Osage Orange (Maclura pomifera) and Spearmint (Mentha spicata) Leaf Extracts Exhibit Antibacterial Activity and Inhibit Human Respiratory Syncytial Virus (hRSV)

Abstract

The increasing prevalence of antibiotic resistance and the limited availability of antiviral therapeutics for pathogens such as human respiratory syncytial virus (hRSV) underscore the need for novel, plant-derived antimicrobial substances. In this study, we evaluated the antiproliferative, antibacterial, and antiviral activities of aqueous leaf extracts from two plants commonly found in North America, Osage orange (M. pomifera) and spearmint (M. spicata). Both extracts exhibited no significant cytotoxic or morphologic impact on HEp-2 human cancer cells up to 25 mg/mL. However, both extracts demonstrated strong dose-dependent antibacterial activity, significantly inhibiting replication of E. coli and S. aureus at concentrations ≥ 1 mg/mL. Antiviral assays revealed that both extracts inhibited hRSV infectivity, with spearmint extract showing higher potency (EC₅₀ = 1.01 mg/mL) compared to Osage orange (EC₅₀ = 3.85 mg/mL). Gas chromatography-mass spectrometry (GC-MS) identified three major extract constituents: 3-hydroxybenzyl alcohol, 4-hydroxybenzyl alcohol (Osage orange), and R-(-)-carvone (spearmint). Among these, only carvone significantly inhibited hRSV in vitro, suggesting its key role in spearmint's antiviral activity. These findings highlight the therapeutic potential of Osage orange and spearmint leaf extracts, particularly as sources of water-soluble compounds with antimicrobial properties, and support further investigation into their mechanisms of action and broader clinical relevance.

Keywords: antibacterial; antiviral; cancer; phytochemistry; respiratory syncytial virus.

Figure 1

Cell viability and morphology of HEp-2 cancer cells following treatment with leaf extracts from Osage orange (M. pomifera) and spearmint (M. spicata). (A) MTS assay was performed to assess HEp-2 cell viability after 24 h of treatment at 37 °C with extracts or a water control at concentrations up to 25 mg/mL. The percent viability (±SEM) was calculated by comparing activity at each concentration to the 0 mg/mL treatment. (B) Representative images of HEp-2 cells after 24 h of exposure to 10 mg/mL of extracts of Osage orange or spearmint, water, or a colchicine control. The cells were fixed and stained with DAPI (DNA; cyan), TRITC-Phalloidin (actin; magenta), and FITC-conjugated anti-tubulin (tubulin; yellow). Scale bar = 100 μm. (C) Quantification of the nuclear morphology (left, middle) or cytoskeletal disruption (right) from six images from each treatment. The average percent apoptotic and mitotic nuclei (±SEM) and average cytoskeleton score (±SD) based on blinded scoring from 1 (normal) to 4 (complete cytoskeletal destruction). An ANCOVA was conducted to evaluate significance for the cell viability assay and a one-way ANOVA was used to evaluate the IF scoring. Significant differences relative to water controls are indicated (***, p < 0.001).

Figure 2

Antibacterial activity of Osage orange (M. pomifera) and spearmint (M. spicata) extracts. (A) Cultures of tryptic soy broth (TSB) were co-inoculated with E. coli (left) or S. aureus (right) in the presence of doses of Osage orange or spearmint extracts ranging from 0 to 5 mg/mL. Bacterial growth was assessed following growth at 37 °C through measurement of the optical density at 600 nm at 0, 1, 3, and 5 h post-inoculation. The average fold increase (±SEM) in OD₆₀₀ of three experimental replicates relative to the 0 h absorbance is shown. An ANCOVA was used to evaluate significance relative to the untreated 0 mg/mL control. Significant differences are indicated (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (B) Disk diffusion tests were performed using 6 mm disks infused with varying concentrations of Osage Orange or spearmint overlaid on cultures of E. coli or S. aureus on Mueller–Hinton agar (MHA) plates. The average zone of inhibition (±SD; N = 3) was measured after growth at 37 °C for 24 h.

Figure 3

Inhibition of hRSV in Osage orange (M. pomifera) and spearmint (M. spicata) extracts. (A) HEp-2 cells were infected for 24 h with a recombinant fluorescent reporter strain (A2-mKate2) of respiratory syncytial virus (hRSV) in the presence of doses ranging from 0 to 2.5 mg/mL of Osage orange, spearmint, water, or a sage extract control. Red fluorescent foci were visualized (left) and quantified to determine the average percent (±SEM, N = 3) of detected virus remaining relative to the untreated control. (B) Replication analysis performed in HEp-2 cells infected at a multiplicity of infection (MOI) of 0.05 FFU/mL over 3 d in the presence of 0, 0.1, or 1 mg/mL of Osage orange, spearmint, or a sage control. The average titers (±SEM; N = 3) of the supernatant are shown as fluorescent focus units (FFU) per mL. Images were taken after 2 d of infection are shown at left. Scale bar = 200 μm. An ANCOVA was used to evaluate overall significance relative to the untreated control. Significant differences are indicated (**, p < 0.01; ***, p < 0.001).

Figure 4

Cell viability and nuclear and cytoskeletal morphology after treatment with chemical constituents identified in Osage orange (M. pomifera) and spearmint (M. spicata) leaf extracts. (A) Chemicals identified by GC-MS screen (left) and their corresponding cell viability determined by an MTS assay performed on HEp-2 cell viability after 24 h of treatment at 37 °C (right). The average percent viability (±SEM) was calculated by comparing activity at each concentration to the 0 mM treatment. (B) Representative images of HEp-2 cells after 24 h of exposure at 10% of the concentration in the parent extracts (390 μM 3-hydroxybenzyl alcohol, 390 μM 3-hydroxybenzyl alcohol, and 170 μM carvone). The cells were fixed and stained with DAPI (DNA; cyan), TRITC-Phalloidin (actin; magenta), and FITC-conjugated anti-tubulin (tubulin; yellow). Scale bar = 100 μm. (C) Quantification of the nuclear morphology (left, middle) or cytoskeletal disruption (right) from six images from each treatment. The average percent apoptotic and mitotic nuclei (±SEM) and average cytoskeleton score (±SD) based on blinded scoring from 1 (normal) to 4 (complete cytoskeletal destruction). An ANCOVA was conducted to evaluate significance for the cell viability assay and a one-way ANOVA was used to evaluate the IF scoring. Significant differences relative to DMSO controls are indicated (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 5

Antibacterial and antiviral activity of chemical constituents derived from Osage orange (M. pomifera) and spearmint (M. spicata) leaf extracts. (A) Cultures of TSB were co-inoculated with E. coli (left) or S. aureus (right) in the presence of the indicated doses of 3- or 4-hydroxylbenzyl alcohol or R-(-)-carvone. Bacterial growth was assessed through measurement of the optical density at 600 nm. The average fold increase (±SEM) in OD₆₀₀ of three experimental replicates relative to the 0 h absorbance is shown. (B) HEp-2 cells were infected for 24 h with a recombinant fluorescent reporter strain (A2-mKate2) of respiratory syncytial virus (hRSV) in the presence of doses of the chemical constituents or a DMSO control. The average percent (±SEM, N = 3) of detected virus remaining relative to the untreated control is shown for all doses relative to their respective concentrations in their parent Osage orange and spearmint extracts. An ANCOVA was used to evaluate significance relative for both antibacterial and antiviral assays to the untreated 0 mg/mL control. Significant differences are indicated (*, p < 0.05; **, p < 0.01; ***, p < 0.001).