Fig 1.
Gibberellic acid germination experiments.
Two A. mexicana seeds were planted per soil pod and watered with equal amounts of either the negative control (no GA), 100 mg/L or 1000 mg/L gibberellic acid solutions, with 10 total pods used per group. Ethanol (the solvent used to prepare the stock GA solution) was used in place of GA in the negative control and 100 mg/L GA water solutions. Seedlings were kept in small greenhouses under a 16/8 light cycle and scored after 30 days. n = 10 pods, with mean germination rate per pod displayed with associated SEM. A two-tailed T-test was used to determine statistically significant differences between means, with significance set at P ≤ 0.05.
Fig 2.
Antimicrobial disc diffusion assay.
1 mg of either methanol or hexane extract for leaf, seed, inner root or outer root was plated against six different microorganisms (S. aureus, B. cereus, E. coli, P. mirabilis, C. albicans or S. cerevisiae). The mean zone of inhibition in millimeters for five biological replicates is shown with associated SEM. Vancomycin, streptomycin and/or fluconazole were used as positive controls, and solvents alone were used as negative controls.
Fig 3.
Antimicrobial effect comparison of immature vs. mature root.
Immature vs. mature roots were harvested from A. mexicana plants either without or with reproductive structures (panel A, photo taken by Sheffield Seed Company, Inc.). Equal amounts of either immature or mature root (pictured in panel B) was then extracted in methanol, and either 1 mg (panel C, top) or 3 mg (panel C, bottom) of the extract was plated against S. aureus, using streptomycin and vancomycin as positive controls and methanol alone as the negative control.
Fig 4.
Viability assay of cancer cells.
T84 human colon cancer cells were treated with 1 mg of either methanol or hexane extract for leaf, seed, inner root or outer root for 1 h. The MTT colorimetric assay was then used to determine cell metabolic activity after treatment with extracts. The mean percentage of viable cells normalized to the control (solvent alone) for at least three biological replicates is shown with associated SEM.
Fig 5.
Viability assay of root methanol extract against cancer cells.
Human colon cancer RKO cells were treated with 0.0625 μg/μL, 0.125 μg/μL, 0.25 μg/μL, 0.50 μg/μL or 1.0 μg/μL dehydrated root methanol extract or no treatment (evaporated methanol alone, labelled as ‘NEG’) and assessed using the MTT assay for cell viability after 72 h. The mean percentage of viable cells for three biological replicates is shown with associated SEM.
Fig 6.
Effect of root methanol extract on c-MYC and APC transcript levels.
Human colon cancer RKO cells were treated with or without 0.0625 ug/uL dehydrated A. mexicana root methanol extract for 24, 48 or 72 h. RNA was then extracted, converted to cDNA and subsequently used in qPCR to quantify the transcript levels of the colon cancer oncogene c-MYC (panel A) or the tumor suppressor gene APC (panel B). The mean transcript level for three biological replicates is shown here with associated SEM and normalized to mRNA levels of the housekeeping gene Actin. The transcript level in the negative control (no treatment) for each condition was set to 1.0. Statistical significance among transcript means was determined through a one-way analysis of variance (ANOVA), with significance set at P ≤ 0.05.
Fig 7.
Root and leaf column chromatography S. aureus plates.
Based on the order of column elution, the root methanol fractions (panel A) were arbitrarily labeled A-F, and the leaf methanol fractions (panel B) were arbitrarily labeled 1–10. After multiple separations by column chromatography, these fractions were tested for antimicrobial activity against S. aureus, with streptomycin and vancomycin used as positive controls and methanol alone as the negative control. (Several representative full plates can be seen in S4 Fig) The fractions with strongest antimicrobial activity (root methanol D and E and leaf methanol 7.3 and 10.5) were then evaluated for purity (S3 Fig) and used for chemical characterization (Table 1).
Table 1.
Summary of mass spectrometry results from active sub-fractions of plant extracts.
Fig 8.
Expected structures of key active fractions of the root and leaf methanol extract.
A) Structure of chelerythrine, consistent with root fraction ‘D’. B) Structure of berberine, consistent with root fraction ‘E’ and leaf fraction ‘10.5’. C) Proposed structure of the impurity observed in root fraction ‘E’. D) Proposed structure of the impurity observed in leaf fraction ‘10’.
Fig 9.
Comparison of known compounds to antimicrobial root and leaf methanol fractions.
Commercially available chelerythrine was compared to root extract fraction ‘D,’ while berberine was compared to and root fraction ‘E’ and leaf fraction ‘10.5’. Antimicrobial effects against S. aureus are shown in Panel A, with streptomycin and vancomycin used as positive controls and methanol alone as a negative control. A representative TLC plate (under 365 nm exposure) is shown in Panel B.