Place of collection of Gymnopodium floribundum material

The study was carried out in the period between December 18, 2017 and December 21, 2018. It was performed in an experimental area of TDF of 12,000 m² (50 x 240 m) located in the Faculty of Veterinary Medicine and Zootechnics of the Autonomous University of Yucatan, Mexico (20°51'93.2'' N and 89°37'11'' W, at 10 m asl). The experimental area has an AW₀ climate (warm subhumid with rains in summer). The soil type is classified as cambisol and luvisol. The average maximum temperature was 32 °C and the minimum 16 °C with an annual rainfall ranging from 984.4 mm to 1,092 mm, distributed from June to November¹⁸.

Collection and production of extracts from Gymnopodium floribundum leaves

Vegetative material was harvested by hand quarterly on the following dates: (a) December 18-21, 2017 and 2018, (b) March 18-21, 2018, (c) June 18-21, 2018, and (d) September 18-21, 2018. Three composite samples were formed in the different harvest months. Each sample was formed with all the leaves of four trees. Samples of leaves of varied age (VA) included the leaves of specimens not previously defoliated. Samples of 90-d-old leaves (A90) were obtained from the same specimens at 90 d postharvest. The fresh leaves of each sample were added methanol:water (70:30 v/v) and homogenized with a blender (Oster®, Mexico) for < 1 min, until a homogeneous particle size was achieved. Ascorbic acid was added to the mixture, and it was left to macerate for 24 h. Subsequently, the mixture was filtered using gauze and No. 50 large pore filter paper (Tequimec SDRL, Mexico). To obtain the extract, the solvent (methanol) was evaporated at 50 °C using reduced pressure (rotavapor Ika®, Germany). Chlorophyll and lipids were removed from the aqueous fraction using methylene chloride (1:1 v/v, 3-7 washes). Finally, the rest of the fraction was lyophilized, bottled and kept in refrigeration at 4 °C until use.

Determination of polyphenols in extracts

The quantity of total phenols (TP), total tannins (TT) and condensed tannins (CT) of each extract obtained, of each age and harvest month, was quantified. The Folin-Ciocalteu technique was used to determine TPs¹⁹. The TT content was determined using the Folin-Ciocalteu technique + PVPP¹⁹. The CT content of the extracts was determined by the vanillin test²⁰.

Production of Haemonchus contortus eggs and larvae

The eggs and infective larvae (L₃) of H. contortus were obtained from donor animals artificially infected with H. contortus (Paraíso isolate, Yucatán, Mexico). Fresh eggs were collected from the feces of each donor animal. The donors’ feces were collected directly from their rectum, using new plastic bags and the feces were processed within 3 hours after collection. Approximately 10 g of feces were macerated in 100 ml of purified water. The suspension was filtered with gauze. The filtered material was centrifuged (168 xg/5 min/21 °C) using 15 ml conical tubes. The supernatant was discarded, and the sediment was mixed with a saturated solution made from commercial cane sugar (relative density 1.28). Once mixed, the sediment was homogenized by a vortex. The suspension was centrifuged (168 xg/5min/21 °C). The surface layer of the solution was recovered with an inoculation loop. The eggs were washed three times with purified water to remove the remaining sugar and were resuspended in 15 ml tubes containing 10 ml of phosphate saline solution (PBS 0.01 M: NaCl 0.138 M, KCl 0.0027 M, KH₂PO₄ 0.001M, Na₂HPO₄ 0.0081M; pH 7.4; Sigma® USA). Egg concentration was determined and the suspension was diluted to 150 eggs/ml of PBS for use in the egg hatch (EH) test¹².

For the larval exsheathment inhibition (LEI) assay, feces were collected from the donor animals and rinsed in a strainer with running water to remove grass or other debris. The feces were placed in Petri dishes (15 cm in diameter), incubated for 5 d at 28 °C and hydrated daily manually with a water spray. L₃ larvae were harvested using Baermann’s technique and stored at 4 °C until use. The age of the larvae used in LEI was between 2 and 5 wk^12,21.

In vitro anthelmintic activity against Haemonchus contortus eggs

Stock solutions (10,000 μg/ml of PBS) were prepared for each extract tested. PBS was used as a negative control. Respective 0.5 ml aliquots of the different dilutions (3,600, 2,400, 1,200, 600, 300 and 150 μg/mL of PBS) were prepared from the stock solution of each plant extract in 24-wells plates. Point five milliliters of the egg suspension (150 eggs/mL) were added to each well until a final volume of 1 mL was achieved. Six replicates were used for each extract concentration. The multi-well plates were incubated at 28 °C (48 h). At the end of this process, two drops of Lugol were added to each well to stop hatching, in addition to staining the eggs and larvae^22,23.

The non-larvated eggs, larvated eggs and L₁ larvae of each well were counted, and the percentage of hatching was calculated with the formula:

To determine the role of polyphenols in the AH effect of extracts, a tannin inhibitor, polyvinylpolypyrrolidone (PVPP), was used^11,19. These bioassays included only the concentration of 3,600 μg of extract / ml of PBS (with and without PVPP) and their respective PBS controls²⁴.

Haemonchus contortus larval exsheathment inhibition (LEI) test

One thousand microliters of L₃ suspension (∼1,000 /ml) were added to each tube to obtain the final extract concentrations (1,200, 600, 400, 200, 100, 30 μg/ml) from the respective stock solutions of G. floribundum. A tube containing 1,000 μl of PBS without extract was used as a negative control. The larvae were incubated for 3 h (24 °C). Aliquots of each larval suspension were placed in microvials (200 μl in each.) with four repetitions for each concentration and PBS control. The exsheathment of L₃ was artificially induced with a solution of hypochlorite (2.2 %) and sodium hydroxide (0.7 %) (Clorox®) diluted to 1/300, 1/343, 1/400 and 1/480. The kinetics of the exsheathment was estimated by counting sheathed and unsheathed larvae with a microscope (10x), and the exsheathment was recorded at 0, 20, 40 and 60 min²³. The percentage of L₃ larval exsheathment for each measurement point was calculated using the following formula:

To determine the role of polyphenols in the AH effect of extracts, a tannin inhibitor, PVPP^12,19, was used. For each extract, only the dose of 1200 μg/ml of PBS (with and without PVPP) and their respective PBS controls were included.

Protein precipitation using the radial diffusion technique

The PP was determined as an indicator of the biological activity of polyphenols. It was performed with the radial diffusion assay²⁵. The technique identifies the ability of polyphenols to bind to protein molecules (e.g., bovine hemoglobin) on a plate with agar. One percent agarose gel (Baker®, Germany) was prepared in acetate and bovine hemoglobin buffer (Sigma®, Germany) (100 mg/L agar). The pH was adjusted to 5.0 with NaOH. Ten milliliters of agar were placed in Petri dishes 10 cm in diameter. Five wells of 4 mm in diameter each were formed in the agar of each Petri dish (one in the center and the remaining four in the positions of 0, 90, 180 and 270 degrees). In the latter, 15 μl of a solution of each extract were added and incubated for 48 h at 25 ºC. At the end of that time, the halo that was formed around each well was measured. This halo is the result of the precipitation of hemoglobin by the action of the polyphenols of each extract. PP was weighted by the concentration of TT, TP and CT contained in each extract evaluated. For this, the formula described by Hagerman²⁵ was used:

PP= ((D2²-D1²) /T); where: D1: smaller diameter of the well (mm); D2: larger diameter (mm); T: Total phenols or total tannins or condensed tannins (mg).

Data processing and statistical analyses

The effect of leaf age (VA or A90) and harvest month, as well as their interaction on polyphenol composition (TP, TT, CT) were determined using respective generalized linear models (GLM). Subsequently, the comparison of means was performed using Tukey’s test with α<0.05²⁶.

For the EH test, the number of eggs that remained in the morula stage (MOE), eggs that developed a larva but did not hatch (LNH), and the number of larvae that emerged from the eggs as a result of their exposure to different extracts at the respective concentration previously described were recorded. This information was used to determine egg hatching rate (%EH) and egg hatching inhibition (%EHI) as follows ^24,27:

The percentage of morulated eggs that did not form larvae (ovicidal effect) was calculated as follows:

The percentage of eggs with larva that did not hatch (%LNH) was calculated as follows:

The percentage of exsheathment (%E) and that of exsheathment inhibition (%LEI) were determined with the following formulae²⁸:

EH inhibition and LEI results obtained for the different extracts were analyzed with the respective generalized linear models (GLM) to evaluate the differences between the PBS control and the different extract concentrations analyzed. Data obtained from PVPP incubations of each extract were analyzed using a completely randomized design (GLM with comparisons made with the respective control group for each extract)²⁶.

The effective concentration required to inhibit 50 % of egg hatching, or 50 % of L₃ exsheathment (effective concentration 50 %; EC₅₀) was estimated with data obtained from EH and LEI tests, respectively, for each plant extract tested using PoloPlus 1.0 software²⁹.

The Shapiro-Wilk test was performed to assess the normality of the PP, EH and LEI data. The respective biological activity (PP, EH and LEI) was analyzed by means of a GLM and the main effects of leaf age (VA and A90) and harvest month (four harvest months), as well as their interaction. The comparison of means was performed using Tukey’s test with α<0.05. Additionally, respective Pearson correlations were performed to determine the association between the content of polyphenols (TP, TT and CT) and PP, as well as the EC₅₀ of EH and LEI, respectively²⁶.

Egg hatching (EH) test

The extract from VA leaves harvested in December was the only one that showed activity on the EH of H. contortus (EC₅₀ = 374.4 μg/ml). In Table 2, it can be seen that A90 leaf extracts of G. floribundum of December, June and September showed low activity on EH (EC₅₀ > 1,500 μg/ml), while for the extract of March, it was not possible to calculate the EC₅₀.

Table 3 shows the effect of blocking polyphenols with PVPP on the proportion of MOE, LNH and L₁ of eggs incubated with the different G. floribundum extracts. The different extracts showed an activity more oriented to retain the L₁ larvae inside the eggs (LNH). However, by blocking polyphenols, increased ovicidal activity was observed for VA leaf extracts (December and March). Correlation analyses showed no association between the content of TP, TT or CT, and the EC₅₀ of egg hatching inhibition (P>0.05).

Larval exsheathment inhibition (LEI) test

The EC₅₀ obtained for the different extracts of G. floribundum from VA and A90 leaves with the LEI test is presented in Table 4. A significant effect of the interaction between leaf age and harvest month was observed. In the case of VA leaf extracts, all harvest months showed different activity, with the extract of June being the most active and that of March being the least active (P<0.05). On the other hand, the extracts from A90 leaves were also different for each month (P<0.05), with that of September being the most active and that of June being the least active.

Table 5 presents the effect of extracts of G. floribundum from leaves of different age and harvest month on the LEI percentages of H. contortus L₃, with or without the addition of PVPP to block polyphenols. The use of PVPP showed that inhibition of exsheathment is partially due to polyphenols and makes it evident that other SCs participate in LEI. In addition, correlation analyses showed no association between TP, TT or CT contents, and the EC₅₀ of LEI.

Radial diffusion test to measure protein precipitation (PP)

The PP obtained with G. floribundum leaf extracts showed no difference due to leaf age or harvest month (Table 6). Correlation analysis showed that a higher content of TP, TT or CT in the extracts did not influence PP.

Anthelmintic activity of methanol:water extracts