The present investigation demonstrated that the compounds of
P. incanum present trypanocidal activity. In addition, it was shown that the bioactive compounds, even from different extractions, maintained their biological activity. The plant material under study was sought according to the physical factors of its distribution. It refers to the geographical location of
P. incanum with shrubs, mesquites, and sandy soil, with a fine texture and eroded by the action of the wind, in rocky places along roads; these characteristics correspond to the collection point. The presence of
P. incanum has been recorded in different states of Mexico, such as Aguascalientes (municipality of Asientos, Tepezalá and the community of Juan El Grande), Chihuahua, Coahuila, Durango, Hidalgo, Guanajuato, State of Mexico, Nuevo León, Michoacán, San Luis Potosí, Querétaro, Sonora, Tamaulipas, and Zacatecas, finding
P. incanum around 2 km from the municipality of Asientos [
6,
26]. Regarding the methanolic extractions, these were selected for the search of metabolites with antimicrobial activity because it has been reported that methanol is commonly used for the extraction of polar bio-active components since it can dissolve and extract most of the active compounds of interest in search of phytochemicals in medicinal plants [
27]. The difference between the extraction yield may be due to the successive extraction using the Soxhlet apparatus since nonpolar compounds were previously eliminated from methanolic extract with hexane and chloroform [
28]; this did not occur in the extraction by maceration since only a single solvent was used. Notably, the yield of the total Soxhlet extraction (hexane + chloroform + methanol) was 12.2% (data not shown). Qualitative phytochemical analyses of both methanolic extracts from
P. incanum showed similar results to two previously reported methanolic extracts from
Parthenium hysterophorus leaves [
28,
30]. Fractionation by column chromatography and their subsequent comparison of the fractions obtained by TLC allows us to obtain some specific regions and the compound of the plant under study [
16]. Regarding this, column chromatography techniques have been widely used as purification techniques for successful fractionation and isolation of desired bioactive compounds from complex extract material; this is due to their simplicity, convenience, specificity, economy, and availability to be used in multiple mobile phases with different polarity. They are used to isolate and purify the active compounds that are responsible for the bioactivity, such as antimicrobial, antioxidant or cytotoxicity [
31,
32]. Following the previously mentioned processes and according to the results of the active fractions identified by disc-diffusion and bioautographic methods, we decided to carry out the detection of parthenin by eluting the compounds using TLC with a mixture of chloroform:acetone (3:1). When sprayed with the vanillin reagent with sulfuric acid, they showed a violet-blue band with an Rf value of around 0.6 after heating at 70 °C for 5 min, which is something characteristic of parthenin [
21]. Subsequently, parthenin was recovered from the active fractions by PTLC with the help of a vanillin reagent. This was carried out because this methodology is used to isolate some compounds of interest, as in the case of isolated sterols of
Lipidium meyenii Walp [
22], obtained by PTLC. The high antimicrobial potential of parthenin has been previously demonstrated [
33]; because of this, the antimicrobial activity was used as a biological indicator for the isolation of parthenin. The data obtained by the FT-IR analysis for the isolated compounds showed the presence of the main absorbance peaks (cm
-1) of parthenin, the hydroxyl group (OH), carbonyl group (C=O), and the C-O that belongs to the lactone ring; these data agree with those obtained for different samples (seeds and leaves) of
Parthenium hysterophorus [
34,
35,
36]. Fourier Transform Infrared Spectroscopy offers a fast, valuable tool to identify and characterize the chemical constituents or functional groups present in the sample to elucidate the structural compounds [
10,
33], as was the case in identifying the isolated compounds. Our results of UHPLC-MS analyses showed the presence of major pseudo molecular ions in both samples,
m/z 263 and 265, corresponding to parthenin and coronopolin, respectively; the molecular mass of the compounds of interest was used (262.31
g/mol for parthenin and 264.32
g/mol for coronopolin), along with an analysis in positive mode using electrospray ionization [M+H]
+ for identification [
21,
37,
38]. The product ions from both samples of main pseudo molecular ions with
m/z 263 show the fragments 245 [M+H-18]
+, 227 [M+H-36]
+, 209 [M+H-54]
+, 199 [M+H-64]
+, 181 [M+H-82]
+ and 149 m/z [M+H-114]
+, which have been reported for the fragmentation of the parthenin by electrospray ionization in positive mode [
38]. Meanwhile, main pseudo molecular ions with
m/z 265 from both samples were obtained; these were the fragments 247 [M+H-18]
+, 229 [M+H-36]
+, 219 [M+H-46]
+, 205 [M+H-60]
+, 201 [M+H-64]
+, 187 [M+H-78]
+, 173 [M+H-92]
+, 159 [M+H-106]
+ and 139 [M+H-126]
+. These types had not been previously reported. Loss of H
2O [M+H-18]
+ and loss of H
2O with CO (carbonyl) [M+H-36]
+ were observed for all analyses [
39], giving characteristic ions for the compounds. The data obtained from the trypanocidal activity suggest that the isolated compounds need a lower concentration to inhibit
T. cruzi than the crude extracts. However, our results are higher than those obtained by Muñoz et al. (2012), which determined, in vitro, the effect of the antiparasitic drugs Nifurtimox and Benznidazole; those results showed an IC 50 of 2.34 ppm ± 0.72 ppm and 13.12 ppm ± 2.45 ppm, respectively [
40]. Furthermore, the data obtained by Acosta et al. (2020), in which the same antiparasitic agents were evaluated against several clones of
T. cruzi, obtained a maximum IC
50 of 15.22 ± 3.10 ppm [
41]. The drugs mentioned above are approved and recognized as trypanocidal by PAHO/WHO. Nifurtimox is produced only in El Salvador, and benznidazole is produced only in Brazil and Argentina; unfortunately, both have a series of adverse effects [
41]. These differences between data may be due to the type of strain used and the concentrations of parasites used.