R 205

MA aerial parts were air-dried in the shade then crushed into a powder (3.45 kg). The MA powder was successively extracted as described by (Pérez-Bonilla et al., 2013 (link)). In a Soxhlet apparatus (500 ml), four different solvents were used in ascending order of polarity (n-hexane, dichloromethane, ethyl acetate, and ethanol (95%)). All extracts of various organic solvents were dried by evaporating each solvent at 40°C using a rotary evaporator (R-205-Buchi, Germany), yielding 102.4 g of ethanol 95% extract, 24 g of ethyl acetate extract, 27.9 g of dichloromethane extract, and 45.2 g of hexane extract, respectively. In this study, two doses of 100 and 200 mg/kg were selected and used (Minaiyan et al., 2011 (link); Kaseb et al., 2018 (link); Hajizadeh-Sharafabad et al., 2020 (link)). The two doses were prepared by dissolving an appropriate amount of the dried extracts in 1 ml of Tween 20. Following that, 9 ml of 0.9% NaCl was added to each mixture. To prepare the vehicle, 1 ml of Tween 20 was dissolved in 9 ml of 0.9% NaCl.

MPF (n = 3) and MPL (n = 3) of each plant (I, II and III) were manually separated and processed according to Smeriglio et al. [31 (link)] to obtain three independent hydroalcoholic extracts for each plant material (n = 9 leaf extracts and n = 9 flower extract for each sampling site). Briefly, fresh MPF and MPL samples were powdered by a blade analytical mill (A11, IKA^®-Werke GmbH & Co. KG, Staufen, Germany) with liquid nitrogen to block the enzymatic activity and preserve the native phytochemical features. One hundred milliliters of a hydroalcoholic mixture (ethanol:water, 80:20 v/v) were added to each powdered flower and leaf sample (10 g), vortex-mixed for 3 min and sonicated in an ice-cold bath for 5 min using a 3 mm titanium probe set to 200 W and 30% amplitude (Vibra Cell™ Sonics Materials, inc., Danbury, Connecticut, USA). Extracts were centrifugated at 3000× g 15 min at 4 °C, and the supernatants, filtered on Whatman filter paper no. 1, were recovered in a round-bottom flask. Extraction was repeated two more times, and the supernatants were pooled and dried by rotary evaporator (Büchi R-205, Cornaredo, Italy). Dry extracts (DEs) were stored in a vacuum glass desiccator for 48 h with anhydrous sodium sulfate. After extraction and yield calculation (MPFE 18.11–22.23% and MPLE 15.47–20.31%), DEs were stored at −20 °C until subsequent analyses.

The aerial parts of E. alba were separated from wild plants, washed thoroughly 5 to 6 times with running tap water and shade dried at room temperature for 15 days, then pulverized to a fine powder. Thirty grams of the powdered sample was placed in soxhlet extractor and sequentially extracted with 250 mL each of hexane, ethyl acetate, methanol, and water (50–85 °C). Extracts were concentrated under low vacuum by using rotary evaporator (Buchi Rotavapor, R-205). The dried extracts were collected and stored as aliquots at 4 °C for further analysis.

M. guilliermondii culture supernatant (100 ml) was extracted three times with ethyl acetate (High Purity, Mexico). Briefly, the culture was mixed with one volume of ethyl acetate and strongly shaken by hand in an extraction funnel to homogenize the mixture. The funnel was then placed in a universal mount until two phases were observed (approximately 3 to 5 min). The upper phase was recovered and further extracted twice.
The final extraction volume (approximately 300 ml) was evaporated in a rotary evaporator (R-205, Buchi, Switzerland) at 80 rpm and 41 °C. The extract was then recovered in amber vials, dried for 4 days in an extractor hood, weighed and dissolved in approximately 20 to 30 μl of dimethyl sulfoxide (DMSO, J.T. Baker, USA). Two milligrams of the supernatant extract was used in anticoccidial activity bioassays. Non-inoculated YPD was also extracted with ethyl acetate and concentrated by evaporation in the same conditions. 2 mg of the product of this extraction resuspended in DMSO were used as negative control in bioassay reactions. In order to assess the nature of the compound extracted with ethyl acetate we performed incubation with trypsin (aqui no se las condiciones de tiempo y cantidad de enzima en que lo hizo Mayra).

The three herbs, Z. officinale, C. asiatica, and B. nivea, were purchased from DAMAONYAKCHO (Yeongcheon-si, Gyeongsangbuk-do, Republic of Korea). To prepare the extracts from 100 g of pulverized powder, each powder was suspended in 2 L of 70% aqueous ethanol and ultrasonicated for 1 h in a 750-W ultrasonic processor (VCX 750, Sonics and Materials, Inc., Newtown, CT, USA). Extraction in the ultrasonic processor was repeated three times. The undissolved residue was filtered out using quantitative Whatman No. 1 filter paper (Whatman, Maidstone, UK) and centrifugation. The extracts were evaporated on a rotary vacuum evaporator (R205, Buchi, Fostfach Switzerland) and lyophilized to yield a dry powder.

For each sample, 1 g of peel and pulp powder were separately mixed with 10 ml of methanol:water (80:20, v/v). The mixture was sonicated using an ultra-sonicator UP 400St Hielscher’s (400 W, 24 kHz) and then macerated for 60 min at 4^°C. Afterward, it was centrifuged for 10 min, 8,000 g at 4^°C (Eppendorf Centrifuge 5804, Eppendorf, Hamburg, Germany) and the supernatant was collected and the sediment was mixed with 10 ml of acetone:water (70:30, v/v). The same steps (sonication, maceration, and centrifugation) were repeated three times, and the supernatants were mixed together and then evaporated using a rotary evaporator (Büchi R-205, Switzerland) under a speed of 1,500 rpm and reduced pressure, at 40^°C. Then, 5 ml of methanol was added to the residue, and the mixture was well shaken in a Vortex for 2 min. The samples were filtered through a Sep-Pak (c-18) to remove the sugar content and then were stored at −20^°C until further use.