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Comparative chemical and antimicrobial evaluation of the essential oils from Callistemon and podocarpus species supported by in-silico molecular simulations against bacterial LacY protease

BMC Complementary Medicine and Therapies volume 25, Article number: 144 (2025) Cite this article

Abstract

Background

Myrtaceae and Podocarpaceae botanical families include several aromatic species that have been proven to have diverse pharmacological potential, especially antimicrobial effects. Additionally, plants of both families were reported for their benefits in traditional medicine. The current study demonstrated the chemical profile, antimicrobial of four investigated plant species (C. subulatus, C. rigidus, P. gracilior, and P. elongatus) leaves cultivated in the same place in Egypt and propose in-silico modeling for the antibacterial mechanistic action.

Method

The essential oils samples were prepared via hydrodistillation and headspace extraction protocol and GC analysis was conducted to obtain a comparative chemical profile. The antimicrobial activity of the obtained hydrodistillation essential oil samples was screened via agar diffusion, and the MIC was calculated via broth microdilution assays. An in silico molecular docking study was performed to investigate the inhibition of the LacY protease efflux pump.

Results

GC results revealed that the percentage of oxygenated monoterpenes was highest in the oil samples from Callistemon species (60.38 and 82.68%). In contrast, sesquiterpene hydrocarbons constituted the highest percentage of volatile classes in the oil samples from Podocarpus species (57.37 and 43.16%). C. rigidus-EO shows significant inhibitory activity against Gram-negative and Gram-positive pathogens, especially E. coli and S. viridans, with a calculated MIC of 0.878 ml, whereas P. elongatus EO shows notable activity against coagulase-negative Staphylococcus and the activity was comparable to that of the positive control antibiotics used (ciprofloxacin & doxycycline). Ultimately, an in silico molecular docking study on the binding site of the LacY protease enzyme revealed a significant binding affinity of the major docked volatile constituents.

Conclusion

The plant species investigated are considered a vital source of safe antimicrobial volatile constituents that are recommended as bioactive entities for controlling microbial infection topically or systemically. The proposed mechanistic action encourages further modification of the major EOs chemical structure by adding more polar substitutions to improve the binding affinity and produce more active semisynthetic analogues.

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Introduction

Plants are considered a treasure filled with different libraries of chemical entities that have several benefits for humans, ranging from nutritional to medicinal significance. The secondary metabolites produced by plants are a product of physiological functions inside the plant cell or as a response to external stimulants as a defense mechanism [1]. Essential oils (EOs) The odoriferous components produced by plant organs, which are liquid and volatile with different scents according to their composition. Several factors influence the number of EOs extracted, such as climate, geographical aspects, collection time, and extraction procedures [2, 3]. The most common techniques used in the extraction of EOs are hydrodistillation, effleurage, steam distillation, solvent extraction, supercritical fluid, and microwave-assisted extraction [4]. The major constituent of EOs is terpenes. The building unit of terpenes is an isoprene unit (C5H8). The number of isoprene units results in different classes of terpenes and volatile oils [4]. The importance of EOs ranges from medicinal benefits to the cosmetic industry, as they are used as raw materials in the production of perfumes, cosmetics, and aromatherapy [5, 6]. With respect to EO samples, modern methods have been established to obtain oil samples with high purity to overcome the limitations of traditional methods and enhance extraction outcomes. Headspace microextraction is a new strategy for microextraction EOs directly from fresh plant samples and offers a rapid and efficient extraction method, especially when the sample amount is limited [6, 7]. Therefore, the optimal extraction technique is selected according to the desired EO quality and constituents, which ultimately support the desired pharmacological benefits [8].

Increasing microbial resistance is urgently needed for the continuous search for new antimicrobial agents with notable effectiveness [9]. The global prevalence of antimicrobial-resistant bacteria has reached alarming levels, posing a significant threat to public health on a global scale, such as during a silent pandemic. This urgent situation necessitates immediate intervention. Infections caused by antimicrobial-resistant bacteria present a serious challenge, as therapeutic options are limited, leading to substantial morbidity, mortality, and a significant economic burden. The scarcity of new and innovative antimicrobial agents to address life-threatening infections caused by resistant pathogens contrasts completely with the growing demand for effective treatments [10]. For decades, plants have offered the opportunity for the discovery of many structurally diverse phytochemicals. The antimicrobial potential of EOs has been well reported, and reports have correlated their efficacy with the chemical nature of the EOs on the basis of their hydrophobic nature, which is mainly hydrocarbons that can sometimes be oxygenated, leading to an increase in the potential for strong hydrophobic interactions with protein-substrate binding sites as a mechanism of action and facilitating membrane transport and permeability [11, 12].

Among plant families, the family Podocarpaceae is well known to include several aromatic trees and shrubs. The family Podocarpaceae consists of evergreen trees or shrubs, including approximately 20 genera and more than 200 species. Warm regions are considered the native sources of these plants, especially in the Southern Hemisphere, and some of these plants are grown as ornamental plants [13]. Podocarpus is a genus that comprises approximately 100 species and is widely spread throughout Mexico, South Africa, southern China, and southern Japan [14]. A wide range of phytochemicals, including terpenes [15,16,17,18,19,20,21,22,23], essential oils [24], phenolics [16, 20, 25], and sterols [26], have been isolated and identified from the genus Podocarpus. Species of this genus were reported to possess several pharmacological importance, such as anti-inflammatory, antioxidant [27, 28], antiseptic, for the treatment of chest complaints [29], carminative, antiulcer, stomachic [29, 30], and cytotoxic effects [20, 31]. Podocarpus gracilior and Podocarpus elongatus were cultivated Podocarpus species in Egypt. P. gracilior (Pilger) is a tree that natively grows in Central and East Africa and is known as African Fern Pine [16, 32]. Several reports have highlighted the pharmacological importance of P. gracilior, such as the anticancer and cytotoxic activities of its ethanol extract [33, 34]. P. elongatus Aiton L'Hér. ex Pers is a tree that natively grows in South Africa and is known as de River yellowwood. Additionally, several reports have indicated the medicinal importance of P. elongatus, such as its crude extracts, which exhibit broad-spectrum antimicrobial activity [23, 35].

The family Myrtaceae is a well-known plant family that includes approximately 140 genera, including many important trees and shrubs. Callistemon is one of the Myrtle family members and comprises approximately forty species native to Australia [36]. Callistemon species are well known for their use in folk medicine, as they have valuable medicinal value. These species have been used for the treatment of lung diseases, skin problems, and GIT manifestations [37]. C. sabulatus Cheel and C. rigidus R.Br have been reported several times for their pharmacological benefits in terms of their phenolic content and essential oils [36, 38].

Ongoing research has focused on the comparative exploration of essential oil (EO) chemical profiles derived from four plant species belonging to the Callistemon and Podocarpus genera in Egypt. Identification of essential oil constituents is performed through gas chromatography (GC) analysis. This study aims to assess and compare the chemical compositions and major components of essential oils extracted using two different extraction methods, hydodistillation as example of conventional old extraction technique and headspace as example of advanced extraction method using two species of two different plant genera growing in the same place and collected at the same seasonal period. Additionally, research has focused on evaluating the antimicrobial potential of the extracted essential oils using conventional hydrodistillation technique, since it is the most available method of extraction. Finally, in order to gain more insight into the potential impact of the identified oils on a membrane transporter, in silico molecular docking was employed as a supplementary analytical approach.

Materials and methods

Plant material

The aerial parts of Callistemon subulatus Cheel, Callistemon rigidus R.Br., Podocarpous gracilior Pilg., and Podocarpous elongatus Aiton L'Hér. ex Pers were collected from Orman Garden, El-Giza, Egypt (February 2022), according to the garden guidelines, and the botanical sample collection rules stated in Egypt. Taxonomic authentication of the samples was performed by Dr. Trease Labib, a plant taxonomist at Mazhar Garden, El-Giza, Egypt. A voucher sample was deposited at the official Herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Cairo, Egypt, under official serial numbers 39Pgr1/2024, 39Pel2/2024, 16Csa2/2024 and 16Cri4/2024 for P. gracilior and P. elongatus, C. subulatus and C. rigidus, respectively.

Essential oil extraction

Hydrodistillation technique

Fresh leaves (750 g) of the mentioned species (Plant material section) were subjected separately to the traditional hydrodistillation method following a previously reported procedure [38]. The leaves were comminuted, distilled water was added until they covered the surface, and the EO hydrodistillation method was performed via a Clevenger apparatus. The distillate was allowed to cool at room temperature, the essential oil was allowed to separate from the water, and each essential oil sample was weighed on an analytical scale (3.75 ml 0.83% v/w, 2.5 ml 0.559% v/w, 0.25 ml 0.025 and 0.2 ml 0.02% v/w) from C. rigidus, C. subulatus, P. gracilior, and P. elongatus, respectively. The obtained EOs were passed over anhydrous Na2SO4 for dehydration and then stored in amber-colored vials at 4 °C for GC/MS analysis and biological activity investigation.

Head-space solid-phase microextraction

Headspace solid-phase (HS) microextraction of the EOs from the collected fresh samples was carried out according to the procedure reported in the literature [39]. Approximately 2 g of collected fresh leaves was placed separately into a glass vial (5 mL). The vial's optimum temperature was maintained at 60–70 °C to create a saturated headspace of the solid samples in the vial with the EO vapor. The solid-phase microextraction syringe was mounted in the headspace, and then, the EO vapors were absorbed by the needle of the syringe (silica phase). The saturated silica fiber of the syringe was fixed to the GC/MS injection unit for the estimation of the EO components.

Gas chromatography analysis of the EOs

For estimation of the obtained EO components, the mass spectrum was generated via a Shimadzu GC/MS-QP2010 system (Kyoto, Japan) equipped with a mass spectrometer (quadrupole) (Shimadzu Corporation, Kyoto, Japan). GC separation was performed on a column [Rtx- (30 m × 0.25 mm i.d. × 0.25-μm film thickness] 5MS column from Restek, United States. All the obtained EOs were subsequently estimated according to the same conditions and as reported previously [38, 40].

Identification of essential oil components

The obtained oil samples were estimated individually via GC analysis. The identification of the EO constituents was determined tentatively on the basis of the component retention indices (RIs) in comparison with those of authentic n-alkanes (C8–C28); then, NIST and Wiley mass spectra library databases and previously reported data were used for comparison of their mass spectra for more confirmation (similarity index > 90%) [41, 42]. The retention indices were obtained by using the GC/MS solution program.

Antimicrobial activity of the obtained EOs

Microbes, positive controls, and culture media

In this study, the following microorganism strains were used to assess the antibacterial and antifungal activities of the essential oils. Gram-positive bacteria such as methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus viridans, Enterococcus faecalis, coagulase-negative Staphylococcus (CoNS), Gram-negative bacteria Escherichia coli, and Candida albicans fungi. All of these are clinical isolates that were recovered from hospitalized patients and those strains were multidrug resistant to commonly used antibiotics. The isolates were cultured on Mueller–Hinton broth and streaked on blood agar. The Candida albicans isolate was maintained on a potato dextrose agar (PDA) slant. All the isolates were identified via VITEK II (bioMerieux, Marcy l’E´toile, France). The bacterial suspensions were adjusted to the desired concentration equivalent to that of a 0.5 McFarland standard and incubated overnight under aerobic conditions at 37°C.

Agar well diffusion assay

Das et al. (2013) reported a protocol in which the agar well diffusion method was used to evaluate the antibacterial activity of essential oils [43]. Water and Tween 80 (less than 10%) were added in a 1:1 ratio (v/v) to the oil. The mixture was mixed and then subjected to 30 min of sonication and vortex homogenization for 8 min to obtain a stable essential oil emulsion, which was subsequently stored at 4○C for further use. The culture plates were allowed to solidify, and then, a volume of 100 μL (1.5 × 108 CFU/mL) of fresh inoculum of the tested bacteria (MRSA, CoNS, S. viridans, E. faecalis, E. coli) was streaked onto Mueller–Hinton agar. The plates were subsequently punched with a sterile cork borer, and the opened wells were inoculated with 50 µL of the tested essential oil mixture. The standard antibiotics doxycycline (30 µg) and ciprofloxacin (5 µg) were used as controls. All the plates were incubated at 37°C for 24 h. For C. albicans, the resulting inhibition zones (ZOIs) were measured after the plates were incubated at 28°C for 24 h. The experiment was performed in triplicate, and the average inhibition zone size was determined.

Minimum Inhibitory Concentration (MIC) determination

The MIC was tested via broth microdilution via a resazurin microtiter plate-based antibacterial assay [44]. Stable essential oil emulsions were used as described above. In a 96-well microtiter plate, 100 µL of sterile nutrient broth and 100 µL of stable essential oil emulsion were added to the first well. Eleven-fold serial dilutions were performed by transferring 100 mL from well to well (in rows). To each well, 10 µL of inoculum (1.5 × 108 CFU/mL) was added. The concentrations reached ranged from 225 µL to 0.878 µL EO/mL. The microplates were incubated for 18–24 h at 37○C. Thirty microliters of resazurin aqueous solution were added to each well. The microtiter plates were incubated at 37 °C for 2 h. The concentration at which the blue color did not turn pink was considered the MIC. Three replicates were performed for each essential oil.

Molecular docking studies

Preparation of protein structure

Lactose permease enzyme (LacY), a membrane protein that is a member of the facilitator family of transporters, was selected from the Protein Data Bank (www.rscb.org). This enzyme (PDB ID: 1PV7 [45]), which consists of C-terminal and N-terminal domains, has 6 symmetrically positioned transmembrane helices. This transmembrane has a large hydrophilic cavity to the inside that opens toward the cytoplasm, indicating that the membrane transporter has an inward facing conformation. The Molecular Operating Environment 2022 (MOE 2022) program was employed to decrease the tension of the crystal structure and to prepare a stable protein structure. MOE was used to delete the hydrogens from the macromolecules and again added to make the enzyme suitable as a protonated macromolecule at a convenient pH and temperature. Energy minimization through the omission of water molecules and ligands. The whole protein was scanned, and optimization steps were performed to allocate the active sites on the protein.

Ligand preparation

The 2D structures of the ligand molecules were sketched via ChemDraw 19.1 (Perkin Elmer Informatics) software, and a database was generated, transferred to MOE software, and converted into a 3D structure. Then, the file was saved as a mdb file extension. Here, ten volatile oil molecules (Table 4) were optimized and introduced into the ligand dataset.

Protein–ligand modeling simulation

The MOE 2020 program was used to create the design, locate the active site, and generate the modeling files. Here, the ligand-binding site is in the hydrophilic cavity at the same distance from both sides of the membrane. The modeling study was adjusted to yield 30 poses of the most stable conformers for each ligand with different torsion angles. The data generated from the modeling study were interpreted according to the S (Score) value, which indicates the binding energy and the possible binding affinity.

Results and discussion

Essential oils (EOs) are mixtures of volatile components that are characterized by their diverse chemical entity, notable aroma, and valuable pharmacological potential [46]. In general, each aromatic species yields a “fingerprint” of EOs, which varies according to the extracted organ and its geographical origin. These parameters usually affect the biological potential of EOs in either a synergistic, positive, or antagonistic way [47]. Another parameter that affects the overall potential and composition of EOs is the method of extraction [48]. First, the hydrodistillation (HD) technique is the most frequently employed method, but the resulting EOs can fluctuate in response to several factors, such as polymerization, saponification, and/or isomerization. Accordingly, in this work, we report the extraction of the EOs of the aerial parts of four different species growing in Egypt (C. subulatus, C. rigidus, P. gracilior, and P. elongatus) via HD and HS methods to investigate the differences in their analysis outcomes in terms of chemical composition and antimicrobial potential.

The extraction techniques affect the composition of the obtained EO in many ways. For example, regarding the EOs obtained from Callistemon species, a total of 22 and 9 volatile constituents were estimated in HD- and HS-derived oils, respectively, from C. subulatus, whereas a total of 14 and 11 volatile constituents were estimated in HD- and HS-derived oils, respectively, from C. rigidus (Table 1, Supplementary Figure S1–S2). On the other hand, in the EOs obtained from Podocarpous species, a total of 32 and 7 volatile constituents were estimated in HD- and HS-derived oils, respectively, from P. gracilior, whereas a total of 17 and 7 volatile constituents were estimated in HD- and HS-derived oils, respectively, from P. elongatus (Table 2, Supplementary Figure S3–S4). Moreover, variability in the percentage of the EO chemical class of the identified compounds was also observed.

Table 1 Percent concentration (%) of the volatile content estimated in the EOs of C. subulata and C. rigidus aerial parts extracted using hydro distillation (HD), and headspace solid-phase micro-sampling
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Table 2 Percent concentration (%) of the volatile content estimated in the EOs of P. gracilior and P. elongatus aerial parts extracted using hydro distillation (HD), and headspace solid-phase micro-sampling
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Based on the GC results for the EOS samples obtained from C. subulatus and C. rigidus, oxygenated Monoterpenes were the major component of all screened callistemon samples. Eucalyptol represents the major EO component in both Hydrodisillation (HD) and headspace (HS) EOs of C. subulatus and C. rigidus. In the case of C. subulatus HD-EO, eucalyptol (38.29%) and α-pinene (22.42%) representing the main EO components and were the major constituents of HS-EO, but the percentages were 59.53% and 27.96% for eucalyptol and α-pinene, respectively. In the case of C. rigidus HD-EO, eucalyptol (69.93%) represented the main EO component and represented the major component in C. rigidus HS EO, with 93.32% of the EO. From this we conclude that head space extraction technique was more efficient in isolation of higher percentage of the major EOs components from both plants.

On the other hand, the GC results for the EOS samples obtained from P. gracilior and P. elongatus, monoterpene and sesquiterpene hydrocarbons were the major component of all screened Podocarpus samples. P. gracilior HD-EO, β-caryophyllene (16.14%), germacrene D (15.66%), and α-pinene (12.2%) represented the main EO components, but α-pinene was the major constituent in HS-EO with a percentage of 82.01%. For the EO component of P. elongatus, the major component of the HD-EO was bicyclogermacrene (17.3%) and germacrene D (12.83%), whereas the HS-EO was rich in myrcene, with a percentage of 79.05%.

The percentages estimated for each chemical class varied, as in the case of oxygenated monoterpenes, the major chemical class in the samples of Callistemon species was 41.28 (HD), 39.62 (HS), 11.44 (HD) and 34.49 (HS) for C. subulatus and C. rigidus, respectively, whereas in the case of the Podocarpus species, the major chemical class of monoterpenes hydrocarbons was 18.23 (HD), 96.16 (HS), 7.16 (HD) and 97.53 (HS) for P. gracilior and P. elongatus, respectively. The HD results of the Callistemon and Podocarpous species coincided with previously published reports [36, 49, 50]. On the other hand, the observed variation in the percentages of some of the identified volatile constituents could be due to seasonal and geographical fluctuations [49].

Through the continuous search for a novel antimicrobial chemical entities and also for having safe food products free from pathogens, there is an increasing demand of active natural compounds due to their effectiveness against various pathogens and limited side effects if compared to synthetic chemicals and preservatives. So, natural products are considered as a safer option than synthetic ones. In this study, we investigated the potential of HD-EOs against methicillin-resistant Staphylococcus aureus (MRSA) (Gram-positive bacteria), which is one of the most common nosocomial pathogens responsible for severe mortality worldwide [51]. The antibacterial activities of the obtained EOs against coagulase-negative Staphylococcus (CoNS), Streptococcus viridans, Enterococcus faecalis, and Escherichia coli were also investigated, and also their antifungal activities against Candida albicans were tested using disc diffusion assay and MIC. The results revealed that the hydrodistillation essential oils extracted from P. elongatus, P. gracilior and C. subulatus demonstrated uniform efficacy against MRSA, as evidenced by a zone of inhibition measuring 15 mm. In the case of the tested HD-EOs activities against Gram-positive bacteria (E. faecalis, CoNS, and S. viridans) and Gram-negative (E coli), the HD-EOs of C. subulatus and C. rigidus exhibit robust efficacy, resulting in a zone of inhibition measuring 20 mm, whereas the HD-EOs of P. elongatus, P. gracilior were active only against CoNS. Regarding the efficacy against CoNS, the HD-EOs of C. subulatus and P. elongatus displayed activity, with zones of inhibition measuring 10 mm, whereas the HD-EOs. C. rigidus and P. gracilior showed no discernible activity (Supplementary Figure S5). Minimal inhibition zone was observed against E. faecalis, specifically with the HD-EOs. of C. subulatus and C. rigidus, whereas no activity was observed with the HD-EOs of P. gracilior and P. elongatus. In the case of S. viridans, both C. rigidus and C. subulatus exhibited activity, with zones of inhibition measuring 16 mm, whereas the HD-EOs of P. gracilior and P. elongatus displayed no observed inhibitory effects. Table 3 illustrates the calculated MIC of the tested samples against different clinical isolates revealing the higher potency of HD-EOs against S. viridans with MIC 0.88 and 1.75 µl/ml of C. rigidus and C. subulatus respectively and the higher potency of P. elongatus HD-EOs against CoNS with MIC 0.88 µl/ml. The observed variations in the activities are generally attributed to differences in the peptidoglycan layer located outside the outer cell membrane of the bacteria. On the other hand, the outer membrane of Gram-negative bacteria consists of phospholipids (double layer) connected with lipopolysaccharides (inner membrane). Therefore, large hydrophobic molecules, such as HD-EOs components, can pass through the double membrane [52]. The potent activity of HD-EO P. elongatus is due to the high percentage of nonoxygenated sesquiterpene hydrocarbons represented by germacrene D, which reportedly has antimicrobial activity in the skimmed literature [53], is closely related to the composition of the oil sample. However, the HD-EO of C. rigidus displays potent antimicrobial activity in E. coli and S. viridans because of the high percentage of eucalyptol (oxygenated monoterpene), which has been extensively reported to have high antimicrobial activity [54]. Finally, the synergistic or antagonistic interaction between the HD-EO components could play a vital role in the antimicrobial activity of the tested oil samples.

Table 3 Minimum inhibitory concentrations (MICs) of the extracted EOs against different clinically isolated pathogens using microdilution broth assay
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Cellular transport proteins are integrated membrane proteins that are classified into two major classes. The first class comprises major facilitator transporters (MFS) [55], including lactose permease protein isolated from E. coli [56], which is responsible for the creation of a solute concentration gradient from the energy of the proton gradient. The second class of transport proteins comprises ATPases and ABC transporters, which utilize the energy (Pi) released from ATP to cause solute assembly or efflux. LacY is responsible for the reactions of translocation triggered by the system of galactoside transport in E. coli as one of the oligosaccharide/proton exchanges of the MFS transporters [56]. The inhibition of the efflux pump is considered one of the strategies that is used efficiently in the control of bacterial resistance. Efflux pump inhibitors can work through several mechanisms, such as energy uncoupling, direct binding, inhibition of gene expression, iron chelation, and changes in membrane fluidity, and these inhibitors could constitute the core of antibiotic analogs [57]. Therefore, in this study, direct binding to the efflux protein was assumed to be one of the mechanisms responsible for the observed antibacterial effect. Figure 1 illustrates all the interactions with the amino acids present at the docking site of lactose permease transport protein for ten of the identified volatile oils from all the examined plants. This figure shows the orientation of the examined ligands inside the binding site, which allows the hydroxy group of the examined oxygenated monoterpenes (α-cadinol, tau-muurolol, cadina-3,9-diene, and α-terpineol and eucalyptol) to extend into the hydrophilic cavity and form hydrogen bonds with the side chain amino acid Gln 359 Asn 119 and Trp 151, thereby increasing the stability of the ligand/lactose permease complex. For the examined nonoxygenated hydrocarbons (bicyclogermacrene, germacrene D, cymene, β-caryophyllene, and α-pinene), the results suggest that van der Waals, π-allyl and hydrophobic interactions with Trp151, and His322 amino acids also help stabilize the ligand/lactose permease complexes. Therefore, an interaction with a binding energy range of − 5.53 to -4.05 kcal/mol was observed in volatile oils, with significant binding affinity and stability (Table 4).

Fig. 1
figure 1

a Docking demonstrating the possible positions for the interaction between the Lactose permease and identified volatile oils. b Docking demonstrating the possible positions for the interaction between the Lactose permease and identified volatile oils. c Docking demonstrating the possible positions for the interaction between Lactose permease and identified volatile oils

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Table 4 Binding energies (∆G) of the identified volatile constituents using hydro-distillation (HD), and headspace (HS), within the active sites of NorA efflux pump through molecular docking and the data expressed in kcal/mol
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Conclusion

The quantitative GC analysis of the EOs obtained from C. subulatus, C. rigidus, P. gracilior, and P. elongatus leaves revealed that headspace extraction is more efficient than conventional hydrodistillation in extraction of the major volatile component from each plant. α-Eucalyptol is considered the major discriminating marker of Callistemon species, whereas caryophyllene, germacrene and myrcene are considered the major discriminating marker of Podocarpous species. The antimicrobial investigation of the tested HD-EOs of the four plants showed higher potential against both G-ve and G + ve clinical isolates, more specifically against MRSA. The obtained results are due to the synergism that occurs based on the odoriferous mixtures of oils, which is one of the possible explanation for the obtained efficacy. Another explanation for the efficacy was delivered through in-silico docking screening of the major identified EOs for the first time against Lactose permease transporter and the results revealed moderate binding affinity of the recognized compounds with the target efflux pump enzyme opening the gate for future chemical modification of the pure compounds to improve their binding affinity and increase their antibacterial effect. Overall, this comprehensive study not only provides valuable insights into the diverse EOs chemical compositions and their antimicrobial activities, but also lays the foundation for potential applications in combating bacterial resistance through interactions with membrane transport proteins. These findings contribute to the understanding the mechanism of natural EOs as antimicrobial agents and offer prospects for the development of novel chemical entities.

Data availability

All data generated or analyzed during this study are included in the manuscript and/or its supplementary information files.

Abbreviations

Eos:

Essential oils

GC/MS:

Gas chromatography‒mass spectrometry

HD:

Hydrodistillation

HS:

Head-space solid phase

MH:

Muller Hinton

MIC:

Minimum inhibitory concentration

ZOI:

Zone of inhibition

Lacy:

Lactose permease transporter

MRSA:

Methicillin-resistant Staphylococcus aureus

CoNS:

Coagulase-negative Staphylococcus

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Funding

Open access funding provided by The Science, Technology & Innovation Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank (EKB). This research did not receive any specific grant from funding agencies in the public, commercial, or non-profit sectors.

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  1. Heba A. M. Ezzat and Nermin A. Younis contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacognosy, Faculty of Pharmacy, Ahram Canadian University, Giza, Egypt

    Heba A. M. Ezzat & Nermin A. Younis

  2. Department of Pharmacognosy, Faculty of Pharmacy, Helwan University, Ein Helwan, Cairo, 11795, Egypt

    Amel M. Kamal, Mohamed I. S. Abdelhady & Mohamed S. Mady

  3. Department of Microbiology and Immunology, Faculty of Pharmacy, Ahram Canadian University, Cairo, Egypt

    Mai M. Zafer

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Contributions

NY, HE, MZ, AK, MA and MM designed the study protocol and research points. NY, HE and MM extracted and prepared the essential oils. NY, HE, AK, MA and MM identify the essential oil components. MM carried out the in silico molecular docking study MZ evaluated the in vitro biological studies. All authors have contributed in the writing and revision of the manuscript to be suitable for publication.

Corresponding author

Correspondence to Mohamed S. Mady.

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The study was conducted according to the relevant guidelines of the Ethics Committee at Ahram Canadian University, which approved the study with number REC0824. Experimental research and field studies on plants.

We confirm that all methods were performed in accordance with the relevant guidelines and regulations and complied with relevant institutional, national, and international guidelines and legislation.

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Ezzat, H.A.M., Younis, N.A., Zafer, M.M. et al. Comparative chemical and antimicrobial evaluation of the essential oils from Callistemon and podocarpus species supported by in-silico molecular simulations against bacterial LacY protease. BMC Complement Med Ther 25, 144 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12906-025-04826-w

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Keywords

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

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