Comparative Antibacterial Activity of Ethanolic Extracts of Fenugreek (Trigonella foenum‑graecum L.), Cinnamon (Cinnamomum sp.) and Carob (Ceratonia siliqua L.) Against Escherichia coli and Staphylococcus aureus

النشاط المضاد للبكتيريا المستخلصات الإيثانولية للحلبة (Trigonella foenum graecum L.) والقرفة (Cinnamomum sp.) والخروب (Ceratonia siliqua L.) مقارنةً ضد الإشريكية القولونية (Escherichia coli) والمكورات العنقودية الذهبية (Staphylococcus aureus)

Abdalrhman Hamza Alkhedir Hamza¹

DOI: https://doi.org/10.53796/hnsj67/42

Arabic Scientific Research Identifier: https://arsri.org/10000/67/42

Volume (6) Issue (7). Pages: 655 - 661

Received at: 2025-06-07 | Accepted at: 2025-06-15 | Published at: 2025-07-01

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1 Introduction

Antimicrobial resistance (AMR) poses one of the most pressing global health challenges of the 21st century. The World Health Organization estimates that, without urgent action, AMR could result in up to 10 million deaths annually by 2050 (O’Neill, 2016). The diminishing efficacy of conventional antibiotics has intensified efforts to explore alternative antimicrobial sources, particularly those derived from medicinal plants known for their ethnopharmacological significance and minimal side effects.

Medicinal plants contain structurally diverse secondary metabolites such as tannins, saponins, flavonoids, alkaloids, and essential oils, which exhibit a range of antimicrobial mechanisms including disruption of bacterial membranes, inhibition of nucleic acid and protein synthesis, and interference with microbial signaling pathways (Cowan, 1999; Cushnie & Lamb, 2005). Importantly, these phytochemicals may act synergistically, enhancing antimicrobial potency and reducing the likelihood of resistance development (Wagner & Ulrich-Merzenich, 2009).

In Sudanese traditional medicine, fenugreek (Trigonella foenum-graecum), cinnamon (Cinnamomum sp.), and carob (Ceratonia siliqua) are commonly used for both nutritional and therapeutic purposes. Fenugreek is known for its hypoglycemic, anti-inflammatory, and antimicrobial properties, attributed to its saponin and flavonoid content (Srinivasan, 2006). Cinnamon, rich in cinnamaldehyde, has demonstrated antimicrobial effects against a wide range of pathogens, including antibiotic-resistant strains (Tomaino et al., 2005). Carob pods, though more often consumed for their high fiber and sugar content, also contain polyphenols and furan derivatives withdocumented antioxidant and antibacterial activity (Simsek, 2017).

Despite their individual bioactivities, few studies have systematically compared the antibacterial efficacy of these three botanicals against common bacterial pathogens. Escherichia coli, a Gram-negative bacterium, and Staphylococcus aureus, a Gram-positive organism, are frequently implicated in gastrointestinal, skin, and soft tissue infections, and are recognized for their increasing resistance to conventional antibiotics (Prestinaci et al., 2015).

This study aims to comparatively evaluate the antibacterial activity of ethanolic extracts of fenugreek, cinnamon, and carob against E. coli and S. aureus using standardized microbiological assays. By quantifying their inhibition-zone diameters and bactericidal capacity, this research contributes to the evidence base supporting phytotherapeutic strategies for infection control.

2 Literature Review

A substantial body of literature underscores the antimicrobial properties of fenugreek (Trigonella foenum-graecum), cinnamon (Cinnamomum spp.), and carob (Ceratonia siliqua), although comparative studies remain limited. Fenugreek seeds are rich in steroidal saponins, alkaloids (trigonelline), and polyphenols such as quercetin. Several studies have documented its efficacy against microbial strains. For example, Randhir et al. (2004) found that sprouted fenugreek seeds exhibited enhanced antimicrobial activity due to increased phenolic content. Additionally, Al-Habori and Raman (2002) reported that fenugreek extracts exhibited dose-dependent inhibition against E. coli and S. aureus, suggesting potential for therapeutic use.

Cinnamon bark has been extensively studied for its essential oil content, especially cinnamaldehyde, which demonstrates strong antibacterial and antifungal effects. Friedman et al. (2004) demonstrated that cinnamaldehyde effectively inhibited Salmonella enterica and Staphylococcus aureus, with lower minimum inhibitory concentrations (MICs) than several synthetic antibiotics. Another study by Chang et al. (2001) observed that cinnamon essential oil not only disrupted bacterial membranes but also altered intracellular pH, indicating multiple mechanisms of action. These findings support cinnamon’s role in food preservation and natural medicine.

Carob pods, though traditionally valued for their nutritional content, contain bioactive compounds such as gallic acid, catechins, and 5-hydroxymethylfurfural (HMF) with antimicrobial potential. Daglia (2012) emphasized that polyphenolic-rich extracts like those found in carob possess antimicrobial properties via enzyme inhibition and membrane damage. More recently, Benković et al. (2018) demonstrated that carob pulp extract inhibited both Gram-positive and Gram-negative bacteria, including S. aureus and E. coli, and proposed its application in functional food formulations.

Despite these findings, inconsistencies in extraction techniques, bacterial strains tested, and assay conditions have made it difficult to draw direct comparisons. This study addresses this gap by evaluating the antibacterial effects of fenugreek, cinnamon, and carob extracts under uniform experimental conditions, targeting E. coli and S. aureus as representative Gram-negative and Gram-positive pathogens, respectively (Prestinaci et al., 2015).

3 Materials and Methods

3.1 Plant Material and Extraction

Market‑purchased fenugreek seeds, cinnamon bark and carob pods (Wad Medani, August 2021) were authenticated and shade‑dried. Each 50 g sample was milled (0.5 mm) and refluxed with 500 mL 50 % ethanol for 2 h. Filtrates were concentrated (40 °C) to 10 mL (100 % stock). Serial dilutions yielded 25 %, 50 %, 75 % and 100 % (v/v) working concentrations.

3.2 Test Microorganisms

Clinical isolates of Escherichia coli and Staphylococcus aureus were obtained from the University of Gezira Microbiology Laboratory, confirmed by biochemical tests and maintained on NA slants at 4 °C.

3.3 Colony‑Count Assay

Approximately 2 × 10⁵ CFU mL⁻¹ bacterial suspensions were mixed with molten NA (for S. aureus) or EMB agar (E. coli) and poured into Petri dishes. Sterile glass‑fiber discs (6 mm) impregnated with 20 µL extract were applied. Plates were incubated (37 °C) and colonies enumerated at 6‑h intervals up to 24 h.

3.4 Inhibition‑Zone Assay

IZDs were measured on pre‑seeded agar plates using the same extract concentrations. Measurements were recorded on days 2, 4, 6 and 8 with a digital caliper. Ciprofloxacin (5 µg) served as positive control; solvent discs were negative controls.

3.5 Statistical Analysis

All experiments were performed in triplicate. Data were expressed as mean ± SD. One‑way ANOVA followed by Tukey’s post‑hoc test determined significant differences (p < 0.05). IC₅₀ values were estimated by nonlinear regression (GraphPad Prism 9).

4 Results

This study evaluated the antibacterial activity of ethanolic extracts from fenugreek, cinnamon, and carob using three complementary microbiological assays: CFU enumeration, inhibition zone measurement, and IC₅₀ estimation. The findings demonstrate differential potency among the extracts against both Escherichia coli and Staphylococcus aureus.

4.1 Colony‑Count Reduction

Table 1. Antibacterial Efficacy of Ethanolic Extracts on Colony-Forming Units (CFU) After 24 Hours

Extract (100 %)	E. coli CFU plate⁻¹ (24 h)	Reduction vs. control	S. aureus CFU plate⁻¹ (24 h)	Reduction vs. control
Fenugreek	1 ± 0.2	99 %	4.5 ± 0.3	91 %
Cinnamon	2.5 ± 0.4	97 %	9.9 ± 0.5	75 %
Carob	10.2 ± 0.6	71 %	12.7 ± 0.7	58 %

Fenugreek extract exhibited the strongest bactericidal activity, reducing E. coli and S. aureus counts by 99% and 91%, respectively. Cinnamon also showed strong inhibition, though less potent, while carob had comparatively moderate effects. These results suggest differential phytochemical efficacy against Gram-negative and Gram-positive bacteria.

4.2 Inhibition‑Zone Diameters

Table 2. Inhibition Zone Diameters (mm) of Plant Extracts at Varying Concentrations

Concentration (%)	Fenugreek IZD (mm)	Cinnamon IZD (mm)	Carob IZD (mm)
25%	7.4 ± 0.2	8.1 ± 0.3	8.0 ± 0.4
50%	9.0 ± 0.3	10.5 ± 0.2	10.0 ± 0.3
75%	11.2 ± 0.4	11.9 ± 0.3	11.6 ± 0.4
100%	10.2 ± 0.3	10.7 ± 0.2	12.7 ± 0.5

All extracts demonstrated a dose-dependent increase in inhibition zone diameter up to 75%, after which fenugreek and cinnamon plateaued, while carob continued to show increased efficacy at 100%. This may reflect the differential release and diffusion of active compounds at higher concentrations.

4.3 Dose–Response and IC₅₀

Extract potency against E. coli followed the order Fenugreek > Cinnamon > Carob with IC₅₀ values of 0.38, 0.52 and 0.67 (fraction of full strength), respectively. Similar ranking occurred for S. aureus (0.42, 0.55, 0.70).

5 Discussion

The results of this study highlight the differential antibacterial potency of fenugreek, cinnamon, and carob ethanolic extracts against E. coli and S. aureus. The superior activity of fenugreek aligns with its well-documented phytochemical composition, notably rich in steroidal saponins, flavonoids, and unsaturated fatty acids such as linoleic and palmitic acids. These compounds are known to compromise bacterial membrane integrity, disrupt metabolic pathways, and induce oxidative stress (Srinivasan, 2006; Thomas et al., 2011; Al-Habori & Raman, 2002; Pandey & Awasthi, 2015). The observed near-complete reduction in E. coli CFUs (99%) and significant inhibition of S. aureus confirms fenugreek’s broad-spectrum efficacy.

Cinnamon’s notable antibacterial activity is consistent with the high concentration of trans-cinnamaldehyde, its principal volatile constituent. Cinnamaldehyde has been shown to inhibit bacterial quorum sensing and biofilm formation, mechanisms essential for pathogenicity and antibiotic resistance (Gill & Holley, 2004; Matan et al., 2006; Chang et al., 2001). The inhibition zone data and CFU reduction strongly support its efficacy, particularly against E. coli, which is in line with prior findings (Friedman et al., 2004).

Carob exhibited the weakest activity among the three, though it still demonstrated measurable antibacterial effects. This result is consistent with its phytochemical profile, which includes high sugar content and comparatively lower levels of polyphenolics and flavonoids. However, the presence of tannins and 5-hydroxymethylfurfural (HMF) may contribute to its moderate inhibitory effects through membrane disruption and oxidative damage (Simsek, 2017; Daglia, 2012; Benković et al., 2018). This suggests possible synergistic action despite the less aggressive antibacterial profile.

Interestingly, all three extracts showed greater activity against E. coli than S. aureus. This could reflect inherent structural differences between Gram-negative and Gram-positive bacteria. While S. aureus possesses a thick peptidoglycan layer, E. coli has an outer membrane rich in lipopolysaccharides that may be more susceptible to phytochemical disruption. Furthermore, efflux pump mechanisms in S. aureus may reduce intracellular accumulation of active compounds (Cowan, 1999).

The observed non-linear dose–response curve, particularly the reduced inhibition zone diameter for fenugreek at 100% concentration, could suggest solubility constraints, compound precipitation, or antagonistic interactions between certain metabolites at higher concentrations (Wagner & Ulrich-Merzenich, 2009). This highlights the importance of considering formulation dynamics and compound synergy when developing plant-based antimicrobial therapies.

Overall, the study reinforces the antimicrobial promise of traditional Sudanese botanicals and supports their potential application as natural antibacterial agents in food safety and therapeutic formulations.

6 Conclusions

All three Sudanese natural products possess antibacterial properties, with fenugreek showing the greatest bactericidal effect and cinnamon a strong bacteriostatic action. Carob, though less potent, still offers measurable inhibition and could serve as a functional food preservative. Future work should (i) fractionate extracts to pinpoint active compounds, (ii) evaluate synergy with standard antibiotics, and (iii) assess cytotoxicity to ensure safety for therapeutic use.

Acknowledgements

The author thanks the Food Analysis and Microbiology Laboratories, University of Gezira, for technical support, and Dr M. Saeed for statistical guidance.

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