Obtaining the extract

Samples of plant material from the aerial part of Croton draco were collected during the summer (June-August) in the municipality of Huatusco (19 ° 08′56 ″ N 96 ° 57′58 ″ W) belonging to Veracruz state; for the identification of the plant, the herbarium of the National Autonomous University of Mexico was consulted, and the plant specimen was identified as Croton draco subsp. draco (IBUNAM: MEXU: 501697).

The plant material was dried at room temperature in the absence of light, 250 g of the dried material (crushed) was macerated in a liter of hydroalcoholic solution (70:30, water: methanol), for 48 hours at room temperature in the absence of light. The liquid extract from the maceration was filtered through filter paper and cotton; subsequently the liquid obtained was concentrated under reduced pressure in a rotary evaporator (BÜCHI™ R-210, Flawil, Germany), according to the methodology described by (^{Rivero-Pérez et al., 2016}). The resulting extract was kept refrigerated until further evaluation.

Chemical characterization of the extract

To the hydroalcoholic extract of Croton draco, the qualitative chemical profile was performed according to the procedure described by Bañuelos-Valenzuela et al., (2018), in 10 ml Pyrex test tubes. The tests were as follows: KMnO4 test for unsaturations, FeCl3 test for phenolic oxydryls (vegetable tannins), Liebermann-Burchard test for sterols and triterpenes, Salkowski test for sterols and triterpenes, coumarins test, Baljet test for sesquiterpenlactones, H₂SO₄ test for flavonoids, Shinoda test for flavonoids, Dragendorff test for alkaloids, tannin test, floratanino test, steroid test, agitation test, sodium bicarbonate test and Salkowski test for saponins.

The chemical composition of the hydroalcoholic extract of Croton draco was determined according to the methodology described by ^{Bañuelos-Valenzuela et al., (2018)}, using a gas chromatograph (CG; Agilent Tecnologies series 6890N manufactured in the USA), with a polar column DB_WAXetr, at 250 °C and 12.13 psi, with a flow of He 36.5 ml min^-1 after injection. The conditions for the column were: initial temperature 50 ° C from zero to two min, increasing from 10 in 10 °C until reaching 250 °C, keeping the temperature constant for 5 min and then descending to 50 °C for two min with a flow of He of 1.6 ml min^-1 at a pressure of 12.13 psi and an average speed of 25 cm s^-1, using an ionizing flame detector (FID), at a temperature of 210 °C, with a flow of H2 of 40 ml min^-1 and an air flow of 450 ml min^-1. The standards (Sigma-Aldrich) were used in different concentrations (Table 1).

Antimicrobial activity

The antibacterial activity was determined by the Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) for the extract, following the CLSI specifications. (2012). Antimicrobial activity tests were carried out with the ATCC strains, Staphylococcus aureus ATCC ⁶⁵³⁸, Escherichia coli ATCC ³⁵²¹⁸, Pseudomona aeruginosa ATCC ⁹⁰²⁷, Salmonella typhi ATCC ¹⁴⁰²⁸, Salmonella cholerasuis ATCC ¹⁰⁷⁰⁸, Listeria monocytogenes ATCC ¹⁹¹¹³ and Bacillus subill ATCC ⁶⁶³³ which were reactivated in Muller Hinton Agar (BD Bioxon), the purity of the strains was corroborated by Gram staining.

Once the morphology of the bacteria was confirmed, a colony was inoculated in nutrient broth (BD Bioxon), which was incubated under constant agitation (70 rpm) for 24 hours at 37 °C. After the incubation time, the inoculum was adjusted with nutrient broth to 0.5 of the McFarland turbidity pattern (Remel, R20421), which corresponds to 150 x 10⁶ cell/ml.

For the determination of the Minimum Inhibitory Concentration (MIC), the plate microdilution method was used; the concentrations evaluated were: 400, 200, 100, 50, 25, 12.50, 6.25, 3.12 mg/ml, for the strains to be evaluated. Each concentration was prepared with nutrient broth (BD Bioxon). The treatment was carried out in triplicate in a 96-well plate, 100 μl of each of the dilutions plus 10 μl of the bacterial suspension previously adjusted to 0.5 McFarland were placed. After inoculation, the plate was incubated at 37 °C for 24 hours at 70 rpm under constant stirring. The positive control was Kanamycin (AppliChem 4K10421) at concentrations of 64, 32, 16, 8.0, 4.0, 2.0, 1.0 and 0.5 µg/ml. Nutrient broth was used as a negative control.

To determine the end point of the MIC, a colorimetric method was used based on the use of tetrazolium salts, as described by ^{Balouiri et al., (2016)}. After the incubation time elapsed, 20 µl of a 0.04% (w / v) solution of p-iodonitrotetrazolium (Sigma-Aldrich I8377) was added to each well; it was incubated for 30 minutes at 37 ° C and the reading was performed, determining as the minimum inhibitory concentration; that to which the solution turns pink (^{Kaewpiboon et al., 2012}; ^{Mothana et al., 2009}).

To determine the Minimum Bactericidal Concentration (MBC), after adding p-iodonitrotetrazolium, 5 µl of each well was inoculated into Mueller Hinton agar, then incubated at 37 °C for 24 hours. After the incubation time, the growth of the bacteria was verified to determine the minimum bactericidal concentration of the extract, fraction or metabolite; considering as MBC, the concentration at which no bacterial growth was observed in the plaque.

Statistical analysis

The data obtained were normalized and analyzed by means of an analysis of variance and a comparison of means by Tukey, at a confidence level of 95 %; with the statistical package Minitab 18.

RESULTS

Chemical characterization of the hydroalcoholic extract of Croton draco

Qualitative characterization tests carried out on the hydroalcoholic extract of Croton draco, indicate the presence of phenolic compounds (coumarins, flavonoids and flavones), terpenes (sesquiterpenes), saponins and alkaloids.

The analysis in the gas chromatograph of the hydroalcoholic extract of Croton draco determined the presence of thymol and carvacrol in concentrations of 0.5340 mg/ml and 0.4206 mg/ml respectively; without determining the presence of linalool, terpinen and limonene.

Antibacterial activity

In the Minimum Inhibitory Concentration (MIC) of the Croton draco hydroalcoholic extract, significant statistical differences (P≤0.05) were determined between the bacteria evaluated, the MIC for E. coli, S. typhi, S. cholerasuis, P. aeruginosa was 100 mg/ml; without finding statistical differences, for L. monocytogenes of 50 mg/ml with significant differences with respect to the rest of the bacteria evaluated and of 25 mg/ml for S. aureus, B. subtillis; showing statistical differences with the aforementioned bacteria. On the other hand, the MIC for the bacteria evaluated showed significant statistical differences with respect to the MIC of the control with Kanamycin, as shown in Table 2.

In the Minimum Bactericidal Concentration (MBC) of the Croton draco hydroalcoholic extract, significant statistical differences (p≥0.05) were determined between the bacteria evaluated, the MBC for E. coli, S. typhi, S. cholerasuis, P. aeruginosa was 200 mg/ml; without finding statistical differences between them, for L. monocytogenes of 100 mg/ml with significant differences with respect to the rest of the bacteria evaluated, and of 50 mg/ml for S. aureus, B. subtillis; showing statistical differences with the aforementioned bacteria. On the other hand, the MBC for the bacteria evaluated showed significant statistical differences (P≤0.05) with respect to the MBC of the positive control (Kanamycin), as shown in Table 2.