Influence of Sequential Solvent Extraction on the Antibacterial Activity of Phyla lanceolata Leaf Extracts ^†

Abstract

Natural products constitute an important source of bioactive compounds with potential for the development of new antimicrobial agents. Phyla lanceolata is a species of the Verbenaceae family whose chemical composition and biological activity have been scarcely studied. In this work, the antibacterial activity of P. lanceolata leaf extracts obtained by sequential extraction with solvents of increasing polarity (n-hexane, ethyl acetate, and methanol) was evaluated against Escherichia coli and Staphylococcus aureus. The crude ex-tracts were tested at concentrations of 200, 100, 50, 25, and 12.5 μg/μL using the disk diffusion method on Mueller–Hinton agar. Doxycycline and ceftriaxone were used as positive controls for E. coli, and amikacin and amoxicillin for S. aureus. The methanolic extract showed antibacterial activity against E. coli at 100 μg/μL, whereas the hexane extract was active against S. aureus at the same concentration. The ethyl acetate extract showed no inhibitory activity against either strain at the concentrations tested. These results suggest that the antibacterial activity of P. lanceolata extracts depends on solvent polarity and sup-port further studies involving phytochemical characterization and bioactivity-guided fractionation.

1. Introduction

Medicinal plants have been used since ancient times to prevent or treat a wide range of diseases, long before the development of modern medicine. This knowledge remains relevant, and numerous plant species continue to be used for medicinal purposes in different regions of the world, particularly in communities where traditional medicine still plays an important role [1].

Antimicrobial resistance (AMR) occurs when microorganisms no longer respond to medicines used to prevent and treat infections. This phenomenon represents one of the major threats to global public health and is associated with a substantial burden of disease and mortality [2,3]. Although antibiotics have been essential for the treatment of bacterial infections, their inappropriate use promotes the selection and spread of resistant micro-organisms. During exposure to an antibiotic, susceptible bacteria may be eliminated, whereas those carrying resistance mechanisms survive and may multiply [2].

Escherichia coli (E. coli) is a Gram-negative bacillus that forms part of the gastrointestinal microbiota, although certain strains can act as pathogens and cause, among other conditions, gastrointestinal disease and urinary tract infections. Its ability to express diverse virulence factors and acquire antibiotic resistance contributes to its clinical im-portance in both community-acquired and healthcare-associated infections [4].

Staphylococcus aureus (S. aureus) is a Gram-positive, facultatively anaerobic coccus that can form part of the human microbiota and behave as an opportunistic pathogen. Its ability to colonize different anatomical sites, evade the immune response, and express multiple virulence factors enables it to cause a wide variety of infections [5]. S. aureus be-longs to the ESKAPE group of pathogens, which are characterized by their clinical relevance and capacity to develop antimicrobial resistance [6,7]. Its resistance mechanisms include the production of β-lactamases and, in methicillin-resistant strains, acquisition of the staphylococcal cassette chromosome mec (SCCmec), which carries the mecA gene and encodes the penicillin-binding protein PBP2a, which has low affinity for β-lactam antibiotics [6,7].

Given the increasing prevalence of antimicrobial resistance, it is necessary to explore new sources of compounds with antibacterial activity. In this context, natural products, particularly those derived from plants, represent a source of bioactive metabolites with potential for the identification of new molecules or mixtures with biological activity. Therefore, this study focused on Phyla lanceolata (P. lanceolata), a species whose antibacterial activity has been scarcely investigated.

Phyla lanceolata belongs to the family Verbenaceae and the genus Phyla. It is a perennial plant that can reach approximately half a meter in height and has erect or creeping stems and leaves with serrated margins. It is distributed mainly in the central and eastern United States and is commonly found in moist environments, although it can also grow in relatively dry areas [8]. The taxonomic delimitation of species within the genus Phyla has recently been revised [9]. Because of the taxonomic proximity among species of this genus, reports on related species, such as Phyla nodiflora, are of comparative interest. Various traditional uses have been attributed to the latter, and several biological activities, including anti-inflammatory, antipyretic, and antimicrobial properties, have been re-ported [10].

Therefore, the search for new plant-derived antimicrobial alternatives remains scientifically relevant. Accordingly, the aim of the present study was to evaluate the antibacterial activity of P. lanceolata leaf extracts obtained by sequential extraction against two clinically important bacteria: E. coli (Gram-negative) and S. aureus (Gram-positive).

Given the limited phytochemical information available on Phyla lanceolata, this study aimed to provide an initial assessment of the antibacterial activity of its leaf extracts obtained through sequential extraction with solvents of increasing polarity. This preliminary approach was intended to identify extracts of potential interest for subsequent phytochemical investigations.

2. Materials and Methods

2.1. Preparation of Extracts

The plant material was dried at room temperature for 24 h under dark and moisture-free conditions. The leaves were then ground and stored in opaque containers. Extraction was performed by sequential maceration in triplicate using 2 L of each solvent in order of in-creasing polarity: n-hexane, ethyl acetate, and methanol. Each extraction was allowed to stand for 24 h, and the resulting extracts were concentrated using a rotavapor (BUCHI-R100, Flawil, Switzerland) at a temperature ≤ 42 °C.

2.2. Antibacterial Assay

Antibacterial activity was evaluated using the disk diffusion method on Mueller–Hinton agar. Each extract was tested at concentrations of 200, 100, 50, 25, and 12.5 μg/μL against two bacterial strains: Escherichia coli and Staphylococcus aureus. Doxycycline and ceftriaxone were used as positive controls for E. coli, and amikacin and amoxicillin for S. aureus; dimethyl sulfoxide (DMSO) and water were used as negative controls. Each of the three extracts (hexane, ethyl acetate, and methanolic) was evaluated on a separate plate with each positive control, for a total of six plates per bacterial strain (12 plates in total). Each plate was divided into eight sections: five corresponding to the tested concentrations, one to the positive control, and two to the negative controls (DMSO and water). A volume of 10 μL of the corresponding extract or control was applied to each disk.

Note: The bacterial strains were provided by the División Académica de Ciencias Básicas, Universidad Juárez Autónoma de Tabasco (DACB-UJAT), Cunduacan, Tabasco, Mexico.

3. Results

In the present study, the antibacterial activity of the hexane (EHex), ethyl acetate (EtOAc), and methanolic (MeOH) extracts was evaluated at concentrations of 200, 100, 50, 25, and 12.5 μg/μL against Escherichia coli and Staphylococcus aureus. As shown in Table 1 the methanolic extract exhibited antibacterial activity against E. coli at a concentration of 100 μg/μL. The hexane extract showed activity against S. aureus at 100 μg/μL, whereas the ethyl acetate extract showed no inhibitory activity against either strain within the concentration range tested. The positive controls used for E. coli were doxycycline (30 μg/10 μL) and ceftriaxone (3 μg/μL); amikacin (30 μg/μL) and amoxicillin (10 μg/μL) were used for S. aureus.

Table 1. Antibacterial activity of Phyla lanceolata extracts against Escherichia coli and Staphylo-coccus aureus.

Microorganism	EHex	AcOEt	MeOH
Escherichia coli	n/a	n/a	100 μg/μL
Staphylococcus aureus	100 μg/μL	n/a	n/a

Positive controls: doxycycline, ceftriaxone, amikacin, and amoxicillin. Negative controls: DMSO and H₂O. No activity: n/a.

4. Discussion

In the present study, the antibacterial activity of P. lanceolata extracts against E. coli and S. aureus was evaluated. The results showed differences according to the extraction solvent. The methanolic extract exhibited activity against E. coli at 100 μg/μL, whereas the hexane extract showed activity against S. aureus at the same concentration. In contrast, the ethyl acetate extract showed no inhibitory activity against either bacterium within the concentration range tested.

The activity observed for the methanolic extract against E. coli could be related to the extraction of more polar metabolites, such as phenolic compounds and flavonoids [11]. In related species of the Verbenaceae family, such as Phyla nodiflora, antibacterial activity has been reported for extracts obtained with polar solvents against both Gram-negative and Gram-positive bacteria, and this activity has been associated with the presence of phenolic compounds and flavonoids [12]. Nevertheless, phytochemical studies of P. lanceolata are required to determine which metabolites contribute to the observed activity.

In contrast, the hexane extract showed inhibitory activity against S. aureus. n-Hexane mainly extracts low-polarity compounds, including terpenoids and lipids, some of which may affect bacterial membrane integrity [13]. This finding is relevant because of the clinical importance of S. aureus and its inclusion in the ESKAPE group. Various plant-derived compounds can exert antibacterial effects through mechanisms such as inhibition of efflux pumps, disruption of biofilm formation, interference with quorum sensing, and mem-brane permeabilization [14]. Activity against S. aureus has also been reported for hexane extracts from related plant species or species used for comparative purposes [15].

The differences observed between E. coli and S. aureus could be related, among other factors, to the characteristics of their cell envelopes. E. coli possesses an outer membrane that can limit the penetration of various antimicrobial compounds, whereas S. aureus lacks this structure, which may influence its susceptibility to certain metabolites present in plant extracts [16].

Given the limited phytochemical information available on Phyla lanceolata, the present study provides preliminary evidence of its antibacterial potential and establishes a starting point for further investigation of this species. The differences in antibacterial activity observed among the extracts suggest that solvent polarity influences the recovery of constituents potentially associated with the biological effects. However, because the chemical composition of the extracts was not determined, these activities cannot be attributed to specific metabolites or compound classes. Further phytochemical profiling, followed by bioassay-guided fractionation and structural characterization of the active constituents, will be necessary to establish the relationship between chemical composition and antibacterial activity.

5. Conclusions

The results show that the antibacterial activity of P. lanceolata extracts varied according to the extraction solvent and the bacterium tested. The methanolic extract exhibited activity against E. coli at 100 μg/μL, whereas the hexane extract showed activity against S. aureus at the same concentration. The ethyl acetate extract showed no inhibitory activity under the experimental conditions.

These findings provide preliminary evidence of the antibacterial potential of P. lanceolata and support further studies aimed at phytochemical characterization and fractionation of the active extracts. The activity should also be confirmed using quantitative assays, the number of microorganisms evaluated should be expanded, and the compounds responsible for the observed effect should be identified before any potential usefulness against clinically relevant bacteria can be established.

These preliminary findings support further phytochemical investigations aimed at identifying the constituents of the active extracts and establishing their contribution to the observed antibacterial effects.