Coefficient of variation LDI: 0.98%. * LPGF: number of infective larvae per gram of faeces in coproculture. Efficacy: % efficacy = 100 x (1 -LPGF of the treated group / LPGF of the control group). Source: Authors (2020).

In vivo anthelmintic activity
Following 14 days of AE administration, the EPG in the treated group was significantly decreased as compared to the non-treated group, displaying a 54.57% efficiency and 37.5% variation coefficient (Table 4). Additionally, clinical signs of toxicity in the mucosas or animals behavior were not observed.  (Kotze & Prichard, 2016).
In the present study, M. flexuosa leaves presented 100% anthelmintic efficiency to inhibit hatchability with both extracts (AE and EE) at 75 mg/ml. However, the EE displayed better results, because in the lowest concentration (9.4 mg/ml) presented 92.36% efficiency, while the AE in the same concentration presented only 75.4% efficiency (Table 2). Oliveira et al., (2009) reported in a study performed with a palm tree from the same family as buriti (Arecaceae), that the EE from the fruits peels presented 100% efficiency at 5.1 mg/ml.
The tests performed with the tannin-free extracts evidenced a decreased anthelmintic effect on the hatchability inhibition for the ethanolic extract, but for the aqueous extract, this reduction was only observed for the lowest concentrations, indicating that the tannins are not the only responsible for the inhibition of hatchability, as suggested by Morais-Costa et al. (2016).
Although M. flexuosa have increased amounts of total tannins (33.23%), higher than the values displayed for C. nucifera (25.87%) Oliveira et al., (2009), these compounds removal did not inhibit completely the anthelmintic effect, suggesting that the presence and/or combination of other secondary metabolites in the plant, such as catechins, flavonoids, and amino acids may contribute to the anthelmintic activity, as proposed by Klongsiriwet et al. (2015).
In our study, M. flexuosa leaves AE oral administration presented a moderate efficiency, however, these results are promising considering that the administered dosage (62.1 mg/kg/pc) was low and applied only twice. In another study that used a higher dosage of Piptadenia viridiflora AE (283 mg/kg/pc), the authors reported a 47.2% EPG reduction in the first week and 32.9% in the third week, after 21 days of treatment (Morais-Costa et al., 2016).

Conclusion
Mauritia flexuosa AE and EE presented increased tannins levels, and the chromatographic analysis revealed the presence of catechins. These extracts were effective to inhibit H. contortus eggs hatchability and larval development. A moderate anthelmintic efficiency was observed when M. flexuosa AE was applied in vivo and the animals did not display any clinical signs of toxicity. It is necessary that bioactive compounds from M. flexuosa are isolated and tested. In this perspective, the present findings highlight the potential of M. flexuosa leaves in the alternative control of this nematode in ruminants.

Conflicts of interest
The authors of this manuscript have no financial or personal relationships with individuals or organizations that may influence this manuscript content.