Tetradecylamine: A Newly Identified Biogenic Amine Compound from the Venom of Vespa affinis

TL;DR

Scientists found a new compound in Asian hornet venom that can fight bacteria and may work better than some antibiotics.

Contribution

Identification of tetradecylamine in Vespa affinis venom and its potential as an antibacterial agent with high binding affinity.

Findings

Tetradecylamine showed inhibitory effects against E. coli, S. aureus, B. cereus, and K. pneumoniae at low concentrations.

Molecular docking studies revealed tetradecylamine binds strongly to PBP2X and MurA proteins, better than some antibiotics.

Tetradecylamine's binding affinity was comparable to or higher than doxycycline and gentamycin in tests.

Abstract

The venom of the Asian hornet (Vespa affinis) contains a diverse array of biologically active compounds that contribute to its defensive and predatory functions. This study aimed to confirm the presence of tetradecylamine in V. affinis venom using the computational analysis software MetFrag, as well as to predict its biosynthetic pathway and potential biological functions. Bioinformatic analysis suggested that tetradecylamine may be involved in antibacterial activity, which was subsequently validated through in vitro antibacterial assays. The compound exhibited significant inhibitory effects against Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Klebsiella pneumoniae at low concentrations. Furthermore, molecular docking studies demonstrated that tetradecylamine interacts favorably with penicillin-binding protein 2x (PBP2X) and UDP-N-acetylglucosamine enolpyruvyl transferase (MurA), a key enzyme involved in bacterial cell wall synthesis. Notably, tetradecylamine showed a higher predicted binding affinity than several commonly used antibiotics.

The venom of the Asian hornet (Vespa affinis) comprises a complex mixture of biologically active substances, including various enzymes such as phospholipase A and hyaluronidase; amines such as histamine, serotonin, and catecholamines; peptides such as mastoparan and vespakinin; and other components including acetylcholine and antigen 5. This complexity reflects the highly evolved nature of V. affinis as a venomous insect. The composition of animal venoms often exhibits a certain degree of variability, making the study of biogenic amines particularly intriguing. The objective of this research was to confirm and identify the presence of tetradecylamine in the venom of Vespa affinis using the scientific computational analysis software MetFrag. In addition, the study aimed to construct the biosynthetic pathway of this compound and to predict its potential biological roles. The predicted biosynthetic route of tetradecylamine suggested its possible involvement in antibacterial activity. Antibacterial assays were performed against four bacterial strains Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Klebsiella pneumoniae. The results revealed that tetradecylamine exhibited notable inhibitory effects, with minimum inhibitory concentration (MIC) values of 2, 4, 8, and 4 µg/mL, and minimum bactericidal concentration (MBC) values of 2, 4, 8, and 4 µg/mL, respectively. Furthermore, molecular docking studies were conducted using penicillin-binding protein 2x (PBP2x, PDB ID: 5OIZ) as the target protein. Among eight tested ligands, streptomycin exhibited the highest binding affinity with a docking score of 64.76. In contrast, biogenic amines such as 2-phenylethylamine and tetradecylamine showed docking scores of 33.74 and 48.2, respectively. In the MurA protein (PDB ID: 3VCY), the biogenic amine ligand tetradecylamine exhibited a binding affinity comparable to that of certain reference drugs. Specifically, tetradecylamine achieved a GOLD score of 52.58, whereas ampicillin showed a higher score of 61.53. Notably, tetradecylamine demonstrated a higher binding affinity to the target protein compared with certain conventional antibiotics such as doxycycline and gentamycin.

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1. Introduction

The Asian giant hornet (Vespa affinis), a member of the order Hymenoptera and the family Vespidae, is widely distributed throughout tropical and subtropical regions of Southeast Asia. Morphologically, it is characterized by a black body with yellow or orange bands and measures approximately 2 cm in length. The venom apparatus, located at the posterior end of the abdomen, comprises paired acid and alkaline glands connected to a retractable chitinous stinger. Unlike honeybees, V. affinis can sting multiple times without losing its stinger, thereby enabling both defensive and predatory functions [1,2].

The venom of V. affinis is a complex biochemical cocktail and well-known to contain a mixture of high-molecular-weight (e.g., phospholipase A_1_, hyaluronidase, antigen 5, dipeptidyl peptidases, and other enzymes) and low-molecular-weight compounds (biologically active amines). These components are related in tissue degradation, promote venom diffusion, and potentiate cytotoxicity at the envenomation site. Collectively, they can induce diverse physiological and pathological effects, including inflammation, pain, and hypersensitivity reactions. Multiple stings may result in severe systemic effects and can be fatal [3,4,5].

These findings reflect the biochemical complexity and diverse pharmacological roles of V. affinis venom. Consequently, it has attracted interest as a model system for understanding venom composition within the order Hymenoptera. However, not only macromolecules but also low-molecular-weight constituents particularly biogenic amines and neurotransmitters are important components of the venom. To date, the research on V. affinis venom has focused predominantly on proteins and peptides [3,4,5,6].

The chemical diversity of non-protein and non-peptide venom components remains incompletely studied. One class of low-molecular-weight venom constituents (molecular weight < 200 Da) is biogenic amines (BAs), which are derived from amino acid decarboxylation and include histamine, dopamine, octopamine, and polyamines [1,7]. Several BAs remain insufficiently studied. Previous research on V. affinis venom has identified common wasp venom BAs, such as histamine, serotonin, and acetylcholine, which contribute to vasodilation, pain sensation, and neuromuscular excitation [8,9].

Notably, long-chain aliphatic amines have not yet been investigated in V. affinis venom. Among these compounds, tetradecylamine is an amphiphilic primary amine with well-documented membrane-active and antimicrobial properties. To date, no publications have reported the presence of tetradecylamine or related long-chain aliphatic amines in V. affinis venom. This lack of information represents a significant gap in the current understanding of the venom’s chemical complexity and biological function. Salem et al. reported that tetradecylamine exhibits significant antimicrobial activity in vitro and reduces viable bacterial counts in saliva, with efficacy comparable to that of chlorhexidine [10]. Despite this functional potential, the presence of tetradecylamine or other long-chain aliphatic amines in V. affinis venom has not been explored, highlighting an important area for further investigation. However, molecular docking was used to study possible molecular interactions between small compounds and host biological targets, providing mechanistic insight that supports experimental findings [11,12].

The aim of this study is to study and identify the presence of tetradecylamine in V. affinis venom using in vitro and in silico approaches. By investigating its biochemical and physicochemical properties, this work focuses on expanding the current understanding of venom chemical diversity and providing novel insights into the contribution of small aliphatic amines to venom bioactivity. These findings may be valuable for elucidating the pharmacological and toxicological significance of such compounds.

2. Materials and Methods

2.1. Venom Sample Collection

Venom from V. affinis was collected by carefully excising the terminal abdominal segment to expose the venom sac or gland. The venom sac was immediately transferred into phosphate-buffered saline (PBS) to preserve bioactive components. The sac was homogenized using a high-speed homogenizer until a uniform suspension was obtained, followed by filtration through a sterile 0.45 µm syringe filter. The filtrate was subsequently lyophilized to obtain venom in powdered form and stored at −80 °C for long-term preservation [13].

2.2. Identification of Biogenic Amines in Vespa affinis Venom

Biogenic amines (BAs) in V. affinis venom were HPLC-MS/MS, adapted from Zhang [14]. Lyophilized venom powder (0.5 mg) was dissolved in 500 µL of deionized water and mixed with 500 µL of 6% trichloroacetic acid (TCA). The solution was centrifuged at 12,000× g for 10 min, and the supernatant was collected into a 1.5 mL centrifuge tube, yielding a final concentration of 0.5 mg/mL. The solution was filtered using a 0.45 µm nylon membrane syringe filter and analyzed via HPLC-MS/MS, columns C_18_ (Intertsil OSD-4 C_18_, 250 mm × 3.0 mm, 5 μm), flow rate 0.3 mL/min, injections 20 µL, ESI+ ionization mode. The mobile phase consisted of (A) 0.1% formic acid in water and (B) methanol. The system was initially maintained at 100% mobile phase A for the first 2 min. This was followed by a rapid change to 55% mobile phase A over the next 2 min. Subsequently, the proportion of mobile phase A was gradually decreased to 2% over an additional 2 min. The analysis was then continued until a total run time of 12 min.

Confirmed using high-performance liquid chromatography (HPLC), adapted from Figueiredo et al. [15], the following HPLC grade reference standards were procured from Tokyo Chemical Industry (TCI) Metropolis, Japan: HPLC (98.0% purity). The certified purity levels were confirmed by the manufacturer’s certificates of analysis (CoA). Spike in V. affinis venom sample 0.6 mg. Agilent 1260, fortis C18 reversed-phase HPLC column (4.6 mm × 250 mm i.d., 5 µm), flow rate 1 mL/min, injections 20 µL, detected by UV detector 254 nm. The mobile phase consisted of (A) acetonitrile and (B) water. The mobile phase gradient began with 60% phase A, increased to 75% over 9 min, rapidly shifted to 80% in 1 min, increased to 95% over 2 min, and reached 100% over 3 min, then gradually decreased back to 60% over 5 min, maintained at 60% for an additional 5 min, with a total runtime of 25 min.

2.3. Antibacterial Activity Assay

The antibacterial activity of 2-phenylethylamine and tetradecylamine derived from V. affinis venom was evaluated against Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Klebsiella pneumoniae to determine the minimum inhibitory concentration (MIC). Stock solutions of the test compounds, along with standard antibiotics (ampicillin, chloramphenicol, streptomycin, gentamicin, and doxycycline) were prepared at concentrations ranging from 1 to 4096 µg/mL. Serial two-fold dilutions were performed in 96-well microplates with 20 µL of each test solution added per well containing 10^6^ CFU/mL bacterial suspensions. Plates were incubated at 37 °C for 24 h, and MIC was defined as the lowest concentration exhibiting a clear, non-turbid well. All experiments were performed in triplicate [16,17,18].

2.4. Outer Membrane Permeability Assay

The outer membrane permeability of bacteria treated with biogenic amines was assessed using the fluorescent probe 1-N-phenylnaphthylamine (NPN) following overnight bacterial cultures grown in 5 mL nutrient broth at 180 rpm were harvested and centrifuged at 10,000× g for 2 min. Cells were washed and resuspended in HEPES buffer containing 20 mM glucose at pH 7.4 to an optical density of 0.5 at 600 nm. Bacterial suspensions (50 µL) were mixed with 50 µL of HEPES buffer containing 20 µM NPN in black 96-well plates. Biogenic amines were added to each well, and fluorescence was monitored immediately at an excitation wavelength of 350 nm and emission at 420 nm for 10 min at 30-s intervals using a microplate reader. NPN uptake was calculated using the equation: [16,19]

$$[eqn]$$

where

Fobs is the fluorescence of treated bacteria;

Fcontrol is the fluorescence of untreated bacteria; and

FB is the fluorescence of NPN in buffer alone.

2.5. Scanning Electron Microscope

Bacterial strains used in this study were cultured in liquid nutrient broth until reaching the logarithmic growth phase. The cultures were then diluted to achieve a final cell density of 1 × 10^8^ CFU/mL (corresponding to an optical density at 600 nm of 0.1). Aliquots of 900 µL of bacterial suspension were incubated with 100 µL of biogenic amine at half of the minimum inhibitory concentration (half MIC) at 37 °C for 16 h. After incubation, samples were centrifuged at 12,000× g for 10 min to collect bacterial pellets. The pellets were washed twice with phosphate-buffered saline (PBS) and fixed in 4% glutaraldehyde for 2 h.

Following fixation, bacterial cells were dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and 100%), then coated with gold particles. The structural effects of the biogenic amines on bacterial cells were subsequently examined using scanning electron microscopy (SEM). Untreated bacteria, not exposed to biogenic amines, served as the negative control for comparison.

2.6. Molecular Docking

Molecular docking studies were performed using GOLD Suite v.5.2.2 (Genetic Optimization of Ligand Docking), which employs a genetic algorithm (GA) to predict ligand-protein binding interactions. Necessary files, including target protein structures (.pdb or .mol2), ligand structures (.mol2 or .sdf) preparation using PyMOL (version 3.1.6.1) software. Docking parameters, including GA population size, number of generations, and scoring functions (GoldScore, ChemScore, ASP, or PLP), were set via GUI or command line. Parameters and scoring functions could be adjusted iteratively to optimize docking accuracy and predictive reliability. Docking runs generated output files that were visualized in BIOVIA Discovery Studio 2024 Client to analyze ligand conformations and binding affinities.

3. Results

3.1. Computational Analysis of Tetradecylamine in Vespa affinis Venom by Metfrag

Among all biogenic amines identified in hornet venom in this study, tetradecylamine accounted for 5.46% of the total composition as shown in Supplementary Table S1. Raw data from HPLC-MS/MS analysis of Vespa affinis venom revealed that tetradecylamine produced eight fragment ions with m/z values of 55.0542, 57.0699, 71.0855, 77.0855, 81.0699, 102.0006, 114.9470, and 214.253 (Figure 1A). The m/z values and corresponding peak intensities (Height) of each fragment were subsequently analyzed using the MetFrag software (https://msbi.ipb-halle.de/MetFrag/ accessed on 8 September 2023), incorporating the NORRMANSusDat_20Nov2019 database for candidate compound identification. MetFrag ranked tetradecylamine as the first candidate, yielding a final score of 1.0 and the highest library score within the platform, indicating a precise match between the observed fragments and the predicted fragmentation pattern. This computational workflow confirms the identity of tetradecylamine in the venom matrix and demonstrates the utility of integrating HPLC-MS/MS data with in silico metabolite annotation for accurate compound verification (Figure 1B). In addition, MetFrag analysis revealed that the fragment peaks observed in the HPLC–MS/MS spectra could be assigned to specific fragmentation sites within the parent tetradecylamine structure. MetFrag analysis enabled the annotation of three fragment ions that matched the parent compound, tetradecylamine. These fragments (highlighted in green on the left side of the figure) represent alkyl-chain–derived ions generated from characteristic cleavages along the linear hydrocarbon backbone of the molecule. Specifically, the detected fragment ions were assigned as [C_4_H_8_]^+^ (m/z 55.05), [C_4_H_9_]^+^ (m/z 57.07), and [C_5_H_11_]^+^ (m/z 71.09), which originate from stepwise cleavage of the alkyl chain. Such fragmentation patterns are typical for long-chain primary amines and are consistent with the proposed chemical structure of tetradecylamine, comprising a linear C14 alkyl chain terminated with a primary amine (–NH_2_) group (Figure 1C).

3.2. Identification of Tetradecylamine in Vespa affinis Venom by HPLC

High-performance liquid chromatography (HPLC) was employed to analyze the chemical constituents present in the venom samples, comparing chromatograms of unspiked samples (Sample Chromatogram without Standard; Figure 2, bottom) and samples spiked with authentic standards (Sample Chromatogram with Standard Spiked In; Figure 2, top). In the unspiked sample, peaks of moderate to high intensity were observed, but their identities could not be conclusively determined. In contrast, upon spiking with reference standards, the chromatogram exhibited peaks at defined retention times corresponding to the added compounds, with significant increases in peak intensity for the pre-existing signals. Comparison of the two chromatograms revealed marked increases in peak heights at approximately 3.09 min and 22.50 min, corresponding to tetradecylamine pose, thereby confirming the presence of these compounds in the venom. The initial ambiguity in the unspiked sample is attributed to their relatively low concentrations, which produced less distinct peaks.

This approach demonstrates the utility of standard spiking in HPLC analysis for unambiguous identification and verification of target compounds in complex biological matrices.

3.3. Antibacterial Activity Assay

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were determined using the broth microdilution and drop plate methods in Table 1, The results revealed that tetradecylamine and the positive control antibiotic ampicillin exhibited inhibitory and bactericidal activity against Escherichia coli, Staphylococcus aureus, Bacillus cereus, and Klebsiella pneumoniae. The experimental results are summarized as follows.

According to Table 1, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of tetradecylamine and the positive control antibiotic ampicillin against E. coli were 0.0625 µg/mL and 2 µg/mL for MIC, and 0.125 µg/mL and 2 µg/mL for MBC, respectively. For S. aureus, the MIC and MBC values of tetradecylamine and ampicillin were 0.25 µg/mL and 4 µg/mL for MIC, and 0.5 µg/mL and 4 µg/mL for MBC, respectively. In the case of K. pneumoniae, both MIC and MBC values of tetradecylamine and ampicillin were found to be 2048 µg/mL and 8 µg/mL, respectively. Similarly, for B. cereus, the MIC and MBC values of tetradecylamine and ampicillin were 2048 µg/mL and 4 µg/mL, respectively.

These results indicate that tetradecylamine exhibits antibacterial activity against both Gram-positive and Gram-negative bacteria, with varying levels of efficacy depending on the bacterial species.

3.4. Outer Membrane Permeability Assay

The fluorescent probe 1-N-phenylnaphthylamine (NPN) was employed to evaluate the ability of biogenic amines to enhance the permeability of the bacterial outer membrane. Under normal conditions, NPN cannot efficiently penetrate the outer membrane of bacteria and therefore exhibits only low fluorescence intensity in aqueous environments. However, NPN displays markedly increased fluorescence when present in hydrophobic environments, such as within phospholipid regions.

In this result, the excitation and emission wavelengths were set at 350 nm and 420 nm, respectively (Figure 3). When biogenic amines affect the permeability of the bacterial outer membrane, NPN can readily associate with the hydrophobic phospholipid components of the bacterial membrane, resulting in a measurable increase in fluorescence intensity.

In this study, the outer membrane permeability of E. coli, S. aureus, K. pneumoniae and B. cereus, was evaluated. Upon exposure to tetradecylamine, a rapid increase in membrane permeability was observed, as evidenced by the immediate rise in fluorescence intensity following the addition of the biogenic amine.

The experimental results demonstrated that tetradecylamine exhibited fluorescence intensity levels comparable to or even equivalent to those of the antibiotic used as the positive control. This finding indicates that tetradecylamine possesses a strong ability to disrupt the integrity of the bacterial outer membrane, suggesting its potential as an effective membrane-permeabilizing agent.

3.5. Scanning Electron Microscope

This method allows high-resolution visualization of morphological alterations induced by biogenic amines, providing insights into their antibacterial mechanisms at the cellular level [16].

The effects of biogenic amines on bacterial cell morphology were examined using scanning electron microscopy (SEM). The observations revealed that all tested biogenic amines induced notable alterations in the surface structure of bacterial cells compared with the untreated control group (Figure 4A–D). After incubation of E. coli, S. aureus, K. pneumoniae and B. cereus with biogenic amines at half of their MIC values for 16 h, the bacterial surfaces exhibited visible pore formation and significant disruption of cell membrane integrity. In several areas, partial rupture of the outer membrane was clearly observed, as indicated by red arrows in the micrographs (Figure 4E–G). Additionally, cytoplasmic membrane blebs were prominently detected in bacterial cells treated with the biogenic amines, suggesting membrane stress and localized damage. Similar morphological alterations were also observed in B. cereus (Figure 4H), where discontinuities in the cell membrane were apparent, accompanied by cytoplasmic leakage from disrupted regions.

These SEM observations provide strong evidence that biogenic amines compromise bacterial membrane integrity, supporting their proposed role in enhancing membrane permeability and inducing bacterial cell damage.

3.6. Molecular Docking

As shown in Table 2, the molecular docking analysis illustrated the binding interactions of four ligands 1S6, UD1, ampicillin, and tetradecylamine—with two bacterial target proteins, penicillin-binding protein 2x (PBP2x, PDB ID: 5OIZ) and UDP-N-acetylglucosamine enolpyruvyl transferase (MurA, PDB ID: 3VCY). The docking results revealed that the ligands exhibiting the strongest binding affinity toward PBP2x (5OIZ) were 1S6, ampicillin, and tetradecylamine, with docking scores of 60.81, 52.86, and 48.11, respectively. In contrast, the ligands with the highest binding affinity to MurA (3VCY) were UD1, ampicillin, and tetradecylamine, with docking scores of 81.06, 61.53, and 52.58, respectively.

The reliability of the docking results was confirmed by the root mean square deviation (RMSD) values of 0.911 Å and 1.893 Å, both of which were less than 2 Å, indicating high accuracy and reproducibility of the docking simulations (Figure 5).

4. Discussion

Venom samples of the Asian hornet (V. affinis) were collected from adult individuals during July to September, corresponding to the rainy season in the northeastern region of Thailand. This period was selected due to increased hornet activity and venom availability. Following collection, the venom was extracted and processed into a powdered form. Previous studies have demonstrated that venom preserved as a dried or powdered sample maintains the stability of its bioactive components for extended periods, making this form suitable for chemical and biological investigations [20,21]. The study of tetradecylamine presence in V. affinis venom was conducted using HPLC-MS/MS to characterize the venom composition. Prior to instrumental analysis, proteinaceous biomolecules were removed from the venom extract, as earlier reports have indicated that proteins constitute approximately 50–60% of the total venom composition and may interfere with the detection of low-molecular-weight compounds [22]. This preprocessing step enhanced the sensitivity and accuracy of small-molecule identification.

Structural confirmation of the selected biogenic amine was performed using computational analysis with the MetFrag software, which identifies compounds based on characteristic fragmentation patterns derived from mass spectrometry data [23]. Fragment ion spectra were matched against curated databases of natural products, including the NORRMANSusDat_20Nov2019 database to increase confidence in compound identification [24,25]. Through this approach, the tetradecylamine was identified with high confidence and selected for further investigation. By analyzing the chemical constituents of V. affinis venom, the chromatogram of unspiked sample showed several-intensity peaks of biogenic amine, which tetradecylamine identity could not be conclusively assigned. Upon spiking with the tetradecylamine standard, a clear increase in peak intensity was detected, corresponding to the tetradecylamine pose. Both chromatograms confirmed that this pre-existing peak split with the standard, thereby verifying the presence of tetradecylamine in the V. affinis venom. These results suggested that standard spiking is an effective HPLC approach for identification of low-abundance small amines in complex venom matrices.

As predictive inhibitory results, it found that tetradecylamine possesses antibacterial properties. Thereby, antibacterial activity was evaluated in vitro against four bacterial strains, namely E. coli, S. aureus, B. cereus, and K. pneumoniae. The results demonstrated that tetradecylamine exhibited strong antibacterial effects against all tested strains. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values were identical for each bacterium, indicating bactericidal activity and preventing mutation in the bacterial strains [26].

To elucidate the antibacterial mechanism of tetradecylamine, molecular docking studies were conducted using penicillin-binding protein 2x (PBP2x; PDB ID: 5OIZ) as the target protein. PBP2x is a key enzyme involved in bacterial cell wall synthesis and is a well-established target of β-lactam antibiotics [27]. Penicillin-binding proteins (PBPs) are essential enzymes involved in the final stages of bacterial cell wall biosynthesis, particularly in the transpeptidation process of peptidoglycan assembly [28]. Notably, tetradecylamine showed strength binding toward both targets, suggesting a potential dual inhibitory profile. These in silico results support the possible contribution of tetradecylamine to the antimicrobial activity observed in the V. affinis venom.

Several studies reported molecular docking and other in silico approaches to investigate the binding of small molecules to the active or allosteric sites of PBP2x, aiming to elucidate ligand–protein interaction patterns and predict inhibitory potential. These computational strategies provide valuable insights into the molecular mechanisms of inhibition and support the rational design of new antibacterial compounds [29,30,31]. Computational approaches targeting MurA have proven to be effective tools for guiding structure-based drug design and prioritizing candidate antibacterial compounds prior to experimental validation [32,33,34].

To further support the proposed mechanism, outer membrane permeability was evaluated using the 1-N-phenylnaphthylamine (NPN) fluorescence assay. The results revealed increased membrane permeability in bacteria treated with tetradecylamine, consistent with the molecular docking predictions [35]. In addition, scanning electron microscopy (SEM) was employed to examine bacterial cell morphology following treatment. SEM images demonstrated pronounced structural damage, including membrane rupture, pore formation, and severe disruption of the outer cell envelope in tetradecylamine-treated bacterial cells [36].

Venoms from insects and other animals are complex mixtures containing a variety of bioactive peptides and small molecules with both antimicrobial and cytotoxic properties. Numerous studies have shown that venom peptides, such as mastoparans identified in hornet venom, exhibit potent antibacterial activity against both Gram-positive and Gram-negative bacteria, suggesting a role for specific venom components in anti-microbial effects observed for whole venom extracts [6]. Similarly, antibacterial, and hemolytic peptides such as crabrolin from V. crabro venom have been reported to disrupt bacterial membranes and contribute significantly to venom’s bioactivity [37]. Beyond peptides, venoms also contain low-molecular-weight amines that may influence irritant effects, and while detailed studies on such compounds remain limited, analogous venom systems (e.g., bee and snake venoms) demonstrate that non-peptide components can modulate both antimicrobial and inflammatory responses. Based on this evidence, it is possible that tetradecylamine or other small venom constituents could be at least partially responsible for the antibacterial activity and irritant effects attributed to whole hornet venom and discussing this possibility may provide additional mechanistic insight into venom bioactivity [38].

Beyond its antibacterial activity, tetradecylamine and related long-chain aliphatic amines associated with lipid bilayers, and longer alkyl chains promote deeper membrane insertion and stronger membrane disruption, resulting in increased cytotoxicity. Zhang et al. showed that long-chain aliphatic amines, including hexadecylamine and related surfactants, induce significant cytotoxic effects in human cell cultures through a chain-length-dependent, membrane-disruptive mechanism [39]. These observations support a possible role for tetradecylamine in V. affinis venom as a local irritant and membrane-active component that may enhance tissue damage and facilitate the entry of co-injected venom peptides and enzymes beyond its antimicrobial activity.

Overall, the antibacterial activity assays, molecular docking analysis, membrane permeability studies, and morphological observations were in strong agreement. These findings indicate that tetradecylamine exerts its antibacterial effects primarily through disruption of the bacterial cell envelope and inhibition of cell wall synthesis. The results suggest that tetradecylamine, a biogenic amine identified from V. affinis venom, represents a promising lead compound for the development of novel antibacterial agents.

5. Conclusions

This study successfully identified and characterized tetradecylamine as a newly detected biogenic amine from the venom of V. affinis. The compound exhibited notable antibacterial activity against both Gram-positive and Gram-negative bacteria, with evidence suggesting that its mechanism of action involves disruption of bacterial cell membrane integrity. Molecular docking and fluorescence permeability assays confirmed its strong interaction with bacterial membrane-associated proteins. These findings provide new insights into the bioactive composition of wasp venom and highlight tetradecylamine as a potential lead molecule for the development of novel antimicrobial agents. Future research should focus on elucidating its biosynthetic pathway, optimizing its antibacterial potency, and assessing its cytotoxicity and pharmacological properties in biological models.