In Vitro Antimicrobial Efficacy Assessment of Ethanolic and Aqueous Extracts of Cinnamon (Cinnamomum Verum) Bark against Selected Microbes

ABSTRACT

To combat pathogens and reduce the major public health problem of antibiotic residues in animal products, scientists are looking for natural antibiotic substitutes that are effective against drug-resistant pathogenic microbes and spoilage fungi. The antimicrobial activity of three Cinnamomum verum extracts prepared with three different solvents (absolute ethanol, 50% ethanol, and aqueous extracts) was determined against two Gram-positive bacteria (Staphylococcus aureus and Listeria monocytogenes) and two Gram-negative bacteria (Salmonella Typhimurium and Escherichia coli) as well as two fungal strains. The antimicrobial activities of various Cinnamomum verum extracts against selected microbes were evaluated using the disc diffusion test, minimum inhibitory concentrations (MIC), minimum bactericidal concentrations (MBC), minimum fungicidal concentrations (MFC), and the poisoned food technique.

Cinnamomum verum bark (CVB) extracts inhibited and killed microbial growth to varying degrees. Our findings also revealed that extracts prepared with alcoholic solvents, particularly absolute ethanol-CVB extract, were more active compared with aqueous solvents, suggesting that the cinnamon plant is a promising natural antimicrobial agent for food preservation.

Keywords:
Antibacterial activity; Antifungal activity; Cinnamomum verum bark; Phytogenic

INTRODUCTION

Antibiotic resistance is a major public health issue caused by antibiotic residue in animal products. Antibiotic-resistant microbes have emerged as a result of excessive and arbitrary antibiotic use (Jayalakshmi et al., 2017; Samanta & Bandyopadhyay, 2019). Thus, researchers have renewed their efforts to reduce the risk of antimicrobial resistance by seeking natural antibacterial agents (phytocompounds) that are effective against drug-resistant pathogenic bacteria (Sharma et al., 2018). Foodborne pathogens including Salmonella, Escherichia coli, and Staphylococcus aureus (St. aureus), as well as Listeria monocytogenes (L. monocytogenes) are among the hazards that cause significant concern in the food industry worldwide (Paswan & Park, 2020). Because of their potential pathogenicity, there is a significant impact on public health, the cost of treating these infections, and economic implications for exporters. L. monocytogenes is associated with a high mortality rate (approximately 20%) and is regarded as one of the most dangerous foodborne illnesses (Rothrock Jr et al., 2017). Between 1999 and 2019, Salmonella was the most common foodborne pathogen in African food exports to the European Union (Somorin et al., 2021). Salmonella Typhimurium has been isolated from eggs and egg-associated cases of human salmonellosis in Europe, and has been linked to outbreaks involving infected laying flocks and contamination of egg contents (Wales & Davies, 2011). Fungal contamination of food, feed, and other agricultural products leads to massive spoilage or decay and a variety of food safety issues (Saladino et al., 2016).

Medicinal plants and spices have received considerable attention in this respect. Active compounds may be extracted from herbal and traditional medicines used to treat disease and to improve health (Sam, 2019). Many plants, including cinnamon (Cinnamomum verum), have medicinal properties. Cinnamon is an aromatic evergreen tree with oval-shaped leaves, thick bark, and berry fruit (Vangalapati et al., 2012). The leaves and bark of the cinnamon tree have aromatic oils. Significant attention has been given to cinnamaldehyde extracted from cinnamon, a major constituent, because of its numerous biological activities including fragrance and a spicy taste produced by the absorption of oxygen (Yeh et al., 2014). The plant has also shown antioxidant activity (Sharma et al., 2016), antibacterial effects (Friedman, 2017; Nematollahi et al., 2020), antiviral activity (Fauvelle et al., 2017), antifungal properties (Rahemi et al., 2015), antidiabetic activity, cholesterol lowering effects (Faghani et al., 2014), and anticancer activity (Hong et al., 2016).

Although cinnamon has been studied for its antimicrobial activity, a comparison of its activity using extracts at high concentration has not been thoroughly investigated. In the present study, cinnamon extracts prepared with hot water, 50:50 ethanol and water, and 100% ethanol were compared for their antimicrobial activity against four bacteria (St. aureus, L. monocytogenes, Escherichia coli, and Salmonella enterica subspecies Typhimurium) and two fungal isolates (Aspergillus fumigatus and Aspergillus niger).

MATERIALS AND METHODS

This manuscript does not require IRB/IACUC approval because there are no human and animal participants.

Proximate Analysis of Cinnamomum verum Bark

Cinnamon (Cinnamomum verum) dry bark was purchased in Riyadh, Saudi Arabia, from a local herb store. The bark was crushed and ground into a fine powder (particle size: 0.25-0.30 mm) when it arrived. The cinnamon bark powder was directly examined. The water content, crude protein, crude fat content, crude fiber content, and ash content of the dried ground cinnamon bark were determined using a drying oven, the Kjeldahl method (N X 6.25) with a 2020 digester and a Velp UDK 140 distillation unit, the Soxhlet apparatus, a Dosi-Fiber, and combustion of the dried samples at 600°C for 6 hours. Based on AOAC-International (2006), the acid detergent fiber content was determined, and the neutral detergent fiber content was analyzed according to Holst (1973). A bomb calorimeter was used to calculate gross energy (kcal/kg). The data were all expressed in terms of dry matter.

Phytochemical Analysis of Cinnamomum verum Bark

The dried CVB (20 g) was macerated with 200 mL of 50% methanol at 25°C for 24 hours, then filtered, and centrifuged. Methanol was evaporated as described by Humeera et al. (2013). The supernatant was then injected into a high-performance liquid chromatography (HPLC) column in the amount of 10µL. Agilent Technologies series HPLC equipped with Zorbax RP-C₁₈ column (1.0 cm long × 4.6 mm i.d., Agilent Shimadzu, Japan) was used for the analysis. Gallic acid, catechin, caffeine, and chlorogenic acid were used as external standards to identify the presence of phenols, flavonoids, alkaloids, and polyphenols, respectively. The ultraviolet absorbance at 280 nm was measured for detection. The mobile phase was a mixture of water, acetonitrile, ethyl acetate, glacial acetic acid, and methanol (89:6:3:1:1 v/v/v/v/v) and flowed at a rate of 1 mL/min. All chromatographic analyses were carried out at a temperature of 25°C ± 2°C.

Cinnamomum verum bark extract was analyzed using GC-MS on an Agilent 6890N gas chromatograph outfitted with an Agilent HP-5MS column and a 5973N selective mass detector. The oven temperature was raised from 60-320°C at a rate of 6°C/min. The constituents were recognized by comparing their mass spectra, retention indices, and bioactive constitutes quality to those in the National Institute of Standards and Technology database library (NIST-based AMDIS software).

Preparation of CVB-derived extracts

The dried CVB powder extracts for antimicrobial testing were prepared using the method described by Muthuswamy et al. (2008). Briefly, CVB extracts were prepared using three solvents: absolute ethanol (C100EOH), 50% ethanol (C50EOH) and pure distilled water (DW) alone (CWA).

Then, 150 g each of CVB powder was weighed and distributed into flasks (50 g/flask). Each container was mixed thoroughly in a tightly sealed flask and separately dissolved 1:10 (w/v) in 500 mL sterile distilled water for water extraction, 500 mL of ethanol/water (2:2, v/v) for ethanol/water extraction, and 500 mL of absolute ethanol for ethanol extraction. The flasks were then boiled for 30 min, 45 min, and 5 h with reflux at 37°C in a water bath. Next, the supernatants were collected and filtered through 0.22 µm Millipore membrane filters (Whatman filter paper No. 1) into new sterile flasks. The filtrate was completely dried or evaporated using a 45°C oven or using a rotary evaporator (Eyela, Irvine, CA). Prior to in vitro antimicrobial testing, the dried cinnamonic extracts were weighed and dissolved in dimethyl sulfoxide (DMSO) to obtain stock concentrations of 500 mg/ mL of each cinnamonic extract.

Determination of extract yield (% Yield)

The percent yield (%, w/w) of each dried extract was calculated by dividing the extract’s weight by the cinnamon bark powder weight: Yield (%) = (W₁ × 100)/W₂, where W₁ is the extract weight after the solvent dried and W₂ is the weight of the powder of cinnamon bark before extraction (Julianti et al., 2017).

Antibacterial activity of CVB extracts

Microorganisms and preparation of bacterial inoculum

The reference strain of two Gram-positive bacteria: St. aureus American Type Culture Collection (ATCC) 29737 and L. monocytogenes ATCC 13932, two Gram-negative bacteria: Escherichia coli ATCC 25922 (E. coli) and Salmonella enterica subsp. Typhimurium (S. Typhimurium) ATCC 14026 were obtained from standard stocks in our Laboratory at King Saud University.

The bacterial inoculate was created using the standard protocol of the Clinical and Laboratory Standards Institute (CLSI, 2006). Bacterial strains were activated for 24 h by suspending in nutrient broth before subculturing on sterile nutrient agar plates. To ensure the bacteria were fully activated, subculturing was repeated three times. The inoculum suspension from each experiment was created by taking four or five pure colonies from a new culture. The suspensions of the colonies were suspended in sterile normal saline and diluted sequentially until the turbidity was comparable to a 0.5 McFarland turbidity standard. This yielded an inoculum density of approximately 10⁸ colony-forming units (CFU)/mL.

Disc-Diffusion Susceptibility Test

Cinnamon bark extracts (absolute ethanol, 50% ethanol, and aqueous extract) were tested for antibacterial activity against several bacteria (St. aureus, L. monocytogenes, E. coli, and S. Typhimurium) using an agar diffusion test based on the standard protocol of the with minor modification. Briefly, after adjusting for the turbidity of the inoculum suspension with McFarland turbidity standards, the adjusted suspensions of microbes were gently swabbed onto sterile nutrient agar plates with a sterile cotton swab. The inoculum was dried in a safety cabinet (Nuaire Class II biological safety cabinet, Plymouth, MN, USA) for 30 min. Whatman filter paper (No. 3) was used to prepare circular antibiotic assay discs with a 6 mm diameter. The sterile dried disks were aseptically applied to the inoculated agar plates after soaking in 40 µL of each cinnamon extract. The plates were left at 4°C for 1 h before incubating at 37°C for 24 h. The diameter of the inhibition zones were then measured (in mm). Inhibition zones with diameters of <12, 12 to 16, and >16 mm were classified as having low, moderately active, and highly active antibacterial activity, respectively (Indu et al., 2006). Microbes were tested for sensitivity to the standard antibiotics, piperacillin (100 mg) and erythromycin (15 mg), as positive controls. Paper disks soaked with the corresponding extraction solvent (DMSO) were used as negative controls. The tests were done in triplicate.

Minimum Inhibitory Concentration Determination

The minimum inhibitory concentration (MIC) of the cinnamon extracts was determined using the CLSI protocol and the microdilution method (a microplate method) (CLSI, 2012). The MIC was calculated by selecting the lowest concentration of each CVB extract that did not result in visible bacterial growth (Zhang et al., 2016). The adjusted inoculum suspensions of bacteria were further diluted (1:100) to 10⁶ CFU/mL. The test was performed using sterile 96-well flat microtiter plates (Nunclon, Denmark). Each well contained 50 µL of sterile Mueller Hinton broth (MHB) as a diluent. Then, twofold serial dilutions were prepared by taking 50 µL from the stock solution (500 mg/mL) of each extract into the first well and mixing the broth with the extract, then transferring 50 µL from the first well to the next and so on and discarding 50 µL from the last well. We prepared 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2,048, and 1:4,096 dilutions in order to obtain 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98, 0.49, and 0.24 mg/mL concentrations of the extract, respectively, using 50% DMSO. Then, 50 µL of the adjusted inoculum (10⁶ CFU) was added to each well except for the negative control wells. Positive controls were adjusted inoculum suspensions without the extract, whereas negative controls consisted of the medium only. The plates were incubated at 37°C for 18 h, and the results were visually read and confirmed through measurements with a spectrophotometer plate reader (Bekkar et al., 2021). Wells with absorbance values comparable to the positive control indicated the presence of turbidity or bacterial growth, whereas wells with the lowest extract concentration and absorbance comparable to the negative control indicated complete growth inhibition and were considered the MIC value.

Minimum bactericidal concentrations (MBC) determination

The MBC is defined as the smallest concentration of antimicrobial agent required to completely inhibit bacterial growth. To calculate the MBC, 10 µL from the wells with no bacterial growth (no bacterial turbidity) was inoculated into sterile nutrient agar plates. Plates inoculated with bacteria served as a positive control, whereas plates containing only nutrient agar served as negative controls. Bacterial growth was measured after 48 h of incubation at 37°C. The experiment was conducted in triplicate.

Antifungal activity

To test the activity of CVB extracts against pure fungal strains Aspergillus niger ATCC 6275 and Aspergillus fumigatus ATCC 28282, the agar disk diffusion method (ADDM) and the poisoned food technique were used. The MIC and MFC were also determined. Aspergillus fumigatus was obtained from our lab’s standard incubator and Aspergillus niger was obtained from the King Saud University’s College of Food and Agriculture’s Laboratory of Food Microbiology.

Preparation fungal spores

The fungal suspension was prepared by the methodology of European Committee on Antimicrobial Susceptibility Testing-Antifungal Susceptibility Testing Subcommittee methodology against Aspergillus spp (EUCAST-AST- ASPERGILLUS) (Lass-Flörl et al., 2006). Briefly, fresh, mature (2 to 5 days old) cultures are grown on sterile potato dextrose agar at 35°C to prepare inoculum suspensions. The colonies were covered with 1 mL of sterile water to which 0.1% Tween 20 was added. The conidia was collected rationally with a sterile cotton swab and transferred to a sterile tube. A rotating vortex blender at 2,000 rpm was used to uniformize the inoculum for 15 seconds. Then the conidia was diluted with sterile water counted in a hemocytometer and examined for hyphae and clumps. In case a significant number of hyphae or clumps were present (> 5% of the fungal structures), the inoculum was filtered through a sterile nylon grid filter with a pore size of 11 mm. Then the suspension was adjusted with sterile DW to 2-5 x 10⁶ CFU/mL by counting the conidia in a hemocytometer. This suspension was then further diluted at 1:10 ratio with sterile DW to give a final working inoculum of 2-5 x 10⁵ CFU/mL. All adjusted suspensions were quantified by plating on SDA plates.

Miconazole nitrate (20 mg/g) was dissolved in DMSO at a concentration of (20mg/mL) and stored at -20°C. The stored solutions were then diluted to the prescribed concentrations used in this study. The final concentration of DMSO in the reaction mixture was limited to less than 1% (v/v) (Oh et al., 1993).

Antifungal susceptibility tested by the disk diffusion method

This technique was used to determine the suppressive activities of CVB extracts against Aspergillus niger and Aspergillus fumigatus according to Naeini et al. (2009). Briefly, 10 µL of an adjusted suspension of Aspergillus niger or Aspergillus fumigatus was spread evenly on a potato dextrose agar plates. The inoculated plates were allowed to dry at room temperature in a safety cabinet. Then, blank paper disks with a thickness of 5 mm were impregnated with 40 µL of selected herbal extracts on the surface of the previously inoculated plates. The plates were parafilm-sealed and incubated at 35°C for 48-72 h. Each plate was examined after 48-72 h of incubation. The Diameter of inhibition zone (DIZ) including the diameter of the disk were both measured. Miconazole nitrate and DMSO-soaked paper disks were used as positive and NC, respectively. The experiment was performed in triplicate.

Determination of minimum inhibitory concentrations (MICs)

The MICs of CVB extracts on Aspergillus niger and Aspergillus fumigatus were determined using a microplate method as described by Lass-Flörl et al. (2006). The adjusted inoculum suspensions (containing conidia or sporangiospores) were mixed with a vortexer (2-5 x10⁵ CFU/mL). The extracts of CVB were dissolved in DMSO (10% of final volume) and diluted to a concentration of 500 mg/mL. Sterile 96-well flat microtiter plates (Nunclon, Denmark) were used for this assay. Each well contained 100µL of sterile RPMI 1640 supplemented with 2% glucose as test medium. Then, serial twofold dilutions (the concentration range obtained was between 250 and 0.24 mg/mL) of each of the stock solutions of the extracts (500 mg/mL) were performed. Then, with the exception of the NC, 100 µL of the adjusted inocula was added to each well. The diluted inoculum suspensions without extracts served as active controls and the medium used without extracts was used as NC. The plates were incubated in the incubator at 35°C for 48 h. The MIC was defined as the lowest concentration of the extracts in the wells that demonstrated no visible growth.

Determination of minimum fungicidal concentrations (MFC)

From the wells that showed no growth 10µL were taken and inoculated onto potato dextrose agar plates to determine MFC. The plates inoculated with fungi were served as positive controls and the plates with only potato dextrose agar were served as NC. The plates were incubated at 35°C for seven days and fungal growth was assessed. The MFC was defined as the highest dilution (lowest concentration of extracts) at which no growth occurred on the plates. The test was carried out in three replicates.

Poisoned food technique

According to Shrestha and Tiwari (2009), the antifungal activity of the extracts was evaluated using the poisoned food procedure. A 0.5 mL of each extract (250 mg/mL concentration) was poured into the Petri plate, followed by the addition of 9.5 mL of sterile melted potato dextrose agar medium to a final volume of 10 mL and then gently swirled to achieve thorough mixing of the contents. After solidification, an Aspergillus niger or Aspergillus fumigatus inoculum disk of 5 mm in diameter was aseptically inoculated upside down in the center of the plates and incubated at 30°C for 7 days. The plates with potato dextrose agar without extracts were served as NC and the plates with miconazole nitrate were served as PC. Subsequently, the average diameters of fungal growth were measured on the 7^th day of incubation and then used to compute an inhibition of mycelial growth (Rao & Srivastava, 1994). The percentage (%) of inhibition of mycelial growth was determined as [(g_c- g_t) / g_c] ×100, where g_c is the average increase in mycelial colony growth in the NC and g_t is the average increase in mycelial colony growth on the plates treated with the selected extracts.

Statistical analysis

The Statistical Analysis System (SAS (2012) software was used to analyze the data. The antimicrobial efficacy was expressed as the mean ± standard deviation (SD), and a one-way variance analysis was used to determine whether there were differences in the parameters between groups. The means of data were separated using Duncan’s new multiple range test for statistical differences. P-values less than 0.05% (p < 0.05) were considered statistically significant.

RESULTS

Proximate analysis and phytochemicals of CVB powder

Proximate analysis revealed the macronutrients in CVB with varying rations, as shown in Table 1. Caffeine concentration was 700 g/g, with a retention time of 5.164 min, an area of 132732, and a height of 2865 measured using HPLC test. In addition, another unknown component was larger than caffeine (Figure 1). Therefore, GC-MS was used to detect the major volatile compounds of CVB extracts. A total of 22 different volatile compounds (Cinnamaldehyde and other 21 volatile compounds) were identified, as shown in Figure 2 and Table 3. Hexadecanoic acid, methyl ester, pentadecanoic acid- 14-methyl- methyl ester, Cinnamaldehyde, (E)-2-propenal, 3-phenyl-, oxime- methoxy-phenyl-, 2-methyl-benzofuran, and Tridecanoic acid, methyl ester were the compounds with the highest quality detected by GC-MS in the CVB extract.

Table 1
Nutrient composition (% DM) of Cinnamomum verum bark powder through approximate analysis. ¹

Item	%
Moisture (% as fed)	10.29
Dry matter (% as fed)	89.71
Crude protein	4.43
Ether extract (Lipid)	4.03
Ash	3.18
Total crude fiber	24.35
2Nitrogen-free extract	64.02
3Organic matter	96.82
Total fiber fractions:
A. Acid detergent fiber	45.75
B. Neutral detergent fiber	65.34
Gross Energy (Kcal/Kg)	4974.52

• ¹
  NB: Average of three replicates. ²NFE (calculated by difference) = 100 - (protein+lipid+ash+fiber), ³Organic matter=100- Ash.

Table 2
Contents of individual phenols and flavonoids (µg/g) of Cinnamon determined by high performance liquid chromatography.

Items1	Retention time (min)	Area	Height	Concentration (µg /g)
Caffeine	5.164	132732	2865	0.003
Catechin	-	-	-	-
Chlorogenic acid	-	-	-	-
Gallic acid	-	-	-	-

• ¹
  External standards have reputable antioxidant, anti-inflammatory, and antimicrobial activities were examined.

Figure 1
Cinnamomum verum chromatogram of flavonoids and phenols mixture at 280 nm obtained by high performance liquid chromatography.

Table 3
Compounds identified in methanolic extract of Cinnamomum verum bark.

Retention Time (RT) (min)	Bioactive chemical constituents	Quality	Molecular weight (amu)	Molecular Formula
4.108	4-Ethylbenzoic acid, 2-butyl ester	78	206.131	C13H18O2
4.108	2-Amino-6-methylbenzoic acid	59	151.063	C8H9NO2
4.211	Oxime-, methoxy-phenyl-	87	151.063	C8H9NO2
4.211	4-Ethylbenzoic acid, hexyl ester	50	234.162	C15H22O2
7.152	Hexamethylcyclotrisiloxane	56	222.056	C6H18O3Si3
7.152	Silane, 1,4-phenylenebis[trimethyl-	50	222.126	C14H26Si2
11.329	Cinnamaldehyde, (E)- 2-Propenal, 3-phenyl-	97	132.058	C9H8O
11.329	Benzofuran, 2-methyl-	87	132.058	C9H8O
11.329	Benzopyrimidine, 3,4-dihydro- 3,4-Dihydroquinazoline	59	132.16	C8H8N2
11.329	2-Propenoyl chloride, 3-phenyl-, (E)-	53	166.6	C9H7ClO
11.329	4-Methylcoumarine-7-cinnamate	53	306.3	C19H14O4
11.329	2-Nitrophenyl cinnamamide	50	268.085	C16H14N2O4
11.329	3-Butenoic acid, 2-oxo-4-phenyl-	50	176.17	C10H8O3
25.536	Hexadecanoic acid, methyl ester	99	270.256	C17H34O2
25.536	Pentadecanoic acid, 14-methyl-, methyl ester	98	270.5	C17H34O2
25.536	Tridecanoic acid, methyl ester	83	228.37	C14H28O2
25.536	Nonadecanoic acid, methyl ester	70	312.303	C20H40O2
25.536	Octadecanoic acid, methyl ester	62	298.287	C19H38O2
25.536	Methyl tetradecanoate	58	242.225	C15H30O2
25.536	Pentadecanoic acid, methyl ester	58	256.24	C16H32O2
25.536	Hexadecanoic acid, 15-methyl-, methyl ester	58	284.272	C18H36O2
25.536	Heptadecanoic acid, methyl ester	58	284.272	C18H36O2

Figure 2
Cinnamomum verum chromatogram of flavonoids and phenols mixture at 280 nm obtained by high performance liquid chromatography.

Cinnamon extracts yield

The yield from grinding 500 g of cinnamon bark was 475 g (95% w/w). The extraction solvent had a significant impact on yield. Fifty grams of cinnamon bark powder was extracted and concentrated using a 45°C oven. The final weight of the CVB extracts was 7.00, 6.75, and 6.24 g for 6.24, 6.75 and 7.00 g for C100EOH, C50EOH and CWA extract resulting in a yield percentage of 12.48, 13.50 and 14.00 (w/w), respectively.

Antibacterial activity

Screening for antimicrobial activity

The antibacterial effects of the three cinnamon bark extracts against four bacterial isolates (St. aureus, L. monocytogenes, E. coli, and S. Typhimurium) were evaluated using the disc-diffusion antibiotic susceptibility test. The growth inhibition zones (mean ± SD) are presented in Table 4. Cinnamon extracts were found to have inhibitory effects against all of the tested isolates. For this test, the inhibition zones with a diameter greater than 6 mm were considered positive.

Table 4
Zone of inhibition (mm) of Cinnamomum verum bark extracts against selected bacteria.

Microorganism	C100EOH	C50EOH	CWA	Piperacillin	SEM	p-value
Salmonella Typhimurium ATCC 14026	14.33±2.1a	10.33±2.1bA	09.0±1.0bA	17.67±1.7a	1.08	0.0018
Escherichia coli ATCC 25922	12.8 ± 2.5b	08.2±0.3cB	09.7±0.6cdA	16.17±1.3a	0.87	0.0008
Staphylococcus aureus ATCC 29737	13.0±1.0b	06.67±0.3cB	06.67±0.3cB	19.33±0.9a	0.456	<0.0001
Listeria monocytogenes ATCC 13932	11.33±1.2b	06.67±0.3cB	06.83±0.3cB	17.83±0.2a	0.363	<0.0001
Standard error mean (SEM)	1.031	0.618	0.354	0.833
p-value	0.307	0.009	0.001	0.142

• Abbreviations: C100EOH, 100% ethanol; C50EOH, 50% ethanol; CWA, water alone cinnamon extraction; SEM. Values are mean±standard deviation of triplicate experiment; ^a-c Rows with different letters indicate statistically significant differences (p<0.05). ^A,B Columns with different letters indicate statistically significant differences (p<0.05).

Compared to standard antibiotics (piperacillin), the extracts had the least significant antibacterial activity, but C100EOH similar effect to piperacillin. Piperacillin has been reported to have similar antimicrobial activity against these organisms when used as a standard. The Piperacillin antibiotic and absolute (100%) ethanol extract treatment resulted in the highest inhibition zone (17.7 and 14.33 mm diameter, respectively) against S. Typhimurium (p < 0.002) compared with the 50% ethanol and aqueous extracts, which exhibited inhibition zones with diameters of 10.3 and 9.0 mm, respectively. When compared with cinnamon extracts, absolute ethanol extract treatment resulted in the largest diameter of inhibition zone (12.8, 14.3, 13.0, and 11.3 mm) against E. coli (p=0.0008), S. Typhimurium (p<0.0008), St. aureus (p<0.0001), and L. monocytogenes (p<0.0001). Therefore, the absolute ethanol extract (100% ethanol) was more active compared to the other Cinnamon extracts.

C100EOH has been reported to have the largest DIZ but showed insignificant effect against tested bacteria (p=0.307). However, C50EOH and CWA extracts of cinnamon bark were found to have different significantly antibacterial activity against the tested bacteria. C50EOH has been reported to have higher significantly (p=0.009) antibacterial activity against S. Typhimurium, and CWA has been reported to have higher significantly (p=0.001) antibacterial activity against S. Typhimurium and E. coli compared to other tested bacteria.

A microplate dilution test

The MIC and MBC values of the cinnamon extracts against St. aureus, L. monocytogenes, E. coli, and S. Typhimurium are summarized in Table 5. The CNB extracts have bacteriostatic and bactericidal effects on tested microbes at those MIC and MBC concentrations. The C100EOH showed antibacterial activity with MIC and MBC values of 6.51-31.25 mg/mL and 13.02-62.50 mg/mL, respectively. The MIC and MBC values for C100EOH were S. Typhimurium = St. aureus ≤ E. coli < L. monocytogenes. While, the C50EOH showed antibacterial activity with MIC and MBC values of 3.26-62.50 mg/mL and 6.51-125 mg/mL, respectively. The MIC and MBC values of C50EOH were in S. Typhimurium ≤ St. aureus = E. coli < L. monocytogenes. Furthermore, CWA extract exhibited the antibacterial activity with MIC and MBC values of 7.81-62.50 mg/mL and 15.62-125 mg/mL, respectively. The MIC and MBC values of CWA were in S. Typhimurium = E. coli ≤ St. aureus < L. monocytogenes.

Table 5
Minimum inhibitory concentrations (MIC) and Minimum bactericidal concentrations (MBC) values for Cinnamomum verum bark extracts on selected bacteria.

Microorganism	C100EOH	C50EOH	CWA	SEM	Probability
	Minimum inhibitory concentrations (MIC)
Salmonella Typhimurium ATCC 14026	6.51±2.3aB	3.26±1.1bB	7.81±0.0aC	0.796	0.021
Escherichia coli ATCC 25922	7.81±0.0B	6.51±2.3B	7.81±0.0C	0.799	0.422
Staphylococcus aureus ATCC 29737	6.51±2.3bB	6.51±2.3bB	15.63±0.0aB	0.957	0.001
Listeria monocytogenes ATCC 13932	31.25±0.0bA	62.50±0.0aA	62.50±0.0aA	1.06	<0.0001
Standard error mean	1.031	0.618	0.354
p-value	<0.0001	<0.0001	<0.0001
	Minimum bactericidal concentrations (MBC)
Salmonella Typhimurium ATCC 14026	13.02 ± 4.5aB	6.51±2.3bB	15.62±0.0aC	1.67	0.021
Escherichia coli ATCC 25922	15.62±0.0B	13.02±4.5B	15.62±0.0C	1.501	0.422
Staphylococcus aureus ATCC 29737	13.02±4.5bB	13.02±4.5bB	31.26±0.0aB	2.123	0.001
Listeria monocytogenes ATCC 13932	62.50±0.0bA	125.0±0.0aA	125.0±0.0aA	0.001	<0.001
Standard error mean (SEM)	1.838	1.945	0.001
p-value	<0.0001	<0.0001	<0.0001

• Abbreviations: Abbreviations: C100EOH: 100 percent ethanol; C50EOH: 50 percent ethanol; and CWA: water alone are used to extract Cinnamomum verum bark. Means of triplicates ±standard deviation. The lowest the value, the better; a,b Rows with different letters indicate statistically significant differences (p < 0.05). A-C Columns with different letters indicate statistically significant differences (p < 0.05). The concentrations of each extract were in the range of 500-0.12 mg.

Based on the MIC and MBC of the selected bacteria, the highest MIC (62.50 mg/mL) and MBC (125 mg/mL) were found in the presence of C50EOH and CWA against L. monocytogenes. On the contrary, the lowest MIC and MBC values were observed when C50EOH was tested with S. Typhimurium. Thus, the result revealed that the MIC values of all the extracts obtained from CVB tested here against selected bacteria were different and found to be the most active extracts. This was because these extracts had the lowest MBC values against tested bacteria except against L. monocytogenes. Very effective antimicrobial activity with bactericidal properties was observed at different concentration ranges against all clinical bacteria tested in this study. The results suggest that the phenols, tannins and flavonoids present in the herbal extracts might have increased toxicity against the pathogenic microorganisms.

Antifungal activity

The antifungal activity of CVB extracts against tested fungi (Aspergillus fumigatus and Aspergillus niger) using ADDM is presented in Table 6. The sensitivity test revealed antifungal inhibitory activity of C100EOH against Aspergillus fumigatus, with a DIZ of 11.33 mm, while the C50EOH and the CWA showed no inhibitory activity on Aspergillus fumigatus. The C100EOH and the CWA had inhibitory activity against Aspergillus niger, while the C50EOH had no inhibitory activity against Aspergillus niger. The CWA showed a stronger effect against Aspergillus niger than the other CVB extracts, with DIZ of 22.00 mm. Although C100EOH had an effect on fungal growth in both Aspergillus fumigatus and Aspergillus niger with a lethal effect with lowest MFC, the effect on these two isolates was less than the DIZ for miconazole. Therefore, C100EOH were the best fungi static and fungicidal compared to other cinnamon extracts.

Table 6
In vitro antifungal activity (Zone of inhibition, minimum inhibitory concentration (MIC), and minimum fungicidal concentration (MFC)) of Cinnamomum verum bark extracts against Aspergillus fumigatus ATCC 28282 and Aspergillus niger ATCC 6275.

Fungal type/Extracts	C100EOH	C50EOH	CWA	Miconazole	SEM	Probability
	Inhibition zone (mm)
Aspergillus fumigatus	11.3±1.7b	6.0±0.0c (R)	6.0±0.0c (R)	15±0.82a	0.667	<0.0001
Aspergillus niger	14.0±1.6b	6.0±0.0c (R)	22.0±1.0a	20±1.87a	1.051	<0.0001
	MIC (mg/mL)
Aspergillus fumigatus	13.0±3.7b	62.5±0.0a	62.5±0.0a	-	2.575	<0.0001
Aspergillus niger	1.3±0.5b	6.5±1.8b	13.1±0.0a	-	1.692	0.008
	MFC (mg/mL)
Aspergillus fumigatus	26.0±7.4b	125.0±0.0a	125.0±0.0a	-	4.515	0.0079
Aspergillus niger	2.6±0.9b	13.0±3.9b	26.5±0.0a	-	13.048	<0.0001

• Abbreviations: C100EOH: 100 percent ethanol; C50EOH: 50 percent ethanol; and CWA: water alone are used to extract Cinnamomum verum bark; Means of triplicates ±standard deviation; The lowest the value, the better. ^a-c Row with different letters indicate statistically significant differences (p<0.05). R, resistance only disk zone “6 mm” without inhibition. (-) no tested. SEM: Standard error mean.

Table 7 showed the inhibitory effects of three CVB extracts on mycelial growth of selected fungi using food poisonous method. The cinnamon extracts showed high inhibitory activity (p<0.05) against Aspergillus fumigatus compared to the control; the degree of inhibition ranged from 59 to 100%, and the highest inhibitory activity was for C100EOH and C50EOH extracts (100% each), as the mycelial growth and speed were zero for both extracts. The CWA extract showed moderate inhibitory activity (59%) and had moderate mycelial growth (16.0±0.0) and mycelial velocity (3.64±0.03).

Table 7
In vitro antifungal activity of Cinnamomum verum bark extracts on mycelial growth of Aspergillus fumigatus (ATCC 28282) and Aspergillus niger (ATCC 6275) using food poisonous method.

Fungal type/ Extracts	C100EOH	C50EOH	CWA	Control (0 mg/mL)	SEM	Probability
	Mycelial growth (mm)
Aspergillus fumigatus	0.0±0.0c	0.0±0.0c	16.0±0.1b	39.00±0.0a	0.001	<0.0001
Aspergillus niger	1.0±0.0c	19.3±0.0b	79.0±0.0a	79.00±1.0a	0.882	<0.0001
	Mycelial growth speed
Aspergillus fumigatus	0.0±0.0b	0.0±0.0b	3.6±0.03a	3.8±0.2a	0.056	<0.0001
Aspergillus niger	1.7±0.1c	4.1±0.6b	21.0±0.0a	21.0±0.0a	0.190	<0.0001
	Growth inhibition (%)
Aspergillus fumigatus	100.0±0.0a	100.0±0.0a	59.0±0.1b	0.0±0.0c	0.001	<0.0001
Aspergillus niger	98.7±0.0a	83.1±3.6b	0.0±0.0c	0.0±0.0c	1.116	<0.0001

• The values are means of three replicates ± standard deviation; C100EOH: 100 percent ethanol; C50EOH: 50 percent ethanol; and CWA: water alone are used to extract Cinnamomum verum bark. a-d Row with different letters indicate statistically significant differences (p<0.05). SEM: Standard error mean.

Similarly, the C100EOH extract exhibited the highest inhibitory activity (p<0.05) against Aspergillus niger. The inhibition threshold was 83.1±3.59 and 98.7±0.0 based on mycelial growth (19.33±0.0 mm and 1.0±0.0 mm) and mycelial velocity (4.13±0.66 and 1.69±0.03) for C50EOH and C100EOH, respectively. The water extract did not illustrate any inhibitory effect against Aspergillus niger as it could not prevent mycelial growth (79.0±0.0 mm diameter) and mycelial velocity (21.0±0.0).

In general, the ethanol extracts of CVB showed the highest (p<0.05) inhibitory effect (≥83.1≤100%) on the growth of the selected fungi, while the water extract showed no or the least effect. In this in vitro experiment, miconazole was also used as a standard for antifungal activity, and it displayed high activity (15±0.82 mm for Aspergillus fumigatus and 20±1.87 mm for Aspergillus niger). The inhibition of mycelial growth of the tested fungi was 98.7-100%, 83.1-100%, and 0.0-59% for the extract of C100EOH, C50EOH, and CWA, respectively.

DISCUSSION

As far as we recognize, the search for herbal extracts with antimicrobial properties has increased due to their potential uses in a variety diseases. The current research focused on well-known medicinal plant, namely Cinnamomum verum. This is the first study to investigate the antimicrobial activity of CVB extracts using three solvents (absolute ethanol, 50% ethanol and water) against Gram-positive bacteria such as St. aureus and L. monocytogenes, Gram-negative bacteria (S. Typhimurium and E. coli) and fungal isolates (Aspergillus fumigatus and Aspergillus niger). Moreover, the extracts were tested for their antimicrobial activity using ADDM and plate dilution manner. The different extracts of CVB showed varying degree of inhibition against chosen bacteria. A positive result was detected when the zone of inhibition had a diameter >6 mm. Based on the obtained results, C100EOH extract had the highest antimicrobial activity against all bacteria. The ethanolic extracts of cinnamon was more effective than aqueous extract on both Gram-negative bacteria and Gram-positive bacteria (Mukhtar & Ghori, 2012). This was consistent with the results of this study based on solvent polarity, and it can be assumed that the lower the polarity of the extracts, the higher the antibacterial activity (Nawaz et al., 2020). Although 100% ethanol is a good organic solvent for the extraction of most compounds, it is not as polar as water.

The extract yields indicated that the extraction solvents affected the recovery of crude powder extract prepared from Cinnamon verum bark.

The extraction yield, antioxidant properties, radical scavenging activity and phytochemical content were affected by the polarity of the extraction solvent because the antioxidant compounds in the plant parts have high affinity for more polar solvents compared to non-polar ones (Nawaz et al., 2020).

They found that extraction in more polar solvents (water extract) resulted in a high yield but low flavonoid and phenolic content compared with that in non-polar solvents. These results suggested that absolute ethanol or a mixture of 50% (v/v) water with ethanol was the solvent of choice for yielding high levels of extractable solids compared with a water solvent alone.

Here in the ethanolic extract of cinnamon inhibited the mycelial growth of Aspergillus fumigatus and Aspergillus niger by 83.1-98.70 percent and 100 percent, respectively. Many studies have shown that cinnamon oil had inhibited numerous fungi including Aspergillus niger, Aspergillus flavus, Aspergillus ochraceus, Aspergillus versicolor, Aspergillus parasiticus, and Fusarium verticillioides (Soliman & Badeaa, 2002; Nasir & Ali, 2020).

Several species of Cinnamon (family Asteraceae) have antimicrobial activity. However, the ADDM findings exhibited that the essential oils (EO) have a wide variety range of antimicrobial activity against pathogenic bacteria based on the presence of different minor and major constituents in the EO of each plant due to the extraction method, plant species, plant part, microbial strains, geographical origin and environmental conditions, test conditions, test procedures and concentrations used … etc. (Oussalah et al., 2007; Ozturk & Ozturk, 2007; Celikel & Kavas, 2008). Studying the antimicrobial activities of 22 EO, discovered discrepancies between ADDM and dilution tests. found that the ADDM is not suitable for samples that are difficult to dissolve in water or for nonpolar samples. In addition, there is no correlation between diffusion power and antimicrobial activity, nor between MIC values and growth inhibition diameter. The ADDM, on the other hand, was ideal for screening because it has a small sample size and can test multiple compounds against a single microorganism. A dilution method was recommended to determine MIC and MBC with the greatest precision and suitability for non-polar samples such as EO (Rios et al., 1988; Khakhanang et al., 2016). Therefore, the microplate dilution assay was used in this study to evaluate the potency of antimicrobial activity. The C50EOH extract had a lower minimal inhibitory effect on S. Typhimurium compared to the C100EOH. This extract was found to be inhibitory as well as bactericidal.

These data indicated that Gram-negative bacteria were more sensitive to CVB extracts than Gram-positive bacteria. Nevertheless, comparisons of bacterial susceptibility may differ. This was not only due to the antimicrobial agents in the EO, but also due to the diversity of microorganisms tested (Oussalah et al., 2007; Kamazeri et al., 2012). Another factor that may have contributed to the different results was the type of media used to test for antimicrobial activity. Bacterial growth conditions may differ when different types of media are used. For example, used nutrient broth medium in their study, used Muller Hinton agar medium in their study, while nutrient agar medium was used here. These aforementioned factors may be responsible for the contradictory results in each case. However, the promising antimicrobial agents and properties of the EO against bacteria and fungi, especially the extracts of CVB reported herein could recommend their use as antibiotics replacements in the cure of infectious diseases in animals.

For the same test strain, C100EOH revealed the highest antibacterial activity and DIZ (11.3 to 14.3 mm), and the C100EOH lowest MIC and MBC range, except for MIC and MBC against S. Typhimurium and E. coli, where C50EOH was lowest. Thus, C100EOH was much more sensitive to foodborne bacteria than the other extracts. These antibacterial activities may be attributed to the presence of the main bioactive compounds, especially (E)-cinnamaldehyde. These results suggest that the alcoholic ethanol extracts are superior to the water extracts and may be effective natural antibacterial agents. Thus, the CVB could be developed as a natural substitute for conventional food preservatives to extend the shelf life of food by preventing bacterial contamination during food production. In vivo experiments still need to be conducted to determine which bioactive compounds of cinnamon extracts are most effective against microbes. In addition, extra research is required to determine the synergistic antibacterial activity of cinnamon in combination with antibiotics or other herb extracts. reported DIZ for ethanol extracts against L. monocytogenes, St. aureus, E. coli, and Salmonella anatum ranging from 7.11 to 10.11 mm, while n-butane extracts yielded DIZ of 18.98 to 37.45 mm.

In this study, the DIZ ranged from 6.7 to 13.0 mm for Gram-positive bacteria and 8.2 to 14.3 mm for Gram-negative bacteria. These results are in agreement with those of Elisha et al. (2017), who found that Gram-negative bacteria were more responsive to extracts than Gram-positive bacteria. In contrast, Gram-positive bacteria were more sensitive to the cinnamon extracts than Gram-negative bacteria (Thirumurugan et al., 2010; Raeisi et al., 2015). These results of reported that both ethanol and n‐butane extracts of cinnamon showed no significant differences between Gram-negative bacteria and Gram-positive bacteria. The difference in susceptibility between Gram-positive bacteria and Gram-negative bacteria may be the result of differences in cell wall structure (Branen & Davidson, 2004; Liang et al., 2019). found that antimicrobial agents have difficulty penetrating the outer layer of Gram-negative bacteria. When antimicrobials such as monolaurin, nisin or EDTA were combined with EO of cinnamon, their effect on the selected bacteria, particularly E. coli, improved.

MICs of CVB ranged from 6.5 to 31.3 mg/mL for the absolute ethanol extract, 3.3 to 62.5 mg/mL for the 50% ethanol extract, and 7.8 to 62.5 mg/mL for the water extract. The MBC of CVB ranged from 13.2 to 62.5 mg/mL for the absolute ethanol extract, 6.5 to 125.0 mg/mL for the 50% ethanol extract, and 15.06 to 125.0 mg/mL for the water extract. This is somehow similar to the reports of who observed MBC values ranging from 20 to 160 mg/mL for ethanol extracts. Moreover, MIC and MBC of CVB extracts ranged from 3.3 to 7.8 for Gram-negative bacteria and from 6.5 to 62.5 mg/mL for Gram-positive bacteria. In contrast, reported that MIC and MBC values ranged from 7.8 to 31.2 mg/mL for both types of bacteria. The C50EOH extract has significant bacteriostatic and bactericidal activity against S. Typhimurium.

The C100EOH extract showed bacteriostatic and bactericidal activity against the tested Gram-positive bacteria (L. monocytogenes and St. aureus), which is confirmed by the sensitivity test results. Based on the obtained results, C100EOH can be used as an effective natural antimicrobial agent for safe preservation of food and drugs against bacterial contamination by L. monocytogenes and St. aureus. However, reported that absolute methanol extract of Chinese star anise had bacteriostatic and bactericidal activity against Gram-positive bacteria and Gram-negative bacteria that was less than or equal to that of 50% methanol and aqueous extracts of star anise. According to (El-Hack et al., 2020), cinnamon oil may be an effective substitute for antibiotics in poultry by reducing fungi and coliform bacteria in the broiler chickens’ intestinal contents (Krauze et al., 2021).

The antimicrobial activity of cinnamon extracts has been associated with bioactive constituents of EOs, especially cinnamaldehyde, which is present in the dried bark (Firmino et al., 2018). Carvacrol, eugenol, thymol, cinnamaldehyde, cinnamic acid, and perillaldehyde, all present in varying amounts in cinnamon, may also be considered for their potential synergistic and antagonistic activities (Burt, 2004). The antibacterial activity and mechanism of action of cinnamon and its derivatives on bacteria can be attributed to filamentous temperature-sensitive protein Z, biofilm inhibition, anti-quorum sensing activity, release of cellular components through loss of cell membrane integrity, permeability, morphological changes, effect on ATP levels and ATPase, and inhibition of bacterial acetyl-CoA carboxylase (López et al., 2007; Doyle & Stephens, 2019). For example, the bacterial membrane integrity, permeability, and cell morphology are damaged when E. coli and St. aureus cells are exposed to MIC of cinnamaldehyde (0.31 mg/mL) (Shen et al., 2015). In addition, the higher the concentration of cinnamaldehyde, the more severe the damage to the bacterial membrane. It is believed that the antibacterial effect of (E)-cinnamaldehyde results from its ability to hinder the cell membrane of bacteria and its structures, leading to ion leakage (Unlu et al., 2010). (E)‐cinnamaldehyde showed stronger antibacterial activity compared to eugenol and linalool (Herman et al., 2016; Marchese et al., 2017). Moreover, reported that 80% methanol extract of Cinnamomum burmannii exhibited antibacterial activity with significantly higher MBC against E. coli and St. aureus compared to water extraction of EO. discovered that the antimicrobial activity of EO of Cinnamomum zeylanicum was more effective compared to 50% ethanol extract. reported that a high concentration of phenolics extracted from the stem bark of Cinnamomum verum with a methanol solvent showed higher inhibitory activity against some organisms compared to an ethyl acetate extract. Thus, Cinnamon and its derivatives have various modes of action that will contribute to the development of new antibacterial agents in the future.

In addition, further research is needed on studying more details of in vitro cinnamaldehyde alone or in combination with other antibacterial products found in cinnamon on other pathogenic bacteria infected broiler chickens. The vast majority of plant extracts have tested positive for antibacterial activity. This confirms previous research that plant pathogenic fungi are more resistant to plant extracts than plant pathogenic bacteria (Heisey and Gorham, 1992). They found that 13 extracts inhibited bacterial growth, but only 5 inhibited fungal growth.

When agar gel inoculated with selected fungi is impregnated with disks of CWA alone or in combination with ethanol, it shows resistance without any inhibitory effect, except the effect of CWA on Aspergillus niger. This may be attributed to the fact that C100EOH extract produces more secondary bioactive components that cannot be extracted by other extracts, and that have an inhibitory effect on fungi. The mean diameter of mycelial growth of the tested fungi in the poisoned food plates was significantly smaller than that of the control plates, indicating that the extracts have antifungal potential. The inhibition was dependent on the alcohol concentration. Among the tested fungi, extract efficacy revealed that ethanolic CVB extracts inhibited the mycelial growth of the isolated fungi more than CWA alone. However, the average diameter of mycelial growth of Aspergillus niger in poisoned food plates with CWA was similar to that of control plates. Thus, the results of the susceptibility test of CWA against Aspergillus niger contradicted the food poisoning method, indicating that CWA had good efficacy to Aspergillus niger, but poor mycelial growth inhibition. The obtained results showed that the C100EOH extract with a much lower concentration compared to C50EOH and CWA extract exerted inhibitory (MIC) and lethal (MFC) effects on Aspergillus fumigatus and Aspergillus niger. Compared to control plates, the antifungal effect of extracts in poisoned plates is demonstrated as a decrease in mycelial growth of selected fungi. Several researchers have used this method to evaluate the antifungal activity of herbs (Debonne et al., 2018; Hu et al., 2019). According to Gwa & Ekefan (2018), some plant extracts (neem, tobacco, ginger, papaya, black pepper, and mancozeb) have fungicidal activity against yam disease caused by Aspergillus niger. Cinnamaldehyde and its derivatives possess activity against a variety of pathogenic fungi such as Aspergillus fumigatus (Abd Rashed et al., 2021), Aspergillus niger (Sun et al., 2020), Aspergillus flavus (Bisceglie et al., 2020), and Candida spp such as Candida albicans and Candida glabrata (Bakhtiari et al., 2019). Interestingly, both geometric isomers of cis-cinnamaldehyde and trans-cinnamaldehyde have potential antifungal properties (Shreaz et al., 2016). They confirmed the potential of cinnamaldehyde derivatives as antifungal alternatives to current remedies. Cinnamon oil was highly effective against Aspergillus fumigatus at a dose of 1 mL/kg ration and can be used in poultry industry to replace other antifungal drugs (Sadiek et al., 2019).

The obtained food poisoning method results partially support the ADDM results suggesting that the C100EOH extract could be used as an effective natural fungicide in the food industry to prevent fungal contamination with Aspergillus fumigatus and Aspergillus niger. As well as the CWA extract against Aspergillus niger.

In the current study, the antifungal activity of cinnamon may be associated with the high content of cinnamaldehyde (97%) and other important bioactive constituents. Which interfere with several biological processes including electron transfer, and may react with nitrogen containing molecules (Gupta et al., 2008). However, the majority of aldehydes and phenols in cinnamon EO can cause severe lesions and secondary damage to the membrane, resulting in increased antifungal activity (Tu et al., 2018; Hu et al., 2019). Consequently, the differences in bioactive substances produced by different CVB solvents may contribute to different antifungal extracts diversity. Moreover, natural antifungal agents are readily biodegradable, highly volatile, low toxic, produce little residue, and are harmless to non-target organisms. The inhibitory effect of plant EOs on many fungi is attributed to the components with high lipophilicity and low molecular weight, which can easily disrupt cell membranes and cause cytoplasm leakage (Chao et al., 2005). Clove and cinnamon EOs as edible spices had the strongest antifungal activity and inhibited mycelial growth and spore germination of Aspergillus niger, Aspergillus ochraceus, and Aspergillus oryzae fungi isolated from moldy wheat bread (Hu et al., 2019). discussed the mechanisms of action of EOs and most of their constituents as antifungal agents, as well as their role in blocking cell communication channels, mycotoxin production, and fungal biofilm formation. The CWA had the least activity on the fungi tested. The presence of more lipid soluble components in CVB could be the cause behind superior antifungal activity of the ethanol extracts. The high inhibitory activity of EOs and cinnamaldehyde against the selected fungi could be due to the presence of cinnamaldehyde in the oil (Hu et al., 2019). Therefore, this can be used as a natural fungicide to control plant diseases and to preserve fruits and vegetables.

CONCLUSION

To summarize, four bacteria were isolated from pure colonies cultured and identified as St. aureus, L. monocytogenes, E. coli, and S. Typhimurium. The edible cinnamon bark spice extracts obtained with different solvents displayed varying degrees of inhibitory activity against these four bacteria. For the same test strain, the absolute ethanol extract of cinnamon exhibited the highest antibacterial activity, the largest diameter inhibition zone, the lowest MIC and MBC range, and the best inhibition of mycelial growth of fungi. Thus, the absolute ethanol extract of cinnamon bark was much more effective against foodborne bacteria than other extracts. CWA exhibited good effectiveness against Aspergillus niger but poor mycelial growth inhibition. The best MIC and MBC values were found with C50EOH against S. Typhimurium.

These antibacterial activities can be attributed to the presence of the principal bioactive constituents, particularly (E)‐cinnamaldehyde. The outcome of this study indicates that ethanol alcoholic extracts are superior to water extracts and may represent effective natural antibacterial agents. Thus, the bark of Cinnamomum verum could be developed as a natural replacement for traditional food preservatives to extend the shelf life of food products by preventing microbial contamination during food manufacturing. In vivo trials need to be performed to determine which bioactive compounds of cinnamon extracts are most effective against microbes. Furthermore, more research is needed to determine the synergistic antibacterial activity of cinnamon when combined with antibiotics or other plant extracts. Field studies recommend testing the bioactive extract against the selected bacteria or fungi. In addition, the results obtained in this study would help to develop new antimicrobial agents in the future.

ACKNOWLEDGMENTS

This work was supported by the Research Supporting Project (RSP-2023R439), King Saud University, Riyadh, Saudi Arabia.

REFERENCES

• Abd Rashed A, Rathi D-NG, Ahmad Nasir NAH, Abd Rahman AZ. Antifungal properties of essential oils and their compounds for application in skin fungal infections: conventional and nonconventional approaches. Molecules 2021;26(4):1093.
• Alhajj M, Qasem M, Nabi A, Al-Mufarrej S. In-vitro antibacterial and antifungal effects of high levels of chinese star anise. Brazilian Journal of Poultry Science 2019;21(1).
• AOAC. Official methods of analysis. Arlington: AOAC International; 2006.
• Bakhtiari S, Jafari S, Taheri JB, Kashi TSJ, Namazi Z, Iman M, et al. The effects of cinnamaldehyde (Cinnamon derivatives) and nystatin on candida albicans and Candida glabrata. Open access Macedonian Journal of Medical Sciences 2019;7(7):1067-70.
• Bekkar NEH, Meddah B, Cakmak YS, Keskin B. Phenolic composition, antioxidant and antimicrobial activities of Zizyphus lotus l. and Ruta chalepensis l. growing in mascara (western algeria). Journal of Microbiology, Biotechnology and Food Sciences 2021;10(5):e3004-e3004.
• Bisceglie F, Degola F, Rogolino D, Giannelli G, Orsoni N, Spadola G, et al. Sisters in structure but different in character, some benzaldehyde and cinnamaldehyde derivatives differentially tune Aspergillus flavus secondary metabolism. Scientific Reports 2020;10(1):1-14.
• Branen JK, Davidson PM. Enhancement of nisin, lysozyme, and monolaurin antimicrobial activities by ethylenediaminetetraacetic acid and lactoferrin. International Journal of Food Microbiology 2004;90(1):63-74.
• Burt S. Essential oils: their antibacterial properties and potential applications in foods-a review. International Journal of Food Microbiology 2004;94(3):223-53.
• Celikel N, Kavas G. Antimicrobial properties of some essential oils against some pathogenic microorganisms. Czech Journal of Food Sciences 2008;26(3):174.
• Chao LK, Hua K-F, Hsu H-Y, Cheng S-S, Liu J-Y, Chang S-T. Study on the antiinflammatory activity of essential oil from leaves of Cinnamomum osmophloeum. Journal of Agricultural and Food Chemistry 2005;53(18):7274-8.
• CLSI - Clinical and Laboratory Standards Institute. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically [standard M7-A7]. 7th ed. Wayne: CLSI; 2006.
• Debonne E, Bockstaele F van, De Leyn I, Devlieghere F, Eeckhout M. Validation of in-vitro antifungal activity of thyme essential oil on Aspergillus niger and Penicillium paneum through application in par-baked wheat and sourdough bread. LWT 2018;87:368-78.
• Doyle AA, Stephens JC. A review of cinnamaldehyde and its derivatives as antibacterial agents. Fitoterapia 2019;139:104405.
• El-Hack MEA, Alagawany M, Abdel-Moneim A-ME, Mohammed NG, Khafaga AF, Bin-Jumah M, et al. Cinnamon (Cinnamomum zeylanicum) oil as a potential alternative to antibiotics in poultry. Antibiotics 2020;9(5):210.
• Elisha IL, Botha FS, McGaw LJ, Eloff JN. The antibacterial activity of extracts of nine plant species with good activity against Escherichia coli against five other bacteria and cytotoxicity of extracts. BMC Complementary and Alternative Medicine 2017;17(1):1-10.
• Faghani M, Rahimian Y, Rafiee A, Namjoo AR. Effect of garlic and cinnamon in comparison to virginiamycin on performance and some haematological parameters in broiler chicks. Research Opinions in Animal & Veterinary Sciences 2014;4(9):504-7.
• Fauvelle C, Lambotin M, Heydmann L, Prakash E, Bhaskaran S, Vishwaraman M, et al. A cinnamon-derived procyanidin type A compound inhibits hepatitis C virus cell entry. Hepatology International 2017;11(5):440-5.
• Firmino DF, Cavalcante TT, Gomes GA, Firmino N, Rosa LD, Carvalho MG de, et al. Antibacterial and antibiofilm activities of Cinnamomum Sp. essential oil and cinnamaldehyde: antimicrobial activities. The Scientific World Journal 2018;2018:7405736.
• Friedman M. Chemistry, antimicrobial mechanisms, and antibiotic activities of cinnamaldehyde against pathogenic bacteria in animal feeds and human foods. Journal of Agricultural and Food Chemistry 2017;65(48):10406-23.
• Gupta C, Garg AP, Uniyal RC, Kumari A. Comparative analysis of the antimicrobial activity of cinnamon oil and cinnamon extract on some food-borne microbes. African Journal of Microbiology Research 2008;2(9):247-51.
• Gupta C, Kumari A, Garg AP. Comparative study of cinnamon oil and clove oil in some oral microbiota. Acta Bio Medica Atenei Parmensis 2012;82(3):197-9.
• Gwa VI, Ekefan EJ. Fungicidal effect of some plant extracts against tuber dry rot of white yam (dioscorea rotundata poir) caused by Aspergillus niger. International Journal of Agriculture and Nutrition 2018;3(3):1-7.
• Heisey R, Gorham BK. Antimicrobial effects of plant extracts on Streptococcus mutans, Candida albicans, Trichophyton rubrum and other micro-organisms. Letters in Applied Microbiology 1992;14(4):136-9.
• Herman A, Tambor K, Herman A. Linalool affects the antimicrobial efficacy of essential oils. Current Microbiology 2016;72(2):165-172.
• Holst DO. Holst filtration apparatus for Van Soest detergent fiber analyses. Journal of the Association of Official Analytical Chemists 1973;56(6):1352-6.
• Hong SH, Ismail IA, Kang SM, Han DC, Kwon BM. Cinnamaldehydes in cancer chemotherapy. Phytotherapy Research 2016;30(5):754-67.
• Hu F, Tu X-F, Thakur K, Hu F, Li X-L, Zhang Y-S, et al. Comparison of antifungal activity of essential oils from different plants against three fungi. Food and Chemical Toxicology 2019;134:110821.
• Humeera N, Kamili AN, Bandh SA, Lone BA, Gousia N. Antimicrobial and antioxidant activities of alcoholic extracts of Rumex dentatus L. Microbial Pathogenesis 2013;57:17-20.
• Indu M, Hatha A, Abirosh C, Harsha U, Vivekanandan G. Antimicrobial activity of some of the south-Indian spices against serotypes of Escherichia coli, Salmonella, Listeria monocytogenes and Aeromonas hydrophila. Brazilian Journal of Microbiology 2006;37(2):153-158.
• Jayalakshmi K, Paramasivam M, Sasikala M, Tamilam T, Sumithra A. Review on antibiotic residues in animal products and its impact on environments and human health. Journal of Entomology and Zoology Studies 2017;5(3):1446-51.
• Julianti E, Rajah KK, Fidrianny I. Antibacterial activity of ethanolic extract of cinnamon bark, honey, and their combination effects against acne-causing bacteria. Scientia Pharmaceutica 2017;85(2):19.
• Kamazeri TSAT, Abd Samah O, Taher M, Susanti D, Qaralleh H. Antimicrobial activity and essential oils of Curcuma aeruginosa, Curcuma mangga, and Zingiber cassumunar from Malaysia. Asian Pacific Journal of Tropical Medicine 2012;5(3):202-9.
• Kasimala MB, Tukue M, Ermias R. Phytochemical screening and antibacterial activity of two common terresterial medicinal plants Ruta chalepensis and Rumex nervosus. Bali Medical Journal 2014;3:166-21.
• Khakhanang R, Khannalao Y, Srirod S. Antimicrobial effect against animal pathogenic bacteria by essential oils. Journal of Mahanakorn Veterinary Medicine 2016;11(2):69-78.
• Krauze M, Cendrowska-Pinkosz M, Matusevicius P, Stepniowska A, Jurczak P, Ognik K. The effect of administration of a phytobiotic containing cinnamon oil and citric acid on the metabolism, immunity, and growth performance of broiler chickens. Animals 2021;11(2):399.
• Lass-Flörl C, Cuenca-Estrella M, Denning D, Rodriguez-Tudela J. Antifungal susceptibility testing in Aspergillus spp. according to EUCAST methodology. Medical Mycology 2006;44(Suppl1):S319-S325
• Liang Y, Li Y, Sun A, Liu X. Chemical compound identification and antibacterial activity evaluation of cinnamon extracts obtained by subcritical n-butane and ethanol extraction. Food Science & Nutrition 2019;7(6):2186-93.
• López P, Sanchez C, Batlle R, Nerín C. Vapor-phase activities of cinnamon, thyme, and oregano essential oils and key constituents against foodborne microorganisms. Journal of Agricultural and Food Chemistry 2007;55(11):4348-56.
• Mahmoodnia L, Aghadavod E, Rafieian-Kopaei M. Ameliorative impact of cinnamon against high blood pressure; an updated review. Journal of Renal Injury Prevention 2017;6(3).
• Marchese A, Barbieri R, Coppo E, Orhan IE, Daglia M, Nabavi SF, et al. Antimicrobial activity of eugenol and essential oils containing eugenol: A mechanistic viewpoint. Critical Reviews in Microbiology 2017;43(6):668-89.
• Mazimba O, Wale K, Tebogo E, Tebogo E, Kwape Shetonde O. Cinnamomum verum: Ethylacetate and methanol extracts antioxidant and antimicrobial activity. Journal of Medicinal Plants Studies 2015;3(3):28-32.
• Mukhtar S, Ghori I. Antibacterial activity of aqueous and ethanolic extracts of garlic, cinnamon and turmeric against Escherichia coli ATCC 25922 and Bacillus subtilis DSM 3256. International Journal of Applied Biology and Pharmaceutical Technology 2012;3(2):131-6.
• Muthuswamy S, Rupasinghe H, Stratton G. Antimicrobial effect of cinnamon bark extract on Escherichia coli O157: H7, Listeria innocua and fresh-cut apple slices. Journal of Food Safety 2008;28(4):534-49.
• Naeini A, Khosravi A, Chitsaz M, Shokri H, Kamlnejad M. Anti-candida albicans activity of some Iranian plants used in traditional medicine. Journal de Mycologie Médicale 2009;19(3):168-72.
• Nasir BF, Ali SM. Efficacy of cinnamon (cinnamomum zeylanicum) extract in reducing toxicity of aspergillus niger isolated from dried figs in the rat bio-system. Plant Archives 2020;20(2):182-88.
• Nawaz H, Shad MA, Rehman N, Andaleeb H, Ullah N. Effect of solvent polarity on extraction yield and antioxidant properties of phytochemicals from bean (Phaseolus vulgaris) seeds. Brazilian Journal of Pharmaceutical Sciences 2020;56:e17129.
• Nazzaro F, Fratianni F, Coppola R, Feo VD. Essential oils and antifungal activity. Pharmaceuticals 2017;10(4):86.
• NCCLS - National Committee for Clinical Laboratory Standards. Method for antifungal disk diffusion susceptibility testing of yeast: proposed guideline M44-P. Wayne: NCCLS; 2003.
• Nematollahi Z, Ebrahimi M, Raeisi M, Dadban Shahamat Y, Ghodsi Moghadam M, Hashemi M, et al. The antibacterial activity of cinnamon essential oil against foodborne bacteria: a mini-review. Journal of Human Environment and Health Promotion 2020;6(3):101-5.
• Oh K, Matsuoka H, Nemoto Y, Sumita O, Takatori K, Kurata H. Determination of anti-Aspergillus activity of antifungal agents based on the dynamic growth rate of a single hypha. Applied Microbiology and Biotechnology 1993;39(3):363-7.
• Oussalah M, Caillet S, Saucier L, Lacroix M. Inhibitory effects of selected plant essential oils on the growth of four pathogenic bacteria: E. coli O157: H7, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes. Food Control 2007;18(5):414-20.
• Ozturk S, Ozturk A. Antibacterial activity of aqueous and methanol extracts of rumex alpinus. and Rumex caucasicus. Pharmaceutical Biology 2007;45(2):83-7.
• Paswan R, Park YW. Survivability of Salmonella and Escherichia coli O157: H7 pathogens and food safety concerns on commercial powder milk products. Dairy 2020;1(3):189-201.
• Raeisi M, Tajik H, Yarahmadi A, Sanginabadi S. Antimicrobial effect of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Health Scope 2015;4(4).
• Rahemi D, Babaee N, Kazemi S, Sefidgar SAA, Moghadamnia AA. An in vitro study of the effect of cinnamaldehyde on the growth of Candida albicans compared to nystatin and fluconazole. Crescent Journal of Medical and Biological Science 2015;2:70-80.
• Rao G, Srivastava A. Toxicity of essential oils of higher plants against fungal pathogens of sugarcane. Current Trend in Sugarcane Pathology 1994;15:347-65.
• Rao PV, Gan SH. Cinnamon: a multifaceted medicinal plant. Evidence-Based Complementary and Alternative Medicine 2014;2014:642942.
• Rios J-L, Recio MC, Villar A. Screening methods for natural products with antimicrobial activity: a review of the literature. Journal of Ethnopharmacology 1988;23(2-3):127-49.
• Rothrock Jr MJ, Davis ML, Locatelli A, Bodie A, McIntosh TG, Donaldson JR, et al. Listeria occurrence in poultry flocks: detection and potential implications. Frontiers in Veterinary Science 2017;4:125.
• Sadiek Y, Abd EL Motelib T, Amen O. Role of essential oil for control of avian aspergillosis in experimentally infected chickens. Assiut Veterinary Medicine Journal 2019;65(163):152-160.
• Saladino F, Luz C, Manyes L, Fernández-Franzón M, Meca G. In vitro antifungal activity of lactic acid bacteria against mycotoxigenic fungi and their application in loaf bread shelf life improvement. Food Control 2016;67:273-7.
• Sam S. Importance and effectiveness of herbal medicines. Journal of Pharmacognosy and Phytochemistry 2019;8(2):354-7.
• Samanta I, Bandyopadhyay S. Antimicrobial resistance in agriculture: perspective, policy and mitigation. London: Academic Press; 2019.
• SAS. User's guide: mathematical programming examples. Cary: SAS Institute; 2012.
• Shan B, Cai Y-Z, Brooks JD, Corke H. Antibacterial properties and major bioactive components of cinnamon stick (Cinnamomum burmannii): activity against foodborne pathogenic bacteria. Journal of Agricultural and Food Chemistry 2007;55(14):5484-90.
• Sharma C, Rokana N, Chandra M, Singh BP, Gulhane RD, Gill JPS, et al. Antimicrobial resistance: its surveillance, impact, and alternative management strategies in dairy animals. Frontiers in Veterinary Science 2018;4:237.
• Sharma UK, Sharma AK, Pandey AK. Medicinal attributes of major phenylpropanoids present in cinnamon. Complementary and Alternative Medicine 2016;16(1):156.
• Shen S, Zhang T, Yuan Y, Lin S, Xu J, Ye H. Effects of cinnamaldehyde on Escherichia coli and Staphylococcus aureus membrane. Food Control 2015;47:196-202.
• Shreaz S, Wani WA, Behbehani JM, Raja V, Irshad M, Karched M, et al. Cinnamaldehyde and its derivatives, a novel class of antifungal agents. Fitoterapia 2016;112:116-31.
• Shrestha AK, Tiwari R. Antifungal activity of crude extracts of some medicinal plants against Fusarium solani (Mart.) Sacc. Ecoprint: an International Journal of Ecology 2009;16:75-8.
• Soliman KM, Badeaa R. Effect of oil extracted from some medicinal plants on different mycotoxigenic fungi. Food and Chemical Toxicology 2002;40(11):1669-75.
• Somorin YM, Odeyemi OA, Ateba CN. Salmonella is the most common foodborne pathogen in African food exports to the European Union: analysis of the rapid alert system for food and feed (1999-2019). Food Control 2021;123:107849.
• Sun Q, Li J, Sun Y, Chen Q, Zhang L, Le T. The antifungal effects of cinnamaldehyde against Aspergillus niger and its application in bread preservation. Food Chemistry 2020;317:126405.
• Thirumurugan K, Shihabudeen M, Hansi P. Antimicrobial activity and phytochemical analysis of selected Indian folk medicinal plants. Steroids 2010;1(7):430-4.
• Tu X-F, Hu F, Thakur K, Li X-L, Zhang Y-S, Wei Z-J. Comparison of antibacterial effects and fumigant toxicity of essential oils extracted from different plants. Industrial Crops and Products 2018;124:192-200.
• Unlu M, Ergene E, Unlu GV, Zeytinoglu HS, Vural N. Composition, antimicrobial activity and in vitro cytotoxicity of essential oil from Cinnamomum zeylanicum Blume (Lauraceae). Food and Chemical Toxicology 2010;48(11):3274-80.
• Vangalapati M, Satya S, Prakash S, Avanigadda S. A review on pharmacological activities and clinical effects of cinnamon species. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2012;3(1):653-63.
• Wales A, Davies R. A critical review of Salmonella Typhimurium infection in laying hens. Avian Pathology 2011;40(5):429-36.
• Yanakiev S. Effects of cinnamon (Cinnamomum spp.) in dentistry: a review. Molecules 2020;25(18):4184.
• Yeh T-F, Lin C-Y, Chang S-T. A potential low-coumarin cinnamon substitute: Cinnamomum osmophloeum leaves. Journal of Agricultural and Food Chemistry 2014;62(7):1706-12.
• Yousef R, Tawil G. Antimicrobial activity of volatile oils. Die Pharmazie 1980;35(11):698-701.
• Zhang Y, Liu X, Wang Y, Jiang P, Quek S. Antibacterial activity and mechanism of cinnamon essential oil against Escherichia coli and Staphylococcus aureus. Food Control 2016;59:282-9.

Publication Dates

• Publication in this collection
  20 Feb 2023
• Date of issue
  2023

History

• Received
  01 June 2022
• Accepted
  28 Nov 2022