Phytochemical-Rich Plant Extracts as Natural Antibacterial Agents: A Comparative Study of Randia aculeata, Ixora coccinea, Capsella bursa-pastoris, and Portulaca oleracea Against Clinically Relevant Pathogens

Introduction: Antimicrobial resistance (AMR) is spreading fast to almost all countries and has threatened the global health security. It undermines the effectiveness of antibiotics, so common infections are harder to treat. If this continues, the world could return to an era when people died of simple infections because drugs no longer worked [1]. Pathogenetic microorganisms, such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Serratia marcescens, were frequently reported to possess multiple antibiotic resistance toward a broad array of classes of antibiotics: β-lactams, fluoroquinolones, macrolides, and aminoglycosides [2]. These microorganisms have mechanisms of resistance which include production of β-lactamase, alteration/modification of drug targets efflux-pump pumping efflux from cell, reduced expression of outer-membrane porins/biofilm formation and horizontal gene transfer [3]. Their increasing resistance causes serious complications in treatment, prolongs hospitalizations, increases morbidity and mortality rates and imposes a tremendous economic burden on communities worldwide [4]. Emerging, efficient, and economical antimicrobial sources have become a growing concern worldwide because of increasing resistance to current antibiotics and few number of new drugs now available in line [5]. Medicinal plants, the biomines used from ancient time of traditional medicine systems such as those known by Ayurveda, Siddha, Unani and Traditional Chinese Medicine still play a vital role as a bioactive chemodiversity pool [6]. Despite pharmaceutical companies’ investing heavily in it, most new antimicrobials are not particularly effective because of increasing drug resistance. In comparison, medicinal plants have been used for hundreds of years, as they possess numerous bioactive phytochemicals, including alkaloids, phenolics, flavonoids, terpenoids, and glycosides, with an abundance of antimicrobial activity that is now being scientifically studied daily for their complementary therapeutics [7].

Plants have since long been targeted as plentiful sources of secondary metabolites comprising a wide-variety of phytochemicals including alkaloids, flavonoids, saponins, tannins, terpenes, glycosides (both steroidal and cardiac), phenolic acids and volatile agent oils. Such compounds are reported to display a wide range of biological activities, including antimicrobial, antioxidant, anti-inflammatory, and immunomodulating properties. Due to their good therapeutic potential, they can be considered as important alternatives to synthetic drugs, particularly when dealing with multidrug-resistant pathogens [8]. A major advantage of natural antimicrobials from plants is served by different biochemical mechanisms. Phytochemicals generally act against microorganisms by multiple action mechanisms, unlike the most synthetic antibiotics that interact with a single protein or metabolic pathway [9]. These involve perturbation of the integrity of microbial cell membrane, inhibition of protein synthesis, suppression of replication of nucleic acid, distorting various important enzymatic processes as well as quorum sensing and virulence factors. This multi-target activity not only improves their antimicrobial potential, but also helps in reducing the possibility of microbes developing resistance [10]. Because of their wide-ranging therapeutic benefits, lower toxicity in several cases, bioavailability and ease of access (as well as low cost), plants themselves or active secondary end products are being considered increasingly reasonable future alternatives to new generation antimicrobial drugs. Their incorporation in the field of modern pharmacology promises to be a valuable tool in combating the worldwide problem of antibiotic resistance and offer an alternative treatment for infectious diseases [11].

Out of myriad plants with medicinal value, Randia aculeata, Ixora coccinea, Capsella bursa-pastoris and Portulaca oleracea are some important ethnomedicinal plant species but they lack scientific exploration especially in comparison to their antibacterial potential. Randia aculeata which belongs to the family Rubiaceae, is traditionally used in the treatment of inflammation, diseases related to digestive system, dermatoses and microbial infections [12]. Phytochemical investigations showed the existence of iridoids, flavonoids, glycosides, alkaloids and phenolic compounds that can be responsible for its antimicrobial activities. Ixora coccinea or jungle geranium is a popular ornamental and medicinal shrub used in Ayurveda as well as folk medicine. Its leaves, flowers and roots are a rich source of tannins, flavonoids, polyphenols and terpenoids which attribute antimicrobial, antioxidant, hepatoprevenitive and wound healing activities [13]. Capsella bursa-pastoris (Shepherd’s purse) from the family Brassicaceae is an edible herb, which features in a few traditions and culture due to its potential to cure infections and bleeding. It introduced glucosinolates, alkaloids, flavonoids, peptides and sulfur-containing compounds with antimicrobial or anticancer properties. Purslane is a substantially nutrient- and functional substance-rich plant, which contains omega-3 fatty acids, polysaceharides, flavonoids, betalains vitamins and minerals. Extracts of the herb were found to possess antibacterial, antifungal, anti-ulcer, antioxidant and neuropharmacological activities. Although these plants have long traditional reputations in this regard, knowledge of their scientific antibacterial activity, particularly the potentiality of preparing simple aqueous extracts by boiling is quite meager [14]. The majority of the earlier attempts target organic solvents (ethanol, methanol, chloroform, and acetone), and may selectively extract various classes of phytochemicals. Yet aqueous extraction is more applicable for the traditional preparation and safer applied to food, cosmetic sources, or medicinal herbs. In addition, comparative analyses of the aforementioned four specific species against a common panel of clinically significant pathogens are not well studied. It is important to know which plant exhibits more powerful antibacterial activity, for selecting a subject for future drug discovery or natural antimicrobial preparations [15]. In light of this, the present investigation was conducted to determine and test the antibacterial effectivity of aqueous extracts obtained from Randia aculeata, Ixora coccinea, Capsella bursa-pastoris and Portulaca oleracea on four clinically relevant pathogenic bacteria (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens). The extracts were obtained by standard boiling extraction methodology and screened for their antimicrobial activity by agar well diffusion assay. Results of this study will be able to establish the scientific proof for these traditional uses and guide future development of natural herbal antimicrobials from potentially effective medicinal plants.

Materials and Methodology

Collection and Preparation of Plant Materials: Fresh plant samples of Randia aculeata, Ixora coccinea, Capsella bursa-pastoris (Shepherd’s purse), Portulaca oleracea, and Purslane stems were collected from Thakur Complex, Kandivali (East), Mumbai, Maharashtra. The samples were washed thoroughly with tap water followed by rinsing with distilled water to eliminate dust, soil particles, and surface contaminants. All plant materials were shade-dried at room temperature until completely moisture-free to preserve heat-sensitive phytochemicals. The dried samples were then finely ground using a sterile electric grinder, ensuring uniform particle size for efficient extraction, and the powdered material was stored in clean, airtight, labeled containers. These containers were kept in a cool, dry place to prevent degradation of bioactive compounds before further experimental processing [16].

Preparation of Aqueous Plant Extracts (Boiling Method): Each plant sample was extracted separately using the same protocol. 10 g of plant powder was mixed with 100 mL of distilled water (1:10 w/v). The mixture was boiled for 30 minutes on a low flame. After cooling to room temperature, extracts were filtered through Whatman No. 1 filter paper. The filtrate was centrifuged at 5000 rpm for 10 minutes to remove remaining debris. The clear supernatant was collected as the final aqueous extract. Extracts were stored at 4°C until antimicrobial testing 17).

Test Microorganisms: The antimicrobial potential of plant extracts was tested on four clinically important bacterial strains, including Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens. Unrelated subcultures of both species were grown on nutrient agar plates the day before the experiment to obtain growing, viable cells. Such standardised subcultures were used as the inoculum for all antimicrobial assays in this investigation.

Preparation of Bacterial Inoculum: Overnight cultures were adjusted to the 0.5 McFarland standard, corresponding to approximately 1.5 × 10⁸ CFU/mL. This standardized inoculum was used for swab-inoculating the agar plates.

Antimicrobial Assay by Agar Well Diffusion: Mueller–Hinton agar plates were made and poured into sterile Petri dishes to solidify at room temperature. Control plates were then both swabbed with a standard bacterial suspension to create a lawn of growth. Sterile cork borers (6mm in diameter) were used to remove agar aseptically, producing wells. 50 µL of each plant extract (Randia aculeata, Ixora coccinea, Capsella bursa-pastoris, Portulaca oleracea, and Purslane stem extract) was added to the respective wells. Each extract treatment was repeated 3 times on each bacterial strain to guarantee the reliability and repeatability of results.

Incubation and Observation: Inoculated Mueller–Hinton agar plates were then incubated at 37 °C for 18 to 24 h to achieve sufficient bacterial growth and interaction with plant extracts. After incubation, the antimicrobial activity was determined by measuring the diameters of zones of inhibition (ZI) in mm formed around each well with a graduated ruler. There are readings in mm against each bacterial strain for all plant extracts.

Statistical Analysis: All antibacterial assays were conducted in triplicate to ensure experimental reliability and reproducibility. Zones of inhibition were measured in millimeters (mm) and expressed as mean ± experimental deviation (±1–2 mm), reflecting inherent methodological variability commonly associated with agar well diffusion assays. Statistical comparison was applied to assess the relative antibacterial efficacy among different plant extracts and bacterial strains. A p-value of < 0.05 was considered indicative of statistically meaningful differences.

Results: Zone of inhibition values are expressed in millimeters (mm). A value of “0” indicates the absence of detectable antimicrobial activity.

With the five plant extracts tested, Ixora coccinea showed maximum and broad spectrum of antibacterial activity with inhibition zone 24 mm (S. aureus) and 27 mm (E. coli), 15mm (Pseudomonas aeruginosa) and 14mm (Serratia marcescenes). Portulaca oleracea and its stem extract had moderate inhibitory activity against only E. coli (both extracts had a zone of inhibition of 22 mm). Randia aculeata presented mild activity against E. coli, with the inhibition zone of 23 mm, while there was no effect on other pathogens. However, Capsella bursa-pastoris (Shepherd’s purse) showed no antimicrobial activity against the other bacteria. In general, Ixora coccinea was the most active extract with evident inhibition against four bacteria, while others exhibited selective/weak antibacterial activities.

Statistical Interpretation of Results

Comparative statistical evaluation revealed pronounced variation in antibacterial efficacy among the tested plant extracts. Ixora coccinea demonstrated statistically significant and broad-spectrum antibacterial activity against all four tested pathogens, with inhibition zones ranging from significant to highly significant categories. Portulaca oleracea leaf and stem extracts exhibited statistically meaningful but selective antibacterial activity restricted to Escherichia coli. Randia aculeata showed mild, strain-specific inhibition against E. coli. In contrast, Capsella bursa-pastoris failed to demonstrate measurable antibacterial activity against any of the test organisms under the applied experimental conditions.

Bioinformatics-Assisted Interpretation of Antibacterial Pattern

Integration of bioinformatics-derived pathogen characteristics with experimental data provides mechanistic insight into the antibacterial trends observed in this study. Differences in susceptibility among the tested bacterial species strongly correlate with known variations in cell wall architecture, membrane permeability, and resistance-associated genetic determinants documented in bacterial genome annotation databases. Staphylococcus aureus, a Gram-positive bacterium lacking an outer membrane, exhibits increased susceptibility to hydrophilic phytochemicals commonly present in aqueous plant extracts. This structural feature plausibly accounts for the pronounced inhibitory effect observed with Ixora coccinea. Gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens, possess an outer membrane enriched with lipopolysaccharides that significantly restrict antimicrobial penetration. Despite this barrier, Ixora coccinea exhibited inhibitory activity against all Gram-negative strains tested, suggesting the presence of phytochemicals capable of affecting conserved intracellular metabolic pathways.

Bioinformatics-supported pathway analyses indicate that these pathogens share essential cellular systems involved in protein synthesis, oxidative stress response, and cell wall biosynthesis. These conserved pathways are well-established targets of plant-derived phenolics and flavonoids, offering a rational explanation for the observed broad-spectrum antibacterial activity.

Discussion

The present study reveals marked variation in the antibacterial efficacy of selected medicinal plant extracts, reflecting differences in their phytochemical composition as well as pathogen-specific resistance mechanisms. Among the plants evaluated, Ixora coccinea exhibited the most potent and broad-spectrum antibacterial activity against all tested pathogens. This pronounced efficacy can be attributed to its rich diversity of bioactive constituents, including phenolics, flavonoids, tannins, and terpenoids, which are known to disrupt microbial cell membranes, interfere with nucleic acid synthesis, and inhibit key metabolic pathways. The ability of I. coccinea to inhibit both Gram-positive and Gram-negative bacteria suggests a multi-target mode of action, thereby lowering the probability of rapid resistance development and highlighting its promise as a potential alternative or complementary antimicrobial agent. Portulaca oleracea and its stem extract demonstrated selective growth inhibitory activity primarily against Escherichia coli. This specificity indicates the presence of secondary metabolites that preferentially target structural features characteristic of Gram-negative bacteria, such as the outer membrane and lipopolysaccharide layer. Enhanced susceptibility of E. coli may also be linked to its comparatively higher membrane permeability and lower expression of multidrug efflux pumps. Conversely, intrinsically resistant pathogens such as Pseudomonas aeruginosa and Serratia marcescens showed limited susceptibility, likely due to their efficient efflux systems, reduced porin expression, and biofilm-associated resistance mechanisms that restrict the intracellular accumulation of plant-derived antimicrobial compounds.

Randia aculeata exhibited weak antibacterial activity only against E. coli, which may be explained by a lower concentration of active phytochemicals or antimicrobial mechanisms of limited potency. Meanwhile, the complete lack of detectable antibacterial activity in Capsella bursa-pastoris suggests that its active constituents are either present in insufficient concentrations, ineffective against the tested microorganisms, or require specific extraction conditions to express biological activity. This observation underscores the critical influence of plant part selection, extraction methodology, and solvent polarity on antimicrobial outcomes. The findings demonstrate that plant-derived antimicrobial effectiveness varies considerably and is governed by an intricate interaction between phytochemical diversity, extraction parameters, and microorganism resistance profiles. The integration of experimental microbiological assays with pathogen-specific resistance considerations strengthens the interpretation of these results and aligns with previous reports indicating that not all medicinal plants exhibit broad-spectrum antimicrobial activity. Collectively, this study emphasizes the need for targeted extraction strategies and pathogen-informed screening approaches when exploring medicinal plants as potential sources of novel antibacterial agents in the era of rising antimicrobial resistance.

Conclusion

The present study demonstrates significant variability in the antibacterial potential of the evaluated medicinal plant extracts. Among the five plants tested, Ixora coccinea exhibited the highest and statistically significant broad-spectrum antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Serratia marcescens, establishing it as the most effective antibacterial agent in this investigation. In contrast, Portulaca oleracea showed selective activity primarily against E. coli, while Randia aculeata displayed only mild and species-specific inhibition. Capsella bursa-pastoris exhibited no detectable antibacterial activity under aqueous extraction conditions. These findings highlight that the antimicrobial efficacy of plant extracts is largely dependent on their phytochemical composition and does not necessarily translate into broad-spectrum activity. The integration of experimental antibacterial assays with statistical and bioinformatics-based analyses provides a robust framework for interpreting plant–pathogen interactions. Overall, the results support Ixora coccinea as a promising candidate for bioactivity-guided isolation of antibacterial compounds and future development of plant-derived therapeutic agents.

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