Aromaticity (H2SO4- CH2O Test)

A mixture of 1 mL of concentrated H2SO<sup>4</sup> was prepared with a drop of CH2O (formaldehyde). An amount of 5 mg of the dissolved extract was dissolved in 1 mL of non-aromatic solvent (ethanol), and three drops of the above mixture were added, and when a red-violet color appeared, the test was considered positive.

### *4.4. Cell Lines*

### 4.4.1. Vero Cell Line

All cell lines were purchased from the American Type Culture Collection (ATCC) (Manassas VA, USA). Non-tumor Vero cells (ATCC: CCL-81) werederived from the kidney of an adult green monkey (*Cercopithecus aethiops*); they are adherent cells and with epithelial morphology. The cells were incubated in Advanced DMEM medium, 1× supplemented with 4% *v*/*v* of inactivated fetal bovine serum (FBS), 1% *v*/*v* of penicillin/streptomycin, and 1% of L-glutamine.

### 4.4.2. HeLa Cell Line

Cells derived from adenocarcinoma of the human cervix (ATCC: CCL-2) from a 31-year-old Black female patient. They are adherent cells, with epithelial morphology and are positive for cytokeratin. They have been reported to contain human papillomavirus 18 (HPV-18) sequences [49]; additionally, these cells express low levels of p53 and normal levels of pRB [50]. The cells were incubated in 1X Advanced DMEM medium supplemented with 4% *v*/*v* of inactivated FBS, 1% penicillin/streptomycin, and 1% L-glutamine.

### 4.4.3. MDA-MB-231 Cell Line

These cells were derived from human mammary adenocarcinoma (ATCC: CRM-HTB-26) from a 51-year-old Caucasian female patient. They are adherent cells of epithelial morphology and express the WNT7B oncogene. They express epidermal growth factor (EGF) and transforming growth factor-alpha (TGF α) [51]. Cells were incubated in 1× DMEM medium supplemented with 10% *v*/*v* of inactivated FBS, pyruvate 1×, 1% *v*/*v* penicillin/streptomycin, and 1% L-glutamine.

All cell lines were incubated at 37 ◦C in a 5% CO<sup>2</sup> atmosphere.

### *4.5. Preparation of Working Solutions*

From the crude extracts, stock solutions of 10 mg/mL were prepared to dissolve this concentration of each extract 50 µL of 100% dimethyl-sulfoxide (DMSO) and 950 µL of culture medium to obtain a DMSO concentration of 5%. Subsequently, solutions of 1 mg/mL were prepared to make a 1:10 dilution of the stock solution, with a DMSO concentration of 0.5%, this is the minimum non-toxic concentration [52]. Finally, from these solutions, the dilutions were prepared at concentrations of 0, 10, 50, 100, 300, and 500 µg/mL for the cell treatments.

### *4.6. Cytotoxicity Test with MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide)*

Mitochondrial function can be evaluated based on the activity of the reductases found in the organelle and are directly related to cell viability, and the MTT assay can be used to measure such activity. This compound is a yellow tetrazolium salt, which is reduced to formazan by the action of mitochondrial enzymes, especially succinate dehydrogenase, resulting in a purple-blue product that can be evaluated by spectrophotometry [53]. For this assay, 5 <sup>×</sup> <sup>10</sup><sup>4</sup> cells per well (*n* = 7) were incubated for 24 h in a 96-well plate to allow adhesion. Subsequently, the treatments of each extract were applied at concentrations of 10, 50, 100, 300, and 500 µg/mL diluted in 100 µL of culture medium. Cell culture medium was used as a negative control (vehicle). The results were analyzed at 12, 24, 40, and 72 h.

Two hours before the end of the corresponding incubation times, representative photomicrographs of each concentration were taken at 40× with a Southern Precision Instrument inverted microscope to observe the effect of the extracts on the cell morphology. Subsequently, 15 µL of MTT (3 mg/mL) were added to each well, and the cells were incubated for 2 h at 37 ◦C; then, the medium was removed, and the MTT developer reagent (4 mM HCl, 0.040% NP40 in isopropanol) was added. The plate was shaken at 125 RPM for 10 min, and an absorbance analysis was performed at 590 nm with a 620 nm reference filter in an iMarkTM microplate reader. The absorbance value is directly proportional to the number of metabolically active cells, which is an indirect measure of cell viability.

### *4.7. DAPI Nucleus Labeling Assay*

To relate the results obtained in the MTT assay, the use of the fluorescent dye DAPI (4',6-diamidino-2-phenylindole) that binds to the nucleic acids of adhered cells, allowed us to estimate the number of cells viable related to cell adhesion. For this, 96-well plate cultures were performed under the same conditions as MTT assay treatments (*n* = 8). At the end of the incubation times, the medium was removed, two washes of 200 µL were performed with 1 × PBS, and subsequently, the cells were fixed with methanol/acetone at a ratio of 1: 1 for 20 min at 4 ◦C. After the fixer was removed, two washes were performed again with 1X PBS, and 100 µL of DAPI (100 ng/mL) were added for 15 min at RT in the darkness. At the end of this period, the excess DAPI was removed, and the plate was dried and observed under fluorescence microscopy. Eight micrographs were taken per concentration at a total magnification of 100×, which were evaluated with ImageJ® software version 1.51, (NIH) which calculated the percentage of the area covered by the adhered cells in each of the wells

### *4.8. Morphological Analysis*

To evaluate the presence of morphological changes in the cells due to exposure to extracts of *A. monostachya*, 1 <sup>×</sup> <sup>10</sup><sup>6</sup> cells were seeded in 60 mm dishes and were incubated for 24 h to allow adherence. Subsequently, the treatments of 0, 10, and 50 µg/mL of EM and EH and 0, 300, and 500 µg/mL of EA were applied and diluted in culture medium at a total of 3 mL per dish and were incubated for 24 and 48 h. At the end of the incubation time, the cells were harvested using 0.25% trypsin, two washes were performed with 1× PBS, and later, they were fixed with 2.5% glutaraldehyde buffered in 0.1 M cacodylate buffer (pH 7.4) for 24 h. Next, the cells were washed two times with cacodylate buffer for 5 min with centrifugation at 14,000 RMP; they were decanted, and a post-fixation was performed with OsO<sup>4</sup> at 2% for 12 h; they were washed again and dehydrated with a gradient of ketones (30%, 50%, 70%, 90%, and 100%) for 5 min and embedded in middle epoxy resin for 72 h at 60 ◦C. From the epoxy blocks, 350 nm thick semi-fine sections were obtained by ultramicrotomy and subsequent staining with 1% toluidine blue for 1 min. One hundred cells were counted per section when analyzing the presence of changes in the nuclear morphology, chromatin condensation, and vacuolization in the cytoplasm to obtain the percentage of positive cells in each treatment.

### *4.9. Statistical Analysis*

For the descriptive statistics, the quantitative variables were summarized by calculating the mean, and as a measure of dispersion, the standard deviation (SD). In the inferential statistics, the Kolmogorov–Smirnov normality test was applied to evaluate the distribution of the data, with all variables having a normal distribution. Subsequently, an ANOVA and a Tukey test were performed to evaluate the presence of the differences between the groups. All graphs, calculations, and statistical analyses were made using GraphPad Prism version 8.0 software for Windows (GraphPad Software, Inc., San Diego, CA, USA).

### **5. Conclusions**

This research provides information on the effect of crude extracts of different polarities obtained from *A. monostachya* on human tumor lines, demonstrating that it possesses timeand concentration-dependent cytotoxic activity. This was evidenced by evaluating the relative viability and the presence of morphological alterations when the extracts were exposed. Likewise, this cytotoxicity is selective for tumor cell lines, highlighting that the HeLa cells presented more cytoplasmic and nuclear morphological changes as well as a lower percentage of relative viability when exposed to extracts compared to MDA-MB-231 cells, which responded in the same way to higher concentrations. This activity can be attributed to the phenolic compounds detected by the phytochemical screening being carried out.

The perspectives of this work are oriented to perform a fractionation guided by bioassay to obtain a fraction with the bioactive compound and thus isolate and purify it. With the data obtained, the IC50 of the crude extracts and subsequently their different fractions can be calculated. Likewise, it is convenient to evaluate the effect of the extracts in other tumor cell lines to compare the induced changes. Finally, it is possible to further evaluate the type of cell death to establish a mechanism by which the bioactive compounds present in *A. monostachya* act.

**Author Contributions:** Conceptualization: G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., M.d.J.L.-A. and A.S.-D.; methodology: G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V.; D.S.-T., M.d.J.L.-A. and A.S.-D.; software, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., D.S.-T., M.d.J.L.-A. and A.S.-D.; validation, G.A.G.- M., S.A.V.-C., R.A.P.-H., U.C.-V., D.S.-T., M.d.J.L.-A. and A.S.-D.; formal analysis, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., D.S.-T., O.S.-C., R.M.-d.-O.-L., C.A.G.-T., M.d.J.L.-A. and A.S.-D.; investigation, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., D.S.-T., O.S.-C., R.M.-d.-O.-L., M.d.J.L.-A. and A.S.-D.; resources, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., O.S.-C., R.M.-d.-O.-L., M.d.J.L.-A. and A.S.-D.; data curation, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., C.A.G.-T., M.d.J.L.-A. and A.S.-D.; writing original draft preparation, G.A.G.-M., S.A.V.-C., R.A.P.-H., M.d.J.L.-A., A.S.-D.; writing—review and editing, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., D.S.-T., O.S.-C., R.M.-d.-O.-L., C.A.G.-T., A.J.G.-A., D.E.Z.-Á., M.d.J.L.-A. and A.S.-D.; visualization, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., D.S.-T., C.A.G.-T., M.d.J.L.-A. and A.S.-D.; supervision, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., D.S.-T., O.S.-C., R.M.-d.-O.-L., M.d.J.L.-A. and A.S.-D.; project administration, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., O.S.-C., R.M.-d.-O.-L., M.d.J.L.-A. and A.S.-D.; funding acquisition, G.A.G.-M., S.A.V.-C., R.A.P.-H., U.C.-V., O.S.-C., R.M.-d.-O.-L., A.J.G.-A., D.E.Z.-Á., M.d.J.L.-A. and A.S.-D. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was partially supported by PAICYT 2021. UANL. México, grant SA1864-21, and GAGM is a recipient of the CONACyT (Consejo Nacional de Ciencia y Tecnología) México scholarship 1006252.

**Institutional Review Board Statement:** The research protocol was approved by the Ethics Committee in the Faculty of Medicine, UANL with approval number HT21–00002. We did not perform studies involving humans or animals.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data and materials supporting the conclusions of this article are included within the article.

**Acknowledgments:** We acknowledge the work of Eduardo Estrada-Castillón and Marco A. Guzmán-Lucio for their assistance in plant identification and to R. Smith for reviewing the English manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

### **References**

