Abstract
The increasing prevalence of antimicrobial resistance and metabolic disorders necessitates the exploration of natural products with multifunctional biological activities. This study investigated the chemical composition and pharmacological properties of essential oil obtained from the dried flower buds of Syzygium aromaticum (L.) Merr. & L.M. Perry (clove). The essential oil was extracted by hydrodistillation and analyzed by gas chromatography–mass spectrometry (GC–MS). Biological activities were evaluated through antibacterial, membrane-disruptive, antiplatelet aggregation, acetylcholinesterase and metabolic enzyme inhibitory assays. GC–MS analysis revealed the predominance of phenylpropanoids (92.8%), with eugenol (91.7%) as the major constituent. The essential oil exhibited broad-spectrum antibacterial activity against selected Gram-positive and Gram-negative bacteria, producing significant growth inhibition. Mechanistic studies demonstrated that the oil compromised bacterial membrane integrity, leading to increased membrane permeability and leakage of intracellular constituents. In addition, the oil significantly inhibited platelet aggregation induced by common agonists (IC50 = 1.05 to 3.55) compared to aspirin (IC50 = 1.88 to 4.20), indicating potential antithrombotic properties. The essential oil also showed marked inhibitory activity against acetylcholinesterase, suggesting possible neuroprotective relevance in the management of cholinergic dysfunction. Furthermore, the oil inhibited key metabolic enzymes associated with carbohydrate and lipid metabolisms, supporting its potential role in the management of metabolic disorders. These findings highlight the multifunctional pharmacological potential of S. aromaticum flower bud essential oil as a natural source of antibacterial, antiplatelet, neuroactive, and metabolic enzyme inhibitory agents. Further in vivo studies and toxicological evaluations are warranted to validate its therapeutic applications and elucidate the underlying molecular mechanisms.
1. Introduction
Syzygium aromaticum (L.) Merr. & L.M. Perry, commonly known as clove, is an important medicinal and culinary plant whose dried flower buds are a rich source of essential oil. Clove essential oil is characterized predominantly by phenylpropanoids, particularly eugenol, which contributes substantially to its diverse biological activities [1,2]. Recent studies have reported antibacterial, anti-inflammatory, antioxidant, antiplatelet, and other pharmacological properties of clove essential oil and eugenol [2].
The antibacterial activity of clove essential oil has been associated with disruption of bacterial membrane integrity, increased membrane permeability, leakage of intracellular constituents, and subsequent loss of cellular viability [3]. Eugenol has also demonstrated inhibitory effects on platelet activation and aggregation, while clove essential oil has shown inhibitory activity against acetylcholinesterase and enzymes involved in carbohydrate metabolism [4,5,6]. These findings suggest that clove essential oil may possess multifunctional biological properties extending beyond its established antimicrobial activity. Therefore, this study investigated the chemical composition and selected pharmacological activities of essential oil obtained from the flower buds of clove. The oil was characterized by gas chromatography–mass spectrometry (GC–MS) and evaluated for antibacterial, membrane-disruptive, antiplatelet aggregation, acetylcholinesterase, and metabolic enzyme inhibitory activities. This integrated assessment provides further insight into the multifunctional biological potential of clove bud essential oil.
2. Materials and Methods
2.1. Chemicals, Media, Reagents and Devices
Analytical-grade chemicals, reagents, microbiological media and reference standards were obtained from certified suppliers. Mueller–Hinton agar No. 2 (pH 7.4) and Mueller–Hinton broth (pH 7.4) were manufactured by Oxoid Laboratories (Thermo Fisher Scientific, UK) and supplied through Merck (Sigma-Aldrich, South Africa). Reference antibiotic discs, including Ciprofloxacin susceptibility-testing discs (30 µg per disc) and Whatman No. 1 filter-paper discs (6 mm) were obtained from Merck (Sigma-Aldrich, South Africa). Mueller–Hinton agar and broth used for bacterial cultivation were also supplied by Merck (Sigma-Aldrich, South Africa). 2,2-diphenyl-1-picrylhydrazyl (DPPH), trichloroacetic acid (TCA), thiobarbituric acid (TBA), Griess–Ilosvay reagents (N-(1-naphthyl) ethylenediamine dihydrochloride and sodium nitroprusside), sulphanilic acid reagent (0.33% in 20% glacial acetic acid), assay salts and antioxidant standards (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ascorbic acid and α-tocopherol) were purchased from Merck (Sigma-Aldrich, South Africa). Artemia salina cysts (brine shrimp eggs) were obtained from Fish Designs, Mtunzini, South Africa. The lactate dehydrogenase (LDH) release assay reagent was manufactured by Sigma-Aldrich (USA). Triton X-100, p-iodonitrotetrazolium violet (INT) and glycerol were purchased from Merck (Sigma-Aldrich, South Africa). Platelet-activation agonists—adenosine diphosphate (ADP), collagen and epinephrine—as well as calcium chloride (CaCl2), aspirin and dimethyl sulfoxide (DMSO), were sourced through the same supplier. Enzymes, substrates and reference inhibitors, including acarbose, orlistat, simvastatin, tacrine, p-nitrophenyl-α-D-glucopyranoside, p-nitrophenyl palmitate and p-nitrophenyl butyrate, were also obtained from Merck (Sigma-Aldrich, South Africa). Assay-preparation reagents and materials including dinitrosalicylic acid (DNS), maltose, Tris–HCl buffer, bovine serum albumin, α-naphthyl acetate and Fast Blue B salt were purchased from Merck (Sigma-Aldrich, South Africa). High-purity hydrogen and helium carrier gases and the n-alkane (C9–C24) reference standards were sourced from Afrox (Linde Group, South Africa). The HP 6820 gas chromatograph, HP 6890 gas chromatograph and HP 5973 mass spectrometer were manufactured by Hewlett-Packard/Agilent Technologies (USA). The BioTek ELx808 microplate reader was manufactured by BioTek Instruments (USA). Dried flower buds of Syzygium aromaticum were purchased from Checkers in Empangeni, KwaZulu-Natal, South Africa.
2.2. Plant Material and Essential-Oil Extraction
Dried flower buds of S. aromaticum (L.) Merr. & L.M. Perry were purchased from Checkers Supermarket, Empangeni, KwaZulu-Natal, South Africa. The material was authenticated by Prof. Helen de Wet, Department of Botany, University of Zululand. The dried flower buds (300 g) were crushed and subjected to hydrodistillation for 3 h using a Clevenger-type apparatus following the British Pharmacopoeia procedure [7]. The recovered S. aromaticum essential oil (SaEO) was collected, sealed and stored under refrigeration until analysis.
2.3. GC-FID and GC–MS Analysis
GC-FID analysis was performed using an HP 6820 gas chromatograph equipped with a flame ionization detector and HB-5 capillary column (60 m × 0.25 mm i.d.; 0.25 µm film thickness). The oven temperature was programmed from 50 °C (2 min) to 240 °C at 5 °C/min and held for 10 min. Injector and detector temperatures were 200 and 240 °C, respectively. Hydrogen was used as carrier gas at 1 mL/min, with a split ratio of 1:25. Retention indices were determined using n-alkanes analyzed under the same conditions. GC–MS analysis was performed on an HP 6890 GC coupled to an HP 5973 mass spectrometer fitted with an HP-5MS column (30 m × 0.25 mm i.d.; 0.25 µm film thickness). The oven temperature was programmed from 70 to 240 °C at 5 °C/min; helium was used at 1 mL/min, electron ionization was 70 eV, and the scan range was 35–425 amu. Constituents were identified by comparison of retention indices and mass spectra with authentic standards, Wiley library spectra and published data [8,9].
2.4. Antibacterial Activity
The essential oil was evaluated against thirteen selected bacterial strains comprising Gram-positive and Gram-negative bacteria, obtained from the culture collection of the Applied and Environmental Microbiology Research Group (AEMREG), Department of Biochemistry and Microbiology, University of Fort Hare, Alice, South Africa. The microorganisms included Bacillus cereus (ATCC 10702), Staphylococcus aureus (ATCC 25925), Aeromonas hydrophila (ATCC 7966), Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Serratia marcescens, Listeria grayi, Listeria ivanovii, Vibrio vulnificus, Vibrio fluvialis, and Vibrio parahaemolyticus. Stock cultures were preserved in 20% (v/v) glycerol at −80 °C and maintained under these conditions until required for antimicrobial susceptibility assays.
Briefly, overnight cultures were grown in Mueller–Hinton broth at 37 °C and adjusted to approximately 0.5 McFarland (about 1 × 108 CFU/mL). Agar disc diffusion was performed using Mueller–Hinton agar. Sterile 6 mm Whatman No. 1 discs were loaded with 10 µL of the essential-oil preparation. Plates were incubated at 37 °C for 24 h, and inhibition zones were measured in millimeters [10]. Ciprofloxacin susceptibility-testing discs (30 µg /disc) was reference antibiotic. MIC was determined by broth microdilution using the INT method [11], with concentrations ranging from 5 to 0.04 mg/mL. After 24 h at 37 °C, 0.2 mg/mL INT was added, and color development was assessed after 30–60 min. The MIC was the lowest concentration showing complete or near-complete inhibition of visible growth. MBCs were determined from the MIC assay according to the experimental protocol [11].
2.5. Membrane Damage by Cytosolic LDH Release
Membrane damage was evaluated by cytosolic lactate dehydrogenase (LDH) release, based on the enzyme-release principle as described [12], with modifications. Standardized bacterial cultures were exposed to the essential oil at 4 × MIC for 18–24 h and centrifuged at 5000× g for 5 min. Fifty microliters of supernatant was mixed with 50 µL of LDH release assay reagent (Sigma-Aldrich) and incubated at room temperature for 30 min, and absorbance was measured at 492 nm using a BioTek ELx808 microplate reader. Cultures treated with 3% Triton X-100 served as the maximum membrane-damage control. Membrane damage was expressed relative to the Triton X-100 control.
2.6. Antiplatelet Aggregation Activity
Antiplatelet activity was evaluated using washed rat platelets [12], with modifications. Platelet suspensions were diluted 1:20, and CaCl2 was added to a final concentration of 1.3 mM. Aggregation was induced separately with ADP (5 mM), collagen (5 mM) and epinephrine (10 mM). Platelets (100 µL) were pre-incubated with essential oil (1–10 mg/mL) for 5 min before addition of the agonist. Aggregation was monitored at 415 nm for 20 min at 30 s intervals. Aspirin was the positive reference, and 1% DMSO was the negative control. IC50 values were calculated from concentration–response data.
2.7. Acetylcholinesterase (AChE) Inhibitory Activity
AChE inhibition was determined using the TLC-bioautographic method [5]. Different concentrations of essential oil were applied to silica-gel TLC plates, which were treated with acetylcholinesterase solution prepared in Tris-HCl buffer (pH 7.8) containing 0.1% bovine serum albumin. After incubation at 37 °C for 20 min under humid conditions, plates were sprayed with α-naphthyl acetate, followed by Fast Blue B salt. Inhibition was indicated by clear or bright zones against the colored background. Tacrine was used as the reference inhibitor.
2.8. Metabolic Enzyme Inhibitory Activities
The essential oil was evaluated against pancreatic α-amylase, intestinal α-glucosidase (maltase and sucrase), pancreatic lipase and pancreatic cholesterol esterase using established spectrophotometric methods with appropriate modifications. α-amylase inhibition was determined by the DNS method [13], with maltose used for calibration and acarbose used as the reference inhibitor. Intestinal α-glucosidase inhibition was evaluated using p-nitrophenyl-α-D-glucopyranoside [1], with maltase and sucrase activities assessed separately. Pancreatic lipase and pancreatic cholesterol esterase inhibitions were determined spectrophotometrically using p-nitrophenyl palmitate and p-nitrophenyl butyrate [14]. Appropriate enzyme, substrate and sample controls were included. Percentage inhibition and IC50 values were calculated from concentration–response data.
2.9. Statistical Analysis
All experiments were performed in triplicate, and results were expressed as mean ± standard deviation (SD). IC50 values were calculated from concentration–response curves using Origin 6.0. Data were subjected to one-way analysis of variance (ANOVA), followed by Tukey’s multiple-comparison test where appropriate. Differences were considered statistically significant at p ≤ 0.05 and highly significant at p ≤ 0.01.
3. Results and Discussion
3.1. Chemical Composition
Hydrodistillation of 300 g of dried S. aromaticum flower buds yielded 1.39% (w/w) of pale-yellow essential oil. GC–MS analysis identified four constituents accounting for 94.8% of the total oil composition, with eugenol (91.7%) as the predominant compound (Table 1). The strong dominance of phenylpropanoids (92.8%) is consistent with the characteristic chemical profile reported for clove-bud essential oil [1] and provides a plausible chemical basis for several of the biological activities observed [1,2].
Table 1.
Chemical composition of essential oil of S. aromaticum a.
3.2. Antibacterial and Membrane-Disruptive Activities
Syzygium aromaticum essential oil inhibited 10 of the 12 tested bacteria (Table 2), producing inhibition zones of 10.0–12.0 mm among susceptible organisms. No inhibition was observed against A. hydrophila, P. vulgaris or V. parahaemolyticus under the assay conditions. MIC values ranged from 2.5 to >5 mg/mL. Where both MIC and MBC were determined, the MBC/MIC ratio was 1, indicating bactericidal activity according to the adopted criterion. The activity against both Gram-positive and Gram-negative bacteria agrees with the broad-spectrum antibacterial potential previously reported for clove essential oil [1,3]. LDH release provided experimental evidence of membrane injury, with release values of 51% and 63% recorded for B. cereus and S. aureus, respectively. Additional LDH release was detected in A. hydrophila and L. monocytogenes, as well as in selected Listeria and Vibrio isolates. These findings support membrane injury as an important component of the antibacterial action of SaEO, consistent with the use of LDH release as an indicator of bacterial membrane damage [12]. However, the absence of detectable LDH release in some organisms despite antibacterial activity suggests that membrane injury is unlikely to be the sole mechanism underlying the observed antibacterial effects.
Table 2.
Antibacterial activity of essential oil of Syzygium aromaticum.
3.3. Metabolic Enzyme Inhibitory Activities
The oil exhibited selective inhibition of carbohydrate-digestive enzymes (Table 3). IC50 values were 263.84 µg/mL for pancreatic α-amylase, >250 µg/mL for maltase and 245.30 µg/mL for sucrase, with sucrase showing the lowest measured IC50. No detectable inhibition of pancreatic lipase or pancreatic cholesterol esterase was observed within the tested concentration range. The observed α-amylase inhibition agrees with previous reports of inhibitory activity of clove essential oil against carbohydrate-digestive enzymes [6,14]. The inhibition of maltase and sucrase observed in the present study further indicates that SaEO may affect multiple stages of carbohydrate digestion, whereas its lack of detectable activity against pancreatic lipase and cholesterol esterase suggests greater selectivity toward carbohydrate-digestive enzymes under the conditions tested.
Table 3.
Inhibitory activities of Syzygium aromaticum essential oil (SaEO) against carbohydrate-, lipid- and cholesterol-metabolizing enzymes.
3.4. Acetylcholinesterase and Antiplatelet Aggregation Activities
Syzygium aromaticum essential oil produced 12.39 ± 0.78% inhibition of AChE compared with 96.90 ± 0.63% for tacrine, indicating weak AChE inhibitory activity under the present assay conditions (Table 4). AChE-inhibitory activity has previously been reported for clove-bud essential oil and its major constituent eugenol [14]. The oil produced concentration-dependent inhibition of platelet aggregation induced by ADP, collagen and epinephrine. Over the concentration range of 1–10 mg/mL, inhibition increased from 57.1 ± 0.48 to 74.5 ± 1.47% for ADP, from 53.0 ± 1.15 to 67.4 ± 0.84% for collagen, and from 41.1 ± 0.94 to 53.9 ± 0.55% for epinephrine. The corresponding IC50 values were 1.05, 1.85 and 3.55 mg/mL, respectively, compared with 4.20, 1.88 and 4.15 mg/mL for aspirin. Thus, SaEO showed lower apparent IC50 values than aspirin against ADP- and epinephrine-induced aggregation and a comparable value against collagen under the present in vitro conditions. These findings agree with earlier reports of antiplatelet activity of clove constituents, particularly eugenol [4], and with more recent evidence of eugenol-mediated inhibition of platelet activation [15].
Table 4.
Acetyl cholinesterase and antiplatelet aggregation activities of S. aromaticum essential oil a.
4. Conclusions
The essential oil obtained from S. aromaticum flower buds was dominated by eugenol (91.7%) and exhibited multiple in vitro biological activities. The oil showed bactericidal activity against several susceptible bacteria, while LDH release supported membrane injury as one component of its antibacterial action. It also selectively inhibited carbohydrate-digestive enzymes and substantially inhibited platelet aggregation, whereas AChE inhibition was weak. Overall, these findings highlight eugenol-rich clove essential oil as a promising multifunctional natural-product candidate. However, the activities observed were in vitro and should not be interpreted as evidence of clinical efficacy.
Author Contributions
O.A.L., K.O.A. and M.M.E.E. conceived and designed the structure of the manuscript. O.A.L. isolated the essential oil and carried out the antibacterial activity. K.O.A., S.A.A. and O.E.O. worked on antibacterial and membrane-disruptive data and wrote part of the manuscript. F.O.O. worked on antiplatelet aggregation activity and analyzed the data. R.A.M. worked on metabolic enzyme inhibitory activities and analyzed the data. V.A.D. reviewed, edited and contributed additional literature to the final manuscript. A.R.O., O.A.L., K.O.A., S.A.A. and M.M.E.E. revised the final manuscript. A.R.O. supervised the research work. All authors have read and agreed to the published version of the manuscript.
Funding
This research received no external funding.
Institutional Review Board Statement
Not applicable.
Informed Consent Statement
Not applicable.
Data Availability Statement
The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.
Conflicts of Interest
The authors declare no conflict of interest.
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